Consumption of complement in a 26-year-old woman with severe thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination.

Extremely rare reactions characterized by thrombosis and thrombocytopenia have been described in subjects that received ChAdOx1 nCoV-19 vaccination 5-16 days earlier. Although patients with vaccine-induced thrombotic thrombocytopenia (VITT) have high levels of antibodies to platelet factor 4 (PF4)-polyanion complexes, the exact mechanism of the development of thrombosis is still unknown. Here we reported serum studies as well as proteomics and genomics analyses demonstrating a massive complement activation potentially linked to the presence of anti-PF4 antibodies in a patient with severe VITT. At admission, complement activity of the classical and lectin pathways were absent (0% for both) with normal levels of the alternative pathway (73%) in association with elevated levels of the complement activation marker sC5b-9 (630 ng/mL [n.v. 139-462 ng/mL]) and anti-PF4 IgG (1.918 OD [n.v. 0.136-0.300 OD]). The immunoblotting analysis of C2 showed the complete disappearance of its normal band at 110 kDa. Intravenous immunoglobulin treatment allowed to recover complement activity of the classical pathway (91%) and lectin pathway (115%), to reduce levels of sC5b-9 (135 ng/mL) and anti-PF4 IgG (0.681 OD) and to normalize the C2 pattern at immunoblotting. Proteomics and genomics analyses in addition to serum studies showed that the absence of complement activity during VITT was not linked to alterations of the C2 gene but rather to a strong complement activation leading to C2 consumption. Our data in a single patient suggest monitoring complement parameters in other VITT patients considering also the possibility to target complement activation with specific drugs.

Clinical relevance of membrane attack complex deposition in children with IgA nephropathy and Henoch-Schönlein purpura.

BACKGROUND: IgA nephropathy (IgAN) and Henoch-Schönlein purpura are common glomerular disorders in children sharing the same histopathologic pattern of IgA deposits within the mesangium, even if their physiopathology may be different. Repeated exposure to pathogens induces the production of abnormal IgA1. The immune complex deposition in the renal mesangium in IgAN or potentially in small vessels in Henoch-Schönlein purpura induces complement activation via the alternative and lectin pathways. Recent studies suggest that levels of membrane attack complex (MAC) in the urine might be a useful indicator of renal injury. Because of the emerging availability of therapies that selectively block complement activation, the aim of the present study is to investigate whether MAC immunostaining might be a useful marker of IgA-mediated renal injury. METHODS: We conducted immunohistochemistry analysis of the MAC on renal biopsies from 67 pediatric patients with IgAN and Henoch-Schönlein purpura. We classified their renal biopsies according to the Oxford classification, retrieved symptoms, biological parameters, treatment, and follow-up. RESULTS: We found MAC expression was significantly related to impaired renal function and patients whose clinical course required therapy. MAC deposits tend to be more abundant in patients with decreased glomerular filtration rate (p = 0.02), patients with proteinuria > 0.750 g/day/1.73 m2, and with nephrotic syndrome. No correlation with histological alterations was observed. CONCLUSIONS: We conclude that MAC deposition could be a useful additional indicator of renal injury in patients with IgAN and Henoch-Schönlein purpura, independent of other indicators.

Sulfated Dendritic Polyglycerol Is a Potent Complement Inhibitor.

The complement system is a powerful mechanism of the innate immune defense system. Dysregulation may contribute to several diseases. Heparin is a known regulator of the complement system, but its application is limited due to its anticoagulative activity. A promising alternative is the synthetic analogue dendritic polyglycerol sulfate (dPGS). Although dPGS-mediated inhibition of the classical and alternative pathway has been roughly described previously, here we analyzed the effects of dPGS regarding the three pathways at different levels of the proteolytic cascades for the first time. Regarding the final outcome (membrane attack complex formation), IC50 values for dPGS varied between the alternative (900 nM), the classical (300 nM), and the lectin pathway (60 nM). In a backward approach, processing of proteins C5 and C3 via the respective convertase was analyzed by ELISA to narrow down dPGS targets. A dose-dependent reduction of C5a and C3a levels was detected. Further, the analysis via surface plasmon resonance revealed novel dPGS binding proteins; the pro-inflammatory anaphylatoxins C3a and C5a and the classical pathway activator C1q showed down to nanomolar binding affinities. The fully synthetic multivalent polymer dPGS seems to be a promising candidate for the further development to counteract excessive complement activation in disease.

