Biomedical subjects

U R Nilsson

Publications and source records attributed to U R Nilsson.

At least 19 recordsLinked to original sources

PubMed1992

Conformational differences between surface-bound and fluid-phase complement-component-C3 fragments. Epitope mapping by cDNA expression.

In previous studies a subset of complement-component-C3 (C3) epitopes, C3(D), expressed in denatured and surface-bound C3 and C3 fragments, has been described. These epitopes were detected by antibodies raised against denatured C3. In the present study we used a cDNA expression strategy to localize epitopes recognized by monoclonal and polyclonal anti-C3(D) antibodies. First, DNAse I digestion of C3 cDNA was used to generate 200-300 bp fragments. These cDNA fragments were expressed as beta-galactosidase-C3 fusion proteins using the lambda gt11 vector. The fusion proteins were tested by Western-blot analysis for reactivity with monoclonal and polyclonal anti-C3 antibodies, and the location of the epitopes were determined by sequencing the cDNA fragments. Affinity-purified polyclonal anti-C3(D) antibodies specific for denatured C3 reacted strongly with the C3 fusion fragments corresponding to segments of the 40 kDa subunit of C3c (residues 1477-1510) and the C3d fragment (residues 1117-1155 and 1234-1294) of C3. Adsorption of the polyclonal antibodies with a mixture of EAC3b and EAC3bi (degradation fragments of C3 bound to sheep erythrocytes) abolished binding to fusion proteins spanning the C3d region, but not the 40 kDa fragment of C3c. No effect was seen with the corresponding soluble C3 fragments. The monoclonal anti-C3(D) antibodies (mAbs) 7D326.1 and 7D331.1, specific for EAC3b and EAC3bi, bound to a fusion protein corresponding to amino acid residues 1312-1404, whereas mAb 7D9.2, specific for EAC3d, reacted with a fusion protein spanning amino acid residues 1082-1118. mAbs 4SD11.1 and 4SD18.1, which did not bind to any physiological C3 fragment, detected a fusion protein covering residues 1477-1510. In summary, the segments of C3 represented by amino acid residues 1082-1118, 1117-1155, 1234-1294 and 1312-1404 accommodate C3(D) epitopes that are expressed by erythrocyte-bound C3 fragments, but not by the corresponding fluid-phase fragment, whereas the segments spanning residues 973-1026 and 1477-1510 contain C3(D) epitopes that are exposed exclusively in denatured C3 and therefore hidden in physiological fragments of the protein.

Animals↗

PubMed1992

Hereditary dysfunction of the third component of complement associated with a systemic lupus erythematosus-like syndrome and meningococcal meningitis.

OBJECTIVE: We describe a dysfunction of C3 in a patient with a systemic lupus erythematosus (SLE)-like syndrome. Alternative pathway complement function was absent, but classical pathway complement function was partially intact. METHODS: We used functional, preparative, and immunochemical techniques in the study. RESULTS: The patient's C3 proved normally susceptible to trypsin proteolysis and partially resistant to classical pathway, but completely resistant to alternative pathway, convertase-dependent cleavage. CONCLUSION: The dysfunction, thus, was caused by a failure of C3 to interact with the C3 convertases, rather than by a lack of a proteinase-sensitive cleavage site in the deficient protein.

Adult↗

PubMed1992

Purification and characterization of porcine C3. Studies of the biologically active protein and its split products.

Separation techniques for obtaining pure and biologically active swine C3 have been improved in this study. Using these procedures and through the further characterization of porcine C3, the possibilities for developing more specific techniques for the analysis of the complement system in swine have been improved. Plasma was initially treated with protease inhibitors, polyethylene glycol (PEG)-fractionation, plasminogen-depletion and a rapid chromatographic desalting step. The essential fractionation was carried out by DEAE-Sephacel chromatography. Contaminants were removed by size-exclusion (Sepharose CL-6B)- and hydroxylapatite-chromatography. The final recovery reached 56% with 73% retaining specific hemolytic activity. The amino acid composition (98.33%), the functional compatibility and the secondary structure of fragments and intact protein indicate a high degree of homology with human C3. In contrast with the findings of earlier studies was the considerable immunologic cross-reactivity observed with human C3, and the size difference between the human and the swine C3-beta subunit, which was found to be 10 kDa lighter than the human analogue. The finding that the swine C3b/iC3b/C3c fragments do not separate from C3 by agarose electrophoresis, unlike the human analogues, demonstrated that this commonly used simple parameter for the detection of complement activation cannot be used in the porcine model.

