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The role of complement in the pathogenesis of IgA nephropathy has been heavily explored over the past 50 years. This has led to the general acceptance that complement plays an important role in the clinical presentation and risk for progression of disease in patients with IgA nephropathy. Herein, we review the evidence for complement activation in IgA nephropathy, focusing on evidence that the lectin and alternate pathways are the main actors. We are entering an era of intense investigation of various inhibitors of complement, which should ultimately be the best indicator of contributions of the lectin, alternate and common complement pathways to disease burden. More importantly, we will see if these efforts result in the discovery of clinically relevant options in managing this important disease., (© 2024 Asian Pacific Society of Nephrology.)

Considering the fact that many retinal diseases are yet to be cured, the pathomechanisms of these multifactorial diseases need to be investigated in more detail. Among others, oxidative stress and hypoxia are pathomechanisms that take place in retinal diseases, such as glaucoma, age-related macular degeneration, or diabetic retinopathy. In consideration of these diseases, it is also evidenced that the immune system, including the complement system and its activation, plays an important role. Suitable models to investigate neuroretinal diseases are organ cultures of porcine retina. Based on an established model, the role of the complement system was studied after the induction of oxidative stress or hypoxia. Both stressors led to a loss of retinal ganglion cells (RGCs) accompanied by apoptosis. Hypoxia activated the complement system as noted by higher C3 + and MAC + cell numbers. In this model, activation of the complement cascade occurred via the classical pathway and the number of C1q + microglia was increased. In oxidative stressed retinas, the complement system had no consideration, but strong inflammation took place, with elevated TNF , IL6 , and IL8 mRNA expression levels. Together, this study shows that hypoxia and oxidative stress induce different mechanisms in the porcine retina inducing either the immune response or an inflammation. Our findings support the thesis that the immune system is involved in the development of retinal diseases. Furthermore, this study is evidence that both approaches seem suitable models to investigate undergoing pathomechanisms of several neuroretinal diseases.

The underlying mechanisms of complement activation in Stargardt disease type 1 (STGD1) and age-related macular degeneration (AMD) are not fully understood. Overaccumulation of all-trans-retinal (atRAL) has been proposed as the pathogenic factor in both diseases. By incubating retinal pigment epithelium (RPE) cells with atRAL, we showed that C5b-9 membrane attack complexes (MACs) were generated mainly through complement alternative pathway. An increase in complement factor B (CFB) expression as well as downregulation of complement regulatory proteins CD46, CD55, CD59, and CFH were observed in RPE cells after atRAL treatment. Furthermore, interleukin-1β production was provoked in both atRAL-treated RPE cells and microglia/macrophages. Coincubation of RPE cells with interleukin-1 receptor antagonist (IL1Ra) and atRAL ameliorated complement activation and downregulated CFB expression by attenuating both p38 and c-Jun N-terminal kinase (JNK) signaling pathways. Our findings demonstrate that atRAL induces an autocrine/paracrine IL-1/IL-1R signaling to promote complement alternative pathway activation in RPE cells and provide a novel perspective on the pathomechanism of macular degeneration., (© 2020 Wiley Periodicals LLC.)

The research described here presents data on the effect of galactans of red algae, carrageenans (λ/μ/ν-, κ-, κ/β-, and ι/κ-types), and agar on complement system activation in normal human serum. The experiments were based on well surfaces coated with triggering agents for binding initiating complement components -C3 and C4. The sulfated galactans inhibited C3 binding to lipopolysaccharide with direct dependence on the sulfation degree of polysaccharides. Sulfation degree was also important in carrageenans' capacity to reduce C4 binding to mannan. However, C4 binding to antibodies was considerably activated by carrageenans, especially with 3,6-anhydrogalactose. The gelling carrageenans were able to block antigen binding centers of total serum IgM and with more intensity than non-gelling. No structural characteristics mattered in ameliorating C5 cleavage by plasmin in extrinsic protease complement activation, but λ/μ/ν- and κ/β-carrageenans almost completely inhibited C5 cleavage. Thus, galactans participated in cell surface biology by imitating surface glycans in inhibition of C3 binding and mannose binding lectin, but as to the tthe heclassical pathway these substances stimulated complement, probably due to their structure based on carrabiose., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Approved for the treatment of autoimmune diseases, hematological malignancies, and solid cancers, several monoclonal antibodies (mAb) make use of complement in their mechanism of action. Such an assessment is based on comprehensive investigations that used mouse models, in vitro studies, and analyses from patients at initiation (basal level to highlight deficiencies) and after treatment initiation (mAb impact on complement), which have further provided key insights into the importance of the complement activation and/or complement deficiencies in mAb activity. Accordingly, new approaches can now be developed with the final objective of increasing the clinical efficacy of mAb. These improvements include (i) the concurrent administration of fresh frozen plasma during mAb therapy; (ii) mAb modifications such as immunoglobulin G subclass switching, Fc mutation, or IgG hexamerization to improve the fixation and activation of C1q; (iii) optimization of the target recognition to induce a higher complement-dependent cytotoxicity (CDC) and/or complement-dependant cellular cytotoxicity (CDCC); and (iv) the control of soluble and cellular complement inhibitors.

Eculizumab inhibits the terminal, lytic pathway of complement by blocking the activation of the complement protein C5 and shows remarkable clinical benefits in certain complement-mediated diseases. However, several reports suggest that activation of C5 is not always completely suppressed in patients even under excess of eculizumab over C5, indicating that residual C5 activity may derogate the drug's therapeutic benefit under certain conditions. By using eculizumab and the tick-derived C5 inhibitor coversin, we determined conditions ex vivo in which C5 inhibition is incomplete. The degree of such residual lytic activity depended on the strength of the complement activator and the resulting surface density of the complement activation product C3b, which autoamplifies via the alternative pathway (AP) amplification loop. We show that at high C3b densities required for binding and activation of C5, both inhibitors reduce but do not abolish this interaction. The decrease of C5 binding to C3b clusters in the presence of C5 inhibitors correlated with the levels of residual hemolysis. However, by employing different C5 inhibitors simultaneously, residual hemolytic activity could be abolished. The importance of AP-produced C3b clusters for C5 activation in the presence of eculizumab was corroborated by the finding that residual hemolysis after forceful activation of the classical pathway could be reduced by blocking the AP. By providing insights into C5 activation and inhibition, our study delivers the rationale for the clinically observed phenomenon of residual terminal pathway activity under eculizumab treatment with important implications for anti-C5 therapy in general., (© 2017 by The American Society of Hematology.)

