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Biomedical subjects

N H Heegaard

Publications and source records attributed to N H Heegaard.

At least 37 records · Page 2Linked to original sources
PubMed1997

Antibodies to cardiolipin may increase the risk of failure of peripheral vein bypasses.

OBJECTIVES: To assess the association between antibodies to cardiolipin and infrainguinal vein graft patency. MATERIALS AND METHODS: Plasma levels of antibodies to cardiolipin, haemostatic factors, lipids and the smoking marker carboxyhaemoglobin were determined preoperatively and 6 weeks postoperatively in 80 patients undergoing infrainguinal vein bypass surgery. Bypass patency was assessed by ankle blood pressure measurements and ultrasound duplex scanning at 1 week, 6 weeks, 3, 6, 9 and 12 months. A localised increase in the graft peak systolic velocity by a factor of 2.5 or more was considered to indicate a significant stenosis. RESULTS: Antibodies to cardiolipin were identified in seven (9%) patients preoperatively. In four of these seven patients the bypasses thrombosed within 3 months after surgery and another two developed stenoses. At 6 months the primary bypass patency, i.e. patency without stenosis, was 14% (95% confidence interval (CI) 0-33%) in patients with antibodies to cardiolipin, as opposed to 57% (95% CI 45-69%) in patients without these antibodies (log rank test: p = 0.03). Diabetes mellitus was also associated with a reduced 6 months primary bypass patency (38% (95% CI 16-60%) vs. 58% (95% CI 45-71%), p = 0.006). A Cox regression analysis showed that both the presence of antibodies to cardiolipin and diabetes independently contributed towards predicting the overall risk of bypass failure. CONCLUSION: Antibodies to cardiolipin were identified in 9% of patients undergoing infrainguinal vein bypass surgery and appeared to be associated with increased risk of bypass failure.

Aged↗
PubMed1997

Specificities of anti-neutrophil autoantibodies in patients with rheumatoid arthritis (RA).

The objective of this study was to characterize antigens recognized by neutrophil-specific autoantibodies from patients with RA. Sera from 62 RA patients were screened by indirect immunofluorescence (IIF). Positive sera were further tested by ELISAs for antibodies against various granule proteins and by immunoblotting of electrophoretically separated cell, granule or nuclear extracts. Forty-two sera (68%) reacted with ethanol-fixed neutrophils. In the ELISAs 32% of the 28 medium to strongly IIF-positive sera were negative, while 43% were weakly positive for more than one antigen. Immunoblots of whole neutrophils showed IgG reactions at 25-35 kD, in the 55-kD region, at 80 kD, and at 110 kD. Most sera reacted with more than one band. Except for the 55-kD antigen, none of the antigens appeared in lymphocytes. The most notable reactivity in subcellular fractions was with lactoferrin and with bands of 25-35 kD from nuclei. In conclusion, anti-neutrophil autoantibodies from RA patients recognize different antigens in the cytoplasm and in the nucleus. Lactoferrin is one of the common antigens recognized, but also unknown nuclear antigens of 25-35 kD mol. wt are involved.

Adult↗
PubMed1996

Immuno-capillary electrophoresis for the characterization of a monoclonal antibody against DNA.

A monoclonal antibody against DNA established from a mouse strain that spontaneously develops systemic lupus erythematosus was characterized by migration shift immuno-capillary electrophoresis. The minimal size for DNA binding antibody was > 16 bases and the interaction with a double-stranded 32-mer oligonucleotide was almost one order of magnitude stronger than the interaction with a single-stranded oligonucleotide. The binding was highly dependent on the ionic strength conditions with an increase in binding with a decrease in ionic strength. The estimate of the dissociation constant for the antibody binding of a single stranded 32-mer oligonucleotide was 0.62 microM at pH 7.90. This value was in good agreement with the value of 0.44 microM measured by an independent method using biosensor (surface plasmon resonance) technology.

Animals↗
PubMed1996

Ligand-binding sites in human serum amyloid P component.

