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Background: The complement system is a central component of the innate immune system. Constitutive biosynthesis of complement proteins is essential for homeostasis. Dysregulation as a consequence of genetic or environmental cues can lead to inflammatory syndromes or increased susceptibility to infection. However, very little is known about steady state levels in children or its kinetics during infection., Methods: With a newly developed multiplex mass spectrometry-based method we analyzed the levels of 32 complement proteins in healthy individuals and in a group of pediatric patients infected with bacterial or viral pathogens., Findings: In plasma from young infants we found reduced levels of C4BP, ficolin-3, factor B, classical pathway components C1QA, C1QB, C1QC, C1R, and terminal pathway components C5, C8, C9, as compared to healthy adults; whereas the majority of complement regulating (inhibitory) proteins reach adult levels at very young age. Both viral and bacterial infections in children generally lead to a slight overall increase in complement levels, with some exceptions. The kinetics of complement levels during invasive bacterial infections only showed minor changes, except for a significant increase and decrease of CRP and clusterin, respectively., Interpretation: The combination of lower levels of activating and higher levels of regulating complement proteins, would potentially raise the threshold of activation, which might lead to suppressed complement activation in the first phase of life. There is hardly any measurable complement consumption during bacterial or viral infection. Altogether, expression of the complement proteins appears surprisingly stable, which suggests that the system is continuously replenished. FUND: European Union's Horizon 2020, project PERFORM, grant agreement No. 668303., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

During a study of the effect of inulin on human complement, we found that complements were partially activated after incubation with inulin for one hour and completely activated after incubation for three hours at 37 degrees C. SC5b-9 was purified by DEAE-Sephacel chromatography, linear sucrose density gradients centrifugation and anti-IgM-protein A affinity chromatography. Purified SC5b-9, as assayed by immunoelectrophoresis, was a single fraction and had C5b, C6, C7/C8 alpha gamma, C8 beta, C9, C92, and S-protein bands appearing in the 10% SDS-PAGE.

The major characteristics of human atherosclerotic lesions are similar to those of a chronic inflammatory reaction, namely fibrosis, mesenchymal cell proliferation, the presence of resident macrophages, and cell necrosis. Atherosclerosis exhibits in addition the feature of lipid (mainly cholesterol) accumulation. The results of the present report demonstrate that a specific cholesterol-containing lipid particle present in human atherosclerotic lesions activates the complement system to completion. Thus, lipid could represent a stimulatory factor for the inflammatory reaction, whose underlying mechanistic basis may be, at least in part, complement activation. The complement-activating lipid was purified from saline extracts of aortic atherosclerotic lesions by sucrose density gradient centrifugation followed by molecular sieve chromatography on Sepharose 2B. It contained little protein other than albumin, was 100-500 nm in size, exhibited an unesterified to total cholesterol ratio of 0.58 and an unesterified cholesterol to phospholipid ratio of 1.2. The lipid, termed lesion lipid complement (LCA), activated the alternative pathway of complement in a dose-dependent manner. Lesion-extracted low density lipoprotein (LDL) obtained during the purification procedure failed to activate complement. Specific generation of C3a desArg and C5b-9 by LCA indicated C3/C5 convertase formation with activation proceeding to completion. Biochemical and electron microscopic evaluations revealed that much of the C5b-9 present in atherosclerotic lesions is membraneous, rather than fluid phase SC5b-9. The observations reported herein establish a link between lipid insudation and inflammation in atherosclerotic lesions via the mechanism of complement activation.

The interaction between the complement components in human serum and the dye, Cibacron Blue F3GA, immobilized on cross-linked agarose (Affi-Gel Blue) has been studied. All nine components of the classical complement pathway bound to the dye and could be recovered using a linear salt gradient. With the exception of C5 and C8, all the components were eluted over a narrow NaCl concentration range, with the following yields: C1, 17%; C2, 69%; C3, 92%; C4, 87%; C6, 105%; C7, 109%; C9, 128%. C5 and C8 eluted throughout the NaCl gradient with yields of 103% and 14%, respectively. Since all components could be eluted without substantial contamination by albumin or IgG, this procedure may prove valuable as an initial step in the purification of complement components. In addition, the ability of immobilized Cibacron Blue F3GA to physicallly remove complement components may prove useful for both the decomplementation of serum and in elucidating the role of complement in immunological reactions.

