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Understanding how human complement proteins interact with human antibodies is important for the development of antibody therapies and understanding autoimmune diseases. At present, many groups use baby rabbit serum as a source of complement because, in contrast to human serum, it lacks preexisting antibodies. However, for characterization of human (monoclonal) antibodies, human serum would be a preferred source of complement. To prevent complement activation via naturally occurring antibodies, this human serum ideally lacks IgG and IgM. Here we describe how to deplete human serum of naturally occurring IgG and IgM using fast protein liquid affinity chromatography (FPLC) while minimizing the loss of serum complement activity. We also describe assays that can be used to validate depletion of IgG and IgM (IgG, IgM, and C1q sandwich ELISAs) and functionally assess remaining serum complement activity (hemolytic assays CH50 and AH50). Finally, we demonstrate how captured IgG and IgM can be purified.

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.)

C5 plays a major role in complement activation; C5 convertase cleaves C5 into the pro-inflammatory C5a, and C5b, the nidus for the formation of the lytic membrane attack complex. C5 is a major target for anti-complement drugs, necessitating better methods for the study of C5 function. Purification of C5 is complicated; classical methods involve precipitation or pH shifts that result in functional loss and low yield. We here present a method for C5 purification using a novel anti-C5 monoclonal antibody (mAb); RO7112689 (C5i mAb, SKY59), pH-switch engineered to induce antibody-antigen dissociation in the acidic endosome (~ pH 5·5). RO7112689 was bound on an affinity column; applied serum was completely depleted of C5. Elution at pH 5 produced fully active C5 at 98% yield. The mAb also bound C5b in the C5b6 complex, preventing C5b6 binding to target membranes and enabling purification of C5b6 from activated serum. RO7112689 inhibited C5 in mouse serum and efficiently purified mouse C5. Used as capture, RO7112689 produced sensitive and specific assays for human and mouse C5. This novel antibody enables efficient production of intact, fully active, pure human and mouse C5, and quantification of C5 in these species. The demonstration that RO7112689 binds C5b6 adds an additional mechanism of membrane attack complex inhibition by this mAb., (© 2018 The Authors. Immunology Published by John Wiley & Sons Ltd.)

To explore the mechanism of psoriasis vulgaris (PV), serum protein expression profiles between PV patients with blood-heat syndrome and healthy volunteers were detected by isobaric tags for relative and absolute quantitation (iTRAQ). First, sera from 15 PV patients with blood-heat syndrome and 10 healthy volunteers were collected; then, serum proteins were separated and hydrolyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and a specific iTRAQ marker enzyme respectively after further purification and protein abundance treatment. Compared with the control group, differentially expressed proteins in PV patients with blood-heat syndrome were identified and analyzed by tandem mass spectrometry. A total of 787 proteins were identified and 718 proteins had a functional annotation with gene ontology (GO) by iTRAQ in the current study. Significant differences (P <0.05) and great differences (P <0.01) were found in 681 proteins and 536 proteins respectively between the patient group and healthy group. ). Different protein expression profiles in serum existed between PV patients with blood-heat syndrome and healthy volunteers; the differences largely involved immune-related proteins and lipoproteins. The proteins specific for PV with blood-heat syndrome deserves further investigation.

The pentraxin serum amyloid P component (SAP) is secreted by the liver and found in plasma at a concentration of approximately 30 mg/L. SAP is a 25 kDa homopentamer known to bind both protein and nonprotein ligands, all in a calcium-dependent manner. The function of SAP is unclear but likely involves the humoral innate immune system spanning the complement system, inflammation, and coagulation. Also, SAP is known to bind to the generic structure of amyloid deposits and possibly to protect them against proteolysis. In this study, we have characterized the SAP interactome in human plasma containing the physiological Ca 2+ concentration using SAP affinity pull-down and co-immunoprecipitation experiments followed by mass spectrometry analyses. The analyses resulted in the identification of 33 proteins, of which 24 were direct or indirect interaction partners not previously reported. The SAP interactome can be divided into categories that include apolipoproteins, the complement system, coagulation, and proteolytic regulation.

