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M Harboe

Publications and source records attributed to M Harboe.

At least 37 records · Page 2Linked to original sources
PubMed1999

Demonstration of expression of six proteins of the mammalian cell entry (mce1) operon of Mycobacterium tuberculosis by anti-peptide antibodies, enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction.

Polyclonal rabbit antibodies were generated to synthetic peptides corresponding to predicted B-cell epitopes of six proteins of the mce1 operon of Mycobacterium tuberculosis. Antipeptide antibodies reacted with Mce1A and Mce1E fusion proteins in sonicates of recombinant Escherichia coli as well as with distinct bands in sonicates, but not in culture fluids of M. tuberculosis and M. bovis bacillus Calmette-Guérin (BCG). Polyvalent rabbit antibodies generated by immunization with sonicates of BCG bacilli reacted with synthetic peptides from the six Mce1 proteins on the solid phase in enzyme-linked immunosorbent assay (ELISA), albeit with different frequencies. The Mce1A peptide (p124-140) reacted most frequently, with seven of the nine antibodies tested, while the Mce1F peptide (p329-343) reacted with two. Used as a control, 20 polyclonal rabbit antibodies to 12 isolated proteins of M. tuberculosis and M. bovis BCG did not react with any of the six synthetic peptides, except in one case. mRNA expression of the six mce1A-mce1F genes of M. tuberculosis was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR). These data indicate that all Mce1A-Mce1F proteins of the mce1 operon are expressed by in vitro-grown M. tuberculosis and M. bovis BCG.

Amino Acid Sequence↗
PubMed1998

Secreted proteins of Mycobacterium leprae.

In mycobacteria, secreted proteins represent a distinct group, probably of particular importance for development of immune responses following infection. Quantification of individual proteins in culture fluid and corresponding disrupted bacilli permits determination of a localization index for identification of secreted proteins. This procedure cannot be applied to Mycobacterium leprae because secreted proteins are lost during isolation of bacilli from tissues. The DNA sequences of secreted proteins of Mycobacterium tuberculosis were compared with sequences of M. leprae. Genes for homologues of the 85a, 85b, 85c, mpt32 (apa), mpt51, erp, mtc28, Rv2376c, Rv3354 and Rv0526 genes were identified. All of these contain signal sequences typical for secretion in M. leprae. In several instances the local distance between marker genes and occurrence on the same or the complementary DNA strand was similar in these two species. The genomic organisation of genes for secreted proteins is thus very similar in M. leprae and M. tuberculosis, the homology being higher for the mature polypeptide chains than for the corresponding signal peptides.

Acyltransferases↗
PubMed1998

In situ complement activation in porcine membranoproliferative glomerulonephritis type II.

Pigs genetically deficient in complement factor H all develop lethal membranoproliferative glomerulonephritis (MPGN) type II characterized by massive glomerular deposits of complement, intramembranous dense deposits, and mesangial hypercellularity. To elucidate the chronological relationship between these glomerular changes, and to precisely determine the localization of glomerular complement deposits, we studied kidney specimens from factor H-deficient piglets at different ages from fetal life until terminal kidney failure had developed. Deposits of C3 and the terminal complement complex localized within the glomerular basement membrane (GBM) were present already in factor H-deficient fetuses, without concurrent intramembranous dense deposits or mesangial hypercellularity. Incipient subendothelial dense deposits containing complement appeared no earlier than four days after birth, and intramembranous dense deposits in older piglets with established MPGN type II also contained large amounts of complement as detected by immune electron microscopy. Onset of kidney failure coincided with pronounced mesangial hypercellularity and expansion, compromising glomerular capillary patency. Formation of glomerular capillary wall double contours coincided with electron microscopic evidence of laminar disintegration of intramembranous dense deposits. Complement was also deposited in the mesangial matrix, but not on glomerular cells. We conclude that all components of the alternative and terminal pathways of complement have access into the GBM and the mesangial matrix. In the absence of factor H, complement is spontaneously activated and deposited in situ in these locations resulting in dense deposit formation. It is proposed that factor H dysfunction may play an essential role even in human MPGN type II.

Animals↗
PubMed1998

Longitudinal trends in serum levels of mycobacterial secretory (30 kD) and cytoplasmic (65 kD) antigens during chemotherapy of pulmonary tuberculosis patients.