The Structural Basis for Complement Inhibition by Gigastasin, a Protease Inhibitor from the Giant Amazon Leech.

Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway.

The Group B Streptococcus-Secreted Protein CIP Interacts with C4, Preventing C3b Deposition via the Lectin and Classical Complement Pathways.

The group B Streptococcus (GBS) is a leading cause of neonatal invasive disease. GBS bacteria are surrounded by a thick capsular polysaccharide that is a potent inhibitor of complement deposition via the alternative pathway. Several of its surface molecules can however activate the classical and lectin complement pathways, rendering this species still vulnerable to phagocytic killing. In this study we have identified a novel secreted protein named complement interfering protein (CIP) that downregulates complement activation via the classical and lectin pathways, but not the alternative pathway. The CIP protein showed high affinity toward C4b and inhibited its interaction with C2, presumably preventing the formation of the C4bC2a convertase. Addition of recombinant CIP to GBS cip-negative bacteria resulted in decreased deposition of C3b on their surface and in diminished phagocytic killing in a whole-blood assay. Our data reveal a novel strategy exploited by GBS to counteract innate immunity and could be valuable for the development of anti-infective agents against this important pathogen.

Critical Role and Therapeutic Control of the Lectin Pathway of Complement Activation in an Abortion-Prone Mouse Mating.

The abortion-prone mating combination CBA/J × DBA/2 has been recognized as a model of preeclampsia, and complement activation has been implicated in the high rate of pregnancy loss observed in CBA/J mice. We have analyzed the implantation sites collected from DBA/2-mated CBA/J mice for the deposition of the complement recognition molecules using CBA/J mated with BALB/c mice as a control group. MBL-A was observed in the implantation sites of CBA/J × DBA/2 combination in the absence of MBL-C and was undetectable in BALB/c-mated CBA/J mice. Conversely, C1q was present in both mating combinations. Searching for other complement components localized at the implantation sites of CBA/J × DBA/2, we found C4 and C3, but we failed to reveal C1r. These data suggest that complement is activated through the lectin pathway and proceeds to completion of the activation sequence as revealed by C9 deposition. MBL-A was detected as early as 3.5 d of pregnancy, and MBL-A deficiency prevented pregnancy loss in the abortion-prone mating combination. The contribution of the terminal complex to miscarriage was supported by the finding that pregnancy failure was largely inhibited by the administration of neutralizing Ab to C5. Treatment of DBA/2-mated CBA/J mice with Polyman2 that binds to MBL-A with high affinity proved to be highly effective in controlling the activation of the lectin pathway and in preventing fetal loss.

New insight into the effects of heparinoids on complement inhibition by C1-inhibitor.

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.

Finding off-targets, biological pathways, and target diseases for chymase inhibitors via structure-based systems biology approach.

Off-target binding connotes the binding of a small molecule of therapeutic significance to a protein target in addition to the primary target for which it was proposed. Progressively such off-targeting is emerging to be regular practice to reveal side effects. Chymase is an enzyme of hydrolase class that catalyzes hydrolysis of peptide bonds. A link between heart failure and chymase is ascribed, and a chymase inhibitor is in clinical phase II for treatment of heart failure. However, the underlying mechanisms of the off-target effects of human chymase inhibitors are still unclear. Here, we develop a robust computational strategy that is applicable to any enzyme system and that allows the prediction of drug effects on biological processes. Putative off-targets for chymase inhibitors were identified through various structural and functional similarity analyses along with molecular docking studies. Finally, literature survey was performed to incorporate these off-targets into biological pathways and to establish links between pathways and particular adverse effects. Off-targets of chymase inhibitors are linked to various biological pathways such as classical and lectin pathways of complement system, intrinsic and extrinsic pathways of coagulation cascade, and fibrinolytic system. Tissue kallikreins, granzyme M, neutrophil elastase, and mesotrypsin are also identified as off-targets. These off-targets and their associated pathways are elucidated for the effects of inflammation, cancer, hemorrhage, thrombosis, and central nervous system diseases (Alzheimer's disease). Prospectively, our approach is helpful not only to better understand the mechanisms of chymase inhibitors but also for drug repurposing exercises to find novel uses for these inhibitors.

