Dual function of insoluble polyanions. Formation of C1 and C42 sites in addition to activation of the alternative pathway of complement.
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Animals↗
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Activation of factor B and C3 in guinea pig serum by zymosan or by activated properdin depends on the presence of factor D.
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Animals↗
Mouse monoclonal antibodies to the human C3b receptor.
Mouse monoclonal antibodies were raised against the human C3b receptor (CR1) molecule that had been purified from solubilized erythrocytes membranes. Four hybridomas were selected, cloned and expanded because their supernatants reacted strongly with insolubilized CR1 by ELISA and intensely stained B-dependent areas of the spleen and glomerular podocytes by indirect immunofluorescence. The four monoclonal antibodies, named J3D3, J8B10, J3B11 and J7C2, were IgG1 immunoglobulins. J3D3 immunoprecipitated two protein bands of apparent mol. wts 200,000 and 220,000 from 125I-surface-labeled human erythrocytes, which correspond to the two major allotypic forms of CR1. By indirect immunofluorescence, monoclonal antibodies stained polymorphonuclear leucocytes (PMN), most peripheral blood B-cells and a small subset of peripheral blood T-cells. J3D3 bound to CR1 on erythrocytes, PMN and lymphocytes with an affinity of 1-3 X 10(9) M-1 and recognized 170-1330 antigenic CR1 sites with an average of 740 sites/erythrocyte in 100 healthy individuals, approx. 50,000 sites/PMN and 15,000 sites/lymphocyte. There was a bimodal distribution of CR1 numbers on erythrocyte in the normal population. The four monoclonal antibodies similarly inhibited CR1-mediated decay of preformed cell-bound alternative- and classical-pathway C3 convertase sites. Two antibodies, J3D3 and J3B11, inhibited C3b-dependent rosette formation with lymphocytes, although much less efficiently than F(ab')2 polyclonal anti-CR1 antibody. Differences that were observed in the relative capacity of the antibodies to inhibit some of the functions of CR1 and in their ability to compete for binding of 125I-J3D3 to CR1 on erythrocytes, suggested that they are directed against different epitopes on CR1. Monoclonal antibodies provide useful means to assess and analyze the biological and immunoregulatory functions of the C3b receptor.
Animals↗
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↗
Isolation of two forms of decay-accelerating factor (DAF) from human urine.
Decay-accelerating factor (DAF) was purified from human pooled urine by conventional techniques. The urine DAF was separated into two peaks, pool I and pool II, by gel chromatography. DAF-U1 was isolated from pool I by hydrophobic chromatography, and DAF-U2 from pool II by anti-DAF IgG column. The specific activities of DAF-U1 and DAF-U2 to decay membrane-phase C5 convertase were about 3% and 70% of membrane form DAF, respectively. However, both urine DAFs revealed a similar activity to each other and slightly higher activity than that of membrane form DAF in decay-accelerating fluid-phase C3 convertase of the alternative pathway.
Amino Acid Sequence↗
Biochemical characterization of a factor produced by trypomastigotes of Trypanosoma cruzi that accelerates the decay of complement C3 convertases.
Infective- and vertebrate-stage trypomastigotes of Trypanosoma cruzi resist serum killing by the alternative complement pathway, whereas noninfective vector-stage epimastigotes, from which trypomastigotes derive, are serum-sensitive. This form of developmental preadaption is commonly observed in protozoan parasites, but its mechanisms are poorly understood. We have demonstrated previously that trypomastigotes spontaneously shed molecules which interfere with formation and accelerate the intrinsic decay of complement C3 convertases, a finding which may explain the evasion of complement lysis by trypomastigotes. We now describe the partial purification and characterization of the T. cruzi C3 convertase inhibitor from the supernatant of culture metacyclic and tissue culture trypomastigotes. Decay-accelerating activity for both classical and alternative pathway C3 convertases copurifies on anion-exchange fast protein liquid chromatography and chromatofocusing with 35S-labeled molecules of 87-93 kDa, pI 5.6-5.8. The labeled components are destroyed by papain and retained on concanavalin A-Sepharose, procedures which remove functional decay-accelerating activity from the supernatant. The 87-93-kDa components are immunoprecipitated by sera from patients chronically infected with T. cruzi, but not by antisera to any known regulatory proteins of the human complement cascade. Lytic activity for tissue culture trypomastigotes in chagasic sera is associated with antibody reactivity against the 87-93-kDa 35S-labeled components and with inhibition of decay-accelerating activity. The T. cruzi factor is the first developmentally regulated microbial complement inhibitor to be biochemically characterized.