The complement system has been studied for about 120 years. Progress in defining this large and complex system has been dependent on the research technologies available, but since the introduction of protein chromatography, electrophoresis, and antibody-based assay methods in the 1950s and 60s, and sequencing of proteins and DNA in the 70s and 80s, there has been very rapid accumulation of data. With more recent improvements in 3D structure determination (nmr and X-ray crystallography), the structures of most of the complement proteins have now been solved. Complement research since 1990 has been greatly stimulated by the discoveries of the multiple proteins in the lectin pathway, the strong association of Factor H, C3, Factor B allelic variants with adult macular degeneration and atypical haemolytic uremic syndrome, and the introduction of the anti-C5 monoclonal antibody as a therapy for paroxysmal nocturnal hemoglobinuria and atypical haemolytic uremic syndrome. Potential new roles for complement in tissue development and the search for novel therapeutics suggest a very active future for complement research., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

C3 glomerulopathies (C3G) are a group of severe renal diseases with distinct patterns of glomerular inflammation and C3 deposition caused by complement dysregulation. Here we report the identification of a familial C3G-associated genomic mutation in the gene complement factor H–related 1 (CFHR1), which encodes FHR1. The mutation resulted in the duplication of the N-terminal short consensus repeats (SCRs) that are conserved in FHR2 and FHR5. We determined that native FHR1, FHR2, and FHR5 circulate in plasma as homo- and hetero-oligomeric complexes, the formation of which is likely mediated by the conserved N-terminal domain. In mutant FHR1, duplication of the N-terminal domain resulted in the formation of unusually large multimeric FHR complexes that exhibited increased avidity for the FHR1 ligands C3b, iC3b, and C3dg and enhanced competition with complement factor H (FH) in surface plasmon resonance (SPR) studies and hemolytic assays. These data revealed that FHR1, FHR2, and FHR5 organize a combinatorial repertoire of oligomeric complexes and demonstrated that changes in FHR oligomerization influence the regulation of complement activation. In summary, our identification and characterization of a unique CFHR1 mutation provides insights into the biology of the FHRs and contributes to our understanding of the pathogenic mechanisms underlying C3G.

Sepsis is one of the main causes of death in critically ill patients. The pathophysiology of sepsis is complex and not completely understood. The proinflammatory and anti-inflammatory response leads to cell and organ dysfunction and, in many cases, death. Thus, the goal of the intervention is to restore the homeostasis of circulating mediators rather than to inhibit selectively the proinflammatory or anti-inflammatory mediators. Blood purification has been reported to remove a wide array of inflammatory mediators. The effects are broad-spectrum and auto-regulating. Blood purification has also been demonstrated to restore immune function through improving antigen-presenting capability, adjusting leukocyte recruitment, oxidative burst and phagocytosis, and improving leukocyte responsiveness. A great deal of work has to be done in order to find and optimize the best extracorporeal blood purification therapy for sepsis. New devices specifically target the pathophysiological mechanisms involved in these conditions. High-volume hemofiltration, hemoadsorption, coupled plasma filtration adsorption, and high cutoff membrane are now being tested in septic patients. Preliminary data indicate the feasibility of these modified techniques in sepsis. Their impact on patient prognosis, however, still needs proof by large randomized clinical trials. Finally, the emerging paradigm of sepsis-induced immune suppression provides additional rationale for the development of extracorporeal blood purification therapy for sepsis., (2010 S. Karger AG, Basel.)

Global nucleotide excision repair is greatly attenuated in terminally differentiated mammalian cells. We observed this phenomenon in human neurons and in macrophages, noting that the transcription-coupled repair pathway remains functional and that there is no significant reduction in levels of excision repair enzymes. We have discovered that ubiquitin-activating enzyme E1 complements the repair deficiency in macrophage extracts, and although there is no reduction in the concentration of E1 upon differentiation, our results indicate a reduction in phosphorylation of E1. In preliminary studies, we have identified the basal transcription factor TFIIH as the potential target for ubiquitination. We suggest that this unusual type of regulation at the level of the E1 enzyme is likely to affect numerous cellular processes and may represent a strategy to coordinate multiple phenotypic changes upon differentiation by using E1 as a "master switch."

The complement system has been thought to originate exclusively in the deuterostomes. Here, we show that the central complement components already existed in the primitive protostome lineage. A functional homolog of vertebrate complement 3, CrC3, has been isolated from a 'living fossil', the horseshoe crab (Carcinoscorpius rotundicauda). CrC3 resembles human C3 and shows closest homology to C3 sequences of lower deuterostomes. CrC3 and plasma lectins bind a wide range of microbes, forming the frontline innate immune defense system. Additionally, we identified CrC2/Bf, a homolog of vertebrate C2 and Bf that participates in C3 activation, and a C3 receptor-like sequence. Furthermore, complement-mediated phagocytosis of bacteria by the hemocytes of horseshoe crab was also observed. Thus, a primitive yet complex opsonic complement defense system is revealed in the horseshoe crab, a protostome species. Our findings demonstrate an ancient origin of the critical complement components and the opsonic defense mechanism in the Precambrian ancestor of bilateral animals.

