Complement Activation In Patients Research Articles

Erythropoietin Effect on Complement Activation in Chronic Kidney Disease

The complement system is an important part of innate immunity. Despite its known protective role, the complement system may contribute to increased inflammation and tissue injury in cases where its balanced activation is disrupted. The kidneys have been shown to be largely affected by complement dysregulation. The aim of the present study was to investigate the effect of erythropoietin administration, on the complement system, in chronic kidney disease patients. The study involved 20 patients with CKD who received erythropoietin and measurements of levels of complement factors C3a and C5a and complement regulatory proteins (CregPs) CD55, CD46, and CD59. An increase in serum C3a and C5a levels was observed in response to EPO therapy. The increase in C3a was statistically significant (p < 0.05) and concurrent with a statistically significant decrease in CD55 in CD4+ T cells (p < 0.05) and B cells (p < 0.05) and CD59 levels in CD4+ and CD8+ T cells (p < 0.05) at completion of EPO therapy compared with healthy controls. The above observations demonstrate that EPO induces complement activation in patients undergoing EPO therapy with a simultaneous restriction of CRegPs expression, thus possibly allowing the uncontrolled complement activation, which may contribute to tissue injury and disease progression.

A new alternative: inhibiting complement activation in patients with IgA nephropathy

Mesangial complement C3 deposits, reflecting alternative and possibly lectin pathway activation, are characteristic in biopsies of patients with IgA nephropathy (IgAN). A recent randomized controlled trial tested the efficacy and safety of iptacopan, a factor B inhibitor, in patients with IgAN. Iptacopan dose-dependently reduced proteinuria, and there was a pronounced decrease of urinary C5b-9. This offers the perspective of "personalizing" therapy, which would be a unique feature of this novel approach to IgAN. A phase III clinical trial (APPLAUSE-IgAN) is ongoing.

Complement involvement in sickle cell disease

Sickle Cell Disease (SCD) is a hereditary blood disorder characterized by the presence of abnormal hemoglobin, leading to the formation of sickle-shaped red blood cells, causing vaso-occlusion. Inflammation is a key component of the pathophysiology of SCD, contributing to the vascular complications and tissue damage. This review is centered on exploring the role of the inflammatory complement system in the pathophysiology of SCD. Our goal is to offer a comprehensive summary of the existing evidence regarding complement activation in patients with SCD, encompassing both steady-state conditions and episodes of vaso-occlusive events. Additionally, we will discuss the proposed mechanisms by which the complement system may contribute to tissue injury in this pathology. Finally, we will provide an overview of the available evidence concerning the effectiveness of therapeutic interventions aimed at blocking the complement system in the context of SCD and discuss the perspective of complement inhibition.

210 Complement activation in patients with ANCA-associated vasculitis – focusing on complement activation markers in remission

210 Complement activation in patients with ANCA-associated vasculitis – focusing on complement activation markers in remission

Platelets and the Lectin Pathway of Complement Activation in Patients with Systemic Lupus Erythematosus or Antiphospholipid Syndrome.

Background Patients with systemic lupus erythematosus (SLE) have an increased risk of thrombosis even when they do not have antiphospholipid syndrome (APS). Interactions between complement activation and activated platelets have been suggested in SLE and APS and could play a role in the increased thrombosis risk. Objectives To explore factors potentially related to the prothrombotic pathophysiology in patients with SLE, primary APS, and healthy controls, by investigating lectin pathway proteins (LPPs), complement activation, platelet aggregation, and platelet activation. Methods This cross-sectional cohort study included 20 SLE patients, 17 primary APS, and 39 healthy controls. Flow cytometry and light transmission aggregometry were used to assess platelet activation and aggregation. Using time-resolved immunofluorometric assays, the plasma concentrations of 11 LPPs and C3dg, reflecting complement activation, were measured. Results H-ficolin plasma concentrations were higher in SLE and APS patients than in controls ( p = 0.01 and p = 0.03). M-ficolin was lower in SLE than in APS ( p = 0.01) and controls ( p = 0.03). MAp19 was higher in APS patients than in SLE patients ( p = 0.01) and controls ( p < 0.001). In APS patients, MASP-2 and C3dg correlated negatively with platelet activation. Platelet-bound fibrinogen after agonist stimulation and C3dg concentrations correlated negatively with platelet activation. Conclusion We observed significant differences between SLE and APS patients regarding complement proteins and platelet activation. Particularly the negative correlations between MASP-2 and C3dg with platelet activation only observed in APS patients suggest that interactions between complement activation and platelets differ in SLE and APS.