Animals↗
Rosmarinic acid: a new inhibitor of complement C3-convertase with anti-inflammatory activity.
Rosmarinic acid (RA) is a naturally occurring compound, isolated from Rosmarinus officinalis or Melissa officinalis which inhibits the in vitro immunohaemolysis of antibody-coated sheep erythrocytes by guinea pig serum. In further experiments this reduced immunohaemolysis was found to be due to inhibition of the C3-convertase of the classical complement pathway. The threshold concentration for inhibition of C3-convertase was 10(-6) mol/l. RA with an optimal inhibitory concentration between 5 and 10 mumol/l., resulting in about 70% inhibition of haemolysis. However, higher concentrations of RA were less effective at inhibiting C3-convertase. The inhibition may not be specific for C3-convertase, since another serine protease, elastase, was also weakly inhibited by RA in vitro. RA also exhibited inhibitory activity in three in vivo models in which complement activation plays a role. Thus, RA (0.316-3.16 mg/kg i.m.) reduced paw oedema induced by cobra venom factor (CVF) in the rat, and at 1-100 mg/kg p.o. inhibited passive cutaneous anaphylaxis in the rat. In addition, at 10 mg/kg i.m. RA impaired in vivo activation by heat-killed Corynebacterium parvum (i.p.) of mouse macrophages, as measured by the decreased capacity of the activated macrophages to undergo the oxidative burst. RA (0.1-10 mg/kg i.m.) did not inhibit t-butyl hydroperoxide-induced paw oedema in the rat, indicating selectivity for complement-dependent processes.
Animals↗
[Effectiveness and tolerance of the C3 convertase inhibitor, N-acetyl-aspartyl-magnesium glutamate with anti-allergic action. Results of a double-blind study].
N-Acetyl-aspartyl magnesium glutamate (Rhinaaxia, NAAGA) is a topically active antiallergic dipeptide. The compound acts in two different ways. On the one hand NAAGA inhibits the mast cell-degranulation, on the other hand this compound blocks the activation of the C3-convertase, subsequently followed by a blocked cleavage of the fragments C3a and C5a, respectively. 20 patients suffering from pollinosis were treated for 2 weeks according to a randomized double-blind placebo-controlled study. Besides subjective complaints nasal obstruction was objectively documented via rhinomanometria. 9 out of 10 patients under placebo had to use the rescue drug tritoqualine, a histidine decarboxylase inhibitor, compared to none in the verum group (p less than 0.01). After 14 days of treatment with NAAGA the nasal peak flow rate increased by 21.5 l/min and 21.8 l/min in the tritoqualine/placebo group, respectively (not significant). Nasal obstruction improved statistically significantly after 7 and 14 days of treatment in both groups. Tolerance was reported to be good in either group.
Adolescent↗
Additional forms of human decay-accelerating factor (DAF).
Decay-accelerating factor (DAF) of human erythrocytes is a glycoprotein with a Mr of 65,000 that is anchored in the membrane via a glycolipid tail. During the purification of DAF, two lower m.w. forms were noted. DAF-A had an Mr of 63,000, and DAF-B had an Mr of 55,000. In a fluid phase assay, both forms accelerated the decay of the classical and the alternative C3 convertases with a specific activity similar to that of DAF. However, the decay-accelerating activity for the cell-bound C3 convertases was abolished, suggesting that neither could insert into E membranes and therefore that the glycolipid tail is altered. Analysis by molecular sieve high-pressure liquid chromatography demonstrated that DAF-A eluted with a Mr of approximately 450,000, similar to native DAF, and was thus in an aggregated form. In contrast, DAF-B eluted as a monomer with a Mr of approximately 60,000. DAF-A, but not DAF-B, bound to a hydrophobic column. To further characterize these two forms, surface-labeled human erythrocytes were incubated with phosphatidyl inositol-specific phospholipase C or papain. The phospholipase inefficiently released a form of DAF that was slightly larger (Mr of 64,000) than DAF-A. Papain efficiently released a 55,000 fragment that had the same Mr as DAF-B. To determine if DAF was cleaved by endogenous enzymes, surface-labeled erythrocytes were incubated with leukocytes. The kinetics of the leukocyte-induced degradation was similar to those observed with papain, and the released fragment aligned on seizing gels with the papain-derived fragment. We hypothesize that endogenous phospholipases and proteases cleave DAF to produce fragments similar to DAF-A and DAF-B, respectively.
