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Background Atypical hemolytic uremic syndrome (aHUS), a severe thrombotic microangiopathy, is often related to complement dysregulation, but the pathomechanisms remain unknown in at least 30% of patients. Researchers have described autoantibodies to complement factor H of the IgG class in 10% of patients with aHUS but have not reported anti-factor H autoantibodies of the IgM class. Methods In 186 patients with thrombotic microangiopathy clinically presented as aHUS, we searched for anti-factor H autoantibodies of the IgM class and those of the IgG and IgA classes. We used immunochromatography to purify anti-factor H IgM autoantibodies and immunoenzymatic methods and a competition assay with mapping mAbs to characterize interaction with the target protein. Results We detected anti-factor H autoantibodies of the IgM class in seven of 186 (3.8%) patients with thrombotic microangiopathy presented as aHUS. No association was observed between anti-factor H IgM and homozygous deletions involving CFHR3-CFHR1. A significantly higher proportion of patients with bone marrow transplant-related thrombotic microangiopathy had anti-factor H IgM autoantibodies versus other patients with aHUS: three of 20 (15%) versus four of 166 (2.4%), respectively. The identified IgM autoantibodies recognize the SCR domain 19 of factor H molecule in all patients and interact with the factor H molecule, inhibiting its binding to C3b. Conclusions Detectable autoantibodies to factor H of the IgM class may be present in patients with aHUS, and their frequency is six-fold higher in thrombotic microangiopathy forms associated with bone marrow transplant. The autoantibody interaction with factor H's active site may support an autoimmune mechanism in some cases previously considered to be of unknown origin.

The pathophysiology of the severe complications of COVID-19 is still unclear. We report preliminary data providing evidence of complement activation in patients with COVID-19 with different degrees of respiratory failure.

Atypical hemolytic uremic syndrome (aHUS) is characterized by platelet consumption, hemolysis, and renal injury. Eculizumab, a humanized antibody that blocks complement activity, has been successfully used in aHUS, but the best treatment schedule has not yet been clearly defined.Herein we report our experience with eculizumab maintenance treatment, in which the interval between subsequent doses was adjusted based on classical complement pathway (CCP) activity, targeted to 30% for the prevention of relapses. Trough circulating levels of free eculizumab were determined by an immunoenzymatic method. Genetic and serologic characteristics of the patients were also assessed.We report on 38 patients with aHUS with a median age of 25.0 years (range 0.5-60.0 years) treated with eculizumab. Once stable disease remission was obtained, the interval between eculizumab doses was extended based on target CCP activity. With this approach, presently, 22 patients regularly receive eculizumab infusion every 28 days and 16 receive it every 21. During a median observation period of 32.3 months (range 4.0-92.4 months) and a cumulative period of 1295 months, no patient relapsed. An inverse correlation between CCP activity and eculizumab circulating levels was present (r = - 0.690, p = 0.0001), with CCP activity being inhibited as long as free eculizumab was measurable in serum.In patients with aHUS on eculizumab maintenance treatment, complement activity measurement can be used as a proxy for circulating levels of the drug. Monitoring complement activity allows for safe tailoring of the frequency of eculizumab administration, thus avoiding excessive drug exposure while keeping the disease in remission.

Background Genetic variation at a multigene cluster at chromosome 3p21.31 and the ABO blood group have been associated with the risk of developing severe COVID-19, but the mechanism remains unclear. Complement activation has been associated with COVID-19 severity. Objective The aim of this study was to examine whether chromosome 3p21.31 and the ABO variants are linked to the activation of the complement cascade in COVID-19 patients. Methods We considered 72 unrelated European hospitalized patients with genetic data and evaluation of circulating C5a and soluble terminal complement complex C5b-9 (SC5b-9). Twenty-six (36.1%) patients carried the rs11385942 G>GA variant and 44 (66.1%) non-O blood group associated with increased risk of severe COVID-19. Results C5a and SC5-b9 plasma levels were higher in rs11385949 GA carriers than in non-carriers (P = 0.041 and P = 0.012, respectively), while C5a levels were higher in non-O group than in O group patients (P = 0.019). The association between rs11385949 and SC5b-9 remained significant after adjustment for ABO and disease severity (P = 0.004) and further correction for C5a (P = 0.018). There was a direct relationship between upper airways viral load and SC5b-9 in carriers of the rs11385949 risk allele (P = 0.032), which was not observed in non-carriers. Conclusions The rs11385949 G>GA variant, tagging the chromosome 3 gene cluster variation and predisposing to severe COVID-19, is associated with enhanced complement activation, both with C5a and terminal complement complex, while non-O blood group with C5a levels. These findings provide a link between genetic susceptibility to more severe COVID-19 and complement activation., Highlights • The rs11385942 G>GA variant and ABO blood group are linked to severe COVID-19. • Complement activation parallels severity and disease activity in COVID-19 patients. • In COVID-19, we found that rs11385949 G>GA predispose to complement activation. • Non-O blood group is also associated with complement activation in COVID-19 patients.

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.