We have devised an enzyme-linked immunosorbent assay (ELISA) to quantitate fluid phase terminal complement pathway activation. Upon activation to form C5b-9, terminal complement components express neoantigens not present in the unassembled individual components. Expression of one of these neoantigens occurs at the step of C9 activation. C9 neoantigen is present in fluid phase SC5b-9 complexes, membrane-bound MC5b-9 complexes, and in in vitro polymerized C9. Under physiologic conditions, the presence of C9 neoantigen indicates that the terminal complement pathway is activated through the terminal component C9. In our assay for C9 neoantigen, we used rabbit antiserum to polymerized C9 rendered specific for C9 neoantigenic determinants by serial absorption with human serum, human C9, and other terminal complement components bound to Sepharose. Using the IgG from this antiserum, we devised a sandwich ELISA to bind SC5b-9 from solution onto polystyrene plates. The ELISA plates were developed with the use of goat antiserum to native C9 epitopes followed by a swine anti-goat IgG-alkaline phosphatase conjugate. Quantitation of SC5b-9 in solution was performed by comparing sample OD to a standard curve generated with human SC5b-9 that was purified from zymosan-activated serum. The assay was sensitive to as little as 100 ng of SC5b-9/ml and should be useful for screening plasma, serum, cerebrospinal fluid, or other biological fluids for the presence of terminal complement pathway activation.

A new type of activation of the terminal route of complement is described. It occurs in precipitates of mouse serum prepared with a critical amount of polyethylene glycol. The activation proceeds in the presence of 10 mM EDTA and in the absence of C1q and functional C2, C3, C4 and factor B, which suggests that it is classical and alternative pathway-independent. The activation is demonstrated by the generation of both thermo-labile haemolytic and thermo-stable chemotactic activity. For both activities C5 seems to be essential. The haemolytic activity does not show species restriction towards sheep erythrocytes, which suggests a similarity with C56-initiated (reactive) but not with (S)C5-9-mediated (deviated) haemolysis. The identity of the haemolytic complex and the chemoattractant and the possible use of the new activation for the functional analysis of the mouse terminal complement route will be the subject of further study.

The known cytolytic function of the alternative pathway in serum was quantitatively reproduced by combining 11 isolated plasma proteins at their respective serum concentrations. These proteins are: C3, Factor B, Factor D, C3b inactivator, beta1H, native properdin, C5, C6, C7, C8, and C9. In absence of activators of the alternative pathway, this mixture was stable at 37 degrees C as evidenced by lack of consumption of Factor B, C3, and C5. Upon addition of either rabbit erythrocytes or neuraminidase-treated sheep erythrocytes, cell lysis ensued and the extent of lysis was dependent on dose of the component mixture. The dose-response curves obtained with the isolated component mixture and with C4-depleted serum were virtually indistinguishable. Nonactivator erythrocytes (untreated sheep erythrocytes) were not lysed by the component mixture. Deletion of properdin resulted only in a twofold diminution of the hemolytic activity of the component mixture. No immunoglobulin requirement was apparent. These results indicate that the cytolytic systems studied are internally sufficient and capable of coupling the initiation and amplification sequence with the cytolytic membrane attack sequence.

A simple three-step method was described for purification of murine beta 1H, one of the essential regulatory proteins of complement system. The method consists of heparin-Sepharose affinity chromatography; gel filtration on a Sepharose 6B column, and DNA-cellulose affinity chromatography. By this method over 10 mg of beta 1H can be purified by more than 200-fold from 100-ml of EDTA serum of various strains. Overall yield of beta 1H was about 45%. The purified beta 1H was homogeneous as judged by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis. The purified mouse beta 1H showed physicochemical properties very similar to those described for human beta 1H: mouse beta 1H is a beta-globulin consisting of a single polypeptide chain of molecular weight of 160,000. Purified mouse beta 1H retained its functional activity as the essential cofactor for the cleavage of fluid-phase human C3b by the human C3b inactivator. Immunization of rabbits with the purified mouse beta 1H resulted in the production of the potent and monospecific antibody.