Butanols↗
Study of the idiotypic response to autoantibody to the alternative pathway C3/C5 convertase in normal individuals, patients with membranoproliferative glomerulonephritis, and experimental animals.
We studied the fluctuation of autoantibody to the alternative pathway C3/C5 convertase (C3NeF) and its autoantidiotypic antibodies, Ab2 alpha and Ab2 beta, in normal individuals, patients with membranoproliferative glomerulonephritis (MPGN), and New Zealand white rabbits. In normal individuals, serum levels of Ab2 alpha (anti-id Ab without internal imagery to the native antigen) are several times higher than those for Ab2 beta (anti-id Ab bearing the internal image of the native antigen). When normal rabbits are immunized with Ab2 beta, Ab3 is produced which has strong C3NeF activity. Ab3 acts to stimulate the prompt production of Ab4 alpha. Ab3 (C3NeF) titers then fall, followed by the appearance of Ab4 beta. Patients with MPGN and C3NeF activity were also studied. Untreated patients at the time of diagnosis have a two- to fourfold predominance of Ab2 beta which is a direct reversal of the normal situation. When the patients were treated with prednisone, C3NeF levels fell and Ab2 alpha again predominated. These data suggest that the idiotypic network acts to control the production of autoantibody in a balanced fashion. Moreover, these data suggest that the patients' response to Ab1 is quite different than that found in normal individuals.
Antibodies, Anti-Idiotypic↗
Is primary biliary cirrhosis an immune complex disease?
Large immune complexes are present in the circulation of patients with primary biliary cirrhosis and result in the activation of complement by the classical pathway. Such large complexes are capable of producing tissue damage. The granulomatous lesions surrounding the small bile-ducts within the liver of patients with primary biliary cirrhosis and the vasculitis, rheumatoid arthritis, and associated lesions are all compatible with immune complex injury. It is postulated that such large complexes could be formed in the vicinity of the bile-ducts by an antigen absorbed from the bile or biliary epithelium. Complexes reaching the systemic circulation might be responsible for the associated extra-hepatic diseases.
Antibody Formation↗
Nonimmunoglobulin C3 activating factor in membranoproliferative glomerulonephritis.
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Adult↗
Different activation patterns in the plasma kallikrein-kinin and complement systems during coronary bypass surgery.
Components of the plasma kallikrein-kinin and complement systems were determined in patients undergoing open heart surgery with cardiopulmonary bypass. Spontaneous kallikrein activity (KK), plasma prekallikrein (PKK), functional kallikrein inhibition capacity (KKI), C3 activation products (C3-act), and the terminal complement complex (TCC) were measured. A marked, transitory increase in KK and a decrease in PKK were found prior to cardiopulmonary bypass just after heparin injection. An additional decline in PKK and KKI during bypass with a return to near control levels in the postoperative period was observed. C3-act increased in all patients during bypass, reaching a peak value at wound closure. The TCC concentration also increased significantly during cardiopulmonary bypass, returned to control levels in the early postoperative period, and then increased again in the late postoperative period. It is concluded that activation of the kallikrein-kinin system started after injection of heparin, prior to cardiopulmonary bypass. Activation of both the initial and the terminal complement cascade, however, started only after onset of cardiopulmonary bypass.
Adult↗
[Description of an unusual enzymic activity cleaving the third component of complement].
A large enzymatic complex cleaving C3 (C3'ase) in the absence of magnesium (EDTA) has been partially characterized in two patients. On gel filtration EDTA-C3'ase was found in an 800 000 fraction containing antigenic C4, IgA and IgG in addition to alpha-2-macroglobulin and IgM normally present. EDTA-C3'ase was specifically neutralized by antibodies to IgG, IgA and C4. The characteristics of this unusual enzymatic complex are compatible with that of a C4b2a complex stabilized by its specific binding to an auto-antibody of the IgG and/or IgA class.