Mechanisms of complement activation by dextran-coated superparamagnetic iron oxide (SPIO) nanoworms in mouse versus human serum.

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.

Endogenous and natural complement inhibitor attenuates myocardial injury and arterial thrombogenesis.

BACKGROUND: Coagulation disorders and reperfusion of ischemic myocardium are major causes of morbidity and mortality. Lectin pathway initiation complexes are composed of multimolecular carbohydrate recognition subcomponents and 3 lectin pathway-specific serine proteases. We have recently shown that the lectin pathway-specific carbohydrate recognition subcomponent mannose-binding lectin plays an essential role in the pathophysiology of thrombosis and ischemia/reperfusion injury. Thus, we hypothesized that the endogenous mannose-binding lectin (MBL)/ficolin-associated protein-1 (MAP-1) that inhibits complement activation in vitro also could be an in vivo regulator by attenuating myocardial schema/reperfusion injury and thrombogenesis when used at pharmacological doses in wild-type mice. METHODS AND RESULTS: In 2 mouse models, MAP-1 preserves cardiac function, decreases infarct size, decreases C3 deposition, inhibits MBL deposition, and prevents thrombogenesis. Furthermore, we demonstrate that MAP-1 displaces MBL/ficolin-associated serine protease (MASP)-1, MASP-2, and MASP-3 from the MBL complex. CONCLUSIONS: Our results suggest that the natural, endogenous inhibitor MAP-1 effectively inhibits lectin pathway activation in vivo. MAP-1 at pharmacological doses represents a novel therapeutic approach for human diseases involving the lectin pathway and its associated MASPs.

Serglycin inhibits the classical and lectin pathways of complement via its glycosaminoglycan chains: implications for multiple myeloma.

Serglycin (SG) is a proteoglycan expressed by hematopoietic cells and is constitutively secreted by multiple myeloma (MM) cells. SG participates in the regulation of various inflammatory events. We found that SG secreted by human MM cell lines inhibits both the classical and lectin pathways of complement, without influencing alternative pathway activity. The inhibitory effect of SG is due to direct interactions with C1q and mannose-binding lectin (MBL). C1q-binding is mediated through the glycosaminoglycan moieties of SG, whereas MBL binds additionally to SG protein core. Interactions between SG and C1q as well as MBL are diminished in the presence of chondroitin sulfate type E. In addition, we localized the SG-binding site to the collagen-like stalk of C1q. Interactions between SG and C1q as well as MBL are ionic in character and only the interaction with MBL was found to be partially dependent on the presence of calcium. We found the serum levels of SG to be elevated in patients with MM compared to healthy controls. Moreover, we found that SG expressed from myeloma plasma cells protects these cells from complement activation induced by treatment with anti-thymocyte immunoglobulins. This might protect myeloma cells during immunotherapy and promote survival of malignant cells.

Mannose-binding lectin has a direct deleterious effect on ischemic brain microvascular endothelial cells.

Mannose-binding lectin (MBL), an initiator of the lectin pathway, is detrimental in ischemic stroke. MBL deposition on the ischemic endothelium indicates the beginning of its actions, but downstream mechanisms are not clear yet.We investigated MBL interactions with the ischemic endothelium by exposing human brain microvascular endothelial cells (hBMECs) to protocols of ischemia. Cells were exposed to hypoxia or oxygen-glucose deprivation (OGD), and re-oxygenated with human serum (HS) or recombinant MBL (rhMBL). Hypoxic hBMECs re-oxygenated with HS showed increased complement system activation (C3c deposition, +59%) and MBL deposition (+93%) than normoxic cells. Super-resolution microscopy showed MBL internalization in hypoxic cells and altered cytoskeletal organization, indicating a potential MBL action on the endothelial structure. To isolate MBL effect, hBMECs were re-oxygenated with rhMBL after hypoxia/OGD. In both conditions, MBL reduced viability (hypoxia: -25%, OGD: -34%) compared to conditions without MBL, showing a direct toxic effect. Ischemic cells also showed greater MBL deposition (hypoxia: +143%, OGD: +126%) than normoxic cells. These results were confirmed with primary hBMECs exposed to OGD (increased MBL-induced cell death: +226%, and MBL deposition: +104%). The present findings demonstrate that MBL can exert a direct deleterious effect on ischemic brain endothelial cells in vitro, independently from complement activation.