Antigen 85 (mol. wt 30,000) (30 kD), secreted by actively growing mycobacteria under axenic conditions, and mol. wt 65,000 (65 kD), a cytoplasmic antigen released during mycobacterial lysis, were used to monitor the efficacy of chemotherapy in previously untreated pulmonary tuberculosis (UPTB) patients using enzyme-linked immunosorbent assay. Sera from 125 UPTB patients were examined for each of the 2 antigens individually and for the ratio of secretory (30 kD) to cytoplasmic (65 kD) antigen (SCR), before commencement of treatment, after intensive phase (IP), completion of optimum period of treatment (COPT) and 6 months post-COPT. 116 controls (normals and contacts) were also checked for these antigens. The detection of 30 kD and 65 kD antigens in UPTB patients had a sensitivity ranging from 50-57% (mean 30 kD value: 0.64 +/- 1.24 ngs/ml) to 20-22% (mean 65 kD value: 0.51 +/- 1.87 ngs/ml), respectively, whereas in controls it ranged from 2-8% (0.05 +/- 0.28 ngs/ml) to 14-47% (0.09 +/- 0.22 ngs/ml), respectively. Although the decline in 30 kD positivity was more evident at COPT, computation of the SCR denoted efficacy of chemotherapy more readily at IP. Similarly, SCR resolved the ambiguity between individual antigen levels and the clinical status of a patient. Since significant numbers of patients demonstrated 30 kD at IP it may be computed that the lifespan of circulating 30 kD in serum could be at least 2 months after the start of treatment, declining gradually thereafter. Although seromonitoring for secretory antigen generally reflects the efficacy of chemotherapy, the interpretation of findings clearly requires further elucidation.

Antibodies, Bacterial↗
PubMed1998

Involvement of antilipoarabinomannan antibodies in classical complement activation in tuberculosis.

We examined alternative and classical complement activation induced by whole bacilli of Mycobacterium bovis BCG and Mycobacterium tuberculosis products. After exposure to BCG, there were higher levels of the terminal complement complex in sera from Indian tuberculosis patients than in sera from healthy controls. The addition of BCG with or without EGTA to these sera indicated that approximately 70 to 85% of the total levels of the terminal complement complex was formed by classical activation. Sera from Indian tuberculosis patients contained more antibody to lipoarabinomannan (LAM) than sera from healthy Indians. Levels of anti-LAM immunoglobulin G2 (IgG2), but not anti-LAM IgM, correlated positively with classical activation induced by BCG in the sera. By flow cytometry, deposition of C3 and terminal complement complex on bacilli incubated with normal human serum was demonstrated. The anticomplement staining was significantly reduced in the presence of EGTA and EDTA. Flow cytometry also revealed the binding of complement to BCG incubated with rabbit anti-LAM and then with factor B-depleted serum. This indicates that classical activation plays a major role in complement activation induced by mycobacteria and that anti-LAM IgG on the bacilli can mediate this response. Classical complement activation may be important for the extent of phagocytosis of M. tuberculosis by mononuclear phagocytes, which may influence the course after infection.

Adolescent↗
PubMed1998

MPB70 and MPB83 as indicators of protein localization in mycobacterial cells.

Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates of Mycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.

Antibodies, Monoclonal↗
PubMed1998

B-cell epitopes and quantification of the ESAT-6 protein of Mycobacterium tuberculosis.

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.

Amino Acid Sequence↗
PubMed1998

Immunochemical characterization of the MPB70/80 and MPB83 proteins of Mycobacterium bovis.

MPB70 and MPB80 (MPB70/80) and MPB83 are closely related antigens which are highly expressed in Mycobacterium bovis. MPB70/80 are soluble secreted antigens, while MPB83 is an exported lipoprotein associated with the bacterial surface. In the present study, these antigens had different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. These differences may be explained by the fact that MPB70 and MPB83 both have two internal cysteine residues which would create ring structures by disulfide bonding. We analyzed the structures of MPB70/80 and MPB83 by using monoclonal antibodies (MAbs) raised against bovine purified protein derivative or whole M. bovis cells. MAb 1-5C reacted specifically with MPB70 and MPB80, and MAb MBS43 reacted specifically with MPB83, while the other antibodies, including several previously described MAbs, bound all three antigens. MAbs and polyclonal antibodies reacted strongly with reduced protein and less well with nonreduced protein, indicating involvement of linear epitopes. Epitopes of MAbs Bov-1, 2-6B, 1-5C, and 1-1D were mapped by using synthetic peptides of MPB70. Sequence comparison showed the peptide with the 1-5C-reactive epitope to have three residues different from those in the homologous region of MPB83. Exchanges of A for S in position 112 or Q for E in position 116 abolished the reactivity of MAb 1-5C. Polyclonal rabbit antibodies to native purified MPB70 reacted strongly with peptides 6, 7, and 8 of the N-terminal half of mature MPB70. Cattle sera of experimentally M. bovis-infected animals recognized a broader spectrum of peptides. These findings indicate that there is diagnostic potential for these proteins and that there is also a possible role for antibodies in elucidation of the host-mycobacterium relationship involving a surface-bound and exposed lipoprotein, MPB83, and its highly homologous soluble secreted MPB70/80 counterparts.