Amyloid P component (AP) is a naturally occurring glycoprotein that is found in serum and basement membranes. AP is also a component of all types of amyloid, including that found in individuals who suffer from Alzheimer's disease and Down's syndrome. Because AP has been found to bind strongly and specifically to certain glycosaminoglycans that are components of amyloid deposits, AP may play an important role in the maintenance of amyloid. In the present work, we isolated and identified two proteolytic fragments of AP that are responsible for its heparin-binding activity. Neither fragment corresponds to published heparin-binding sequences. The structural requirements for activity of the peptides (amino acid residues 27-38 and 192-203 of AP) were examined by means of solid-phase inhibition assays with synthetic peptides. AP-(192-203)-peptide inhibits the Ca(2+)-dependent binding of AP to heparin with an IC50 of 25 microM, while the IC50 of AP-(27-38)-peptide and AP-(33-38)-peptide are 10 microM and 2 microM, respectively. The understanding of the structure and function of active AP peptides will be useful for development of amyloid-targeted diagnostics and therapeutics.

Amino Acid Sequence↗
PubMed1996

Comparison of heparin-binding to lactoferrin from human milk and from human granulocytes by means of affinity capillary electrophoresis.

Human lactoferrin from two different sources: milk and secondary granules of neutrophil granulocytes, was compared with respect to heparin binding by means of affinity capillary electrophoresis. This method is based on analysis of migration pattern shifts induced by various amounts of ligand present in the electrophoresis buffer. Since lactoferrin is a basic molecule, analyses were performed at pH 8 in a capillary with a neutral hydrophilic coating. However, analyzable peaks were only obtained in the presence of heparin in the electrophoresis buffer. Proportionally to the amount of heparin present, these peaks exhibited shape changes and strong migration shifts. In the ensuing analysis it could be demonstrated that the two lactoferrins had comparable migration shifts and peak shape changes. This indicates that also in this respect the two forms of lactoferrin are identical. Affinity capillary electrophoresis is a convenient and fast method to investigate functional characteristics of low amounts of unmodified biomolecules.

Electrophoresis, Capillary↗
PubMed1996

Multiple autoimmune manifestations in monoclonal gammopathy of undetermined significance and chronic lymphocytic leukemia.

In 18 cases of monoclonal gammopathy of undetermined significance, MGUS (monoclonal gammopathy of undetermined significance), admitted for diagnosed or suspected peripheral neuropathy, 11 patients showed other co-existing autoimmune manifestations. Two had POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, M-component, and skin symptoms), the others mainly endocrinopathy and polyclonal pseudolymphoma. There were 13 cases of sensorimotor neuropathy, two cases of neuritis, while neuropathy could not be confirmed in three cases. Compared with a retrospective review of autoimmunity in a randomly selected CLL (chronic lymphocytic leukemia) cohort of 115 patients, 13 out of 42 patients with clinical and/or laboratory features of autoimmunity showed co-expression of autoimmune signs, the dominating traits being Coombs positive AIHA (auto-immune hemolytic anemia), platelet autoantibodies, endocrinopathy mainly associated with the thyroid gland, serological and/or rheumatological symptoms, but only one case of sensorimotor neuropathy. Viewed from a current model of acquired autoimmunity it is perhaps not surprising that such autoimmunity is seen predominantly in patients with monoclonal gammopathy. Thus, a high concentration of cross-reacting polyreactive autoantibodies related to the M-component might be present in these patients. Furthermore, quantitative defects of the immunoglobulins including the hypogammaglobulinemia associated with M-components can presumably give rise to a defect of the anti-idiotypic network's regulation of natural autoantibodies and autoimmune manifestations in vivo. Such autoimmune manifestations, which are easily overlooked in CLL may call for additional treatment with immunosuppression and/or intravenous, polyclonal IgG.

Adult↗
PubMed1995

Demonstration of a heparin-binding site in serum amyloid P component using affinity capillary electrophoresis as an adjunct technique.

Linear heparin-binding sites in the DNA- and heparin-binding serum protein amyloid P component were investigated using affinity capillary electrophoresis and reversed-phase HPLC in conjunction with affinity chromatography. Peptide fragments were generated from amyloid P component by treatment with Glu-C and Asp-N endoproteinases. This peptide mixture was separated by HPLC before and after passage through a column of immobilized heparin. In addition, the proteolytic digest was separated by capillary electrophoresis in the presence of various amounts of heparin in solution. Migration shift patterns in the presence of heparin were in agreement with one of the components shown by HPLC to interact with immobilized heparin. The identity of this fragment was established by mass spectrometry after preparative HPLC and represents a novel heparin-binding sequence. The results illustrate the potential synergy in the combination of the two high-resolution separation techniques HPLC and CE. HPLC has the advantages of high recovery and preparative power while capillary electrophoresis is noted for highly efficient separations under physiological conditions. The possibility of using unmodified ligands in the study of biological activities of protein substructures while consuming very little material makes CE further attractive.