Amino Acids↗

PubMed1992

Lack of evidence for altered complement and immunoglobulin levels in patients with sickle cell anaemia.

Forty-three patients with homozygous sickle cell disease (haemoglobin SS), 24 heterozygous (AS) subjects and 24 controls (AA) from Sudan were investigated by haemolytic activation for complement function via the classical and the alternative pathways, respectively, and by determination of the plasma levels of C3, C4 and factor B as well as of immunoglobulins IgG, IgA and IgM. The study failed to reveal any direct involvement of the complement system in sickle cell disease and nor was any alteration of the immunoglobulin levels registered as a possible cause of increased susceptibility to infections in patients with homozygous sickle cell anaemia.

Adolescent↗

PubMed1992

Detection and characterization of immunoconglutinins in patients with systemic lupus erythematosus (SLE): serial analysis in relation to disease course.

The levels of IgA, IgG and IgM immunoconglutinins (IK) were assessed in sera from 20 patients with SLE which were followed for 8-month periods. At the time of the exacerbation, IgG IKs were significantly increased to 226 +/- 90 arbitrary units (mean +/- s.e.m.) compared with both the minimum value of 75 +/- 28 in the SLE patients and with 31 +/- 2 in healthy controls (P < 0.05). There was no difference between SLE patients and controls in the levels of IgM and IgA IKs. Most of the SLE patients in this material showed maximal IgG IK levels before exacerbation, but there was no correlation between the clinical disease index and the levels of IgG IK. The specificity of IgG IKs showed a broad diversity for microtitre-fixed C3b, iC3b, C3c and C3dg. The antibodies were of IgG1, IgG3 and in two patients, IgG4 subclass. IgG IKs were correlated to the C3d/C3 ratio which suggested that the IK responses were secondary to C3 activation. In summary, unlike other conditions associated with complement activation where elevated IgM IKs are common, an increase in IgG IK levels was observed. It is possible that this diverging IK response contributes to the pathophysiology of the disease.

Antibody Specificity↗

PubMed1992

Generation of iC3 at the interface between blood and gas.

Earlier studies have shown that C3 can be denatured when blood comes in contact with a polystyrene surface. This study was undertaken to see if similar denaturation of C3 occurs at the gas-plasma interface which is found in all kinds of oxygenator used during cardio-pulmonary operations. An in vitro system consisting of gas bubbling through human blood, serum or plasma was used. The generation of C3a, as an indicator of complement activation, and iC3 and iC3 fragments were monitored. Both C3a and iC3/iC3 fragments levels were increased during bubbling. In contrast to the C3a level, no reduction in iC3/iC3 fragments formation was seen in the presence of EDTA, indicating that it was independent of complement activation. The rate of iC3/iC3 fragments generation was unaffected by the composition of the gas (pure oxygen, pure nitrogen or air), suggesting that the denaturation of C3 indeed occurred at the serum-gas interface. C3 and iC3/iC3 fragments were isolated from bubbled EDTA-chelated serum by PEG precipitation and chromatography on FPLC, using a Mono S column and detected by two ELISAs, specific for native C3 and iC3/iC3 fragments. After 240 min approximately 20% of the total amount of C3 consisted of intact iC3 and it was confirmed that this population bound to human erythrocytes.

Blood↗

PubMed1992

RF-plasma-modified polystyrene surfaces for studying complement activation.

Five different plasma modified surfaces were made for studying different aspects of biocompatibility. These surfaces were: 1,2-diaminocyclohexane (DACH), acrylic acid (AA), Hydroxyethylmethacrylate (HEMA), methane and hexamethylene-disiloxane (HMDSO). In addition a polyethylene-glycol (PEG) was made by grafting aldehyde functional PEG to the DACH surface. PEG and HMDSO which are the most hydrophilic and the most hydrophobic surface shows the lowest amount of adsorbed protein of the three proteins studied here (albumin, IgG and C3). Methane, HMDSO and HEMA was found to activate via the classical (complement activation) pathway while the others activated via the alternative pathway.

