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Mutations in fibrillin 1 (FBN1) is the main cause of Marfan syndrome (MFS) with thoracic aortic aneurysm (TAA) as the main complication. Activation of the complement system plays a key role in the formation of thoracic and abdominal aortic aneurysms. However, the role of the complement system in MFS-associated aortic aneurysms remains unclear. In this study, we observed increased levels of complement C3a and C5a in the plasma of MFS patients and mouse, and the increased deposition of the activated complement system product C3b/iC3b was also observed in the elastic fiber rupture zone of 3-month-old MFS mice. The expression of C3a receptor (C3aR) was increased in MFS aortas, and recombinant C3a promoted the expression of cytokines in macrophages. The administration of a C3aR antagonist (C3aRA) attenuated the development of thoracic aortic aneurysms in MFS mice. The increased inflammation response and matrix metalloproteinases activities were also attenuated by C3aRA treatment in MFS mice. Therefore, these findings indicate that the complement C3a/C3aR inhibition alleviates the formation of aortic aneurysm in Marfan syndrome mice., (© 2024. The Author(s).)

The novel Coronavirus SARS-CoV-2 is the viral pathogen responsible for the ongoing global pandemic, COVID-19 (Coronavirus disease 2019). To date, the data recorded indicate 1.62 Mln deaths and 72.8 Mln people infected (WHO situation report Dec 2020). On December 27, the first anti-COVID-19 vaccinations started in Europe. There are no direct antivirals against SARS-CoV-2. Understanding the pathophysiological and inflammatory/immunological processes of SARS-CoV-2 infection is essential to identify new drug therapies. In the most severe COVID-19 cases, an unregulated immunological/inflammatory system results in organ injury that can be fatal to the host in some cases. Pharmacologic approaches to normalize the unregulated inflammatory/immunologic response is an important therapeutic solution. Evidence associates a non-regulation of the "complement system" as one of the causes of generalized inflammation causing multi-organ dysfunction. Serum levels of a complement cascade mediator, factor "C5a", have been found in high concentrations in the blood of COVID-19 patients with severe disease. In this article we discuss the correlation between complement system and COVID-19 infection and pharmacological solutions directed to regulate., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Ulcerative colitis (UC) is a serious digestive system disease. Furthermore, the activation of C3a/C3aR axis promoted the expression of caspase-11. And higher levels of caspase-11 could induce the pyroptosis and inflammation of cells. However, some studies suggested that caspase-11 could promote and suppress the inflammation during the development of UC. In addition, whether C3a/C3aR axis could affect the development of UC by modulating the expression of caspase-11 is unclear. We established the UC rat model in this study. Next, the C3aR inhibitor was used to treat these rats at diverse stages of UC. Next, the HE staining was performed to detect the intestinal damage. ELISA was performed to reveal the expression of IL-6 and TNF-α in different stages of UC. Western blotting was used to detect the expression of caspase-11 and C3aR in different stages of UC. Stimulation of C3aR inhibitor in early stage of UC promoted the expression of C3aR and caspase-11 in later stage of UC. Treatment of C3aR inhibitor in later stage of UC inhibited the expression of C3aR and caspase-11 in later stage of UC. Furthermore, application of C3aR inhibitor in early stage of UC aggravates the damage of colon tissue and enhanced the secretion of TNF-α and IL-6 in the later stage of UC. Treatment of C3aR inhibitor in later stage of UC relieved the symptoms of UC and suppressed the production of TNF-α and IL-6 in the later stage of UC. Application of C3aR inhibitor in early stage of UC induced the poor prognosis of UC by upregulating the expression of caspase-11. Treatment of C3aR inhibitor in later stage of UC relieved the symptoms of UC and lead to the favorable prognosis of UC by inhibiting the expression of caspase-11.

Complement component 3a (C3a) plays a crucial role in the immune response and host defense, but it is also involved in pro-inflammatory responses, causing many inflammatory disorders. Blockade of C3a has been regarded as a potent therapeutic strategy for inflammatory diseases. Here, we present the development of a human C3a (hC3a)-specific protein binder, which effectively inhibits pro-inflammatory responses. The protein binder, which is composed of leucine-rich repeat modules, was selected against hC3a through phage display, and its binding affinity was matured up to 600 pM by further expanding the binding interface in a module-by-module manner. The developed protein binder was shown to have more than 10-fold higher specificity to hC3a compared with human C5a, exhibiting a remarkable suppression effect on pro-inflammatory response in monocyte, by blocking the interaction between hC3a and its receptor. The hC3a-specific protein binder is likely to have a therapeutic potential for C3a-mediated inflammatory diseases., (© 2020 Wiley Periodicals, Inc.)