Complement activation is of major importance in numerous pathological conditions. Therefore, targeted complement inhibition is a promising therapeutic strategy. C1-esterase inhibitor (C1-INH) controls activation of the classical pathway (CP) and the lectin pathway (LP). However, conflicting data exist on inhibition of the alternative pathway (AP) by C1-INH. The inhibitory capacity of C1-INH for the CP is potentiated by heparin and other glycosaminoglycans, but no data exist for the LP and AP. The current study investigates the effects of C1-INH in the presence or absence of different clinically used heparinoids on the CP, LP and AP. Furthermore, the combined effects of heparinoids and C1-INH on coagulation were investigated. C1-INH, heparinoids or combinations were analysed in a dose-dependent fashion in the presence of pooled serum. Functional complement activities were measured simultaneously using the Wielisa(®) -kit. The activated partial thrombin time was determined using an automated coagulation analyser. The results showed that all three complement pathways were inhibited significantly by C1-INH or heparinoids. Next to their individual effects on complement activation, heparinoids also enhanced the inhibitory capacity of C1-INH significantly on the CP and LP. For the AP, significant potentiation of C1-INH by heparinoids was found; however, this was restricted to certain concentration ranges. At low concentrations the effect on blood coagulation by combining heparinoids with C1-INH was minimal. In conclusion, our study shows significant potentiating effects of heparinoids on the inhibition of all complement pathways by C1-INH. Therefore, their combined use is a promising and a potentially cost-effective treatment option for complement-mediated diseases., (© 2016 British Society for Immunology.)

HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets) is a severe variant of pre-eclampsia whose pathogenesis remains unclear. Recent evidence and clinical similarities suggest a link to atypical hemolytic uremic syndrome, a disease of excessive activation of the alternative complement pathway effectively treated with a complement inhibitor, eculizumab. Therefore, we used a functional complement assay, the modified Ham test, to analyze sera of women with classic or atypical HELLP syndrome, pre-eclampsia with severe features, normal pregnancies, and healthy nonpregnant women. Sera were also evaluated using levels of the terminal product of complement activation (C5b-9). We tested the in vitro ability of eculizumab to inhibit complement activation in HELLP serum. Increased complement activation was observed in participants with classic or atypical HELLP compared with those with normal pregnancies and nonpregnant controls. Mixing HELLP serum with eculizumab-containing serum resulted in a significant decrease in cell killing compared with HELLP serum alone. We found that HELLP syndrome is associated with increased complement activation as assessed with the modified Ham test. This assay may aid in the diagnosis of HELLP syndrome and could confirm that its pathophysiology is related to that of atypical hemolytic uremic syndrome., (Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Atypical haemolytic uraemic syndrome (aHUS) is associated with (genetic) alterations in alternative complement pathway. Nevertheless, comprehensive evidence that the complement system in aHUS patients is more prone to activation is still lacking. Therefore, we performed a thorough analysis of complement activation in acute phase and in remission of this disease. Complement activation patterns of the aHUS patients in acute phase and in remission were compared to those of healthy controls. Background levels of complement activation products C3b/c, C3bBbP and terminal complement complex (TCC) were measured using enzyme-linked immunosorbent assay (ELISA) in ethylenediamine tetraacetic acid (EDTA) plasma. In vitro-triggered complement activation in serum samples was studied using zymosan-coating and pathway-specific assay. Furthermore, efficiencies of the C3b/c, C3bBbP and TCC generation in fluid phase during spontaneous activation were analysed. Patients with acute aHUS showed elevated levels of C3b/c (P < 0·01), C3bBbP (P < 0·0001) and TCC (P < 0·0001) in EDTA plasma, while values of patients in remission were normal, compared to those of healthy controls. Using data from a single aHUS patient with complement factor B mutation we illustrated normalization of complement activation during aHUS recovery. Serum samples from patients in remission showed normal in vitro patterns of complement activation and demonstrated normal kinetics of complement activation in the fluid phase. Our data indicate that while aHUS patients have clearly activated complement in acute phase of the disease, this is not the case in remission of aHUS. This knowledge provides important insight into complement regulation in aHUS and may have an impact on monitoring of these patients, particularly when using complement inhibition therapy., (© 2014 British Society for Immunology.)

Background: The complement system is a key component of innate immunity implicated in the neutralization and clearance of invading pathogens. Dextran coated superparamagnetic iron oxide (SPIO) nanoparticle is a promising magnetic resonance imaging (MRI) contrast agent. However, dextran SPIO has been associated with significant number of complement-related side effects in patients and some agents have been discontinued from clinical use (e.g., Feridex™). In order to improve the safety of these materials, the mechanisms of complement activation by dextran-coated SPIO and the differences between mice and humans need to be fully understood., Methods: 20 kDa dextran coated SPIO nanoworms (SPIO NW) were synthesized using Molday precipitation procedure. In vitro measurements of C3 deposition on SPIO NW using sera genetically deficient for various components of the classical pathway (CP), lectin pathway (LP) or alternative pathway (AP) components were used to study mechanisms of mouse complement activation. In vitro measurements of fluid phase markers of complement activation C4d and Bb and the terminal pathway marker SC5b-C9 in normal and genetically deficient sera were used to study the mechanisms of human complement activation. Mouse data were analyzed by non-paired t-test, human data were analyzed by ANOVA followed by multiple comparisons with Student-Newman-Keuls test., Results: In mouse sera, SPIO NW triggered the complement activation via the LP, whereas the AP contributes via the amplification loop. No involvement of the CP was observed. In human sera the LP together with the direct enhancement of the AP turnover was responsible for the complement activation. In two samples out of six healthy donors there was also a binding of anti-dextran antibodies and C1q, suggesting activation via the CP, but that did not affect the total level of C3 deposition on the particles., Conclusions: There were important differences and similarities in the complement activation by SPIO NW in mouse versus human sera. Understanding the mechanisms of immune recognition of nanoparticles in mouse and human systems has important preclinical and clinical implications and could help design more efficient and safe nano-formulations.

The species sensitivity and mechanism of complement pathway activation by a phosphorothioate oligonucleotide were investigated in monkey and human serum. Increasing concentrations of a phosphorothioate oligonucleotide, ISIS 2302, were incubated in either monkey or human serum. Complement activation in monkey serum was selective for the alternative pathway and occurred at concentrations ≥ 50 μg/mL ISIS 2302. By comparison, complement activation in human serum was absent. A similar difference in sensitivity for activation was also observed for a representative 2'-methoxyethyl (MOE)-modified oligonucleotide. The absence of oligonucleotide-induced complement activation was also observed in dogs. Protein binding with ISIS 2302 and enzyme competition studies suggested that factor H was important in oligonucleotide-mediated complement activation process, and addition of factor H to serum effectively prevented the activation in monkey serum. Furthermore, based on the immunoassay for factor H, there was an apparent decrease in factor H concentration as the ISIS 2302 concentration increased. This result suggests that ISIS 2302 binds to factor H and interferes with the factor H antibody from the immunoassay. Factor H is a regulatory protein that limits alternative pathway activation. Disruption of factor H interaction with C3 convertase by oligonucleotide could promote activation in this pathway.