Adsorption↗

PubMed1991

Defective Fc receptor-mediated clearance in patients with primary biliary cirrhosis.

Fc receptor-mediated clearance of immunoglobulin G-coated autologous erythrocytes was studied in patients with primary biliary cirrhosis (n = 14), alcoholic liver cirrhosis (n = 5) and healthy reference individuals (n = 14). The mean half-life of the sensitized erythrocytes was significantly prolonged in patients with primary biliary cirrhosis (85 +/- 25 minutes; P less than 0.001) compared with the corresponding value in patients with alcoholic cirrhosis (16 +/- 2 minutes) and healthy reference individuals (20 +/- 5 minutes), respectively. No correlation between clearance rate and age, liver histopathology, or serum levels of bilirubin, aminotransferases, immunoglobulin G, immunoglobulin A, and Clq binding or C3-containing immune complexes was found. The results presented here indicate a profound disturbance of Fc receptor-mediated immune clearance function in patients with primary biliary cirrhosis.

Antigen-Antibody Complex↗

PubMed1991

An improved method to study complement receptor-mediated function of the fixed macrophage system in vivo.

A method to coat unsensitized erythrocytes with fragments of C3 and C4 using autologous serum, in order to study complement receptor-dependent function of the fixed macrophage system, is presented. After incubation with serum under optimal conditions, at least 90% of the cells had C3b/iC3b deposited on the surface, with an average of 20 x 10(3) molecules per cell. Elimination of the coated cells by the fixed macrophage system was studied in 12 normal subjects. With a dose of 4.5 x 10(8) red cells injected, 75% of the cells were eliminated with a half-life of approximately 2.4 +/- 0.3 min (n = 7). In subjects receiving ten times more cells, there was a rapid decrease in the amount of C3-coated cells, reaching a nadir with 85% remaining for 4-6 min, after which there was a gradual release of cells for another 20 min (n = 5). In absolute numbers, 3 x 10(8) of labeled cells were eliminated regardless of the dose injected. The coating procedure presented here is simple, does not introduce heterologous blood components and makes it possible to control the amount and the degree of fragmentation of the C3 and C4 deposited on the erythrocyte surface.

Adult↗

PubMed1991

Fc receptor function and circulating immune complexes in gluten sensitive enteropathy--possible significance of serum IgA.

The capacity to clear IgG containing immune complexes from the circulation was studied in patients with coeliac disease (n = 13), dermatitis herpetiformis (n = 8), and coeliac disease with concomitant serum IgA deficiency (n = 4). A small group of patients with active ulcerative colitis (n = 4) was included as a bowel disease control group. Clearance was estimated by measuring the disappearance rate of a bolus dose of intravenously injected IgG coated autologous erythrocytes. The mean T1/2 of clearance was prolonged in both coeliac disease (86 (24) minutes) and dermatitis herpetiformis (111 (35) minutes), compared with healthy subjects (20 (5) minutes) and coeliac patients with concomitant serum IgA deficiency (T1/2 = 17 (6) minutes). Patients with ulcerative colitis had a prolonged clearance, with a T1/2 of 195 (63) minutes. Values of circulating immune complexes were measured by four assays; C1q binding and C3, IgG, and IgA containing immune complexes. C1q binding immune complexes were detected only in IgA deficient gluten sensitive enteropathy. Patients with coeliac disease and dermatitis herpetiformis had higher values of C3, IgG, and IgA containing immune complexes than control subjects and serum IgA deficient patients with coeliac disease. The clearance rate was inversely correlated to the amount of immune complexes for the subgroups of gluten sensitive enteropathy.

Adult↗

PubMed1990

Inhibition of factor I by diisopropylfluorophosphate. Evidence of conformational changes in factor I induced by C3b and additional studies on the specificity of factor I.