Therapeutic apheresis is divided in cytapheresis and plasmapheresis. And plasmapheresis(PP) is divided into three treatments, plasma exchange(PE), double filtration plasmapheresis(DFPP) and immunoadsorption plasmapheresis(IAPP). PE has been applied in the neuroimmunological disorders and the effectiveness of PP has been well established in some neuroimmunological disorders. In this article, PP treatment of multiple sclerosis(MS) was reviewed. PP is an effective means of removing the pathogenic immune-mediated factors, such as inflammatory cytokines, autoantibodies, immune complexes, and complements. PP may affect not only humoral immune responses but also cellular immune responses. Previous clinical reports suggested that PE might be effective in treating acute attacks of MS, but be no effective in patients with chronic progressive MS. IAPP may be superior to PE in the treatment of MS.

The low-density lipoprotein receptor-related protein (LRP) is a large surface receptor that mediates binding and internalization of a large number of structurally and functionally unrelated ligands. The ligand binding sites are located in clusters of complement-type repeats (CR), where the general absence of mutual binding competition suggests that different ligands map to distinct sites. Binding of alpha(2)-macroglobulin-protease complexes to the LRP is mediated by the receptor binding domain (RBD) of alpha(2)-macroglobulin (alpha(2)M). To determine the major binding epitope(s) in the LRP, we generated a complete set of tandem CR proteins spanning the second cluster of CR domains, and identified a binding site for alpha(2)M in the N-terminal part of the cluster comprising CR3-CR6, using ligand blotting and surface plasmon resonance (SPR) analysis. The specific site involved in alpha(2)M recognition resides in the fourth CR domain, CR4, whereas another site is identified in CR5. An acidic epitope in CR4 is identified as important for binding alpha(2)M by mutagenesis and SPR analysis. The formation of the complex between the rat alpha(1)-macroglobulin RBD and CR domain pairs is characterized by analytical size-exclusion chromatography, which demonstrates a sufficiently strong interaction between the alpha(1)M RBD and CR34 or CR45 for the isolation of a complex.

To prevent hyperacute xenograft rejection (HXR) caused by preformed natural antibodies (XNAb) after orthotopic heart xenotransplantation (oXHTx) of landrace pig hearts into baboons, we used immunoadsorption of immunoglobulins IgG, IgM and IgA and complement with the reusable Ig-Therasorb column. In addition to functional data, tissue was sampled for histological, immunohistochemical and electron microscopical analysis. We performed three oXHTx of landrace pig hearts to baboons using extracorporeal circulation (ECC) connected to the immunoadsorption unit. Intraoperative treatment consisted of four cycles of immunoabsorption (IA). One oXHTx of a baboon without IA served as a control. A mismatch of donor and recipient heart size was prevented by selecting a 30-40% lower body weight of donor pigs than recipients. Four cycles of IA removed more than 80% of IgG, IgM and IgA, 86% of antipig antibodies and 66% of complement factors C3 and C4 from plasma. The graft of the control animal failed after 29 min. Orthotopic xenotransplantation with IA was selectively terminated after 100 min, 11 h and 21 h, respectively without any histological signs of HXR in light and electron microscopy. After weaning off from ECC these donor xenografts showed sufficient function with normal ECG and excellent cardiac output in echocardiography and invasive measurement (1.93 +/- 0.035 l/min). The myocardium of the control xenograft demonstrated more deposits of Ig and complement components (C3, C4) than in the IA group. Baboons survive HXR after orthotopic pig heart xenotransplantation due to antibody depletion by reusable Ig-Therasorb column treatment. Long-term survival in an orthotopic baboon xenotransplantation model after IA, especially in combination with transgenic pig organs, could be a reliable preclinical trial for future clinical xenotransplantation programs.