A Role of Immunothrombosis in Acquired Antibody-Mediated Thrombophilia's: Classical Complement and Neutrophil Activation in APS and HIT

Introduction: Heparin induced thrombocytopenia (HIT) and APS (antiphospholipid syndrome) are acquired thrombophilia's characterized by both arterial and venous thromboembolic events (TE). As a common feature APS and HIT share the presence of pathological antibodies, which play a crucial role in disease pathogenesis. In HIT, antibodies are directed to platelet factor (PF) 4 and heparin, respectively, and in APS, antibodies target anti-phospholipids (anti-cardiolipin antibody, anti-Beta2- glycoprotein I and lupus anticoagulant). There is growing evidence that complement- and neutrophil activation in the form of neutrophil extracellular traps (NETs) play an important role in the pathogenesis of thrombosis, the so-called "immunothrombosis". Insufficient control with subsequent unrestrained complement activation results in thrombosis as seen in patients with paroxysmal nocturnal hemoglobinuria (PNH). Therapeutic inhibition of complement activation abrogates thrombosis in these patients, emphasizing the important role of complement activation in the pathogenesis of thrombosis. It has been demonstrated, that complement activation products efficiently induce neutrophil activation, and therapeutic complement inhibition in sepsis and PNH prevents neutrophil activation. Here, we investigate the role of complement activation and neutrophil activation in patients with APS and HIT, respectively. Methods To assess the association of complement activation and NET Formation, we measured complement activation products (C4bc, C3bc) and marks for neutrophil activation, such as nucleosomes and human neutrophil elastase-α1- antitrypsin complexes (HNE) by ELISA in plasma of 34 primary APS- and 26 HIT- patients and 31 healthy controls. Groups have been compared using the Mann- Whitney-Rank Test. Values are indicated as median and range, p<0.05 is considered statistically significant. Results Patients with APS and HIT showed sign. higher levels of complement activation products as compared to healthy controls. Complement activation mainly of the classical (C4b/c) and alternate pathway (C3b/c) in APS (11 nM and 263 nM) and in HIT (33 nM and 324 nM) were sign. higher (p<0.0001) as compared to the healthy controls (7 nM and 19 nM). C4bc levels were sign. higher in HIT as compared to APS patients (p<0001), but there was no difference in C3b/c levels between these two groups (see figure 1). Patients with HIT had a sig. (p<0.0001) higher nucleosomes (440 U/ml) and HNE (121 ng/ml) as compared to healthy controls (12 U/ml and 26 ng/ml). HNE levels were sign. (p<0.001) higher in APS patients (37 ng/ml) as compared to healthy controls (26 ng/ml), however no difference was observed in nucleosome levels (12 U/ml vs 14 U/ml) (see figure 2). Based on the number of positive antibodies, eleven APS patients have been stratified as high-risk and 24 APS patients as low- to intermediate risk. C4bc levels were sign. increased in the high-risk patients as compared to the low- to intermediate risk group, whereas no difference in C3b/c levels could be observed between the 2 groups. Similarly, HNE complexes sign. increased in the high-risk patients as compared to the low- to intermediate risk group, whereas no difference in nucleosome levels could be observed between the 2 groups. Six HIT patients (22%) had a diagnosed TE event whereas 17 patients (63%) had no diagnosed TE. In four patients (15%) no clinical data were available, and they have been excluded from subgroup analysis. Interestingly, the measured markers for complement - and neutrophil activation in HIT patient with and without thrombosis were not different. Conclusions We demonstrate complement activation in patients with HIT and APS. Based on the simultaneous raise in nucleosomes and HNE, there is evidence of NET formation in HIT, whereas only weak neutrophil activation can be found in APS. In APS classical complement activation and neutrophil activation correlates with APS risk stratification. Our results demonstrate classical complement pathway- and neutrophil activation in HIT and APS patients. These findings may form the base for new therapeutic strategies targeting complement and neutrophil activation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Heterogeneity of Acetylcholine Receptor Autoantibody-Mediated Complement Activity in Patients With Myasthenia Gravis.