Age-related macular degeneration (AMD) is a complex disease caused by genetic and environmental factors, including genetic variants in complement components and smoking. Smoke exposure leads to oxidative stress, complement activation, endoplasmic reticulum (ER) stress, and lipid dysregulation, which have all been proposed to be associated with AMD pathogenesis. Here we examine the effects of smoke exposure on the retinal pigment epithelium (RPE). Mice were exposed to cigarette smoke or filtered air for 6 months. RPE cells grown as stable monolayers were exposed to 5% cigarette smoke extract (CSE). Effects of smoke were determined by biochemical, molecular, and histological measures. Effects of the alternative pathway (AP) of complement and complement C3a anaphylatoxin receptor signaling were analyzed using knock-out mice or specific inhibitors. ER stress markers were elevated after smoke exposure in RPE of intact mice, which was eliminated in AP-deficient mice. To examine this relationship further, RPE monolayers were exposed to CSE. Short term smoke exposure resulted in production and release of complement C3, the generation of C3a, oxidative stress, complement activation on the cell membrane, and ER stress. Long term exposure to CSE resulted in lipid accumulation, and secretion. All measures were reversed by blocking C3a complement receptor (C3aR), alternative complement pathway signaling, and antioxidant therapy. Taken together, our results provide clear evidence that smoke exposure results in oxidative stress and complement activation via the AP, resulting in ER stress-mediated lipid accumulation, and further suggesting that oxidative stress and complement act synergistically in the pathogenesis of AMD., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Hyperactivity of the alternative complement pathway is the principle defect in C3 glomerulopathies (C3G). Eculizumab, a monoclonal antibody that binds C5 to prevent formation of the membrane attack complex, has been shown to be beneficial in some patients with this disease., Methods: In this open-label, proof-of-concept efficacy-and-safety study, a patient with the initial diagnosis of dense deposit disease (DDD) and allograft recurrence of C3 glomerulonephritis (C3GN) was treated with eculizumab every other week for 1 year. The patient had pathological evidence of C3GN and proteinuria >1 g/day at enrollment. He underwent graft biopsy before enrollment and repeat biopsy at 6 and 12 months., Results: Although no mutations were identified in complement genes, functional studies were positive for C3 nephritic factors and elevated levels of soluble membrane attack complex (sMAC). On therapy, sMAC levels normalized and although proteinuria initially decreased, it increased reaching pre-treatment levels at 12 months. Although serum creatinine remained stable, repeat allograft biopsies showed progression of disease., Conclusions: Clinical and histopathologic data suggest a partial response to eculizumab in this patient. While eculizumab blocked activation of the terminal complement cascade, persistent dysregulation of the alternative pathway remained, indicating eculizumab alone cannot control disease in this patient. Additional research is required to identify effective anticomplement therapy for this group of C3G patients.

Purpose of Review: The purpose of this review is to discuss emerging nomenclature, review the salient clinicopathological features and describe the therapeutic options available for the treatment of C3 glomerulopathy (C3G)., Recent Findings: C3G is minimally responsive to traditional immune suppression and randomized controlled trials to support therapy are absent. The burgeoning understanding of the role of the alternative complement pathway in C3G combined with animal data supporting the use of terminal complement blockade and a few reports suggesting that the anticomplement drug eculizumab may offer a therapeutic advantage have triggered great interest in the field of complement-mediated renal disease., Summary: Anticellular immune suppression and plasma therapy have limited efficacy in C3G. Data suggest that eculizumab may ameliorate disease in some C3G patients. The limited, recently published cohort data highlight crucial aspects of this group of diseases and support the need for extensive genetic and biomarker research to validate the pathologic mechanisms, delineate the spectrum of disease and guide the design of the rigorous trials to identify effective therapies for the treatment of C3G.

The complement system, which consists of more than 30 plasma and cell surface proteins, is activated by three pathways: the classical, lectin, and alternative pathways, leading to the generation of opsonins and pathogen destruction. In the lectin pathway, mannose-binding lectin (MBL) and ficolins act as pattern recognition molecules for pathogens, resulting in the activation of MBL-associated serine proteases (MASPs: MASP-1, MASP-2, and MASP-3). Among these proteases, MASP-2 is a key enzyme that cleaves C4 and C2 to assemble a C3 convertase (C4b2a). However, the physiological function of MASP-1 and MASP-3 remains unclear. To investigate the roles of MASP-1 and MASP-3, we generated a MASP-1- and MASP-3-deficient (M1/3 KO) mouse model and found that the deficient mice lacked alternative pathway activation because factor D (Df) remained as a proenzyme in the serum. MASP-1 and MASP-3 were able to convert the proenzyme of Df to an active form in vitro. In addition, MASP-1 was able to activate MASP-2 and MASP-3 as C1r activates C1s. Thus, MASP-1 and MASP-3 seem to be involved in activation of both the lectin and alternative pathways.

Hemolytic uremic syndrome (HUS) is commonly associated with Shiga toxin (Stx)-producing Escherichia coli O157:H7 infection. This study examined patient samples for complement activation on leukocyte-platelet complexes and microparticles, as well as donor samples for Stx and lipopolysaccharide (O157LPS)-induced complement activation on platelet-leukocyte complexes and microparticles. Results, analyzed by flow cytometry, showed that whole blood from a child with HUS had surface-bound C3 on 30% of platelet-monocyte complexes compared with 14% after recovery and 12% in pediatric controls. Plasma samples from 12 HUS patients were analyzed for the presence of microparticles derived from platelets, monocytes, and neutrophils. Acute-phase samples exhibited high levels of platelet microparticles and, to a lesser extent, monocyte microparticles, both bearing C3 and C9. Levels decreased significantly at recovery. Stx or O157LPS incubated with donor whole blood increased the population of platelet-monocyte and platelet-neutrophil complexes with surface-bound C3 and C9, an effect enhanced by costimulation with Stx and O157LPS. Both Stx and O157LPS induced the release of C3- and C9-bearing microparticles from platelets and monocytes. Released microparticles were phagocytosed by neutrophils. The presence of complement on platelet-leukocyte complexes and microparticles derived from these cells suggests a role in the inflammatory and thrombogenic events that occur during HUS.

Activation of the complement system significantly contributes to the pathogenesis of various acute and chronic inflammatory diseases. Current strategies to inhibit complement include the replacement or substitution of endogenous soluble complement inhibitors (e.g., C1 inhibitor [C1 inh], recombinant soluble complement receptor 1, TP10), the administration of antibodies to block key proteins of the cascade reaction (e.g., C5) or to neutralize the action of the complement-derived anaphylatoxins, or blockade of complement receptors (e.g., C5aR, CD88). The recent approvals of anti-C5 for the treatment of paroxysmal nocturnal hemoglobinuria as well as of C1 inh for the treatment of hereditary angioedema beyond European countries have provided a resurgence of interest in the potential of complement therapeutics for the treatment of disease., (© Thieme Medical Publishers.)

The complement system forms the first defense line of innate immunity and is activated within seconds upon infection by human pathogenic yeast Candida albicans. In this study, we identified a new complement evasion strategy used by C. albicans. The fungus secretes a potent complement inhibitor, pH-regulated Ag 1 (Pra1), which in the direct surrounding of the pathogen binds to fluid-phase C3 and blocks cleavage of C3 to C3a and C3b, as shown by ELISA, native gel electrophoresis, and Western blotting. Consequently, complement activation via the alternative and classical pathways is inhibited. In addition, the release of the anaphylatoxins C3a and C5a, as well as C3b/iC3b surface deposition, is reduced, as demonstrated by Western blotting, ELISA, confocal microscopy, and flow cytometry. By reducing C3b/iC3b levels at the yeast surface, Pra1 decreases complement-mediated adhesion, as well as uptake of C. albicans by human macrophages, as shown by flow cytometry. Thus, Pra1 is, to our knowledge, the first potent fungal complement inhibitor that favors C. albicans immune escape by inactivating and controlling host complement attack at the level of C3.