Background and Objective: Complement activation as an early and important inflammatory process contributes to multiple organ dysfunction after trauma. We have recently shown that complement inhibition by decay-accelerating factor (DAF) protects brain from blast-overpressure (BOP)-induced damage. This study was conducted to determine the effect of DAF on acute lung injury induced by BOP exposure and to elucidate its possible mechanisms of action., Methods: Anesthetized adult male Sprague-Daley rats were exposed to BOP (120 kPa) from a compressed air-driven shock tube. Rats were randomly assigned to three experimental groups: 1) Control (no BOP and no DAF treatment), 2) BOP (120 kPa BOP exposure), and 3) BOP followed by treatment with rhDAF (500μg/kg, i.v) at 30 minutes after blast. After a recovery period of 3, 24, or 48 hours, animals were euthanized followed by the collection of blood and tissues at each time point. Samples were subjected to the assessment of cytokines and histopathology as well as for the interaction of high-mobility-group box 1 (HMGB1) protein, NF-κB, receptor for advanced glycation end products (RAGE), C3a, and C3aR., Results: BOP exposure significantly increased in the production of systemic pro- and anti-inflammatory cytokines, and obvious pathological changes as characterized by pulmonary edema, inflammation, endothelial damage and hemorrhage in the lungs. These alterations were ameliorated by early administration of rhDAF. The rhDAF treatment not only significantly reduced the expression levels of HMGB1, RAGE, NF-κB, C3a, and C3aR, but also reversed the interaction of C3a-C3aR and nuclear translocation of HMGB1 in the lungs., Conclusions: Our findings indicate that early administration of DAF efficiently inhibits systemic and local inflammation, and mitigates blast-induced lung injury. The underlying mechanism might be attributed to its potential modulation of C3a-C3aR-HMGB1-transcriptional factor axis. Therefore, complement and/or HMGB1 may be potential therapeutic targets in amelioration of acute lung injury after blast injury., Competing Interests: The authors have declared that no competing interests exist.

The mechanisms that connect complement system activation and basal deposit formation in early stages of age-related macular degeneration (AMD) are insufficiently understood, which complicates the design of efficient therapies to prevent disease progression. Using human fetal (hf) retinal pigment epithelial (RPE) cells, we have established an in vitro model to investigate the effect of complement C3a on RPE cells and its role in the formation of sub-RPE deposits. The results of these studies revealed that C3a produced after C3 activation is sufficient to induce the formation of sub-RPE deposits via complement-driven proteasome inhibition. C3a binds the C3a receptor (C3aR), stimulates deposition of collagens IV and VI underneath the RPE, and impairs the extracellular matrix (ECM) turnover by increased MMP-2 activity, all mediated by downregulation of the ubiquitin proteasome pathway (UPP). The formation of basal deposits can be prevented by the addition of a C3aR antagonist, which restores the UPP activity and ECM turnover. These findings indicate that the cell-based model can be used to test potential therapeutic agents in vitro. The data suggest that modulation of C3aR-mediated events could be a therapeutic approach for treatment of early AMD.

Deficiency of ribosomal proteins (RPs) leads to Diamond Blackfan Anemia (DBA) associated with anemia, congenital defects, and cancer. While p53 activation is responsible for many features of DBA, the role of immune system is less defined. The Innate immune system can be activated by endogenous nucleic acids from non-processed pre-rRNAs, DNA damage, and apoptosis that occurs in DBA. Recognition by toll like receptors (TLRs) and Mda5-like sensors induces interferons (IFNs) and inflammation. Dying cells can also activate complement system. Therefore we analyzed the status of these pathways in RP-deficient zebrafish and found upregulation of interferon, inflammatory cytokines and mediators, and complement. We also found upregulation of receptors signaling to IFNs including Mda5, Tlr3, and Tlr9. TGFb family member activin was also upregulated in RP-deficient zebrafish and in RPS19-deficient human cells, which include a lymphoid cell line from a DBA patient, and fetal liver cells and K562 cells transduced with RPS19 shRNA. Treatment of RP-deficient zebrafish with a TLR3 inhibitor decreased IFNs activation, acute phase response, and apoptosis and improved their hematopoiesis and morphology. Inhibitors of complement and activin also had beneficial effects. Our studies suggest that innate immune system contributes to the phenotype of RPS19-deficient zebrafish and human cells.

Candida albicans the most frequently isolated clinical fungal pathogen can cause local as well as systemic and life-threatening infections particularly in immune-compromised individuals. A better and more detailed understanding how C. albicans evades human immune attack is therefore needed for identifying fungal immune-evasive proteins and develop new therapies. Here, we identified Pra1, the pH-regulated C. albicans antigen as a hierarchical complement inhibitor that targets C3, the central human complement component. Pra1 cleaved C3 at a unique site and further inhibited effector function of the activation fragments. The newly formed C3a-like peptide lacked the C-terminal arginine residue needed for C3a-receptor binding and activation. Moreover, Pra1 also blocked C3a-like antifungal activity as shown in survival assays, and the C3b-like molecule formed by Pra1 was degraded by the host protease Factor I. Pra1 also bound to C3a and C3b generated by human convertases and blocked their effector functions, like C3a antifungal activity shown by fungal survival, blocked C3a binding to human C3a receptor-expressing HEK cells, activation of Fura2-AM loaded cells, intracellular Ca 2+ signaling, IL-8 release, C3b deposition, as well as opsonophagocytosis and killing by human neutrophils. Thus, upon infection C. albicans uses Pra1 to destroy C3 and to disrupt host complement attack. In conclusion, candida Pra1 represents the first fungal C3-cleaving protease identified and functions as a fungal master regulator of innate immunity and as a central fungal immune-escape protein., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Regeneration of skeletal muscle following injury is accompanied by transient inflammation. Here we show that complement is activated in skeletal muscle injury and plays a key role during regeneration. Genetic ablation of complement C3 or its inactivation with Cobra Venom Factor (CVF) result in impaired muscle regeneration following cardiotoxin-induced injury in mice. The effect of complement in muscle regeneration is mediated by the alternative pathway and C3a receptor (C3aR) signaling, as deletion of Cfb, a key alternative pathway component, or C3aR leads to impaired regeneration and reduced monocyte/macrophage infiltration. Monocytes from C3aR-deficient mice express a reduced level of adhesion molecules, cytokines and genes associated with antigen processing and presentation. Exogenous administration of recombinant CCL5 to C3aR-deficient mice rescues the defects in inflammatory cell recruitment and regeneration. These findings reveal an important role of complement C3a in skeletal muscle regeneration, and suggest that manipulating complement system may produce therapeutic benefit in muscle injury and regeneration.