Background: Sepsis is associated with enhanced cytokine production. Here, we examined the in vitro removal of plasma cytokines during continuous plasmafiltration coupled with sorbent adsorption., Methods: Proinflammatory (tumour necrosis factor-alpha, interleukins-1, -8) and anti-inflammatory (interleukin 1 receptor antagonist, soluble tumour necrosis factor receptor type I and II) cytokines in whole blood spiked with Escherichia coli endotoxin were determined during 2-h recirculation in the ultrafiltrate (condition A), plasma filtrate (condition B), before and after different sorbents (of the Amberlite-, Amberchrome- Ambersorb -type and charcoal). We studied the maximal adsorbing capacity, the 1% leakage test for cytokines and C3a des Arg and the adsorption of complement-dependent leukocyte chemiluminescence. Plasma proteins eluted from the resins were examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting with an anti-human alpha2-macroglobulin., Results: In condition B, we observed a 40- and 121-fold % increase (vs condition A) in the removed mass and clearance of tumour necrosis factor-alpha. For all other cytokines, the removed mass and the clearance increased from 2.3- up to 6-fold. The Amberchrome but not the Amberlite or Ambersorb resins could remove the highest amount of cytokines and could reduce complement-dependent chemiluminescence. Two protein bands of approximately 400,000 D and 200,000 D were eluted only from Amberchrome resins and immunoprecipitated by anti-human alpha2-macroglobulin and anti-human C3c antibodies, respectively., Conclusions: These studies suggest an efficient removal of cytokines in continuous plasmafiltration with sorbent adsorption. The binding of alpha2-macroglobulin, a carrier of cytokines in plasma, might be a additional mechanism in the removal of cytokines from plasma.

An opsonic molecule that is designated the third component of hagfish complement (HC3), and a fragment of HC3 known as HC3b have recently been identified in the hagfish, Eptatretus burgeri. These proteins were purified from plasma and generated a set of several bands and/or smears during SDS-PAGE under standard, non-reducing conditions. Two-dimensional electrophoretic analysis of the proteins under non-reducing and reducing conditions revealed the breakdown of polypeptides at the site of a thioester bond and the concomitant partial release of a split product, depending on the weak covalent or non-covalent association of polypeptide chains, in a large fraction of molecules of HC3 during SDS-PAGE. Moreover, the heterogeneity of HC3b can be ascribed to the different configurations of subunits. A similar phenomenon was not observed in the case of lamprey C3, even though breakdown of polypeptides at a thioester bond did occur in some molecules.

To investigate whether circulating immune complexes can be used as a disease marker for assessment of the activity of Lyme disease and for monitoring patients response to treatment, we tested 104 sera from patients with different stages of Lyme disease using the C1q enzyme-linked immunosorbent assay (ELISA) and a modified Raji cell test. Among 62 sera of patients with clinically active disease 27 sera (43.5%) reacted positively in the C1q-ELISA and 21 sera (33.9%) positively in the Raji cell test. In contrast, serum circulating immune complexes were found in less than 10% of 42 sera after antibiotic treatment. Similar results were obtained by both tests in 35 cerebrospinal fluid samples from patients with neuroborreliosis. Most importantly, dot blot analysis revealed the presence of both Borrelia burgdorferi-specific antigen(s) and host-derived components in the isolated immune complexes from serum samples of patients with active Lyme disease. These results indicate that detection of circulating immune complexes may be an useful parameter for judging the activity of Lyme disease. Moreover, preliminary characterization of spirochete-specific immune complexes implies new pathophysiological aspects of Lyme disease.

The amnion is a metabolically active tissue that has been identified as a site of synthesis of numerous products. We report that amnion tissue explants and amnion-derived epithelial cells synthesize and secrete six proteins of the complement system, C1r, C1s, C1 inhibitor, factor B, C3, and factor H. Synthesis of C2 was minimal and variable, and C5 was not detected. The six synthesized proteins had size and subunit composition characteristic of proteins synthesized in HEp2, a long term cell line derived from malignant epithelial cells. Constitutive and regulated synthesis of five of the six proteins was similar in amnion tissue and cells. However, synthesis of factor B was different in tissue and cells; constitutive synthesis was 12-fold higher in tissue than in cells, and interleukin-1 did not alter synthesis in tissue, but increased synthesis by 11.7-fold in cells. These results indicate that amnion may be a source of complement proteins present in the amnion fluid and may contribute to local host defense along with endometrial glandular epithelial cells, which synthesize C3. Furthermore, our results suggest that amnion tissue is stimulated in vivo to synthesize factor B and cannot respond to interleukin-1 with a further increase in the synthesis rate.