Amino Acid Sequence↗
PubMed1995

Electrophoretic and chromatographic differentiation of two forms of albumin in equilibrium at neutral pH: new screening techniques for determination of ligand binding to albumin.

Analysis of normal human serum by crossed hydrophobic interaction immunoelectrophoresis with Phenyl-Sepharose revealed a biphasic appearance of the albumin peak. The molecular mechanism behind this apparent albumin heterogeneity was investigated. Analysis of defatted purified albumin showed that a major fraction bound to the Phenyl-Sepharose and that addition of ligands (e.g. long-chain fatty acids, bilirubin, sulfonamides and warfarin) before electrophoresis blocked this binding to different degrees. A quantitative relation between ligand binding and the amount of nonbinding albumin was found. Thus the technique might be suitable for screening of ligand binding to albumin. Analysis of serum samples from newborns with hyperbilirubinemia revealed a positive correlation between the fraction of the nonretarded albumin and the bilirubin concentration. By chromatography on Phenyl-Sepharose, defatted albumin was separated into a binding and a nonbinding form and this technique was subsequently used to determine the kinetics of the intramolecular conversion. After rechromatography, each of the fractions could again be separated into two fractions, indicating the presence of an equilibrium. By varying the passage time for albumin on the column or varying the period between the first and the second separation it was possible to calculate the conversion rates. The half-life for the conversion was found to be as long as 1 1/4 h. It is the first time that a conformational change for albumin with such a long conversion time has been described experimentally.

Bilirubin↗
PubMed1994

Determination of antigen-antibody affinity by immunocapillary electrophoresis.

The use of affinity capillary electrophoresis for the characterization of antigen-antibody interactions (immunocapillary electrophoresis) is shown using monoclonal antibodies against phosphotyrosine as a model system. The influence of the interaction kinetics on the peak profiles was demonstrated in experiments with addition of phosphotyrosine to the electrophoresis buffer. One of the two antibodies that were tested exhibited peak broadening while the other showed no change in peak shape but had a decreased mobility proportional to the amount of phosphotyrosine present. The migration shifts which were of the order 0.05 to 0.15 min at 439 V/cm were a consequence of the antibody-antigen complexes having a slower mobility than the non-complexed antibody. On the basis of measurement of migration shifts at different antigen concentrations, dissociation constants were estimated and shown to be independent on the applied field strength. Thus, when certain requirements are fulfilled, immuno-capillary electrophoresis is a fast and simple method for establishing binding characteristics of unlabelled antigen and antibody molecules under non-denaturing conditions and consumes minute amounts of sample.

Antibodies, Monoclonal↗
PubMed1993

A capillary electrophoresis-based assay for the binding of Ca2+ and phosphorylcholine to human C-reactive protein.

Affinity capillary electrophoresis was performed to quantitate the binding of Ca2+ and phosphorylcholine to human C-reactive protein (CRP). The assay requires no modifications of any of the molecules involved, uses minuscule amounts of protein (8.5 x 10(-15) mol per analysis, i.e., less than 1 pmol for 15 triplicate data points), and the binding could be examined under conditions of physiological ionic strength and pH. The values for the dissociation constants obtained here (KD = 59 microM for Ca(2+)-CRP and 18 microM for the phosphorylcholine-CRP interaction) were in close agreement with previous studies using gel filtration and equilibrium dialysis. As long as one of the reactants can be detected and recovered quantitatively in the capillary electrophoresis system, the method is generally useful to study interactions where complexed molecules display an electrophoretic mobility that is different from that of unbound molecules and where the rates of association and dissociation are sufficiently fast.

Blood Protein Electrophoresis↗
PubMed1993

Interactions of IgG with specific erythrocyte membrane proteins in affinity electrophoresis are highly dependent on low ionic strength conditions.