Inflammatory processes play a key role in pathophysiology of many neurologic diseases/trauma, but the effect of immune cells and factors on neurotransplantation strategies remains unclear. We hypothesized that cellular and humoral components of innate immunity alter fate and migration of human neural stem cells (hNSC). In these experiments, conditioned media collected from polymorphonuclear leukocytes (PMN) selectively increased hNSC astrogliogenesis and promoted cell migration in vitro. PMN were shown to generate C1q and C3a; exposure of hNSC to PMN-synthesized concentrations of these complement proteins promoted astrogliogenesis and cell migration. Furthermore, in vitro, Abs directed against C1q and C3a reversed the fate and migration effects observed. In a proof-of-concept in vivo experiment, blockade of C1q and C3a transiently altered hNSC migration and reversed astroglial fate after spinal cord injury. Collectively, these data suggest that modulation of the innate/humoral inflammatory microenvironment may impact the potential of cell-based therapies for recovery and repair following CNS pathology., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Larvae of the blowfly Lucilia sericata facilitate wound healing by removing dead tissue and biofilms from non-healing and necrotic wounds. Another beneficial action of larvae and their excretions/secretions (ES) is down-regulation of excessive inflammation. As prolonged complement activation is key to excessive inflammation, the aim of this study was to elucidate the mechanisms underlying the anti-complement activities of ES. Results revealed that heat sensitive serine proteases in ES degrade multiple complement proteins in all steps of the three complement activation pathways. Importantly, C3a and C5a-major activators of inflammation-were also degraded by ES and pretreatment of these factors with ES completely blocked their ability to induce activation of human neutrophils. Pre-exposure of the neutrophils to ES did not affect their responsiveness to C3a/C5a and fMLP, indicating that the receptors for these activators on neutrophils were not affected by ES. Surprisingly, heat and serine protease inhibitor pretreatment did not affect the ability of ES to inhibit C5b-9 complex formation despite degrading complement proteins, indicating a second complement-inhibiting molecule in ES. Heated ES was as effective as intact ES in inhibiting C3 deposition upon activation of the alternative pathway, but was significantly less effective in wells with a classical or lectin pathway-specific coating. Unfortunately, the molecules affecting the complement system could not be identified due to an insufficient database for L. sericata. Together, larval ES inhibit complement activation by two different mechanisms and down-regulate the C3a/C5a-mediated neutrophil activation. This attenuates the inflammatory process, which may facilitate wound healing., (© 2016 by the Wound Healing Society.)

Background and Objectives: Carboxypeptidase B2 (CPB2) is a basic carboxypeptidase with fibrin and complement C3a and C5a as physiological substrates. We hypothesized that in polymicrobial sepsis, CPB2-deficient mice would have sustained C5a activity, leading to disease exacerbation., Methods: Polymicrobial sepsis was induced by cecal ligation and puncture (CLP)., Results: Contrary to our hypothesis, Cpb2(-/-) mice had significantly improved survival, with reduced lung edema, less liver and kidney damage, and less disseminated intravascular coagulation. Hepatic pro-CPB2 was induced by CLP, leading to increased pro-CPB2 levels. Thrombomodulin present on mesothelium supported thrombin activation of pro-CPB2. Both wild-type and Cpb2(-/-) animals treated with a C5a receptor antagonist had improved survival, demonstrating that C5a was detrimental in this model. Treatment with a fibrinolysis inhibitor, tranexamic acid, caused a decrease in survival in both genotypes; however, the Cpb2(-/-) animals retained their survival advantage. Administration of a C3a receptor antagonist exacerbated the disease in both wild-type and Cpb2(-/-) mice and eliminated the survival advantage of Cpb2(-/-) mice. C5a receptor is expressed in both peritoneal macrophages and neutrophils; in contrast, C3a receptor expression is restricted to peritoneal macrophages, and C3a induced signaling in macrophages but not neutrophils., Conclusions: While C5a exacerbates the peritonitis, resulting in a deleterious generalized inflammatory state, C3a activation of peritoneal macrophages may limit the initial infection following CLP, thereby playing a diametrically opposing protective role in this polymicrobial sepsis model., (© 2015 International Society on Thrombosis and Haemostasis.)