Normal human sera contained 10-60 ng/ml of soluble membrane cofactor protein (MCP, CD46) whereas sera of > 50% of the cancer patients contained > 60 ng/ml. MCP purified by immunoaffinity chromatography from both normal and cancer patients' sera consisted of three bands of 56, 47 and 29 kDa on SDS-PAGE/immunoblotting. The upper two components were increased in cancer patient sera. The 56 and 47 kDa soluble forms served as a cofactor for factor I-mediated cleavage of C3b. MCP expressed on Chinese hamster ovary (CHO) cells protects host cells from human C3 deposition and complement-mediated cytolysis, especially by activation of the alternative pathway. In this same assay system, exogenously added soluble MCP also protected untransfected CHO cells; however, its potency was much less than that of the endogenous membrane form. For example, 8 micrograms/ml of soluble MCP was equal to 10(4) copies/cell of the expressed MCP. Recombinant soluble forms possessed similar activity to the naturally occurring soluble forms and high doses (> 150 micrograms) blocked Arthus-like reaction induced in guinea-pigs by anti-Forssman antibody. These data establish that soluble forms of MCP are present in human sera that possess cofactor activity and their concentrations, especially the 56 and 47 kDa forms, are increased in sera of cancer patients. High doses of the recombinant soluble forms may be therapeutically useful for suppressing inflammatory responses.

Although the third component of complement, C3, has been isolated and its primary structure determined from most living classes of vertebrate, limited information is available on its structure and function for aves, which represent a significant stage in complement evolution. In this study, we present the complete cDNA sequence of chicken C3, the cDNA sequences of the thioester region for two chicken alpha 2-macroglobulin (alpha 2M)-related proteins, a simplified method for purifying chicken C3, and an analysis of the C3 convertase and factor I-mediated cleavages in chicken C3. Using the reverse-transcriptase PCR, with degenerate oligonucleotide primers derived from two conserved C3 sequences (GCGEQN/TM, TWLTAY/FV) and liver mRNA as template, we isolated three distinct 220-bp PCR products, one with a high degree of sequence similarity to C3 and two to alpha 2M and pregnancy zone protein from other species. The complete cDNA sequence of chicken C3 was obtained by screening a chicken liver lambda gt10 library with the C3 PCR product and probes from the 5' end of the partial-length C3 clones. The obtained sequence is in complete agreement with the protein sequence of several tryptic peptides of purified chicken C3. Chicken pro-C3 consists of an 18-residue putative signal peptide, a 640-residue beta-chain (70 kDa), a 989-residue alpha-chain (111 kDa), and an RKRR linker region. It contains an internal thioester and three potential N-glycosylation sites, all in the alpha-chain. The convertase cleavage site, predicted to be Arg-Ser, was confirmed by sequencing the zymosan-bound C3 fragments generated upon complement activation. NH2-terminal sequencing of the purified C3 chains showed that 1) pro-C3 is indeed cleaved at the RKRR linker sequence to generate the mature two-chain molecule, and 2) the beta-chain of chicken C3 is blocked. The deduced amino acid sequence shows 54, 54, 54, 53, 52, 57, and 55% amino acid identities to human, mouse, rat, guinea pig, rabbit, cobra, and Xenopus C3, respectively, and an identity of 44, 31, and 33% to trout, hagfish, and lamprey C3, respectively. The identities to human C4, C5, and alpha 2M are 31, 29 and 23%, respectively. A phylogenetic tree for C3, C4, C5, and alpha 2M-related proteins was constructed based on the sequence data and is discussed.

In conclusion, the "M(r) 40kD" antigen is expressed on the apical face of colonic enterocytes often in spatial relation to immune complexes in active UC, apparently targeting an IgG1-mediated autoimmune attack. Thus, our findings support the notion that an autoimmune response to the "M(r) 40kD" antigen, with local production of specific IgG1, is a possible immunopathological mechanism(s) in UC.

We have devised a purification scheme for human complement proteins C3, C3u, and C5. Affinity chromatography is employed to isolate each of these three proteins in a highly purified form. Typical yields were 910, 30, and 53 mg for C3, C3u, and C5, respectively, from 3 liters of human plasma. These yields reflect 31 and 32% recoveries for C3 and C5, respectively. The major advantages of this protocol over other published procedures are the application of affinity chromatography to yield high quality purified complement components, and the fact that the purification of C3, C3u, and C5 is part of a broader scheme designed for the isolation of over 20 plasma proteins from a single batch of plasma.