Agarose gel affinity electrophoresis has been used to demonstrate interactions between autologous IgG and specific erythrocyte membrane proteins. These binding phenomena are here further examined by combining affinity electrophoresis with affinity chromatography, absorption experiments, and immunoblotting. It is demonstrated that the interactions are highly dependent on polyreactive IgG binding favored by the low ionic strength conditions of the electrophoretic assay. Thus, about 25% of normal IgG under low ionic strength conditions bound to the purified cytoskeletal protein, spectrin, immobilized on Sepharose. This IgG reacted in affinity electrophoresis in a polyspecific fashion with the same array of membrane proteins as before the low ionic strength-affinity chromatography. Further, the binding seen in affinity electrophoresis, including the interaction with spectrin, was completely abolished by preabsorption of the IgG with spectrin-devoid membranes. The charge characteristics of an IgG subclass might be responsible for the observed binding. However, the observed precipitate formation suggested an interaction involving at least two binding sites on each molecule and the binding appears to require structurally intact IgG because reductive treatment with dithiothreitol diminished the reactivity considerably. Conclusively, under the conditions of affinity electrophoresis with ligand present in the gel, electrostatic interactions are amplified. The degree of binding of IgG to erythrocyte membrane proteins that take place under these conditions does not reflect binding which would occur to the same extent under physiological ionic strength conditions.

Blood Platelets↗
PubMed1992

Use of capillary zone electrophoresis to evaluate the binding of anionic carbohydrates to synthetic peptides derived from human serum amyloid P component.

Capillary zone electrophoresis was used to study interactions between anionic carbohydrates and synthetic peptides derived from the heparin-binding region of human serum amyloid P component. The method involves quantitation of unbound peptides after a charge-dependent electrophoretic separation of the peptide-carbohydrate mixture. The concentrations of free peptide were determined by extrapolating the obtained peak areas of the peptide in the presence of ligand to a standard curve. Dissociation constants in the 10(-5) M range were determined, and differences in binding affinity of various peptide modifications were illustrated. The assay requires minute amounts of material (sample volume is 7-15 nL), and as long as the reactants are soluble at the chosen conditions, no modifications or special characteristics of the interacting molecules are needed for their identification. It should be possible to use electrophoretic separation in capillaries to evaluate the binding of peptides to any ligand as long as the differences in charge/mass ratio between free and complexed peptide are of a sufficient magnitude as in the peptide-heparin binding demonstrated here.

Amino Acid Sequence↗
PubMed1992

Immunochemical characterization of a monoclonal antibody specific for Alzheimer's disease associated protein.

In this study, the monoclonal antibody PHF-1 which recognizes epitopes unique to Alzheimer's disease associated proteins (ADAP) has been characterized. Crossed affinity immunoelectrophoresis was used to estimate the binding constant for the interaction of PHF-1 with ADAP and to estimate the fraction of PHF-1 reactive protein. The binding constant of PHF-1 was determined to be 1.3 x 10(-8) M. Furthermore, the effect of dephosphorylation on the electrophoretic pattern of the PHF-1 reactive protein and the ensuing changes in its immunoreactivity were demonstrated.

Alzheimer Disease↗
PubMed1991

[Use of adsorption methods for plasma component apheresis].

Plasma-apheresis is a nonspecific and wasteful intervention requiring the use of potentially infectious and expensive replacement fluids. Selective removal of the unwanted plasma component circumvents most of the problems. For selective binding and removal of plasma components adsorption methods based on the principles of affinity chromatography have been useful. The ideal adsorption column still does not exist, but the number of clinical applications is increasing. The results vary, but the treatment has been used with success in hypercholesterolemia, and in patients with hemophilia with antifactor antibodies and patients with antibodies directed towards HLA-antigens awaiting renal transplantation. In conclusion selective plasma component-apheresis is an improvement in some diseases as compared to conventional plasma-apheresis. The technique is still being improved but large clinical trials examining the effects of plasma-component-apheresis have not yet been published.

Adsorption↗
PubMed1991

Affinity electrophoresis used for determination of binding constants for antibody-antigen reactions.

The use of affinity electrophoresis in agarose gels for determination of binding constants for the interaction of antigens with monoclonal antibodies is exemplified for monoclonal anti-human serum albumin and anti-alpha 1-fetoprotein antibodies. The calculated binding constants are verified by independent binding assays. The electrophoretic separation of antigen-antibody complexes of different stoichiometry is also demonstrated. Thus, affinity electrophoresis represents an alternative method for both qualitative and quantitative assessment of antigen-antibody interactions.

Antibodies, Monoclonal↗