The factor I-mediated cleavage of C3b, using factor H as a cofactor was completely inhibited by diisopropylfluorophosphate (DFP) when factor I and C3b were incubated with DFP before the addition of factor H. Inhibition, although to a lesser degree, was observed when factor H was present during DFP-exposure. No inhibition in factor I activity was seen when factor I and H were incubated with DFP either alone or together. It was also demonstrated that the 38-kDa subunit of factor I bound radiolabeled DFP when factor I and C3b together were exposed to DFP. These observations suggest that factor I interacts with C3b in a manner that exposes its catalytic site to DFP, an interaction that is independent of factor H. The inhibitory effect by DFP on factor I led us to further investigate the factor I cleavage products of iC3b, inasmuch as previous reports were ambiguous as to whether digestion occurs in the presence of DFP. Digestion of C3b bound to activated thiol Sepharose (ATS-C3b) in the presence of factor H at low pH and ionic strength and in serum by complement activation produced C3d,g-like fragments with apparent molecular mass of 41 and 43 kDa. These fragments were shown to have three different N-terminal and two different C-terminal ends. The major fragments had N-terminal sequences starting with Glu933, as shown by sequence determination. Traces of fragments extending beyond this point were also found, shown by Western blot analysis using a panel of mAb previously shown to bind to epitopes exposed within a region of C3 spanning residues 929 to 943, as well as a shorter fragment starting with Glu938. When digestion of C3b is carried out in the presence of DFP, the factor I level necessary for digestion is elevated and may explain how the first two cleavages producing iC3b but not the following giving C3d,g, can occur. The finding of several factor I cleavage sites in the C3d,g region of C3 demonstrates that factor I has a broad specificity, mainly for arginyl bonds. It has also been shown to digest a lysyl bond exposed in ATS-bound C3b.

Amino Acid Sequence↗

PubMed1990

Neoantigens in complement component C3 as detected by monoclonal antibodies. Mapping of the recognized epitopes by synthetic peptides.

The different fragments of the third complement component, C3, generated upon complement activation/inactivation have the ability to bind to several other complement components and receptors as well as to proteins of foreign origin. These multiple reactivities of C3 fragments are associated with a series of conformational changes occurring in the C3 molecule during its degradation. The conformations acquired by the different C3 fragments are also associated with the exposure of neoantigenic epitopes that are specific for (a) particular fragment(s). In order to study these epitopes and thus the conformational changes occurring in C3, monoclonal antibodies (mAbs) recognizing such epitopes were produced in Balb/c mice after immunization with denatured human C3. Two of the three antibodies (7D84.1 and 7D264.6) presented in this study recognized predominantly surface-bound iC3b, and one mAb (7D323.1) recognized both surface-bound and fluid-phase iC3b. Although none of the mAbs recognized any other fluid-phase C3 fragment, all three antibodies detected micro-titre-plate-fixed C3b and iC3b, but not C3c or C3d. In addition to the reaction with human C3, mAb 7D323.1 also bound to micro-titre-plate-fixed rabbit C3. The epitopes recognized by the three mAbs were further localized by using synthetic peptides that were designed on the basis of the differential binding of the mAbs to the C3 fragments. All three antibodies reacted with C3-(924-965)-peptide, which represents the region of C3 between the kallikrein-cleavage site (923-924) and the elastase-cleavage site (965-966). On the basis of the binding of the mAbs to five different overlapping peptides spanning the region between residues 924 and 965 of the human C3 sequence, and the sequence similarity between human C3 and rabbit C3 within this area, the epitopes recognized by these antibodies are mapped. The contribution of the individual amino acid residues in the formation of the epitopes is discussed.

Antibodies, Monoclonal↗

PubMed1990

In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C. Effects on the classical and alternative pathways.