The complement system is important for host resistance to hematogenously disseminated candidiasis. However, modulation of complement activation by cell wall components of Candida albicans has not been characterized. Although intact yeast display mannan on the surface, glucan, typically located in the interior, becomes exposed during C. albicans infection. We show here the distinct effects of mannan and glucan on complement activation and opsonophagocytosis. Previous studies showed that intact cells are resistant to initiation of complement activation through the alternative pathway, and antimannan antibody reverses this resistance via an Fc-independent mechanism. The present study shows that this mannan-dependent resistance can be overcome by periodate-borohydride conversion of mannose polysaccharides to polyalcohols; cells treated with periodate-borohydride initiate the alternative pathway without the need for antibody. These observations identify an inhibitory role for intact mannan in complement activation. Next, removal of the surface-displayed mannan by acid treatment of periodate-borohydride cells exposes glucan. Glucan-displaying cells or purified beta-glucan initiate the alternative pathway when incubated with the purified proteins of the alternative pathway alone, suggesting that C. albicans glucan is a natural activator of the alternative pathway. Finally, ingestion of mannan-displaying cells by human neutrophils requires anti-mannan antibody, whereas ingestion of glucan-displaying cells requires complement. These results demonstrate a contrasting requirement of natural antibody and complement for opsonophagocytosis of C. albicans cells displaying mannan or glucan. Thus, differential surface expression of mannan and glucan may influence recognition of C. albicans by the complement system.

Complement plays an important role in the immune attack against invading microorganisms. However, blood-contacting medical device biomaterials lacking specific complement inhibitors, with free hydroxyl and/or amino groups, or with absorbed antibodies, may inappropriately activate complement. Inappropriate activation by either the antibody-mediated classical or the antibody-independent alternative pathway may have well-known acute or poorly understood chronic effects on the host or device. This article describes methods for screening biomaterials for functional whole complement activation using normal human serum, or specifically for alternative pathway activation using C4-deficient guinea pig serum, or for classical pathway activation using both sera in combination. Detailed protocols are available as standard methodologies from the American Society for Testing and Materials (ASTM F1984, ASTM F2065, and ASTM F2567). Results obtained with these functional tests are confirmed by detection of classical and alternative pathway-specific markers C4d and Bb, respectively. These methods demonstrate dose and time-course activation of complement to beaded agarose, cellulose acetate, and purified alginate as examples of biomaterials. Significant difference in a functional endpoint denoting specific complement pathways is an appropriate screening method for complement activation by medical device biomaterials which might result in adverse events when the device is implanted or contacts human blood.

This study was designed to determine the complement activation effects of carotenoid-derived aldehydes (CDA) on cultured human umbilical vein endothelial cells (HUVEC). A dose-dependent complement activation upon incubation of HUVEC with CDA was observed. Interestingly, the data showed that the alternative pathway was not activated. In addition, upon CDA treatment a significant number of apoptotic cells was also observed. The results revealed that CDA could activate the complement by way of the classical pathway. The study suggests that high carotenoid supplementation for the treatment of coronary heart disease should be used cautiously., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Phosphorothioate oligodeoxynucleotides can activate complement, and experimental murine studies have revealed differential effects upon simultaneous TLR stimulation and complement activation compared with either event alone. We set out to investigate the immune stimulatory effects of CpG 2006 in fresh non-anticoagulated human blood with or without presence of active complement. We also sought to elucidate the mechanism behind complement activation upon stimulation with phosphorothioate CpG 2006. In a human blood loop system, both backbone and sequence-specific effects by CpG were counteracted by selective inhibition of C3. Furthermore, DNA backbone-mediated CD40 and CD83 expression on monocytes and sequence-specific IL-6 and TNF production were reduced by complement inhibition. CpG-induced complement activation occurred via either the classical or the alternative pathway and deposits of both IgM and properdin, two activators of complement, were detected on CpG after incubation with EDTA plasma. Quartz crystal microbalance with dissipation monitoring demonstrated alternative pathway convertase build-up onto CpG as a likely pathway to initiate and sustain complement activation. Specific inhibition of C3 suppressed CpG 2006 uptake into monocytes indicating that C3 fragments are involved in CpG internalization. The interplay between complement and TLR9 signaling demonstrated herein warrants further investigation.

Animal data strongly support a role for inflammation in myocardial ischemia reperfusion injury. Attempts at cardioprotection by immunomodulation (such as with the specific C5 antibody pexelizumab) in humans have been disappointing. We hypothesized that a broader spectrum antiinflammatory agent might yield successful cardioprotection. The serine protease inhibitor nafamostat (FUT-175), which is already in clinical use, is a potent antiinflammatory synthetic serine protease inhibitor with anticomplement activity that we tested in a well-established rabbit model of 1 hour of myocardial ischemia followed by 3 hours of reperfusion. Compared to vehicle-treated animals, nafamostat (1 mg/kg of body weight) administered 5 minutes before reperfusion significantly reduced myocardial injury assessed by plasma creatine kinase activity (38.1 +/- 6.0 versus 57.9 +/- 3.7I U/g protein; P < 0.05) and myocardial necrosis (23.6 +/- 3.1% versus 35.7 +/- 1.0%; P < 0.05) as well as myocardial leukocyte accumulation (P < 0.05). In parallel in vitro studies, Nafamostat was a significantly more potent broad spectrum complement suppressor than C1 inhibitor. Nafamostat appears to have capability as an inhibitor of both complement pathways and as a broad-spectrum antiinflammatory agent by virtue of its serine protease inhibition. Administration of nafamostat before myocardial reperfusion after ischemia produced significant, dose-dependent cardioprotection. Reduced leukocyte accumulation and complement activity seem involved in the mechanism of this cardioprotective effect.

The fusion (F) protein of measles virus (MeV) activates the alternative pathway of human complement in the presence of both CD46 and CD55 which regulate the complement activation [Devaux, P., Christiansen, D., Plumet, S., Gerlier, D., 2004. Cell surface activation of the alternative complement pathway by the fusion protein of measles virus. J. Gen. Virol. 85, 1665-1673]. The original observation of cold detergent-resistant membranes sedimenting at a higher density than the membrane rafts lead us to analyse the respective distribution of F, CD46 and C55 molecules in what we call heavy rafts (HRs) and in the classical low-density membrane rafts (Rs). Membrane rafts were isolated after cold TX100 solubilization and flotation on a sucrose gradient. The denser fractions collected from the lower part of the gradient could be further separated into a translucent pellet (HR) and a soluble supernatant (S). HR and R were both sensitive to TX100 solubilization after cholesterol depletion and solubilized by octyl-d-glucoside but differed in their lipid and protein composition. A proteomic analysis revealed that the HR fraction was derived from heterogeneous cellular membranes including plasma membrane, early endosomes and rough endoplasmic reticulum. Interestingly, CD55 and CD46 almost exclusively associated with R and S fractions, respectively, while after MeV infection or transient expression, MeV-F distributed almost equally between R, HR and S fractions. However more immature MeV-F(0) than mature MeV-F(1) proteins was associated with the HR fraction whereas this ratio was reverse in R and S fractions. After activation of the alternative pathway of human complement by F expressing cells, both C3b and F protein associated with R, HR and S fractions. When four or five of the five cysteines located in the transmembrane and cytoplasmic tail of F protein were substituted with serine residues, the mutated F distributed almost exclusively in HR fractions and was still efficient in activating the complement. We propose that the partitioning of F, CD46 and CD55 molecules in different membrane microdomains could account for the ability of F to escape complement regulation by the CD55 and CD46 regulators.