Heterogeneity of Acetylcholine Receptor Autoantibody-Mediated Complement Activity in Patients With Myasthenia Gravis.

Thrombotic thrombocytopenic purpura in HIV-infected patients: new twists on an old disease.

Investigate the presence of inflammation, endothelial dysfunction and complement activation in patients with HIV-associated thrombotic thrombocytopenic purpura (HIV-TTP) to support the hypothesis that these processes probably contribute to the development of this thrombotic microangiopathy. A prospective, investigational cohort study of 35 consecutive patients diagnosed with HIV-associated TTP presenting to three academic, tertiary care hospitals in Johannesburg, South Africa over 2 years. The patients with HIV-TTP received therapeutic plasma therapy and supportive treatment. Demographic data, the results of routine investigations and patient outcomes were recorded. Peripheral blood samples were collected prior to and on completion of plasma therapy and the following additional parameters were assessed at both time points: activity of the von Willebrand factor (VWF) cleaving protease, a-disintegrin-and-metalloproteinase-with-thrombospondin-motifs 13 (ADAMTS-13) and the presence of ADAMTS-13 autoantibodies, levels of pro-inflammatory cytokines, interleukin-6 and tumour necrosis factor-alpha, and two endothelial cell adhesion molecules. Complement activation was assessed by sequential measurement of C3 and C4 as well as levels of the complement inhibitor, factor H. The inflammatory and endothelial activation markers were significantly ( P < 0.001) elevated in the cohort of patients prior to plasma therapy compared with levels on discharge. Complement was activated and normalized with therapy. The ADAMTS-13 levels were reduced with significant auto-antibodies to this protease at presentation. Inflammation in HIV mediates endothelial damage and complement activation. This study proposes that these processes are probably contributory to the development of HIV-TTP, which can therefore be characterized in part as a complementopathy, resembling TTP-like syndrome.

Sex-Differences and Associations Between Complement Activation and Synovial Vascularization in Patients with Late-Stage Knee Osteoarthritis.

PurposeSynovial inflammation in knee osteoarthritis (OA) causes disorganized synovial angiogenesis and complement activation in synovial fluid, but links between complement and synovial microvascular pathology have not been established. Since complement causes vascular pathology in other diseases and since sex-differences exist in complement activation and in OA, we investigated sex differences in synovial fluid complement factors, synovial tissue vascular pathology, and associations between complement and synovial vascular pathology in patients with late-stage knee OA.MethodsPatients with symptomatic, late-stage radiographic knee OA undergoing total knee arthroplasty or high tibial osteotomy provided matched synovial fluid and tissue biopsies during surgery. Complement factors (C2, C5, adipsin, MBL, and CFI) and terminal complement complex (sC5b-C9) were measured in synovial fluid by multiplex or enzyme-linked immunosorbent assay, respectively. Features of synovial vascular pathology (vascularization, perivascular edema, and vasculopathy) were assessed by histopathology. Multivariate linear regression models were used to assess associations between synovial fluid complement factors and histopathological features of vascular pathology, with adjustment for age, sex, body mass index, and sex interaction. Sex-disaggregated comparisons were completed.ResultsSynovial fluid biomarker and histopathology data were included from 97 patients. Most synovial fluid complement factors and synovial tissue histopathological features were similar between sexes. Synovial fluid C5 trended to lower levels in males (-20.93 ng/mL [95%CI -42.08, 0.23] p=0.05). Median vasculopathy scores (0.42 [95%CI 0.07, 0.77] p=0.02) were higher in males. In the full cohort, C5 concentration was associated with lower vascularization scores (-0.005 [95%CI -0.010, -0.0001] p=0.04) while accounting for sex*C5 interaction. In sex-disaggregated analyses, increased C5 concentration was associated with lower vascularization scores (-0.005 [95%CI –0.009, -0.0001] p=0.04) in male patients, but not in female patients. Males had higher sC5b-C9 compared to females. Additionally, males with high C5 had a higher synovial fluid concentration of sC5b-C9 compared to males with low C5. No differences were found in females.ConclusionHigher synovial fluid C5 levels were associated with increased complement activation and decreased synovial vascularization in males but not in females with OA. Future studies should test whether synovial fluid complement activation suppresses synovial angiogenesis and identify mechanisms accounting for C5-related sex-differences in synovial fluid complement activation in patients with knee OA.