The viability of the bovine embryo was monitored by measuring the early pregnancy factor (EPF). The EPF activity was measured by the rosette inhibition test before and after artificial insemination (AI) at natural estrus (n = 14), and after superovulatory treatment followed by embryo removal on day 7 after AI (n = 5). In the cows inseminated artificially at natural estrus, there were significant differences (p < 0.01) in the rosette inhibition titer (RIT) between pregnant and non-pregnant cows on day 13-16 and day 20-25 after AI. In the 8 pregnant cows, the RIT remained more than 5 from day 6-9 after AI. In the 6 non-pregnant cows, two patterns were observed. In one pattern, RIT rose transiently to more than 5 and decreased to less than 4 thereafter. In the other pattern, RIT remained less than 4 throughout the experimental period. The former pattern suggested early embryonic death, while the latter suggested that fertilization had not taken place or that early embryonic death had occurred before the first blood collection on day 6-9 after AI. In the cows superovulated followed by embryo removal on day 7 after AI, the RIT values were all less than 4 on the day of AI (day 0), rose to more than 5 on day 3 and thereafter then until the day of embryo removal on day 7. In 4 cows, the RIT decreased to less than 4 by 3 days after embryo removal, and in the remaining one cow, the RIT decreased to less than 4 by 7 days after embryo removal. These findings suggest that the measurement of EPF activity is useful for monitoring the viability of bovine embryos.

Membrane cofactor protein (MCP) regulates C activation by serving as a cofactor for the cleavage of C3b and C4b by the serine protease factor I. An MCP-like molecule on the inner acrosomal membrane of human spermatozoa has been characterized. Three mAb and a rabbit polyclonal antibody against MCP recognized the sperm protein. On SDS-PAGE, it migrated as a single band with a molecular mass of 38,000 and 44,000 Da under nonreducing or reducing conditions, respectively. The molecular mass was 10,000 to 20,000 Da less than the two forms of MCP expressed on others cells. The electrophoretic pattern, by one- and two-dimensional gel analysis, and the isoelectric point profile (4.5 to 5.0) of the sperm protein were similar among multiple individuals. In contrast to MCP of other cells, digestion with endoglycosidases did not alter either the m.w. or the pI of the protein, suggesting that it is a poorly or nonglycosylated form of MCP. The solubilized sperm protein bound C3 with broken thioester bond to Sepharose and possessed cofactor activity for factor I-mediated cleavage of C3 with the broken bond. A mAb that blocks the regulatory function of MCP inhibited the cofactor activity of the sperm lysate. Thus, the sperm protein is an antigenic and functional homologue of MCP but has the distinct structural features of a lower m.w. and an apparent lack of glycosylation. MCP may play an essential role in the survival of the acrosome-reacted spermatozoa by modulating C activation in the female genital tract.

Biodegradable rods made of polyglycolide (Dexon, Biofix) or lactide-glycolide copolymer (Vicryl) have been used for the past 5 years for internal fixation of a variety of fractures and osteotomies (Böstman et al. 1989, 1990b). Experience from using such rods for fixation of intraarticular osteochondral lesions seems to be less extensive. We report a case of severe synovial reaction to biodegradable rods used for fixation of osteochondritis dissecans of the knee and discuss possible reasons for the increased risk of foreign-body reactions when these rods are used intraarticularly.

The effects of sequential additions of purified human complement proteins C5b-6, C7, C8, and C9 to assemble the C5b-9 membrane attack complex (MAC) of complement on electrical properties of planar lipid bilayers have been analyzed. The high resistance state of such membranes was impaired after assembly of large numbers of C5b-8 complexes as indicated by the appearance of rapidly fluctuating membrane currents. The C5b-8 induced conductance was voltage dependent and rectifying at higher voltages. Addition of C9 to membranes with very few C5b-8 complexes caused appearance of few discrete single channels of low conductance (5-25 pS) but after some time very large (greater than 0.5 nS) jumps in conductance could be monitored. This high macroscopic conductance state was dominated by 125-pS channels having a lifetime of approximately 1 s. The high conductance state was not stable and declined again after a period of 1-3 h. Incorporation of MAC extracted from complement-lysed erythrocytes into liposomes and subsequent transformation of such complexes into planar bilayers via an intermediate monolayer state resulted in channels with characteristics similar to the ones produced by sequential assembly of C5b-9. Comparison of the high-conductance C5b-9 channel characteristics (lifetime, ion preference, ionic-strength dependence) with those produced by poly(C9) (the circular or tubular aggregation product of C9) as published by Young, J.D.-E., Z.A. Cohn, and E.R. Podack. (1986. Science [Wash. DC]. 233:184-190.) indicates that the two are significantly different.