Anti-factor D (AFD; FCFD4514S, lampalizumab) is a humanized IgG Fab fragment directed against factor D (fD), a rate-limiting serine protease in the alternative complement pathway (AP). Evaluation of AFD as a potential intravitreal (IVT) therapeutic for dry age-related macular degeneration patients with geographic atrophy (GA) is ongoing. However, it is unclear whether IVT administration of AFD can affect systemic AP activation and potentially compromise host-immune responses. We characterized the pharmacologic properties of AFD and assessed the effects of AFD administered IVT (2 or 20 mg) or intravenous (0.2, 2, or 20 mg) on systemic complement activity in cynomolgus monkeys. For the IVT groups, serum AP activity was reduced for the 20 mg dose group between 2 and 6 hours postinjection. For the intravenous groups, AFD inhibited systemic AP activity for periods of time ranging from 5 minutes (0.2 mg group) to 3 hours (20 mg group). Interestingly, the concentrations of total serum fD increased up to 10-fold relative to predose levels following administration of AFD. Furthermore, AFD was found to inhibit systemic AP activity only when the molar concentration of AFD exceeded that of fD. This occurred in cynomolgus monkeys at serum AFD levels ≥2 µg/ml, a concentration 8-fold greater than the maximum serum concentration observed following a single 10 mg IVT dose in a clinical investigation in patients with GA. Based on these findings, the low levels of serum AFD resulting from IVT administration of a clinically relevant dose are not expected to appreciably affect systemic AP activity., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Early-onset pre-eclampsia is characterized by decreased placental perfusion, new-onset hypertension, angiogenic imbalance, and endothelial dysfunction associated with excessive activation of the innate immune complement system. Although our previous studies demonstrated that inhibition of complement activation attenuates placental ischemia-induced hypertension using the rat reduced uterine perfusion pressure (RUPP) model, the important product(s) of complement activation has yet to be identified. We hypothesized that antagonism of receptors for complement activation products C3a and C5a would improve vascular function and attenuate RUPP hypertension. On gestational day (GD) 14, rats underwent sham surgery or vascular clip placement on ovarian arteries and abdominal aorta (RUPP). Rats were treated once daily with the C5a receptor antagonist (C5aRA), PMX51 (acetyl-F-[Orn-P-(D-Cha)-WR]), the C3a receptor antagonist (C3aRA), SB290157 (N(2)-[(2,2-diphenylethoxy)acetyl]-l-arginine), or vehicle from GD 14-18. Both the C3aRA and C5aRA attenuated placental ischemia-induced hypertension without affecting the decreased fetal weight or decreased concentration of free circulating vascular endothelial growth factor (VEGF) also present in this model. The C5aRA, but not the C3aRA, attenuated placental ischemia-induced increase in heart rate and impaired endothelial-dependent relaxation. The C3aRA abrogated the acute pressor response to C3a peptide injection, but it also unexpectedly attenuated the placental ischemia-induced increase in C3a, suggesting nonreceptor-mediated effects. Overall, these results indicate that both C3a and C5a are important products of complement activation that mediate the hypertension regardless of the reduction in free plasma VEGF. The mechanism by which C3a contributes to placental ischemia-induced hypertension appears to be distinct from that of C5a, and management of pregnancy-induced hypertension is likely to require a broad anti-inflammatory approach., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Antiphospholipid syndrome is a pregnancy related, systemic autoimmune disorder in which antibodies directed against cell membrane phospholipids with multiple venous/arterial thromboses. Though the etiology of antiphospholipid syndrome has not been identified exactly, experimental evidences suggest the possible mechanisms involved in the pathogenicity of this syndrome. Antiphospholipid antibodies mediate deposition of complement proteins in placenta and over expression of tissue factor on the surface of neutrophils which are reported to be the prominent cause of prothrombotic phenotype. The activation complement components C3 and C5 by antiphospholipid antibodies would eventually activates blood coagulation pathway that leads to thrombosis. Inhibition of activated complement components C3a and C5a by anticomplement agents protects from antiphospholipid antibody mediated fetal loss. Since the interference of complement pathway may lead to deleterious effects, we hypothesis that local inhibition of complement proteins C3a and C5a at placenta would reduce the risk of antiphospholipid antibody mediated placental thrombosis and pregnancy complications., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Allergen-specific IgE plays an essential role in the pathogenesis of allergic asthma. Although there has been increasing evidence suggesting the involvement of IL-17 in the disease, the relationship between IL-17 and IgE-mediated asthmatic responses has not yet been defined. In this study, we attempted to elucidate the contribution of IL-17 to an IgE-mediated late-phase asthmatic response and airway hyperresponsiveness (AHR). BALB/c mice passively sensitized with an OVA-specific IgE mAb were challenged with OVA intratracheally four times. The fourth challenge caused a late-phase increase in airway resistance associated with elevated levels of IL-17(+)CD4(+) cells in the lungs. Multiple treatments with a C3a receptor antagonist or anti-C3a mAb during the challenges inhibited the increase in IL-17(+)CD4(+) cells. Meanwhile, a single treatment with the antagonist or the mAb at the fourth challenge suppressed the late-phase increase in airway resistance, AHR, and infiltration by neutrophils in bronchoalveolar lavage fluid. Because IL-17 production in the lungs was significantly repressed by both treatments, the effect of an anti-IL-17 mAb was examined. The late-phase increase in airway resistance, AHR, and infiltration by neutrophils in bronchoalveolar lavage fluid was inhibited. Furthermore, an anti-Gr-1 mAb had a similar effect. Collectively, we found that IgE mediated the increase of IL-17(+)CD4(+) cells in the lungs caused by repeated Ag challenges via C3a. The mechanisms leading to the IgE-mediated late-phase asthmatic response and AHR are closely associated with neutrophilic inflammation through the production of IL-17 induced by C3a.

Background and Purpose: The restoration of blood-flow following cerebral ischemia incites a series of deleterious cascades that exacerbate neuronal injury. Pharmacologic inhibition of the C3a-receptor ameliorates cerebral injury by attenuating post-ischemic inflammation. Recent reports also implicate C3a in the modulation of tissue repair, suggesting that complement may influence both injury and recovery at later post-ischemic time-points., Methods: To evaluate the effect of C3a-receptor antagonism on post-ischemic neurogenesis and neurological outcome in the subacute period of stroke, transient focal cerebral ischemia was induced in adult male C57BL/6 mice treated with multiple regimens of a C3a receptor antagonist (C3aRA)., Results: Low-dose C3aRA administration during the acute phase of stroke promotes neuroblast proliferation in the subventricular zone at 7 days. Additionally, the C3a receptor is expressed on T-lymphocytes within the ischemic territory at 7 days, and this cellular infiltrate is abrogated by C3aRA administration. Finally, C3aRA treatment confers robust histologic and functional neuroprotection at this delayed time-point., Conclusions: Targeted complement inhibition through low-dose antagonism of the C3a receptor promotes post-ischemic neuroblast proliferation in the SVZ. Furthermore, C3aRA administration suppresses T-lymphocyte infiltration and improves delayed functional and histologic outcome following reperfused stroke. Post-ischemic complement activation may be pharmacologically manipulated to yield an effective therapy for stroke.