Heterogeneity of Acetylcholine Receptor Autoantibody-Mediated Complement Activity in Patients With Myasthenia Gravis.

Background and ObjectivesAutoantibodies targeting the acetylcholine receptor (AChR), found in patients with myasthenia gravis (MG), mediate pathology through 3 mechanisms: complement-directed tissue damage, blocking of the acetylcholine binding site, and internalization of the AChR. Clinical assays, used to diagnose and monitor patients, measure only autoantibody binding. Consequently, they are limited in providing association with disease burden, understanding of mechanistic heterogeneity, and monitoring therapeutic response. The objective of this study was to develop a cell-based assay that measures AChR autoantibody–mediated complement membrane attack complex (MAC) formation.MethodsAn HEK293T cell line—modified using CRISPR/Cas9 genome editing to disrupt expression of the complement regulator genes (CD46, CD55, and CD59)—was used to measure AChR autoantibody–mediated MAC formation through flow cytometry.ResultsSerum samples (n = 155) from 96 clinically confirmed AChR MG patients, representing a wide range of disease burden and autoantibody titer, were tested along with 32 healthy donor (HD) samples. AChR autoantibodies were detected in 139 of the 155 (89.7%) MG samples through a cell-based assay. Of the 139 AChR-positive samples, autoantibody-mediated MAC formation was detected in 83 (59.7%), whereas MAC formation was undetectable in the HD group or AChR-positive samples with low autoantibody levels. MAC formation was positively associated with autoantibody binding in most patient samples; ratios (mean fluorescence intensity) of MAC formation to AChR autoantibody binding ranged between 0.27 and 48, with a median of 0.79 and an interquartile range of 0.43 (0.58–1.1). However, the distribution of ratios was asymmetric and included extreme values; 16 samples were beyond the 10–90 percentile, with high MAC to low AChR autoantibody binding ratio or the reverse. Correlation between MAC formation and clinical disease scores suggested a modest positive association (rho = 0.34, p = 0.0023), which included a subset of outliers that did not follow this pattern. MAC formation did not associate with exposure to immunotherapy, thymectomy, or MG subtypes defined by age-of-onset.DiscussionA novel assay for evaluating AChR autoantibody–mediated complement activity was developed. A subset of patients that lacks association between MAC formation and autoantibody binding or disease burden was identified. The assay may provide a better understanding of the heterogeneous autoantibody molecular pathology and identify patients expected to benefit from complement inhibitor therapy.

Complement Activation in Patients With Heat-Related Illnesses: Soluble CD59 Is a Novel Biomarker Indicating Severity of Heat-Related Illnesses.

OBJECTIVES:Although multiple organ dysfunction syndrome (MODS) is the main cause of death in patients with heat-related illnesses, its underlying pathophysiological mechanism remains elusive. Complement activation is considered one of the main causes of MODS in patients with sepsis and trauma. Considering the pathophysiological similarity of heat related-illnesses with sepsis and trauma, the complement system might be activated in patients with heat-related illnesses as well. Our aim was to investigate whether excessive complement activation occurs in patients with heat-related illnesses.DESIGN:Prospective observational study.SETTING:Emergency department in the university hospital.PATIENTS:Thirty-two patients with heat-related illnesses and 15 age-matched healthy controls were enrolled in this study.INTERVENTIONS:Blood samples were collected from the study subjects for the measurement of complement factors.MEASUREMENTS AND MAIN RESULTS:Complement component 3a (C3a), complement component 5a (C5a), C5b-9, complement factor B (Ba), Factor H, and soluble CD59 in plasma were measured. The levels of C3a, C5a, C5b-9, and Ba significantly increased in patients with heat-related illnesses on day 0 compared with those in the healthy controls. Soluble CD59 was significantly high in patients with heat-related illnesses on day 0 and showed a correlation with the severity of the condition (Acute Physiology and Chronic Health Evaluation II, Sequential Organ Failure Assessment, and staging scores), Japanese Association for Acute Medicine disseminated intravascular coagulation scores, and the coagulation system (prothrombin time and fibrin degradation products).CONCLUSIONS:The complement system was activated in patients with heat-related illnesses, suggesting that it is one of the causes of MODS. Soluble CD59 may be a potent biomarker for the severity of heat-related illnesses.