Complement factor C3, recently found to contain covalently bound phosphate, was phosphorylated in vitro by cyclic AMP-dependent protein kinase (protein kinase A) and Ca2(+)-activated, phospholipid-dependent protein kinase (protein kinase C). Both protein kinases phosphorylated the same serine residue(s) located in the C3a portion of the alpha-chain. In addition, protein kinase C phosphorylated the beta-chain to a lesser extent. Protein kinase A gave a maximal incorporation of 1 mol of phosphate/mol of C3 while that value with protein kinase C was 1.5 mol of phosphate/mol of C3. The velocity in pmol of [32P]phosphate/(min x unit kinase) was 20 times higher for protein kinase C than for protein kinase A although a 10 times lower ratio of protein kinase to C3 was used in the former case. The apparent Km for C3 was 2.6 microM when protein kinase C was used. The phosphorylated C3 was found to be more resistant to partial degradation by trypsin than unphosphorylated C3. It was also found that phosphorylation of C3 in the C3a portion of the alpha-chain inhibited both the classical and alternative complement activation pathways on an approximately stoichiometric basis.

Amino Acids↗

PubMed1990

Purification and characterization of IgG immunoconglutinins from patients with systemic lupus erythematosus: implications for a regulatory function.

The levels of IgG immunoconglutinins in plasma from patients with rheumatoid arthritis, systemic lupus erythematosus and primary biliary cirrhosis were monitored by ELISA. High levels of IgG immunoconglutinins were found mainly in plasma from patients with systemic lupus erythematosus. These immunoconglutinins bound to microtitre plate-fixed C3, C3b and C3c but poorly to C3d. This binding was inhibited by particle-bound C3b and iC3b but not by the corresponding soluble fragments. Furthermore, Western blot analysis revealed no immunoconglutinin-binding to reduced C3 peptides and no binding was shown to soluble C3 alpha and beta chain by ELISA. IgG immunoconglutinins were purified from three plasma specimens by affinity chromatography on activated thiol sepharose ATS/C3 fragments. Two immunoconglutinin preparations that preferentially recognize ATS-C3b, inhibited C5-convertase function by 50-100% while one immunoconglutinin that recognized ATS-C3d,g had no effect. The two former immunoconglutinins also inhibited all three factor I cleavages in C3 alpha chain but the latter inhibited only the third cleavage. None of the immunoconglutinins affected the binding of complement-coated anti-BSA/BSA complexes to CR1 (CD35) on human erythrocytes, but the two immunoconglutinins that inhibited all factor I cleavages also inhibited the factor I-induced release of anti-BSA/BSA complexes from CR1. The results show that immunoconglutinins recognize specific epitopes on bound C3 fragments and that they are able to modulate C3-mediated functions.

Antibody Specificity↗

PubMed1989

Distinctive expression of neoantigenic C3(D) epitopes on bound C3 following activation and binding to different target surfaces in normal and pathological human sera.

Binding of C3 to sheep erythrocytes in a serum-free milieu (EAC14oxy2, EAC142) has previously been shown to mimic the antigenic change that occurs upon denaturation of C3 in sodium dodecyl sulphate (SDS), whereby neoantigenic C3(D) epitopes are exposed. The present paper deals with C3 bound to various target surfaces which are known to modulate the functional properties of C3 in different ways. Bound C3 fragments on serum-treated human aggregated gammaglobulin, zymosan, rabbit and sheep erythrocytes, and on circulating immune complexes isolated from sera of patients with rheumatoid arthritis and systemic lupus erythematosus, were shown to be mainly in the iC3b form. By RIAs, employing polyclonal antibodies, the range of C3(D) antigenic epitopes of 125I-labelled SDS denatured C3 expressed by the particle-bound iC3b was monitored. The physiologically bound iC3b on all tested particles expressed wide range of C3(D) epitopes and each type of particle-bound C3 exposed its individual range. By competition ELISA specific C3(D) alpha epitopes were monitored, employing monoclonal antibodies. A distinct difference in the expression of these epitopes was observed in iC3b bound to various test particles in the presence of normal serum and in iC3b present on circulating immune complexes from pathological sera. Considering that the neoantigenic C3(D) epitopes have been shown to be associated with different functions of C3, the distinctive antigenic expression of each type of serum-treated particle might reflect different functional forms of the protein.

Antibodies, Monoclonal↗

PubMed1989

Development of an immunoassay for the detection of minute amounts of IgG-coated erythrocytes in whole blood and its application for the assessment of Fc-mediated clearance of anti-D-coated erythrocytes in vivo.

