U R Nilsson

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 sec 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 il 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.

Modification of the complement binding properties of polystyrene: Effects of end-point heparin attachment

In recent years, conjugation of heparin to biomaterials has been shown to improve its biocompatibility. The purpose of the present work was to compare complement activation and binding of C3 to unmodified and heparin‐treated polystyrene surfaces of microtitre plates. When polystyrene was incubated with human serum, C3 was deposited on the surface by both adsorption and binding dependent on activation of the classical (CPW) and alternative (APW) pathways After end‐point attachment of heparin, significant C3 deposition, although at reduced levels, occurred only by CPW‐mediated mechanisms. while adsorption and APW ‐mediated binding were strongly reduced. Generally, the modified surface bound lower amounts of protein, e.g. serum albumin and IgG, than the unmodified. By contrast, it had increased affinity for Clq which leads to binding of Cl and activation of complement via the CPW. Nevertheless, the net effect of the surface modification on the complement reaction was an overall reduction of C3 binding due to obliteration of APW. This can be related to an enhanced factor H/I‐dependent down‐regulation of C3b and to the lowered protein‐adsorbing property of the surface, both of which have inhibitory effects on APW and on the C3 shunt‐dependent activation of the complement system.

Conformational epitopes of C3 reflecting its mode of binding to an artificial polymer surface

The aim of the study was to investigate the incompletely understood mechanisms of complement (C) activation and binding on artificial biomaterials. Polystyrene in the form of microtitre plates was used as target for C binding, detectable by ELISA using monoclonal anti-C3 antibodies specific for conformational epitopes expressed by bound C3 and C3 fragments. C3 binding in whole blood/plasma/serum is maximal at low dilutions and occurs predominantly by C activation. At higher dilutions, C3 binding occurs at approximately 1/3 of maximal levels and is solely an effect of adsorption. C3 adsorption in the lower serum dilution range, occurs at low but clearly detectable levels. Comparative epitope analysis between C3 fragments, actively bound to polystyrene in the presence of serum, and of iC3b bound to sheep erythrocytes, clearly indicates that C3 binding/activation on polystyrene takes place as a C3 convertase-mediated reaction, which in serum/plasma is followed by a secondary factor I-dependent degradation of the bound C3b into iC3b. The neo-epitope analysis of serum-contacting polystyrene revealed that the adsorbed C3, throughout the entire serum dilution range tested, deposits in a state closely similar to that observed for purified C3 at a high packing density. Polystyrene surfaces with adsorbed purified C3 expressing this epitope profile were found to mediate APW dependent deposition of C3b in pig serum, presumably by forming a hybrid convertase with porcine Bb. These data therefore suggest that adsorbed C3 on serum-contacting polystyrene surfaces may initiate complement activation via the APW.

Cereal fructosans: Part 2—Characterization and structure of wheat fructosans

Abstract Wheat fructosans were prepared from two different flours and separated according to degree of polymerization by gel permeation chromatography. The different fractions were quantified and characterized with regard to monomer carbohydrate composition. The tri-, tetra- and pentasaccharides made up about 50% of the total fructosan content. The structures of the trisaccharides were identified by gas chromatography/mass spectrometry. Two fructosan trisaccharides were found, neokestose and 6-kestose, along with raffinose.

A simplified assay for the detection of C3a in human plasma employing a monoclonal antibody raised against denatured C3.

A monoclonal antibody raised against SDS-denatured C3 was shown to react with both solid-phase C3a and unfragmented C3. However, in the fluid phase the antibody was found to bind only to C3a and not to native C3. These findings indicated that the antibody could be used in an assay to detect C3a in human EDTA-plasma without prior separation of C3a from native C3. A simple and rapid competition ELISA was developed which monitored soluble C3a. 200 microliter of C3a (8 ng) was absorbed to plastic wells over night at 4 degrees C. Thereafter, 50 microliter of sample and 50 microliter of constant amounts of monoclonal antibody conjugated with beta-galactosidase, were incubated for 60 min at 37 degrees C. After washing, the colour reaction was started by adding nitrophenyl-galactopyridine to the wells. The microtitre plate was incubated at 37 degrees C for 30 min and the staining intensity was quantified at 405 nm. The assay detected both C3a and C3ades arg. A strong correlation was obtained between the new technique and an RIA which used an acid precipitation step for the separation of C3a prior to the determination of C3a (r = 0.9). Significantly higher levels of C3a were detected both in plasma from patients with immune complexes (93 +/- 9 ng/ml; P less than 0.1) and in plasma from patients treated in blood oxygenators (140 +/- 19 ng/ml; P less than 0.05) than in plasma from normal subjects (74 +/- 4 ng/ml). The results were not affected by repeated freezing and thawing of the plasma samples.

Cereal fructosans : Part 1 - Isolation and characterization of fructosans from wheat flour

Abstract Wheat fructosans were isolated by two different methods—One, described earlier by Montgomery & Smith (1957), and one new, simpler method. The preparations were characterized by chemical analysis and by gel filtration chromatography. Their carbohydrate composition was practically identical. The ash content was lower in fructosans prepared by Montgomery and Smiths method, since it involves an ion exchange treatment. The yield of total fructose was higher with our method (68% compared with 32%). Acid hydrolysis was performed with wheat fructosans and, for comparison with inulin, to investigate the possibility of degradation of fructosans in the stomach. The results indicated very slow breakdown at physiological conditions. Wheat fructosans were slightly more acid resistant than inulin.

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 α and β 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‐converlase 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 C3α chain but the latter inhibited only the third cleavage. None of the immunoconglutinins affected the binding of complement‐coated anti‐BSA/BSA complexes to CRI (CD 35) 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 CRI. The results show that immunoconglutinins recognize specific epitopes on bound C3 fragments and that they are able to modulate C3‐mcdiated functions.

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.

SDS denaturation of complement factor C3 as a model for allosteric modifications occurring during C3b binding: demonstration of a profound conformational change by means of circular dichroism and quantitative immunoprecipitation.

The antigenic expression of bound but not fluid-phase C3b closely resembles that of sodium dodecyl sulphate (SDS) denatured C3. For this reason, denatured C3 has been used in this study as a model to characterize the conformational changes associated with bound C3b. It was shown in circular dichroism in the far UV spectrum that profound changes in the secondary structure occurred in denatured C3. Furthermore, quantitation by immunoprecipitation of the previously observed antigenic changes during denaturation demonstrated that C3 lost 2/3 of the antigens associated with native C3 whereas 1/3 were stable. The lost antigens were completely replaced by antigens that are specific for denatured and bound C3. We postulate that the binding of C3b is accompanied by a profound conformational change distinctive of that observed in fluid-phase C3b.

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.