Activation of the Complement System in Patients with Cancer Cachexia.

Simple SummaryPatients with cancer often suffer from severe weight loss as a result of the wasting of skeletal muscle and fat tissue. This has a strong negative impact on the patient’s prognosis and quality of life. Inflammation is thought to contribute to weight loss in cancer. To enable the future targeting of inflammation in patients with cancer who experience weight loss, we set out to characterize an important pro-inflammatory component of the immune system: the complement system. The blood levels of several elements of the complement system were analyzed in patients with and without weight loss and inflammation. We found that complement factors were activated specifically in patients with both weight loss and inflammation. Since complement inhibitory drugs are already on the market, these findings may open new opportunities for treating inflammation-mediated weight loss in patients with cancer.Systemic inflammation is thought to underlie many of the metabolic manifestations of cachexia in cancer patients. The complement system is an important component of innate immunity that has been shown to contribute to metabolic inflammation. We hypothesized that systemic inflammation in patients with cancer cachexia was associated with complement activation. Systemic C3a levels were higher in cachectic patients with inflammation (n = 23, C-reactive protein (CRP) ≥ 10 mg/L) as compared to patients without inflammation (n = 26, CRP < 10 mg/L) or without cachexia (n = 13) (medians 102.4 (IQR 89.4–158.0) vs. 81.4 (IQR 47.9–124.0) vs. 61.6 (IQR 46.8–86.8) ng/mL, respectively, p = 0.0186). Accordingly, terminal complement complex (TCC) concentrations gradually increased in these patient groups (medians 2298 (IQR 2022–3058) vs. 1939 (IQR 1725–2311) vs. 1805 (IQR 1552–2569) mAU/mL, respectively, p = 0.0511). C3a and TCC concentrations were strongly correlated (rs = 0.468, p = 0.0005). Although concentrations of C1q and mannose-binding lectin did not differ between groups, C1q levels were correlated with both C3a and TCC concentrations (rs = 0.394, p = 0.0042 and rs = 0.300, p = 0.0188, respectively). In conclusion, systemic inflammation in patients with cancer cachexia is associated with the activation of key effector complement factors. The correlations between C1q and C3a/TCC suggest that the classical complement pathway could play a role in complement activation in patients with pancreatic cancer.

Role of uromodulin and complement activation in the progression of kidney disease.

Uromodulin (UMOD) is a glycoprotein that is selectively expressed on the epithelial cells of the thick ascending limb of Henle's loop and the early distal renal tubule. The present study aimed to investigate whether UMOD was associated with complement activation in patients with renal diseases. In addition, its biological function was examined in vitro. The expression levels of UMOD and complement components, including C1q, C3, C4 and C3a, and membrane attack complex (MAC) in the plasma of patients with IgA nephropathy (IgAN; n=58) and lupus nephritis (LN; n=36) were detected using ELISA, which was used to determine the association between UMOD expression and complement components. In addition, a simulated hypoxia-reoxygenation (H/R) model was used to stimulate UMOD expression in mouse inner medullary collecting duct cells. Additionally, the association between UMOD expression and complement components C1q and C3d at the cellular level was identified using western blotting and immunofluorescence, respectively. It was revealed that the plasma UMOD concentration was significantly decreased in patients with IgAN and LN compared with in healthy controls, and the levels of C3a and MAC were significantly increased in the plasma of patients with IgAN and LN. Furthermore, the plasma levels of C1q, C3 and C4 in patients with LN, but not in patients with IgAN, were significantly decreased compared with in healthy controls. The plasma levels of UMOD were negatively correlated with the plasma C3a and MAC concentrations. However, the plasma levels of UMOD were significantly and positively correlated with the plasma C1q concentration, but not with that of C3 and C4. It was identified that UMOD expression started to increase after 1 h of simulated H/R, and continued to increase at 6 and 12 h. In addition, cells with lower UMOD expression had higher C3d expression in vitro. Collectively, the present results suggested that UMOD was associated with severe complement activation and may be involved in complement-mediated immune protection by inhibiting complement activation in renal disease.