Endothelial injury and thrombotic microangiopathy in COVID-19: Treatment with the lectin-pathway inhibitor narsoplimab.

In COVID-19, acute respiratory distress syndrome (ARDS) and thrombotic events are frequent, life-threatening complications. Autopsies commonly show arterial thrombosis and severe endothelial damage. Endothelial damage, which can play an early and central pathogenic role in ARDS and thrombosis, activates the lectin pathway of complement. Mannan-binding lectin-associated serine protease-2 (MASP-2), the lectin pathway's effector enzyme, binds the nucleocapsid protein of severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2), resulting in complement activation and lung injury. Narsoplimab, a fully human immunoglobulin gamma 4 (IgG4) monoclonal antibody against MASP-2, inhibits lectin pathway activation and has anticoagulant effects. In this study, the first time a lectin-pathway inhibitor was used to treat COVID-19, six COVID-19 patients with ARDS requiring continuous positive airway pressure (CPAP) or intubation received narsoplimab under compassionate use. At baseline and during treatment, circulating endothelial cell (CEC) counts and serum levels of interleukin-6 (IL-6), interleukin-8 (IL-8), C-reactive protein (CRP) and lactate dehydrogenase (LDH) were assessed. Narsoplimab treatment was associated with rapid and sustained reduction of CEC and concurrent reduction of serum IL-6, IL-8, CRP and LDH. Narsoplimab was well tolerated; no adverse drug reactions were reported. Two control groups were used for retrospective comparison, both showing significantly higher mortality than the narsoplimab-treated group. All narsoplimab-treated patients recovered and survived. Narsoplimab may be an effective treatment for COVID-19 by reducing COVID-19-related endothelial cell damage and the resultant inflammation and thrombotic risk.

Poly(ethylene glycol)s generate complement activation products in human serum through increased alternative pathway turnover and a MASP-2-dependent process.

Poly(ethylene glycol) (PEG) is receiving increasing attention as an intravenous therapeutic agent per se in a variety of experimental therapeutics and veterinary settings, such as spinal cord injury and traumatic axonal brain injury. PEG is often perceived to be immunologically safe, but here we demonstrate that near-monodisperse endotoxin-free PEGs, at concentrations relevant to above-mentioned settings, can generate complement activation products in human serum on a time scale of minutes (reflected in significant rises in serum levels of C4d, Bb, C3a-desArg and SC5b-9). With the aid of sera depleted from either C2 or C1q, and devoid of anti-PEG antibodies, we further demonstrate that, depending on PEG concentration and M(wt), generation of complement activation products occur either exclusively through the lectin pathway activation or through both the lectin pathway and increased fluid phase turnover of the alternative pathway. Inhibition of PEG-mediated C4d elevation in C1q-depleted serum by the broad serine protease inhibitor Futhan and anti-MASP-2 antibodies as well as competitive studies with d-mannose and N-acetylglucosamine indicated a likely role for ficolins/MASP-2 in PEG-mediated triggering of the lectin pathway and independent of calcium. PEG-mediated amplification of the alternative pathway is a complex process related to protein partitioning and exclusion effect, but factor H depletion/exclusion seems to play a minor role. Our results are relevant to the proposed potential therapeutic applications of intravenous PEG and warn about possible acute PEG infusion-related reactions in sensitive individuals and animals. PEG-mediated generation of complement activation products further provides a plausible explanation to the previously reported unexplained anaphylaxis or the referred cardiovascular collapse in sensitive animals that have received medicines containing high levels of PEG as solubilizer/carrier.

Effect of supraphysiologic levels of C1-inhibitor on the classical, lectin and alternative pathways of complement.