The complement system plays an important role in eliminating invading pathogens. Activation of complement results in C3b deposition (opsonization), phagocytosis, anaphylatoxin (C3a, C5a) release, and consequently cell lysis. Moraxella catarrhalis is a human respiratory pathogen commonly found in children with otitis media and in adults with chronic obstructive pulmonary disease. The species has evolved multiple complement evasion strategies, which among others involves the ubiquitous surface protein (Usp) family consisting of UspA1, A2, and A2 hybrid. In the present study, we found that the ability of M. catarrhalis to bind C3 correlated with UspA expression and that C3 binding contributed to serum resistance in a large number of clinical isolates. Recombinantly expressed UspA1 and A2 inhibit both the alternative and classical pathways, C3b deposition, and C3a generation when bound to the C3 molecule. We also revealed that the M. catarrhalis UspA-binding domain on C3b was located to C3d and that the major bacterial C3d-binding domains were within UspA1(299-452) and UspA2(165-318). The interaction with C3 was not species specific since UspA-expressing M. catarrhalis also bound mouse C3 that resulted in inhibition of the alternative pathway of mouse complement. Taken together, the binding of C3 to UspAs is an efficient strategy of Moraxella to block the activation of complement and to inhibit C3a-mediated inflammation.

It was investigated whether the C3a-receptor antagonist (C3aRA) SB 290157 was involved in the suppression of anti-OVA pAb-induced arthritis because it is well known that anaphylatoxin C3a plays a crucial role in the development of an effective inflammatory response during complement activation. Anti-OVA pAb-induced arthritis was induced in DBA/1J mice by administration of anti-OVA pAb 0.5 h prior to intra-articular (i.a.) injection of OVA (0 h). Two peaks of joint swelling were observed at 0.5 and 3 h. The role of C3aRA in arthritis was investigated by injection of SB 290157 at concentrations of 10 and 30 mg/kg at 0 and 2 h. The antagonist was able to reduce joint swelling only at 3 h, and about 50% inhibition of joint swelling was observed with the concentration of 30 mg/kg. The C3 level was significantly decreased at 3 h compared with naïve mice showing complement consumption. Furthermore, the C3 activation was observed and increased corresponding to the graded concentration of anti-OVA pAb. The results also revealed that the C3aRA was able to reduce the expression of IL-1beta in synovial tissue. Taken together, the results suggested that C3aRA may be effective in the inhibition of arthritis.

Rationale: Donor brain death (BD) is an unavoidable occurrence in heart transplantation and results in profound physiological derangements that render the heart more susceptible to ischemia/reperfusion injury in the recipient and likely has negative long-term consequences to allograft survival., Objective: We developed a novel mouse model of BD and investigated the role of complement in BD-induced myocardial inflammation and injury., Methods and Results: BD was induced by inflation of a balloon catheter in the cranial cavity. BD in wild-type mice resulted in a significant increase in serum concentrations of the complement activation product complement component (C)3a, and immunohistochemical analysis of heart sections demonstrated C3 deposition on the vascular endothelium and surrounding myocytes. Following induction of BD in complement (C3)-deficient mice, cardiac troponin levels, and histological evidence of injury were significantly reduced compared to wild-type mice. C3 deficiency was also associated with reduced myocardial leukocyte infiltration and reduced or absent expression of P-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, tumor necrosis factor-alpha, and interleukin-1beta., Conclusions: These data indicate an important role for complement in BD-induced inflammation and injury and suggest that a complement inhibitory strategy applied to the donor (in addition to the recipient) may provide graft protection.

In the rat, C5a infusion mediates well-defined effects including hypotension and neutropenia. Conversely, the comparative effect of C3a in the rat is not yet defined. In the current study, we have investigated C3a receptor (C3aR) activation in the rat, using recombinant human C3a, the C3aR agonist WWGKKYRASKLGLAR, which is a C-terminal analogue of C3a, and a nonpeptide C3aR antagonist SB-290157, as pharmacological tools. In vitro, C3a and WWGKKYRASKLGLAR selectively bound to C3aRs and induced degranulation of C3aR-transfected RBL-2H3 cells. C3a or WWGKKYRASKLGLAR-induced degranulation was dose-dependently antagonized in a surmountable fashion by the nonpeptide C3aR antagonist. Intravenous infusion of C3a and WWGKKYRASKLGLAR to rats induced a rapid, transient and concentration-dependent hypertensive response, which was mediated by C3aR-induced prostanoid release. C3a and WWGKKYRASKLGLAR caused a small drop in circulating neutrophils, but a rise in circulating neutrophils was evident after 90-120 min. In contrast to C3a, C5a infusion resulted in hypotension, and rapid and transient neutropenia. These results demonstrate that C3a and C5a mediate distinct effects on blood pressure and circulating polymorphonuclear leukocytes in the rat.