Chromatography, Gel↗
Requirements for the production of high-titre C3 nephritic factor (NEF) antibody in vitro.
C3 nephritic factor (NEF) is an IgG autoantibody directed against neoantigenic determinants of the alternative C3 convertase (C3b.Bb). Structural and functional studies require important amounts of this antibody, which are difficult to obtain from patients' sera. We have developed a method for increasing NEF production in vitro. Epstein-Barr virus is a herpes virus which transforms B lymphocytes. Some authors were able to induce the production of NEF in vitro after infection with Epstein-Barr virus (EBV). These works were preformed without any previous cellular selection of B cells. We have performed a method of preselecting antigen-binding cells prior to EBV transformation. Non-preselected cells yielded 0.16 U/million cells in culture (U/M) of NEF antibody, whereas enriched cells for NEF antibody in eliminated 8 U/M (sheep erythrocytes coated with anti-IgG, A, M). Specific NEF synthesis can be increased, in peripheral blood mononuclear cells (PBMC) from patients by in vitro stimulation with the antigens recognized by NEF [C3b.Bb, 21,000 MW protein from patients' E membranes and 26,000 MW protein from sheep E membranes (ShE)]. The highest stimulation is induced by the C3b.Bb and by 26,000 MW protein, 21,000 MW protein had lowest stimulatory effect. In this work also we have shown that patients having NEF antibody in sera have an increase of the CD5-CD19 subset, when compared with the controls.
Antigens, CD↗
Autoantibody to the alternative pathway C3/C5 convertase and its anti-idiotypic response. A study in affinity.
In an effort to understand the development and control of autoantibody production, we studied the affinity of autoantibody to the alternative pathway C3/C5 convertase (C3 nephritic factor (C3NeF)) and its autoanti-idiotypic antibodies, Ab2 alpha and Ab2 beta. These were isolated and purified from newborns, normal adults, and patients with membranoproliferative glomerulonephritis. In all cases, both IgG and IgM C3NeF were available for study. The affinity of IgG and IgM C3NeF for their natural Ag (10(8) liters/mol) as well as for the internal image of that Ag displayed on Ab2 beta was high (10(10) liters/mol). Furthermore, the affinity of IgG C3NeF was nearly 100-fold higher in patients than in newborns, whereas there were no significant changes with IgM C3NeF. By contrast, there were not differences in the affinity of IgG Ab2 alpha (which does not display any likeness to the native Ag) from normal adults and patients to any C3NeF isolate. There was, however, a progressive increase in affinity between both Ab2 alpha preparations and IgG C3NeF from newborns, adult normal subjects, and patients, implying an alteration in C3NeF to account for the changes in affinity. These data suggest that Ag-driven affinity maturation occurs with autoantibody but may not occur within the idiotypic network. These data also indicate that as autoantibody affinity matures, it appears to modify its idiotype, perhaps in an effort towards autoregulation.
Adult↗
[Effect of Expafusion (HES 40/0.5) on the corpuscular elements of blood and inhibitors of blood coagulation].
The present study reports on an investigation of 32 normal subjects who were given intravenously 500 ml of hydroxyethyl starch (HES) over 30 min. In 16 normal subjects, 500 ml of blood had been previously withdrawn over 20 min. Tolerance proved to be good. Side effects such as hyperergic reactions, influence on circulatory or cardiac function were not observed. Changes in corpuscular elements of blood did not exceed the dilution effect and mostly returned to the initial value 60 min after termination of HES infusion. The change in thrombocyte function, immunoglobulins and the inhibitors alpha 1-antitrypsin, alpha 2-macroglobulin, as well as C1-inactivator and C3-activator only slightly, but not more pronounced than it could have been expected by the dilution effect. A favorable effect could be recorded in erythrocyte filtration according to Schmidt-Schoenbein. Here, we found an improved flow property which exceeded the hemodilution effect and lasted over the 4-hour test period. In addition, thrombocyte aggregation during ADP administration in the Born chamber did not show any change which suggests that a clinically relevant effect on thrombocyte function and thus the danger of hemorrhage during HES administration can be excluded.
Adolescent↗