C1-inhibitor is increasingly used experimentally and clinically in inflammatory conditions like septicemia and ischemia-reperfusion injury. Several mechanisms may account for the anti-inflammatory effects of C1-inhibitor, including inhibition of complement. The aim of the present study was to investigate and compare the supraphysiologic effect of C1-inhibitor on the three complement pathways. Novel assays for specific evaluation of the classical, lectin and alternative pathways were employed using normal human serum supplemented with increasing concentrations of C1-inhibitor. Solid-phase classical- and lectin pathway activation was dose-dependently and significantly reduced up to 85% in the range of 2-28 times physiologic C1-inhibitor concentration. The lectin pathway was more potently inhibited than the classical at low doses. A functional lectin pathway assay demonstrated a significant reduction of C4 deposition up to 86% even at low concentration of C1-inhibitor and documented the effect to be at the level of MBL/MASPs. In contrast, C1-inhibitor had no effect on solid-phase alternative pathway activation, but significantly reduced cobra venom factor-induced fluid-phase activation up to 88%. The negative controls albumin and IgG had no effect on complement activation. The positive inhibitory controls compstatin (C3 inhibition), EDTA- or MBL-deficient sera reduced complement activation by 82-100%. We conclude that C1-inhibitor in high physiologic doses differentially inhibits all three-complement pathways. The inhibition pattern was strikingly different in the classical and lectin pathway, compared to the alternative. Previous studies interpreting the effects of C1-inhibitor as only due to classical pathway inhibition needs reconsideration. The data has implications for the therapeutic use of C1-inhibitor.

The Lectin Pathway of Complement in Myocardial Ischemia/Reperfusion Injury-Review of Its Significance and the Potential Impact of Therapeutic Interference by C1 Esterase Inhibitor.

Acute myocardial infarction (AMI) remains a leading cause of morbidity and mortality in modern medicine. Early reperfusion accomplished by primary percutaneous coronary intervention is pivotal for reducing myocardial damage in ST elevation AMI. However, restoration of coronary blood flow may paradoxically trigger cardiomyocyte death secondary to a reperfusion-induced inflammatory process, which may account for a significant proportion of the final infarct size. Unfortunately, recent human trials targeting myocardial ischemia/reperfusion (I/R) injury have yielded disappointing results. In experimental models of myocardial I/R injury, the complement system, and in particular the lectin pathway, have been identified as major contributors. In line with this, C1 esterase inhibitor (C1INH), the natural inhibitor of the lectin pathway, was shown to significantly ameliorate myocardial I/R injury. However, the hypothesis of a considerable augmentation of myocardial I/R injury by activation of the lectin pathway has not yet been confirmed in humans, which questions the efficacy of a therapeutic strategy solely aimed at the inhibition of the lectin pathway after human AMI. Thus, as C1INH is a multiple-action inhibitor targeting several pathways and mediators simultaneously in addition to the lectin pathway, such as the contact and coagulation system and tissue leukocyte infiltration, this may be considered as being advantageous over exclusive inhibition of the lectin pathway. In this review, we summarize current concepts and evidence addressing the role of the lectin pathway as a potent mediator/modulator of myocardial I/R injury in animal models and in patients. In addition, we focus on the evidence and the potential advantages of using the natural inhibitor of the lectin pathway, C1INH, as a future therapeutic approach in AMI given its ability to interfere with several plasmatic cascades. Ameliorating myocardial I/R injury by targeting the complement system and other plasmatic cascades remains a valid option for future therapeutic interventions.

Be on Target: Strategies of Targeting Alternative and Lectin Pathway Components in Complement-Mediated Diseases.

The complement system has moved into the focus of drug development efforts in the last decade, since its inappropriate or uncontrolled activation has been recognized in many diseases. Some of them are primarily complement-mediated rare diseases, such as paroxysmal nocturnal hemoglobinuria, C3 glomerulonephritis, and atypical hemolytic uremic syndrome. Complement also plays a role in various multifactorial diseases that affect millions of people worldwide, such as ischemia reperfusion injury (myocardial infarction, stroke), age-related macular degeneration, and several neurodegenerative disorders. In this review, we summarize the potential advantages of targeting various complement proteins with special emphasis on the components of the lectin (LP) and the alternative pathways (AP). The serine proteases (MASP-1/2/3, factor D, factor B), which are responsible for the activation of the cascade, are straightforward targets of inhibition, but the pattern recognition molecules (mannose-binding lectin, other collectins, and ficolins), the regulatory components (factor H, factor I, properdin), and C3 are also subjects of drug development. Recent discoveries about cross-talks between the LP and AP offer new approaches for clinical intervention. Mannan-binding lectin-associated serine proteases (MASPs) are not just responsible for LP activation, but they are also indispensable for efficient AP activation. Activated MASP-3 has recently been shown to be the enzyme that continuously supplies factor D (FD) for the AP by cleaving pro-factor D (pro-FD). In this aspect, MASP-3 emerges as a novel feasible target for the regulation of AP activity. MASP-1 was shown to be required for AP activity on various surfaces, first of all on LPS of Gram-negative bacteria.