Erythrocytes identify complement activation in patients with COVID-19.

COVID-19, the disease caused by the SARS-CoV-2 virus, can progress to multisystem organ failure and viral sepsis characterized by respiratory failure, arrhythmias, thromboembolic complications, and shock with high mortality. Autopsy and preclinical evidence implicate aberrant complement activation in endothelial injury and organ failure. Erythrocytes express complement receptors and are capable of binding immune complexes; therefore, we investigated complement activation in patients with COVID-19 using erythrocytes as a tool to diagnose complement activation. We discovered enhanced C3b and C4d deposition on erythrocytes in COVID-19 sepsis patients and non-COVID sepsis patients compared with healthy controls, supporting the role of complement in sepsis-associated organ injury. Our data suggest that erythrocytes may contribute to a precision medicine approach to sepsis and have diagnostic value in monitoring complement dysregulation in COVID-19-sepsis and non-COVID sepsis and identifying patients who may benefit from complement targeted therapies.

Ocular and Systemic Complement Activation during Anti-Vascular Endothelial Growth Factor Treatment and Age-Related Eye Disease Study 2 Dietary Supplementation in Neovascular Age-Related Macular Degeneration

Purpose: The aim of this study was to investigate the influence of dietary supplementation using Age-Related Eye Disease Study 2 (AREDS2) on complement activation in patients with neovascular age-related macular degeneration (nAMD) under ongoing treatment. Methods: In this prospective, single-center, controlled, open-label investigator-initiated trial, eligible nAMD patients were randomized at a ratio of 1:1 in 2 groups: those with and without dietary AREDS2 supplementation for 4 weeks. Zinc, plasma, and aqueous humor (AH) complement levels were quantified via enzyme-linked immunosorbent assays. Results: Fifty of 62 enrolled patients completed the trial (AREDS2 n = 27, controls n = 23). Systemic zinc and complement levels were not different at baseline between the 2 groups (p > 0.1). At the final visit, systemic zinc levels were significantly higher in the AREDS2 group (10.16 ± 2.08 μmol/L; 8.66 ± 1.17 μmol/L; p = 0.007), whereas systemic and AH complement levels were not different (p > 0.1). In both groups, no significant change was observed in systemic levels of C3, C3a, FH, FI, and sC5b-9 (p > 0.1). Only systemic complement component Ba showed an increase from baseline to the end visit (p = 0.01). This increase was higher in the control group (p = 0.02) than in the AREDS2 group (p = 0.23). Conclusions: Short-term dietary AREDS2 supplementation leads to a significant increase in systemic zinc levels without any influence on complement activation levels.

Targeting the Complement Serine Protease MASP-2 as a Therapeutic Strategy for Coronavirus Infections.

MASP-2, mannose-binding protein-associated serine protease 2, is a key enzyme in the lectin pathway of complement activation. Hyperactivation of this protein by human coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2 has been found to contribute to aberrant complement activation in patients, leading to aggravated lung injury with potentially fatal consequences. This hyperactivation is triggered in the lungs through a conserved, direct interaction between MASP-2 and coronavirus nucleocapsid (N) proteins. Blocking this interaction with monoclonal antibodies and interfering directly with the catalytic activity of MASP-2, have been found to alleviate coronavirus-induced lung injury both in vitro and in vivo. In this study, a virtual library of 8736 licensed drugs and clinical agents has been screened in silico according to two parallel strategies. The first strategy aims at identifying direct inhibitors of MASP-2 catalytic activity, while the second strategy focusses on finding protein-protein interaction inhibitors (PPIs) of MASP-2 and coronaviral N proteins. Such agents could represent promising support treatment options to prevent lung injury and reduce mortality rates of infections caused by both present and future-emerging coronaviruses. Forty-six drug repurposing candidates were purchased and, for the ones selected as potential direct inhibitors of MASP-2, a preliminary in vitro assay was conducted to assess their interference with the lectin pathway of complement activation. Some of the tested agents displayed a dose-response inhibitory activity of the lectin pathway, potentially providing the basis for a viable support strategy to prevent the severe complications of coronavirus infections.