Amino Acid Sequence↗
PubMed1998

Protective immunity to intracellular parasites--a focus for vaccine research and a challenge to immunization programmes.

Protective immunity to intracellular parasites is mediated by T cells. This response is complex, requiring activation of several subgroups of T cells and production of a balanced blend of cytokines to obtain protection rather than delayed hypersensitivity leading to tissue damage. To obtain additional, detailed information, on the kind, proper localization and specificity of the required immune response represents a major challenge in current vaccine research. Insufficient knowledge of these features is the major constraint for development as illustrated in tuberculosis and related mycobacterial infections.

Animals↗
PubMed1997

Eradication of porcine factor H deficiency in Norway.

In pigs a hereditary deficiency of the complement-inhibitory protein factor H consistently leads to the development of lethal membranoproliferative glomerulonephritis type II. This autosomal recessive disease has been a common cause of early losses of piglets in the Norwegian Yorkshire breed, but has not been reported in the Norwegian Landrace breed. The aim of the present work was to identify carriers of factor H deficiency and to eradicate the disease from commercial pig populations. Factor H in plasma was measured by an enzyme immunoassay. Sixteen known carriers of the disease (parents of factor H-deficient offspring) had half the level of factor H (median 110, range 87 to 156 mg/litre) recorded in 17 homozygous healthy Yorkshire pigs (median 212, range 183 to 293 mg/litre) and 20 Landrace pigs (median 227, range 200 to 255 mg/litre). Factor H analysis in 397 piglets produced by the mating of known carriers revealed an approximately 1:2:1 distribution of individuals with very low, half-normal and normal levels of factor H representing homozygous deficient, heterozygous and homozygous healthy individuals. Thus, carriers could be identified reliably by measuring the plasma concentration of factor H. Most of the population of Norwegian Yorkshire breeding pigs (490 pigs) was therefore examined, and a half-normal factor H level consistent with the carrier state was found in 13.5 per cent. These animals were prevented from breeding and since then no losses of piglets suspected of being due to factor H deficiency have been reported. No carrier was identified among 102 Norwegian Landrace boars, almost excluding the existence of factor H deficiency in this breed.

Aging↗
PubMed1997

Attenuation of changes in leukocyte surface markers and complement activation with heparin-coated cardiopulmonary bypass.

BACKGROUND: The inflammatory response induced by cardiopulmonary bypass can result in severe organ dysfunction in some patients. This postperfusion response is caused mainly by contact between blood and the foreign surface of the cardiopulmonary bypass equipment and includes adhesion of leukocytes to vascular endothelium, which precedes a series of events that mediate inflammatory damage to tissues. METHODS: Low-risk patients accepted for coronary artery bypass grafting were randomized to operation with the cardiopulmonary bypass surface either completely heparin coated (Duraflo II) or uncoated. There were 12 patients in each group. Blood plasma sampled during cardiopulmonary bypass was analyzed for complement activation (C3bc and terminal SC5b-9 complement complex) and neutrophil activation (lactoferrin and myeloperoxidase). In addition, neutrophils, monocytes, and platelets were counted, and the expression of surface markers on the neutrophils and monocytes (complement receptor [CR] 1, CR3, CR4, and L-selectin) and on the platelets (P-selectin and CD41) was quantified with flow cytometry. RESULTS: Clinical and surgical results were similar in both groups. In the group with the heparin-coated surface, the formation of the terminal SC5b-9 complement complex was significantly reduced, and the counts of circulating leukocytes and platelets were significantly less reduced initially but were higher at the end of cardiopulmonary bypass compared with baseline. Also, the expression of CR1, CR3, and CR4 was significantly less upregulated and the L-selectin, significantly less downregulated on monocytes and neutrophils. CONCLUSIONS: We conclude that heparin coating reduces complement activation and attenuates the leukocyte integrin and selectin response that occurs when uncoated circuits are used.