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.

A case of angioedema due to acquired deficiency of the regulatory protein C1-esterase-inhibitor (C1-INH) is reported. The edematous attack occurred 3 1/2 weeks after initiation of successful therapy for autoimmune-hemolytic anemia in the course of long-standing non-Hodgkin's lymphoma. At the time of acute edema the complement profile was typical: virtual absence of C1-INH function was associated with diminished concentrations of the components of the classical pathway of complement (C1q, C1r, C1s, C2, C4) and reduced complement hemolytic activity (CH50). Anti-C1-INH-autoantibodies were not detected. The angioedema lasted for about one week, and no further attacks occurred during the five-months follow-up period. Although there was only a minor adjustment to the therapy, the C1q, C2, C4 and CH50 values gradually increased to levels close to the lower limit of the normal range, while C1r and C1s showed normal values. In contrast to most other reports, this case was characterized by angioedema which was precipitated only after initiation of appropriate treatment for the underlying disease rather than before therapy or even diagnosis of the underlying disease.

A group of 103 SID victims was compared with a group of 87 clinically treated infants at the same age with respiratory and enteral infections. It was stated that immunological parameters were in agreement in both groups. These findings show that SID victims are ill before sudden death.

Circulating immune complexes (CIC) containing HBsAg and HBeAg were identified in sera of 5 out of 6 children with hepatitis B mediated membranous glomerulonephritis. CIC were precipitated from sera by 3.5% PEG, washed and subsequently analysed after acid dissociation and trypsin digestion. HBsAg, anti-HBs and albumin; HBeAg, anti-HBe and anti-HBc were recovered from the isolated complexes and these findings are discussed. Analysis of 3.5% PEG mediated precipitate of human serum proteins showed the relatively high content of IgG classical pathway complement components: C1q, C4 and C3.

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.

Two forms of activated C1s, a subcomponent of the first component of complement, were present in preparations of C1 specifically purified from rabbit serum by affinity chromatography on IgG-Sepharose 6B and were separated by DEAE-cellulose chromatography in the presence of EDTA. These two activated C1s, designated C1s(I) and C1s(II), were indistinguishable with regard to hemolytic activity as well as C1s esterase activity, though they had different molecular weights. C1s(I) had a molecular weight of 106,000, consisting of H and L chains connected by disulfide bonds; the molecular weights of the chains were 70,000 and 36,000, respectively. On the other hand, C1s(II), with a molecular weight of 72,000, consisted of two chains each with a molecular weight of about 37,000, which were also connected by disulfide bonds. These results suggest that, in the case of rabbit C1s, the primary product of activation with C1r, C1s(I), may be susceptible to further cleavage of its H chain without any loss of C1s activity, resulting in the formation of C1s(II), though the active principle responsible for this conversion remains to be elucidated.

The binding of C8 and C9 from human serum to target erythrocytes was quantified, and the molecular stoichiometries of C9:C8 within terminal C5b-9(m) complexes were determined. Low doses of serum generated terminal complexes with mean C9:C8 ratios of 2 to 3:1, whereas complexes generated by highest serum doses harbored an average of six to eight C9/C8 molecules. From the collective biochemical and ultrastructural data, we concluded that heterogeneous populations of terminal complexes regularly form on target membranes; those containing high numbers of C9 molecules (greater than or equal to six to eight) exhibit the structure of the classical "lesion", whereas those containing low numbers of C9 do not exhibit this typical structure, although they probably still function as small pores. A major cause for this heterogeneity of the lesions derives from shortage of C9, which is naturally present in a 2 to 1 molar ratio relative to C8 in serum. Generation of terminal complexes harboring high numbers of C9 on erythrocyte membranes is possible in spite of this natural shortage because SC5b-9 does not form in the fluid phase to compete for C9 binding. If interrupted, the process of C9-C9 oligomerization cannot be recontinued, and "incomplete" C5b-9 complexes are unable to bind additional C9 upon reincubation with this component. The demonstrated heterogeneity of terminal complexes with respect to their C9 content may explain the functional heterogeneity of complement lesions observed previously by other investigators.