The correlation of anti-β2-glycoprotein I antibody with complement activation in patients with idiopathic membranous nephropathy.

This study evaluated the correlation of anti-β2-glycoprotein I (anti-β2-GPI) antibodies with complement activation in patients with idiopathic membranous nephropathy (IMN). Thirty-two IMN patients with positive anti-β2-GPI antibody were enrolled, and 32 age- and sex-matched IMN patients with negative anti-β2-GPI antibody were randomly enrolled as controls. The frozen serum samples of these 64 patients were collected for detection of anti-phospholipase A2 receptor (PLA2R) antibody and the activity of three complement pathways. Paraffin specimens of the kidney tissues of these 64 patients were collected for immunohistochemical staining of C4d. IMN patients with positive anti-β2-GPI antibody had a significant decline of the residual complement activity of alternative pathway than those with negative anti-β2-GPI antibody (37.4 ± 21.2% vs 65.7 ± 50.5%, P = 0.021). The positive rate of kidney C4d staining in IMN patients with and without anti-β2-GPI was 65.6 and 81.2%, with no significant difference (P = 0.257). Patients with and without anti-β2-GPI had the same positive rate of anti-PLA2R antibody. Anti-β2-GPI antibody was associated with alternative complement activation in patients with IMN.

Thrombotic Microangiopathy Variants Are Independently Associated with Critical Disease in COVID-19 Patients

Background: Several recent studies support the notion of excessive complement activation in patients with severe coronavirus disease-19 (COVID-19), with beneficial results of complement inhibition in case series. In this context, severe COVID-19 shares common characteristics with complement-mediated thrombotic microangiopathy (TMA). TMA is commonly characterized by genetic susceptibility, and presents with thrombocytopenia, anemia, increased lactate dehydrogenase (LDH), and organ damage (renal, neurological, cardiac).Aims: We hypothesized that genetic susceptibility would be also evident in patients with severe COVID-19 and would be associated with disease severity.Methods: We prospectively studied consecutive adult patients hospitalized with COVID-19 in our referral centers (April-May 2020). Diagnosis was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). COVID-19 severity was assessed based on World Health Organization's (WHO) criteria into moderate/severe, and critical disease. Additional data on patients' history and course were recorded by treating physicians that followed patients up to discharge or death. Patients' DNA was obtained from peripheral blood samples. Probes were designed using the Design studio (Illumina). Amplicons cover exonic regions of TMA-associated genes (Complement factor H/CFH, CFH-related, CFI, CFB, CFD, C3, CD55, C5, MCP, thombomodulin/THBD, ADAMTS13) spanning 15 bases into the intronic regions. We used 10ng of initial DNA material. Libraries were quantified using Qubit and sequenced on a MiniSeq System in a 2x150 bp run. Analysis was performed using the TruSeq Amplicon application (BaseSpace). Alignment was based on the banded Smith-Waterman algorithm in the targeted regions (specified in a manifest file). We performed variant calling with the Illumina-developed Somatic Variant Caller in germline mode and variant allele frequency higher than 20%. Both Ensembl and Refseq were used for annotation of the output files. Variants clinical significance was based on ClinVar and the current version of the Complement Database, as we have previously described.Results: We studied 60 patients, 40 with moderate/severe disease hospitalized in COVID-19 general ward (GW) and 20 with critical disease hospitalized in intensive care units (ICU). Among them, 11 patients succumbed due to COVID-19 disease. Patients laboratory characteristics are shown in Figure.In genetic analysis, patients presented heterogeneous variant profiles including pathogenic, benign, likely benign, and variants of unknown significance (median number of variants: 62, range: 51-89). Search in the Complement Database revealed seven patients, each carrying one pathogenic or likely pathogenic variant in C3, CD46, DGKE, and CFH. Based on ClinVar, we found a pathogenic variant of ADAMTS13 (rs2301612, missense) in 28 patients. We also detected two missense risk factor variants, previously detected in complement-related diseases: rs2230199 in C3 (13 patients); and rs800292 in CFH (26 patients). Among them, 22 patients had a combination of these characterized variants. This combination was significantly associated with critical disease that required intensive care (p=0.037), as well as low lymphocyte counts (p=0.021) and high neutrophil-to-lymphocyte ratio (p=0.050). In the multivariate model, critical disease was an independent predictor of double heterozygocity in these variants. Furthermore, one patient had a rare germline missense variant in CFI (rs112534524), previously detected in complement-related diseases. This patient suffered from critical disease but survived after long-term ICU hospitalization. Interestingly, five patients showed a likely protective missense variant in CFB (rs641153).Conclusion: We have detected for the first time rare and pathogenic TMA-associated variants in patients with severe COVID-19. Our findings of variants in complement-regulatory genes and ADAMTS13 suggest genetic susceptibility and define proof-of-concept for proper selection of patients that would benefit from complement inhibition.TableDisclosuresGavriilaki:Omeros Pharmaceuticals: Consultancy.