Antigens, Surface↗
PubMed1997

Effect of whole and fractionated intravenous immunoglobulin on complement in vitro.

Intravenous immunoglobulins (IVIG) are increasingly used for treatment of inflammatory diseases, and the modulation of complement may contribute to some of its beneficial effects. IVIG may bind C1q and activated C3 and C4, and enhance inactivation of C3b. We have previously shown that IVIG inhibited complement-mediated lysis solely via its Fc part through interaction with the classical pathway. In the present study we have investigated whole IVIG (Octagam, and Sandoglobulin) and the monomer, dimer and multimer fractions of Octagam with respect to complement activation in serum and inhibition of complement lysis of red cells. The isolated fractions were found to be stable, homogeneous (monomer, dimer or multimer) and pure (virtually only IgG). Both whole IVIG and its fractions significantly activated complement in serum and inhibited hemolysis compared with human albumin. These effects were most pronounced in the monomer, less in the multimer and least in the dimer fraction. The complement activation was shown to be mediated through the classical pathway since formation of C1rs-C1inh complexes and C4bc were increased, in contrast to Bb. Surprisingly, heat aggregation of Octagam was not followed by a corresponding increase in complement activation, as would be expected, unless it was dialysed before heating, suggesting that it is stabilized to avoid excess activation. In conclusion, the results support the hypothesis that IVIG causes a mild activation of complement in vitro. We suggest that this effect may contribute to the complement inhibitory properties of IVIG by diverting complement deposition from the target to the fluid phase.

Animals↗
PubMed1996

Clinical immunology.

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Allergy and Immunology↗
PubMed1996

Heterogenous expression of the related MPB70 and MPB83 proteins distinguish various substrains of Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv.

MPB70 and MPB83 are homologous cross-reactive secreted mycobacterial proteins with very limited species distribution. The expression of these two proteins was compared between several substrains of Mycobacterium bovis BCG, virulent M. bovis and Mycobacterium tuberculosis H37Rv. A polyclonal antibody specific for MPB70 in Western blotting, and a monoclonal antibody, MBS43, found to be specific for MPB83 in ELISA and Western blotting, were used for the comparison. The previously established pattern of high- and low-producing substrains of BCG for MPB70 is only partially applicable for MPB83. MPB70 low-producing strains are also MPB83 low-producing, but the expression of MPB83 is much more variable than the expression of MPB70 in the MPB70 high-producing strains. Purified MPB83 (23 kDa) was found to be glycosylated. A band in SDS-PAGE at 1-2 kDa lower than that of purified MPB83 may represent unglycosylated MPB83. Furthermore, it was confirmed that purified MPB70 (22 kDa) is unglycosylated. There is cross-reactive antigen at 26 kDa. The MPB83 related antigen at 26 kDa was found to be the most abundant. These findings indicate greater heterogeneity between different substrains of BCG than previously realized. Virulent M. bovis produce and secrete large amounts of MPB70 and MPB83 while both these proteins occur in a far lower concentration in M. tuberculosis.

Antibody Specificity↗
PubMed1996

Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG.

ESAT-6 is a secreted protein present in the short-term culture filtrate of Mycobacterium tuberculosis after growth on a synthetic Sauton medium. ESAT-6 has recently been demonstrated to induce strong T-cell responses in a mouse model of memory immunity after infection with M. tuberculosis. In Western blotting (immunoblotting), the monoclonal antibody HYB76-8, reacting with ESAT-6, gave a 6-kDa region was observed in filtrates from four of eight substrains of M. bovis BCG that produced high levels of MPB64, while no band occurred in the 6-kDa region with any of these BCG substrains. Southern blotting and PCR experiments with genomic mycobacterial DNA showed the presence of the esat-6 gene in reference strains and clinical isolates of M. tuberculosis as well as in virulent M. bovis. The esat-6 gene could not be demonstrated in any of the eight substrains of M. bovis BCG tested by these techniques. Two gene deletions that distinguish M. bovis BCG from virulent M. bovis have thus now been demonstrated. Deletion of mpb64 affects four of the eight substrains tested; deletion of esat-6 affects all of them. The reaction of HYB76-8 AT 26 kDa with four of the BCG substrains was demonstrated to result from cross-reactivity with MPB64. HYB76-8 was also shown to cross-react with the A, B, and C components of the antigen 85 complex and MPT51.

Amino Acid Sequence↗