A short term microcytotoxicity assay system using radioisotope release as an index of target cell damage has been developed to evaluate immune killing of cultured adherent human colon cancer cell lines. Using this assay system, complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and spontaneous cell-mediated cytoxicity of adherent human colon cancer cell lines can be assessed in 4 hr or less. An essential step in the development of this system was the successful 51Cr-labeling of human colon cancer target cells with subsequent low spontaneous release. This was achieved through careful attention to cellular growth phase and medium pH during the labeling and assay period. In this microsystem, labeled colon cancer cells spontaneously released 51Cr at a mean rate of 2% per hr during the assay, a level low enough not to obscure specific cytotoxic responses. Complement-dependent cytoxicity was measured most conveniently over a 2-hr period, whereas antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity were optimal when measured over a 4-hr period.

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.

We recently showed that culture-derived metacyclic trypomastigotes (CMT), but not epimastigotes (Epi), of the Miranda 88 strain of Trypanosoma cruzi evade lysis by the human alternative complement pathway because of inefficient binding of factor B to complement component C3b on the parasite surface. These results suggested that CMT and tissue-culture-derived trypomastigotes (TCT), which also activate the alternative pathway poorly, might produce a molecule capable of interfering with factor B binding to C3b. We now demonstrate that CMT and TCT lysates, as well as molecules spontaneously shed from CMT and TCT but not Epi, accelerate decay of 125I-labeled factor Bb from the alternative-pathway C3 convertase (C3bBb) assembled on zymosan or Epi and also accelerate decay of the classical-pathway C3 convertase (C4b2a) on sheep erythrocytes. Parasites metabolically labeled with [35S]methionine spontaneously shed a limited number of radioactive components ranging in molecular mass from 86 to 155 kDa for trypomastigotes and 25 to 80 kDa for Epi. Decay-accelerating activity within supernatants is inactivated by papain and is coeluted with 35S-containing polypeptides on FPLC anion-exchange chromatography, suggesting that the active constituents are protein molecules. Molecules with decay-accelerating activity may explain the developmentally regulated resistance to complement-mediated lysis in infective and vertebrate stages of the T. cruzi life cycle.

In order to understand immunological responses in psoriatic lesions, immunofluorescent findings have been compared in early psoriatic lesions and fully developed plaque lesions. IgG and IgA were detected as the main antibodies in the horny layer by immunohistology. The IgA at the site of leukocyte accumulation in the horny layer seemed to be associated with secretory component (S-component) and joining chian. Regarding complement, the alternate pathway might be related to the immune reaction as the initial response, because deposits of C3, properdin and glycine rich beta-glycoprotein were found more frequently than C1q-deposit in the both lesions. In the dermis S-component was also detected at the dermal papillae adjacent to the dermoepidermal (D-E) junction of the lesions and no deposit of S-component was observed in uninvolved skin of psoriatic patients. This, however, is not a specific pattern in psoriatic lesions since deposits of S-component were found at the dermal papillae and D-E junction in other dermatoses. These findings might suggest that the immunological response was initiated by secretory IgA in horny layer, which was composed of dimeric IgA and S-component at the D-E junction and which passed easily through the epidermal cells.

Glomerular involvement characterized by mesangial cell proliferation with fibrillar thickening of the axial region and deposits of immune complexes is reported in three human cases of kala-azar. IgG was seen in all 3 and Igm in 2 patients. Complement (C3) was detected in the glomeruli in all cases and fibrinogen in the only case in which it was tested for. The deposits appeared mainly along the mesangium and their staining was particularly strong for complement and IgG. Electron microscopy detected granular electron dense deposits mainly close to mesangial cells. In one case clumps made us of electron dense lamellae were seen in the glomerular basal membrane interpreted as evidence of focal membranolysis. No granulocytes were seen in the glomeruli. Attempts to demonstrate antigen were unsuccessful. The pattern of the lesion resembles that described in the kidney of human cases of hepatosplenic schistosomiasis, and the distribution of the deposits suggests that relatively large, poorly soluble complexes formed either in the presence of excess antigen or, under certain circumstances, in the presence of excess antibody, are trapped in the glomerular capillaries. The aggregates are partially shunted to the mesangial cells, which enlarge and proliferate.