Association of imaging biomarkers and local activation of complement in aqueous humor of patients with early forms of age-related macular degeneration

PurposeTo investigate a possible correlation between established imaging biomarkers for age-related macular degeneration and local complement system activation, measured in aqueous humor (AH) of patients with early stages of age-related macular degeneration (AMD) and controls.MethodsThis analysis included prospectively acquired AH samples of 106 eyes (35 with early/intermediate AMD, 71 controls). The levels of complement protein 3 (C3), 4 (C4), 5 (C5); activation products of complement factor 3a (C3a) and Ba, C3b/iC3b; complement factors B, D, H, I (CFB, CFD, CFH, CFI); and total protein concentration were analyzed. Quantitative levels of complement factors were correlated to the presence of reticular pseudodrusen (RPD), the presence of hyperreflective foci (HRF), and total drusen volume (DV) graded on imaging by spectral-domain optical coherence tomography and using Spearman’s rank correlation test.ResultsDV correlated with C3b/iC3b (r = 0.285; P = 0.034), C3a (r = 0.200; P = 0.047), Ba (r = 0.262; P = 0.009), and C5 (r = 430; P = 0.005), and showed a tendency towards correlation with C3a (r = 0.198; P = 0.057). HRF correlated significantly with C5 (r = 0.388; P = 0.011) and RPD showed a tendency towards correlation with CFB (r = 0.196; P = 0.050).ConclusionIn patients with early AMD, HRF and drusen parameters but not RPD show low to fair levels of correlation with local complement activation in patients’ AH. Better understanding of complement activation could provide some insights into the pathogenesis of AMD. Imaging biomarkers could be useful to identify suitable patients for future clinical trials with complement-modulating therapies.

Systemic complement activation in anti-neutrophil cytoplasmic antibody-associated vasculitis and necrotizing glomerulonephritis.

Due to the accumulating evidence of complement activation in anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV), we decided to investigate the possibility of systemic complement activation in patients with Necrotizing Glomerulonephritis secondary to AAV. Clinical, laboratory and histological findings, and serum levels of complement components, C3a, C5a and Bb fragment of Factor B and C4d, were estimated in patients with AAV and glomerulonephritis, at time of diagnosis, before any treatment had been applied. All patients were treated with the same immunosuppressive protocol and followed up for total 24 months. Twenty age and sex matched healthy individuals served as controls. Serum levels of all complement components were significantly increased in patients, compared to controls; C5a: 19.9(0.02-48) vs 9.06(2.1-16.3)pg/mL, P = .002, Bb: 7.3(0.02-31.4) vs 0.2(0.02-1.6)pg/mL, P < .0001, C3a: 4.7(0.4-7.2) vs 2.4(1.09-5)pg/mL, P = .05 and C4d: 11.6(0.07-70) vs 0.7(0.07-8.2)pg/mL, P = .001, respectively. There was strong correlation between serum Bb levels and eGFR and FFS2009 score at time of diagnosis (r = -.41, P = .002 and r = .41, P = .003 respectively). Also, serum Bb levels were increased in patients with severe interstitial infiltration (P = .04) and focal necrosis (P = .01) on renal biopsy. Serum Bb levels could also predict renal function outcome during the acute phase of disease, but not at the end of follow up. We provided strong evidence of systemic activation of complement alternative pathway in the development and progression of AAV and glomerulonephritis. Serum Bb seem to play a critical role in the induction, also predicting disease activity and outcome, yet activation of classical pathway cannot be excluded.