Tubulointerstitial inflammation and progressive fibrosis are common pathways that lead to kidney failure in proteinuric nephropathies. Activation of the complement system has been implicated in the development of tubulointerstitial injury in clinical and animal studies, but the mechanism by which complement induces kidney injury is not fully understood. Here, we studied the effect of complement on the phenotype of tubular epithelial cells. Tubular epithelial cells exposed to serum proteins adopted phenotypic and functional characteristics of mesenchymal cells. Expression of E-cadherin protein decreased and expression of both alpha-smooth muscle actin protein and collagen I mRNA increased. Exposure of the cells to the complement anaphylotoxin C3a induced similar features. Treating with a C3a receptor (C3aR) antagonist prevented both C3a- and serum-induced epithelial-to-mesenchymal transition. In the adriamycin-induced proteinuria model, C3aR-deficient mice demonstrated less injury, preserved renal function, and improved survival compared with wild-type mice. Furthermore, the kidneys of C3aR-deficient mice had significantly less interstitial collagen I and alpha-smooth muscle actin. In summary, the complement anaphylotoxin C3a is an important mediator of glomerular and tubulointerstitial injury and can induce tubular epithelial-to-mesenchymal transition.

The complement anaphylatoxin C3a contributes to injury after cerebral ischemia in mice. This study assesses the effect of C3a receptor antagonist (C3aRA) on leukocyte infiltration into the ischemic zone. Transient or permanent middle cerebral artery occlusion (MCAO) was induced in wild-type C57Bl/6 mice. Intraperitoneal C3aRA or vehicle was administered 45 mins before or 1 h after occlusion. Twenty-four hours after occlusion, we harvested brain tissue and purified inflammatory cells using flow cytometry. Soluble intercellular adhesion molecule (ICAM)-1 protein levels were assessed using enzyme-linked immunosorbent assays, and ICAM-1 and C3a receptor (C3aR) expression was confirmed via immunohistochemistry. In the transient MCAO model, animals receiving C3aRA showed smaller strokes, less upregulation of C3aR-positive granulocytes, and less ICAM-1 protein on endothelial cells than vehicle-treated animals; no significant differences in other inflammatory cell populations were observed. C3a receptor antagonist-treated and vehicle-treated animals showed no differences in stroke volume or inflammatory cell populations after permanent MCAO. These data suggest that blocking the binding of C3a to C3aR modulates tissue injury in reperfused stroke by inhibiting the recruitment of neutrophils to the ischemic zone. It further establishes antagonism of the C3a anaphylatoxin as a promising strategy for ameliorating injury after ischemia/reperfusion.

Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.

The feeding success of a tick upon a host depends on its ability to suppress host anti-tick responses which include activation of the complement system. We investigated the mechanism of inhibition of the alternative pathway of complement by salivary gland extract (SGE) of the ixodid tick species, Ixodes ricinus. SGE treatment strongly inhibited C3a generation and factor B cleavage in serum when rabbit erythrocytes were used as complement activator, but not when cobra venom factor (CVF) was used as an activator. SGE treatment strongly inhibited C3b deposition on rabbit erythrocytes, and the turnover of C3 (to C3b/iC3b) in serum. However, there was no significant effect upon the formation, stability or activity of C3 convertase (C3bBb) when formed from purified C3b, factor B and factor D. SGE treatment of isolated C3 resulted in a shift in mobility of the alpha-chain (by about 5 kDa). N-terminal sequencing of this species suggests that cleavage occurs at the C-terminus of the alpha-chain of C3. Consistent with this hypothesis, the modified alpha-chain was still a substrate for pre-formed convertase. The activity was specific for the alpha-chain of C3 but not of C3(H2O) nor the alpha'-chain of C3b. It is proposed that SGE-modified C3 does not participate in convertase formation, probably having a reduced affinity for factor B.

Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli-derived protein were obtained and diffracted to 2.2 A, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.

High-dose intravenous immunoglobulin (IVIG) prevents immune damage by scavenging complement fragments C3b and C4b. We tested the hypothesis that exogenous immunoglobulin molecules also bind anaphylatoxins C3a and C5a, thereby neutralizing their pro-inflammatory effects. Single-cell calcium measurements in HMC-1 human mast cells showed that a rise in intracellular calcium caused by C3a and C5a was inhibited in a concentration-dependent manner by IVIG, F(ab)2-IVIG and irrelevant human monoclonal antibody. C3a- and C5a-induced thromboxane (TXB2) generation and histamine release from HMC-1 cells and whole-blood basophils were also suppressed by exogenous immunoglobulins. In a mouse model of asthma, immunoglobulin treatment reduced cellular migration to the lung. Lethal C5a-mediated circulatory collapse in pigs was prevented by pretreatment with F(ab)2-IVIG. Molecular modeling, surface plasmon resonance (SPR) and western blot analyses suggested a physical association between anaphylatoxins and the constant region of F(ab)2. This binding could interfere with the role of C3a and C5a in inflammation.