Prion protein (PrP) is an endogenous protein involved in the pathogenesis of bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Murine PrP has been reported to bind C1q and activate the classical pathway of complement in a copper-dependent manner. Here we show that various conformational isoforms (native, amyloid fibrils, and beta-oligomers) of recombinant human PrP (90-231 and 121-231) bind C1q and activate complement. PrP binds both the globular head and collagenous stalk domains of C1q. Native, beta-oligomeric and amyloid fibrils of PrP all activate the classical and alternative pathways of complement to different extent. However, they do not trigger the lectin pathway. Of the tested PrP conformational isoforms we find that beta-oligomers bind C1q and activate complement most strongly. Membrane attack complex formation initiated by PrP is subdued in comparison to deposition of early complement components. This is most likely attributed to the interaction between human PrP and complement inhibitors factor H and C4b-binding protein. Accordingly, PrP-triggered complement activation in the terminal pathway was increased in serum lacking C4b-binding protein. Taken together the present study indicates that complement activation may be an important factor in human prion diseases, suggesting that complement induced activities may prove relevant therapeutic targets.

The aim of this study was to evaluate the immune responses in hybrid tilapia (Nile tilapia Oreochromis niloticus x Mozambique tilapia O. mossambicus) and Japanese eels Anguilla japonica after treatment with five glycans: barley, krestin, MacroGard, scleroglucan, and zymosan. The effects of the glycans on the innate immune responses of the fish were investigated using the phagocytic index (PI), lysozyme activity, complement opsonization, and activation assay. The results of the lysozyme assay demonstrated that the lysozyme activities increased after treatment with glycans. Moreover, based on the PI, treatment with each of the five glycans resulted in increased phagocytic activities in anterior kidney and peripheral blood phagocytes in both tilapia and Japanese eels. The opsonic effect of complement on phagocytosis in tilapia and Japanese eels were investigated using baker's yeast, which served as the activator in the classical complement pathway (CCP) and in the alternative complement pathway (ACP). Tilapia and Japanese eel sera that were treated with glycans greatly enhanced phagocytosis. The classical pathway--hemolytic complement titer (CH50) of Japanese eels treated with glycans was slightly increased in vitro and in vivo. While glycan treatment enhanced the CCP of both species in vitro and in vivo, the alternative pathway-hemolytic complement titer (ACH50) was only increased in vitro and in vivo in glycan-treated tilapia. Thus, it follows that the ACP must have been activated in tilapia treated with glycans. However, in Japanese eels, the ACH50 of the ACP activation assay was undetected in vitro or in vivo due to possible unknown factors in the Japanese eel serum that caused lysis of the rabbit red blood cells. Our study investigated the effects of glycans used to enhance phagocytosis and activate both of the complement pathways involved in stimulating the innate immune responses of Japanese eels and tilapia.

While the complement system is an essential component of immunity, shutting down all or part of it could be beneficial in a wide range of clinical situations. Designer, small-molecule, protease inhibitors and antagonists of protein-protein interactions are under development, while an approach based on a humanized monoclonal antibody to the C5 component works effectively against the later stages of complement activation and is close to completing clinical trials. The cobra venom factor depletes plasma of essential complement components, and a humanized (nonimmunogenic) version is being sought. Perhaps the most promising approach to comprehensive complement downregulation, however, is the exploitation of innate regulators of complement activation, with two products in clinical trials. The potential for more efficacious complement blockers of this kind is growing because of better targeting, but a deeper knowledge at the atomic level of mechanisms of action of these regulators is needed to underpin a rational approach to design of still more potent complement inhibitors.

The scorpion Tityus serrulatus is considered one of the most dangerous species in Brazil. Its venom evokes an inflammatory response, although the exact mechanism of this effect is still unknown. The aim of the present study was to investigate the effect of Tityus serrulatus venom (TsV) on the complement system (CS) and on leukocyte recruitment. Complement consumption by TsV was evaluated using in vitro hemolytic assays, immunoelectrophoresis and two-dimensional immunoelectrophoresis of complement components (factor B and C3). In order to evaluate neutrophil migration induced in normal human serum (NHS) in the presence of TsV, in vitro chemotaxis assays were performed using the Boyden chamber model. In vitro TsV induced a concentration- and time-dependent reduction in hemolytic activity of the classical/lectin and alternative complement pathways, with samples of 43.0 microg and 43.4 microg, respectively, inhibiting 50% of the lytic activity. Alterations in C3 and factor B electrophoretic mobility after incubation of NHS with TsV, were identical to those obtained with zymosan (positive control). Incubation of NHS with TsV induced neutrophil chemotaxis similar to that observed with zymosan-activated serum. Our results show that TsV activates the CS, leading to factor B and C3 cleavage, to reduction of serum lytic activity and generation of complement chemotactic factors. Therefore, CS may play an important role in the inflammatory response observed upon scorpion envenomation.

Background: An enhanced presence of 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) triggers the alternate pathway (AP) in Indian visceral leishmaniasis (VL). Antibodies directed against these epitopes are present in high titers. The biological relevance of these antibodies, with regard to activation of the classical pathway (CP), was investigated., Methods: Complement activators were affinity purified, complement activation via the CP, AP, and lectin-mediated complement pathway was measured by use of an anti-C3 radio-binding assay, and the number of C3 molecules was quantitated by Scatchard analysis. Cell death induced via the complement pathways was measured by use of MTT (tetrazolium salt 3- [4, 5-dimethylthiazol-2-yl] -2, 5-diphenyltetrazolium bromide) assay, and uptake of propidium iodide (PI) was measured by flow cytometry., Results: Anti-O-AcSGs from both healthy donors and patients with VL elicited C3 deposition as early as 3 min, which triggered parasite lysis, as demonstrated by use of MTT assay and corroborated by the high rate of uptake of PI. Analysis of complement activation by mannan-binding lectin and C-reactive protein demonstrated their negligible contribution during the 3-min time frame., Conclusions: Anti-O-AcSGs were identified as an important source of CP activation under normal physiological conditions, suggesting that they play a role in conferring host protection against parasite infection.

Staphylococcus aureus can cause a variety of acute and chronic diseases. The ability of S. aureus to cause persistent infections has been linked to its ability to evade or inactivate host immune responses. We have identified a secreted 19-kDa protein produced by S. aureus that binds to the complement protein C3. N-terminal sequencing of this protein identified it as the extracellular fibrinogen-binding protein (Efb). In this study, we demonstrate that Efb can bind to the alpha -chain of C3 and inhibit both the classical and alternative pathways of complement activation. In addition, we show that Efb can inhibit complement-mediated opsonophagocytosis in a dose-dependent manner and that Efb inhibits complement activity by blocking deposition of C3 or by preventing further complement activation beyond C3b. These data suggest that Efb is a virulence factor involved in facilitating persistent S. aureus infections by interfering with complement activity in vivo.