Asparaginase inhibits the lectin pathway of complement activation.

Oncological treatment has been associated with an increased risk of infection, most often related to therapy-induced pancytopenia. However, limited research has been conducted on the effect of oncological therapy on the complement system, being part of the non-cellular innate immune system. This became the rationale for an observational clinical study (C2012) in which we have investigated the prevalence of transient complement defects. Once we had observed such defects, a correlation of the complement defects to specific clinical parameters or to specific therapeutic regimens was investigated. A prominent defect observed in C2012 was the inhibition of the lectin pathway (LP) of complement activation during the treatment of acute lymphoblastic leukemia (ALL), which we could directly associate to the use of asparaginase (ASNase). Ex-vivo experiments confirmed a direct dose-dependent inhibitory effect of ASNase on the LP functionality.

Mannan-binding lectin (MBL)-mediated opsonization is enhanced by the alternative pathway amplification loop.

The complement system is a humoral effector in the innate immune system. Three activation pathways exist in the complement system, known as the classical pathway, the lectin pathway and the alternative pathway. Dysfunction of lectin pathway activation is caused by MBL deficiency. MBL deficiency in a cohort of healthy Caucasian blood bank donors was investigated with MBL genotyping and MBL plasma concentration. Recognition of the yeast-derived zymosan by MBL was investigated with Western blot. The involvement of the alternative pathway amplification loop in enhancing MBL-mediated opsonization of zymosan was investigated in a novel opsonophagocytosis assay for flow cytometry. Sera deficient for MBL, factor D or properdin were tested, and purified MBL, factor D or properdin were used to recover opsonization. The optimal receiver-operator characteristic (ROC) cut-off value for dividing the Caucasian cohort in MBL-sufficient and MBL-deficient was calculated at 0.7 microg/ml. Thirty-eight percent of the group had concentrations below 0.7 microg/ml. Zymosan eluates opsonized with MBL-sufficient sera contain high oligomers of MBL, while eluates from MBL-deficient donors contained hardly any MBL. The MBL-, factor D- and properdin-deficient sera showed reduced opsonophagocytosis by human control neutrophils, as compared to normal MBL-sufficient sera. This reduction in opsonization was restored to normal levels by addition of purified MBL, factor D and properdin. The absence of opsonization in the factor D- and properdin-deficient sera, but presence in normal serum after blocking with anti-C1q-F(ab)2 and anti-MBL-F(ab)2, demonstrates the involvement of the amplification loop in MBL-initiated zymosan opsonization, even at very low serum concentrations (up to 3%, v/v). In conclusion, our data demonstrate that the MBL-mediated route of complement activation depends on the alternative pathway amplification loop for optimal opsonization of zymosan.

Collectin-11 detects stress-induced L-fucose pattern to trigger renal epithelial injury.

Physiochemical stress induces tissue injury as a result of the detection of abnormal molecular patterns by sensory molecules of the innate immune system. Here, we have described how the recently discovered C-type lectin collectin-11 (CL-11, also known as CL-K1 and encoded by COLEC11) recognizes an abnormal pattern of L-fucose on postischemic renal tubule cells and activates a destructive inflammatory response. We found that intrarenal expression of CL-11 rapidly increases in the postischemic period and colocalizes with complement deposited along the basolateral surface of the proximal renal tubule in association with L-fucose, the potential binding ligand for CL-11. Mice with either generalized or kidney-specific deficiency of CL-11 were strongly protected against loss of renal function and tubule injury due to reduced complement deposition. Ex vivo renal tubule cells showed a marked capacity for CL-11 binding that was induced by cell stress under hypoxic or hypothermic conditions and prevented by specific removal of L-fucose. Further analysis revealed that cell-bound CL-11 required the lectin complement pathway-associated protease MASP-2 to trigger complement deposition. Given these results, we conclude that lectin complement pathway activation triggered by ligand-CL-11 interaction in postischemic tissue is a potent source of acute kidney injury and is amenable to sugar-specific blockade.