The physiological interaction between glycyrrhizin (GL) and serum complement C3, and the inhibitory effects of GL, glycyrrhetinic acid (GA), and a GA derivative (oGA) on the phosphorylation of C3 by casein kinase 2 (CK-2), were investigated in vitro. C3 was found to be a GL-binding protein (gbP), because (i) of its high affinity for a GL-affinity HPLC column; and (ii) both GL and GA induce conformational changes in C3. At least four trypsin-resistant fragments (p30, p25, p18, and p15) were detected when the (32)P-labeled C3alpha was digested with trypsin in the presence of 100 micro M GA. Two of these (p25 and p15) were immuno-precipitated with anti-C3a serum. Furthermore, it was found that C3a contains GL-binding domains, because (i) C3a (anaphylatoxin) could be selectively purified from the synovial fluids of patients with rheumatoid arthritis by GL-affinity column chromatography (HPLC); and (ii) purified human C3a has a high affinity for a GL-affinity column. In addition, C3alpha (p115) of C3 was effectively phosphorylated by CK-2 in the presence of poly-Arg (a CK-2 activator) in vitro. This phosphorylation was completely inhibited by 10 micro M oGA, 30 micro M GA, or 100 micro M GL. Taken together, these results suggest that the GL-induced inhibition of the physiological activities of C3a and C3alpha may be involved in the anti-inflammatory effect of GL in vivo.

Objective: To assess complement-mediated myocardial injury on isolated guinea pig working hearts and cardioprotective effects of CD59., Methods: Using a modified Langendorff apparatus, isolated guinea-pig working hearts were perfused with a modified Krebs Henseleit buffer containing 3% heat-inactivated human plasma and zymosan (IPZ) (control) (n = 10), 3% normal human plasma and zymosan (NPZ) (n = 10), or 3% normal human plasma and zymosan and 1.5 microg/ml CD59 (NPZC) (n = 10), respectively. Epicardial electrocardiogram (ECG), cardiac output (CO), coronary arterial flow (CF), maximum left ventricular developed pressure (LVP(max)), maximum left ventricular developed pressure increase rate (+ dp/dt(max)), maximum left ventricular developed pressure decrease rate (- dp/dt(max)) and heart rate (HR) were recorded at 0, 15, 30, 45 and 60 min of treatment. After the experiment, immunohistochemical examination was performed to detect the presence of C3a or C5b-9 in the myocardium of the isolated hearts., Results: Compared the IPZ group, hearts treated with NPZ showed a slight depression on ST segments of epicardial ECG at 15 min, a significant elevation between 30 min to 60 min, a decrease in CF, CO, LVP(max), + dp/dt(max) and - dp/dt(max), and an increase in HR at 15 min. The observed alterations in CF, CO, LVP(max), + dp/dt(max) and - dp/dt(max) remained decreased, while the HR remained increased until the end of the protocol. The all above parameters of hearts treated with NPZC were similar to the control group (IPZ) at any given time. Immunohistochemical examination showed positive signals of C3a and C5b-9 in the myocardium of hearts treated with NPZ. C3a was positive in NPZC, and C3a and C5b-9 were negative in IPZ., Conclusions: Activated human complements directly damage isolated guinea pig working hearts, and CD59 offers a significant protection against the injuries.

Pro-carboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), precursor of carboxypeptidase U and plasma carboxypeptidase B is present in plasma and following activation by thrombin/thrombomodulin and/or plasmin can remove arginine from the carboxyterminal of C3a and C5a. We have shown that this enzyme can remove terminal arginine from the C5a octapeptide much more efficiently than the classical anaphylatoxin inactivator, carboxypeptidase N (CPN). Since we have previously demonstrated that proCPR is significantly upregulated in the inflammatory state, this enzyme would appear to significantly contribute to the inactivation of C5a, the most potent of the complement derived anaphylatoxins.

The failure to fully regulate the inflammation response has been linked to diseases such as rheumatoid arthritis, septic shock syndrome, and asthma. The human complement system initiates and regulates the inflammation response through a cascade of regulatory factors. Complement Component 3a (C3a) is an essential regulatory factor and inhibiting its binding to a C3a receptor will diminish the inflammation response by disrupting the cascade. We report the design, synthesis, in vitro and in vivo activity of diiminoisoindolines as C3a antagonists.

Background: We previously demonstrated that inhibiting formation of terminal complement components (C5a and C5b-9) prevents platelet and neutrophil (PMN) but not monocyte activation during simulated extracorporeal circulation (SECC). This study examined whether earlier complement inhibition during SECC, blocking C3a formation, would additionally prevent monocyte activation., Methods and Results: SECC was established by recirculating heparinized whole blood from human volunteers on a membrane oxygenator. CAB-2, a chimeric protein constructed from genes encoding the complement regulatory proteins CD46 and CD55, inactivates the C3/C5 convertases and blocks in vitro generation of C3a, C5a, and C5b-9. CAB-2 was used in 4 experiments at a final concentration of 300 micrograms/mL and 4 experiments at 30 micrograms/mL; 4 control runs used vehicle alone. Samples were assayed for C3a and C5b-9, monocyte activation (CD11b upregulation), PMN activation (CD11b upregulation and elastase release), and platelet activation (P-selectin expression and monocyte-platelet conjugate formation). CAB-2 at both doses significantly inhibited formation of C3a and C5b-9 during SECC. High-dose CAB-2 significantly blocked monocyte and PMN CD11b upregulation and PMN elastase release. CAB-2 also inhibited formation of platelet activation-dependent monocyte-platelet conjugates., Conclusions: Blockade of complement activation early in the common pathway inhibited monocyte CD11b upregulation during SECC, suggesting that early complement components contribute most to monocyte activation during SECC. As expected, PMN and platelet activation were blocked by terminal complement inhibition. This investigation further elucidates the relation between complement and blood cell activation during simulated cardiopulmonary bypass.