A rapid and sensitive immunoassay for the detection of minute quantities of IgG-coated erythrocytes in whole blood was developed. Washed red blood cells were incubated in two steps with anti-human IgG antiserum followed by 125I-labelled protein A. The assay was able to detect amounts of sensitized erythrocytes as small as 0.5 ml of packed erythrocytes in a total blood volume of 5 liters and hematocrit 40%. A linear relation between increasing amounts of IgG-coated red cells in whole blood and the binding of 125I-labelled protein A was obtained. We applied the technique on the assessment of the removal of IgG anti-D-coated erythrocytes from the circulation of test individuals. T1/2 for the elimination of approximately 4 ml packed red cells sensitized with 62 micrograms of anti-D in 14 normal subjects was 20 +/- 5 min (mean +/- SEM). A splenectomized person did not clear the injected cells from the circulation during the test period of 70 min. If a standard curve was constructed the total blood volume in the test subjects could be calculated. This value correlated well (r = 0.99) with the blood volume calculated from the height and weight of the test individuals.

Adult↗

PubMed1987

Anti-idiotypic antibodies in antisera against human C3 and factor H and their application in the enrichment of antibodies specific for H-binding domains of C3.

Antisera separately raised against C3 and factor H should contain antibodies against the binding domains by which these factors interact. Because these interacting domains are likely to be sterically complementary to each other, antibodies specific for these domains should also be sterically complementary or of anti-idiotypic specificity. To test this hypothesis, rabbit anti-human C3 IgG antibodies which bound to Sepharose-coupled rabbit anti-factor H IgG (anti-H-binding anti-C3) were separated by affinity chromatography. In a control experiment, the anti-human factor H column was found to bind 10 times more anti-C3 antibodies than a Sepharose column coupled with nonimmune rabbit IgG. The binding specificity of the control eluate was indistinguishable from the original anti-C3 preparation, whereas the anti-H-binding anti-C3 was shown to be enriched in regard to antibodies directed against the 42 kd fragment of the C3 alpha-chain of C3c, as demonstrated by immunoblotting of electrophoretically separated polypeptides of C3c and C3d in polyacrylamide gels. In competition binding studies, factor H was 25 times more potent as inhibitor of the anti-H-binding anti-C3 than of the original anti-C3 preparation. This similarity in binding specificity also paralleled a functional similarity in that anti-H-binding anti-C3 in the presence of factor I effected a 20% degradation of the alpha'-chain of C3b into fragments of 65, 45, and 42 kd which are the normal degradation products in the presence of factors I and H. These results suggest that the affinity procedure described resulted in the enrichment of anti-C3 antibodies with selective specificities for factor H-binding domains of C3, from a polyclonal antibody source.

Antibody Specificity↗

PubMed1987

Quantitation and antigenic characterization of bound C3 of circulating immune complexes in systemic lupus erythematosus, rheumatoid arthritis, and primary biliary cirrhosis.

In recent years defective function of the complement-mediated clearance of immune complexes (IC) has been reported in patients with immune complex disease. The defect has been found at different levels in the clearance system. An important event in this sequential system is the binding of C3-coated particles to C3 receptors on erythrocytes and phagocytes. This study focuses on immunochemical properties of IC-bound C3 that reflect the functional state of the molecule. Sera from patients with primary biliary cirrhosis (PBC), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) and from normal subjects were analyzed for their level of C3 precipitable in 2.7% (w/v) polyethylene glycol (PEG). The mean levels for the patient categories were significantly higher than that for the normal subjects. The immunochemical study revealed several differences among the different forms of PEG-precipitable C3. All forms expressed C3(D) antigens which are expressed by immune complex-associated and denatured forms but not by soluble physiological forms of C3. The expression of these antigens was proportionately lower for the complex-associated C3 of PBC compared to that of RA and SLE. Furthermore, employing monoclonal anti-C3(D) antibodies against the C3c and the C3d domain, distinct differences could be detected among all forms of PEG-precipitable C3. Sera from RA and SLE, in particular, contained PEG-precipitable C3 that exhibited distinctive immunochemical features with respect to these epitopes.

Antibodies↗