Statin drugs prevent coronary heart disease through anti-inflammatory mechanisms in addition to the well-known reduction of low-density lipoproteins. The complement system plays an essential role in the inflammatory response and has been postulated to be modified by statins. A direct role for statins in complement activation, however, has not been previously investigated. We therefore studied the effect of statins on in vitro complement activation. Pravastatin, atorvastatin and the active metabolite of the latter, ortho-hydroxy atorvastatin, were added to normal human serum and incubated for 1 h in the absence or presence of aggregated immunoglobulin (classical pathway activation) or cobra venom factor (alternative pathway activation). The degree of complement activation, as detected by specific complement-activation products for the classical pathway (C1rs-C1-inhibitor complexes), the combined classical and lectin pathway (C4bc), the alternative pathway (C3bBbP) and the final common pathway (C3bc and TCC), was not affected by pre-incubation of the serum with any of the statins. Statins do not affect complement activation directly, but indirect effects in vivo may well be operative.

The objective of this study was to define the role of complement activation in the acute and transient toxicities associated with administration of phosphorothioate oligonucleotides in monkeys. In the absence of complement inhibitor, complement activation blocker-2 (CAB-2), i.v. infusion of 20 mg/kg ISIS 2302 produced increases in the concentrations of the complement split products Bb and C5a (100- and 7-fold, respectively). Monkeys also experienced marked changes in bloodpressure (hypertension and hypotension), clinical signs of toxicity (lethargy and periorbital edema), fluctuations in circulating neutrophil counts, and elevations in serum cytokine levels (45-, 12-, and 4-fold increases in IL-6, MCP-1, and IL-12, respectively). Changes occurred at or near the end of infusion and returned to normal over time. One of the three animals died approximately 4 h following infusion of 20 mg/kg ISIS 2302 alone. In contrast, prior treatment with CAB-2 effectively blocked complement activation, as well as the ISIS 2302-induced hemodynamic and clinical responses. Importantly, plasma concentration of ISIS 2302 were unaffected by CAB-2 pretreatment. Thus, the protection afforded by CAB-2 was due to its inhibition of complement activation rather than to any impact on the disposition of ISIS 2302. These results clearly demonstrate the causal relationship between activation of the alternative complement pathway and the hemodynamic and clinical responses associated with rapid infusion of phosphorothioate oligonucleotides. Demonstration of this relationship underscores the importance of avoiding complement activation in patients to ensure the continued safe use of phosphorothioate oligodeoxynucleotides.

In vitro studies with miniaturized rotating circuits and heparinized human blood, as well as long-term extracorporeal membrane oxygenation with either heparin coated (HBS) or uncoated surfaces connected to adult sheep, were performed comparing the impact on complement activation in blood and on surfaces. Analysis of surface bound complement proteins revealed significantly reduced binding of activated C3 and C5b-9 to HBS in vitro, compared with uncoated surfaces, which was probably due to more HBS bound complement inhibitors (C1-Inhibitor, factor H) being present. This was reflected by significantly reduced activation of the alternative pathway (C3bBbP) and terminal complex (SC5b-9) by HBS but slightly increased levels of classic pathway complex (C1rs-C1-inhibitor). These results were confirmed during in vivo study by analysis of hemolytic complement function, activation specific C3 derived split products, and surface bound complement proteins. Increased binding of complement regulators to HBS appears to effectively reduce complement activation by biomaterials, leading to improved long-term biocompatibility.

Objectives: Patients undergoing cardiopulmonary bypass frequently manifest generalized systemic inflammation and occasionally manifest serious multiorgan failure. Inflammatory responses of bypass are triggered by contact of blood with artificial surfaces of the bypass circuits, surgical trauma, and ischemia-reperfusion injury. We studied the effects of specific inhibition of the alternative complement cascade by using an anti-factor D monoclonal antibody (166-32) in extracorporeal circulation of human whole blood used as a simulated model of cardiopulmonary bypass., Methods: Five healthy blood donors were used in the study. Monoclonal antibody 166-32 was added to freshly collected, heparinized human blood recirculated in a pediatric cardiopulmonary bypass circuit at a final concentration of 18 microg/mL. An irrelevant monoclonal antibody was used as a negative control with the same donor blood in a parallel bypass circuit on the same day. Blood samples were collected at different time points during recirculation for measurement of activation of complement, neutrophils, and platelets by immunofluorocytometric methods and enzyme-linked immunosorbent assays., Results: Monoclonal antibody 166-32 inhibited the alternative complement activation and the production of Bb, C3a, sC5b-9, and C5a. Upregulation of CD11b on neutrophils and CD62P on platelets was also significantly inhibited by monoclonal antibody 166-32. This is consistent with the inhibition of the release of neutrophil-specific myeloperoxidase and elastase and platelet thrombospondin. The production of proinflammatory cytokine interleukin 8 was also suppressed by the antibody., Conclusions: The alternative complement cascade is predominantly activated during extracorporeal circulation. Anti-factor D monoclonal antibody 166-32 is effective in inhibiting the activation of complement, neutrophils, and platelets. Inhibition of the alternative complement pathway by targeting factor D could be useful in reducing systemic inflammation in patients undergoing cardiopulmonary bypass.

A frequent long-term complication of total joint arthroplasty is aseptic loosening, the end result of wear debris accumulation, synovitis, and osteolysis about the implant-bone or cement-bone interface. Complement, an effector system in plasma, synovial fluid, and tissue, has powerful chemotactic, inflammatory, and osteoclast-activating potentials. This study explored the complement-activating ability of polyethylene, a material used in joint implants. In vitro hemolytic assays using sheep red blood cells (E(sh)), human serum, and particulate polyethylene suggested alternative pathway complement activation, as well as polyethylene adsorption of activated complement components. These results were confirmed by enzyme-linked immunosorbent assay (ELISA) quantification of activated complement factors Bb and C3b. In situ double antibody immunoperoxidase staining for factors Bb, C3a, iC3b, and SC5-9 in synovial tissue from revision hip specimens showed localized alternative pathway activation and component adsorption. These results introduce a likely role for complement activation in particle-mediated recruitment, proliferation, and activation of macrophages during early events in osteolysis and implant loosening., (Copyright 2000 John Wiley & Sons, Inc.)

Biomaterial-mediated complement activation repeatedly has been invoked as a trigger of phagocyte reactions and inflammation. However, a direct correlation between complement activation and inflammatory responses to biomaterial surfaces has yet to be established. Using an animal implantation model and gold surfaces bearing various thiol-linked functionalities, we investigated the potency of different surface groups in prompting complement activation in vitro and surface-mediated accumulation of inflammatory cells in vivo. Among the surfaces tested, mercaptoglycerol- and mercaptoethanol-bearing surfaces engendered the strongest inflammatory responses, as reflected by the accumulation of large numbers of adherent neutrophils and monocytes/macrophages. In contrast, L-cysteine-coated surfaces caused only minor inflammatory responses, and both glutathione-modified and untreated gold implants attracted minimal numbers of inflammatory cells. The accumulation of inflammatory cells on mercaptoglycerol surfaces appears to arise from surface-mediated complement activation because complement-depleted animals failed to exhibit inflammatory responses to mercaptoglycerol-modified implants. Furthermore, there is a close relationship between surface-mediated complement activation (as measured by in vitro iC3b/C5b-9 generation and C3 deposition) and in vivo inflammatory responses. At least in this animal model and with these model surfaces, our results indicate that surface-mediated complement activation can be responsible for the subsequent accumulation of inflammatory cells on implant surfaces.