The complement activation products C3a and C3a desArg are generated in the course of trauma, infection, tissue injury, and ischemia. We have investigated the effects of C3a and C3a desArg on gene expression and protein synthesis of TNF-alpha and IL-1 beta in PBMC. Neither C3a nor C3a desArg alone induced detectable protein or mRNA levels for TNF-alpha and IL-1 beta. C3a modulated LPS-induced TNF-alpha and IL-1 beta synthesis. In nonadherent PBMC, C3a suppressed LPS-induced synthesis of TNF-alpha (20-71% decrease by 0.2-10 microgram/ml of C3a, p less than 0.01) and IL-1 beta (19-57% decrease by 0.5-10 microgram/ml of C3a, p less than 0.01), independently of endogenous production of PGE2. C3a also suppressed LPS-induced mRNA levels for TNF-alpha and IL-1 beta. In contrast, in adherent PBMC, C3a at 5 to 20 microgram/ml enhanced LPS-induced TNF-alpha (75-188% increase, p less than 0.001) and IL-1 beta (119-274% increase, p less than 0.001) synthesis. C3a enhanced TNF-alpha and IL-1 beta mRNA levels in LPS-stimulated adherent cells. Furthermore, C3a desArg shared with C3a the ability to modulate LPS-induced mRNA and protein synthesis for TNF-alpha and IL-1 beta. These results suggest that C3a, thought to be proinflammatory, and C3a desArg, thought to be biologically inactive, are modulators of inflammation. Both C3a and C3a desArg may enhance cytokine synthesis by adherent monocytes at local inflammatory sites, while inhibiting the systemic synthesis of proinflammatory cytokines by circulating cells.

The terpenoid 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydrox y-2',5',5', 8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran] (1a; K-76), a natural product of fungal origin, and its monocarboxylate sodium salt 1c (R = COONa; K-76COONa) inhibit the classical and alternative pathways of complement, and 1c was shown to inhibit the classical pathway at the C5 activation step. In an attempt to elucidate the essential pharmacophore of 1a,c, the natural product was used as a "topographical model" for the design of partial analogs retaining the desired complement inhibiting potency. Therefore, A/C/D-ring analogs have been synthesized, as shown in Scheme 1 using 3-methoxyphenol (3) and limonene chloride (5) as starting materials, which contain functional groups similar to those found on the natural product. The use of (4R)-(+)- and (4S)(-)-limonene chloride (5a,b, respectively) provided two series of compounds differing in the stereochemistry of the C-4 chiral center (limonene moiety numbering). The in vitro assay results of the inhibition of anaphylatoxin production and classical complement-mediated hemolysis revealed that 7-carboxy-2-(R,S)-methyl-2-(1'-methylcyclohexen-(4'R)-yl)-4-met hoxybenzofuran (13a) and 7-carboxy-2-(R,S)-methyl-2-(1'-methylcyclohexen-(4'S)-yl)-4-met hoxybenzofuran (13b) were active in the same range of concentrations as the natural product.

Peptide compounds resembling the receptor-binding C-terminal domain of the anaphylatoxic peptide C3a were synthesized to examine two kinds of C3a antagonism: (a) specific desensitization of C3a-sensitive cells and (b) competitive binding to the C3a receptor. We used guinea-pig platelets, which express a C3a receptor and specifically release ATP upon stimulation, to evaluate the actions of the C3a analogues. The ATP liberation can be inhibited by pretreatment (i.e. desensitization) of the guinea-pig platelets with substimulatory concentrations of C3a or its analogues. Compared to C3a, several peptides were found with at least a tenfold greater difference between the required concentrations for C3a-specific half-maximal desensitization (DD50) and half-maximal platelet activation (ED50). The most potent compounds were YAAALKLAR and Fmoc-EAALKLAR (Fmoc: 9-fluorenylmethoxycarbonyl) with an ED50/DD50 of 140 +/- 28 and 80 +/- 17, respectively (mean +/- standard deviation). The ED50/DD50 of human C3a was found to be only 6 +/- 2. Some C3a derivatives were also tested in competitive binding studies for their ability to compete with C3a for receptor sites on guinea-pig platelets. Three of them were considered partial antagonists [YRRGRCGGLCLAR, YRRGRXCGGLCLAR and YRRGRXCGALCLAR (X = 6-aminohexanoyl)] because their Ki were smaller than their ED50 (Ki/ED50 = 0.6 +/- 0.3, 0.5 +/- 0.1 and 0.4 +/- 0.2, respectively). Interestingly, the last two compounds also had ED50/DD50 values greater than 60. Common to all three peptides are N-terminal arginine-rich sequences and intramolecular disulfide bridges which introduce conformational constraint.

During LDL apheresis, various combinations of heparin and citrate are used for anticoagulation. With an in vitro batch system we examined whether heparin/citrate combinations can be optimized in terms of complement activation inhibition without the loss of anticoagulant potency. Plasma anticoagulated by using six clinically applicable regimens was incubated with anti-apo-B antibody-coupled Sepharose 4B CL, and the anaphylatoxin content of the supernatant was investigated. A significant dose-dependent reduction of complement activation was achieved by anticoagulating whole blood with 10 U/ml heparin (p < 0.05) if compared with serum whereas citrate inhibited more effectively the generation of C5a (desarg) even at a low concentration (ACD-B 1:20) (p < 0.01). The lowest anaphylatoxin level was generated when heparin (10 U/ml) plus citrate (ACD-B 1:10) were applied, although such an approach may be of limited clinical interest. The empirically chosen heparin plus citrate ratio (2 U/ml, 1:20, respectively) provides for an optimal and almost ideal inhibition of complement activation and contributes considerably to the good tolerability of the immunoadsorbent.