The study conducted proved that triombrast (dose dependently) > hexabrics > Ultravist > or = melitrast = omnipac in a concentration interval of 0.03-30.0 mg iodine/ml in vitro and in a dose interval of 0.5-2.0 g iodine/kg in vivo activate the complement system (CS) according to the alternative way in the blood of "sensitive" rats. The degree of CS activation by radiopaque agents (ROA) is significantly determined mathematically by their viscosity and relation of the number of iodine atoms to the number of ions or dissolved particles, and by their hydrophilic (for nonion CS) and osmotic (for ion monomeric CS) properties.

The present study was undertaken to determine whether teicoplanin and vancomycin influence the hemolytic and bactericidal activity of serum obtained from mice (Swiss, aged 15 +/- 2 weeks). Haemolytic activity was measured in CH-50 units (which represents the capacity of serum complement to lyse 50% of sheep red blood cells in the presence of specific antibody) and the bactericidal activity was estimated from the number of colony-forming units (CFU/ml) of Staphylococcus aureus that survived after 24 h of incubation in the presence of serum. The results indicate that (1) teicoplanin and vancomycin increase the haemolytic activity of serum; (2) in the serum from animals treated with both teicoplanin or vancomycin, the number of CFU/ml of Staphylococcus aureus declines; and (3) these antibiotics appear to have a similar effect upon the complement system.

Complement activation and leukocyte stimulation were prospectively studied during and after cardiopulmonary bypass in 16 children receiving sodium nitroprusside--a nitrovasodilator releasing nitric oxide--for vasodilation during the cooling and rewarming periods of extracorporeal circulation. Results were compared with those in 29 patients who were not treated with sodium nitroprusside during the operation. Patients treated with sodium nitroprusside had significantly less C3 conversion during cardiopulmonary bypass as measured by the ratio C3d/C3 (p <0.05) and significantly less C5a liberation immediately after cardiopulmonary bypass (p < 0.005) than patients not treated with sodium nitroprusside. C4 was not overtly consumed in our series. Leukocyte count during the rewarming period of cardiopulmonary bypass, but not leukocyte elastase release during cardiopulmonary bypass, was significantly reduced in patients treated with sodium nitroprusside (p <0.05). In vitro experiments were conducted to analyze the effect of sodium nitroprusside on complement hemolytic activity initiated by the classic and the alternate pathways and on zymosan-induced C3 conversion by the activation of the alternate pathway. The in vitro experiments clearly demonstrate inhibition of complement hemolytic activity by sodium nitroprusside in the sera tested. The 50% inhibitory concentration of sodium nitroprusside on the available complement hemolytic activity was less through the alternate pathway than through the classic one (4.2 +/- 0.8 mmol/L and 14.0 +/- 2.88 mmol/L, respectively). The decrease of complement hemolytic activity measured was dose-dependent and was enhanced by the sodium nitroprusside preincubation of the sera tested. This effect was related to the duration of preincubation. Sodium nitroprusside photodegradation (enhancing nitric oxide release) increased the anticomplementary effect of the drug, reducing the 50% inhibitory concentration on complement hemolytic activity to 0.24 to 0.02 mmol/L for the alternate pathway and 2.74 o 0.3 mmol/L for the classic pathway. the zymosan-induced C3 conversion was inhibited by sodium nitroprusside. Nitroglycerin and isosorbide dinitrate (other nitric oxide donors) had in vitro effects on complement hemolytic activity similar to those of nonphotodegraded sodium nitroprusside at similar concentrations (1 mmol/L). Our results suggest that sodium nitroprusside, both in vitro and in vivo, has an inhibiting effect on complement activation initiated by both classic and alternate pathways and that this effect is mediated by nitric oxide release from sodium nitroprusside. This is the first report on the anticomplementary effect of sodium nitroprusside by nitric oxide release.

Background: Although it is well established that heparin-coated extracorporeal circuits reduce complement activation during cardiac operations, little in vivo information is available on the reduction in alternative and classic pathway activation., Methods: In a prospective, randomized study involving patients undergoing coronary artery bypass grafting with standard full heparinization, we compared heparin-coated circuits (Duraflo II) (10 patients) with uncoated circuits (10 patients) and assessed the extent of initiation of complement activation by detecting iC3 (C3b-like C3) concentrations, classic pathway activation by C4b/c (C4b, iC4b, C4c) concentrations, terminal pathway activation by soluble C5b-9 concentrations, and C3 activation by C3a (C3a desArg) and C3b/c (C3b, iC3b, C3c) concentrations., Results: Heparin-coated extracorporeal circuits significantly reduced circulating complement activation product C3b/c and soluble C5b-9 concentrations at the end of cardiopulmonary bypass and after protamine sulfate administration compared with the uncoated circuits, but not iC3, C4b/c, or C3a concentrations., Conclusions: Heparin-coated extracorporeal circuits reduce complement activation through the alternative complement pathway, probably at the C3 convertase level, and, consequently, the terminal pathway. C3b/c seems to be a more sensitive marker than C3a to assess complement activation during cardiac operations.

We investigated the ability of atheroma-associated liposomes and malondialdehyde (MDA)-modified low-density lipoproteins (MDA-LDL) to activate complement. Complement activation markers C3a, Bb, C4d and SC5b-9 were measured in both normal and complement-deficient sera. We found that MDA-LDL was able to generate C3a and SC5b-9, predominantly by the alternative pathway. High-density lipoproteins modified with MDA were also capable of C3a generation although to a lesser degree. The presence of atheroma-associated liposomes did not result in detectable levels of complement activation markers. We conclude that MDA-modified lipoproteins may represent a possible source for complement activation within atherosclerotic lesions.

In the present study, we demonstrate that natural sulfated polysaccharides (fucans) isolated from brown seaweed are potent inhibitors of human complement activation. A fucan fraction of chromatographic molecular weight 22,600, termed BS8, was found to inhibit classical and alternative pathway activation in whole serum in a dose-dependent fashion. Fucan BS8 inhibited formation of the classical pathway C3 convertase by interfering with C1 activation or by inhibiting C4 cleavage and the interaction between C4b and C2. The fucan also inhibited formation/function of the alternative pathway C3 convertase by suppressing the binding of B to C3b and by interfering with the stabilizing function of Properdin. The inhibitory effect of fucans on formation of the C3 convertases was dependent on the molecular weight of the polysaccharide for compounds of chromatographic molecular weight below 16,600. Fucan had no effect on the function of the terminal complex. Since fucans were more efficient than heparin in inhibiting activation of the classical pathway in whole serum and exhibited a lesser specific anticoagulant activity on a molar basis, our results suggest that these natural sulfated polysaccharides have a potential for use as anti-complementary and anti-inflammatory agents.

Polyacrylic acid-IgG polymer (PAA-IgG) activates the classical and alternative pathway of complement as shown by specific Clq-(IgG-PAA) interaction and the generation of C4d, Bb, C3b, C3a and C5a. This water soluble and stable PAA-IgG polymer represents a model substance for the study of humoral and cellular inflammatory mechanisms, mediated by the complement system.

In immunopathological study of complement activation, we recognised a marked inhibitory effect of sodium selenite on the haemolysis induced by complement fixation in vitro, thereafter on mouse complement activation in vivo induced by endotoxin, inulin or aggregated IgG indicated by a special rocket immunoelectrophoresis test of C3 split products. The effective inhibition usually began at the concentration of 0.002 mol/L of selenite in vitro, and 20 micrograms/25 g BW intravenously in vivo. The inhibitory effect was found evident on alternative pathway. This inhibitory effect was further identified in cases of epidemic haemorrhagic fever (EHF) treated by multiple dosages of 2 mg selenite per day in the first 9 days of hospitalization other than general management of 80 severe cases, including fulminant and moderate grade EHF cases. C3 activation was inhibited accordingly and the mortalities also dropped markedly from 100% of the non-treated group to 36% of the selenite treated group of fulminant type, and from 22% to zero of severe type group.

This study describes activation of serum complement by lipoteichoic acid (LTA) from Streptococcus mutans OMZ 176, while in solution. Serum from 16 healthy students was taken. Test samples were incubated with increasing doses (1-5,000 micrograms/ml) of LTA or lipopolysaccharide (LPS) from Escherichia coli 0111:B4 for 1 h at 37 degrees C; then assayed for degradation of C3, C4 or factor B by crossed immunoelectrophoresis. Each preparation caused a significant (p < 0.05) dose-dependent conversion of C3. The response curves obtained were not statistically different. LPS was a stronger activator of the alternative pathway than LTA, as judged from analysis of C3 degradation in the presence of Mg2+/EGTA, and from their effects on factor B cleavage. LTA caused, however, pronounced alterations in the shape of C4 precipitation in the gels. Functional (hemolytic) assays showed that, when tested at 200 micrograms/ml, LTA and LPS triggered significant (p < 0.05) consumptions of both classical and alternative pathway proteins. LPS was a significantly (p < 0.05) stronger activator than LTA. Apparently, the C3 degradation found for this LTA involved the alternative pathway to a small extent; thus some other mechanism of fluid-phase C3 cleavage seemed also to be operative.

Heparin, a polyion, exerts its main activity to inhibit coagulation through a serine protease inhibitor, antithrombin III. Previous studies have clearly shown that heparin in the absence of antithrombin III also has the capacity to regulate C activity. The present studies examined the ability of purified human antithrombin III to regulate classical and alternative pathways of C, alone and in the presence of heparin. Antithrombin III alone inhibited generation of both pathways in a dose-related manner; antithrombin III at 8 micrograms/10(7) cellular intermediates inhibited generation of the classical and alternative pathway convertases by 60 and 42%, respectively. Antithrombin III and heparin augmented each other's capacity to inhibit generation of both convertases in a dose-related manner. Antithrombin III did not appear to inhibit on the basis of charge because it is only slightly anionic (isoelectric pH value, 5.0); instead, antithrombin III may have acted as a serine protease inhibitor of the proteolytic enzymes of the C cascades. Antithrombin III acted only to inhibit formation of the alternative pathway convertase but had no activity on terminal lysis by this pathway; similarly, antithrombin III inhibited preformed EAC1,4b,2a,3b but had no activity on classical pathway cellular intermediates containing additional components. Finally, antithrombin III inhibited consumption of factor B hemolytic activity in a reaction mixture that also contained factor D and C3b, suggesting that factor D activity was also inhibited. These studies demonstrate the capacity of antithrombin III to regulate C and suggest that, in concert with heparin, antithrombin III may play an important role in the regulation of C in vivo.

Polymer surfaces activate complement pathways resulting in platelet and leucocyte deposition as well as possible release of growth factors. A consequence of these interactions may be early graft failure or intimal hyperplasia leading to late graft failure. C5a generation in human plasma by vascular sutures, both alone and in combination with synthetic vascular prostheses was measured by radioimmunoassay to determine the influence of suture materials on C5a activation. Prolene and ePTFE suture material caused significant activation of C5a (p less than 0.01), while Novafil did not. Both Dacron and ePTFE graft material caused significant activation (p less than 0.01) of C5a. The addition of the suture materials to the ePTFE did not increase the C5a levels above the ePTFE material alone. In contrast, the addition of either Prolene or Novafil suture to Dacron material elevated C5a levels significantly over Dacron alone (p less than 0.01). The combination of Dacron material with ePTFE suture did not increase C5a levels over Dacron alone. The pattern of C5a activation by Prolene, ePTFE and Novafil sutures parallels the relative degree of in-vivo platelet accumulation on these suture materials as previously reported by our group. Since these experiments demonstrate that vascular suture material influences human complement activation, it may be that this interaction contributes to either early or late graft failure by enhancing platelet reactivity or neointimal proliferation, respectively.

Lentinan, a pure polysaccharide, augments many immune responses in vivo but not in vitro. Therefore reaction with any factor is thought to be required for triggering of immunopotentiation by lentinan. To clarify the role of complement in this effect, we investigated whether lentinan could activate alternative pathway of complement or not and the relation with the immunopotentiation in 13 gastric cancer patients. C3, C3a, C5 and C5a levels as parameters of complement activation and interleukin-2 producing activity as parameter of immunopotentiation were assayed before and immediately after intravenous injection of lentinan. Among these complement components, remarkable change was observed only in C3a level. C3a level elevated to more than 1000ng/ml from less than 300ng/ml. And according to the degree and continuation of the elevation, these patients were classified into 3 types-(1) every time little elevation, (2) first remarkable and thereafter little elevation and (3) continuous remarkable elevation. Such an elevated C3a level returned to the level before lentinan injection within one day. When C3a level elevated to more than 1000ng/ml, leukocyte counts of peripheral blood decreased significantly and interleukin-2 producing activity increased concomitantly. Thus, there was diversity in response to lentinan and complement activation was suggested to have contribution in immunopotentiation by lentinan.

Human B cell lines were tested for the capacity to convert C3 to C3b/iC3b through the alternative C pathway (ACP) and to fix the generated C3 fragments. The panel of lines included four Epstein-Barr virus (EBV)-negative Burkitt's lymphomas and their sublines converted with two EBV strains: the prototype B958 (B virus) and the nontransforming P3HR-1 (P virus). Comparison of these groups of parallel lines (derived from a single patient) showed that the EBV-carrying cells were more efficient in activation and fixation of C3. All cell lines activated C3 with higher efficiency when treated with interferon gamma or tumor necrosis factor alpha. After treatment, the comparative relationships between the sublines changed. The treated EBV-negative and the P virus converted sublines showed similar levels of ACP activation, while the B virus carrying sublines were more efficient. The amounts of bound C3 fragments on the activating cells correlated with the ACP efficiencies. In accordance with the elevated ACP activation capacity of the cells after cytokine treatment, the amounts of C3 fragments detected on their surfaces were higher. Among the lines tested, only two (BL28 and Ramos) were sensitive to C-mediated lysis. The lytic sensitivity of these two lines increased after treatment with the cytokines. The results indicate, therefore, that the cytokine-induced increase in ACP activation by Burkitt's lymphoma line is not sufficient to overcome resistance to C lysis.