Patients with atypical hemolytic uremic syndrome (aHUS) have long been considered ineligible for kidney transplantation (KTx) in several centers due to the high risk of disease recurrence, graft loss and life-threatening complications. The availability of Eculizumab (ECU) has now overcome this problem. However, the best approach towards timing, maintenance schedule, the possibility of discontinuation and patient monitoring has not yet been clearly established. This is a single center case series presenting our experience with KTx in aHUS. This study included 26 patients (16 females) with a diagnosis of aHUS, who spent a median of 5.5 years on kidney replacement therapy before undergoing KTx. We compared the aHUS relapse rate in three groups of patients who underwent KTx: patients who received no prophylaxis, patients who underwent plasma exchange, those who received Eculizumab prophylaxis. Complement factor H-related disease was by far the most frequent etiology (n = 19 patients). Untreated patients and patients undergoing pre-KTx plasma exchange prophylaxis had a relapse rate of 0.81 (CI 0.30–1.76) and 3.1 (CI 0.64–9.16) events per 10 years cumulative observation, respectively, as opposed to 0 events among patients receiving Eculizumab prophylaxis. The time between Eculizumab doses was tailored based on classic complement pathway activity (target to

Background Animal models and few clinical reports suggest the involvement of the complement system in the onset of severe manifestations of coronavirus disease-2019 (COVID-19). However, complement contribution to endotheliopathy and hypercoagulability has not been elucidated yet. Objective To evaluate the association among complement activation, endothelial damage and disease severity or activity in COVID-19 patients. Methods In this single-centre cohort study, 148 patients with COVID-19 of different severity were evaluated upon hospital admission and 30 days later. Markers of complement activation (SC5b-9 and C5a) and endothelial perturbation (von Willebrand factor [vWF], tissue-type plasminogen activator [t-PA], plasminogen activator inhibitor-1 [PAI-1], soluble thrombomodulin [sTM], and soluble endothelial selectin [sE-selectin]) were measured in plasma. Results The patients had high plasma levels of SC5b-9 and C5a (p = 0.0001 for both) and vWF, t-PA and PAI-1 (p = 0.0001 for all). Their SC5b-9 levels correlated with those of vWF (r = 0.517, p = 0.0001) and paralleled disease severity (severe vs mild p = 0.0001, severe vs moderate p = 0.026 and moderate vs mild p = 0.001). The levels of sE-selectin were significantly increased only in the patients with severe disease. After 30 days, plasma SC5b-9, C5a and vWF levels had significantly decreased (p = 0.0001 for all), and 43% of the evaluated patients had normal levels. Conclusions Complement activation is boosted during the progression of COVID-19 and dampened during remission, thus indicating its role in the pathophysiology of the disease. The association between complement activation and the biomarkers of endothelial damage suggests that complement may contribute to tissue injury and could be the target of specific therapy.

Genetic variation at a multigene cluster at chromosome 3p21.31 and the ABO blood group have been associated with the risk of developing severe COVID-19, but the mechanism remains unclear. Complement activation has been associated with COVID-19 severity.The aim of this study was to examine whether chromosome 3p21.31 and the ABO variants are linked to the activation of the complement cascade in COVID-19 patients.We considered 72 unrelated European hospitalized patients with genetic data and evaluation of circulating C5a and soluble terminal complement complex C5b-9 (SC5b-9). Twenty-six (36.1%) patients carried the rs11385942 GGA variant and 44 (66.1%) non-O blood group associated with increased risk of severe COVID-19.C5a and SC5-b9 plasma levels were higher in rs11385949 GA carriers than in non-carriers (P = 0.041 and P = 0.012, respectively), while C5a levels were higher in non-O group than in O group patients (P = 0.019). The association between rs11385949 and SC5b-9 remained significant after adjustment for ABO and disease severity (P = 0.004) and further correction for C5a (P = 0.018). There was a direct relationship between upper airways viral load and SC5b-9 in carriers of the rs11385949 risk allele (P = 0.032), which was not observed in non-carriers.The rs11385949 GGA variant, tagging the chromosome 3 gene cluster variation and predisposing to severe COVID-19, is associated with enhanced complement activation, both with C5a and terminal complement complex, while non-O blood group with C5a levels. These findings provide a link between genetic susceptibility to more severe COVID-19 and complement activation.

Background Raynaud9s phenomenon (RP) can be the first manifestation of systemic sclerosis (SSc) or other connective tissue diseases (CTDs), often preceding an overt disease by years. It is not known if markers of endothelial damage are detectable in those RP patients who subsequently develop a CTD. Objectives We studied RP patients at their first evaluation to correlate the levels of endothelial markers with the subsequent development of an overt disease 36 months later. Methods Eighty-two patients with RP at their first evaluation were recruited. We measured plasma levels of tissue-type plasminogen activator (t-PA) and von Willebrand factor (vWF), two markers of endothelial damage, and interleukin-6 (IL-6), a pro-inflammatory cytokine. Thirty healthy subjects served as healthy controls. Results At baseline, 67 patients showed capillaroscopic normal pattern and 15 patients, of which 11 were very early SSc, had scleroderma pattern. Plasma levels of t-PA, vWF and IL-6 were higher in patients with capillaroscopic normal pattern (p=0.0001) than in normal controls and even much higher in patients with scleroderma pattern (p=0.0001). In patients with capillaroscopic normal pattern and RP of recent onset ( Conclusions Our findings indicate that markers of endothelial damage are elevated in RP patients who subsequently develop SSc or other CTDs, even in the absence of capillaroscopic abnormalities. Disclosure of Interest None declared

The pro-inflammatory cytokine interleukin (IL)-6 is involved in the pathogenesis of both rheumatoid arthritis (RA) and cardiovascular events. We evaluated the correlation of prothrombotic biomarkers, in particular those of thrombin generation, with inflammatory and clinical parameters in RA patients treated with tocilizumab, an IL-6 receptor (IL-6R) inhibitor. Naïve and maintenance patients were compared.We studied 15 RA patients undergoing tocilizumab infusions at a University Outpatient Clinic. Eight received tocilizumab for the first time and were evaluated at baseline. Seven were in maintenance therapy (9 to 77 months). All 15 patients were evaluated four weeks after the last administration of tocilizumab. At each time, we assessed disease activity score 28 (DAS28), erythrocyte sedimentation rate (ESR) and plasma levels of C-reactive protein (CRP), IL-6, soluble (s)IL-6R, tumour necrosis factor-alpha (TNF-alpha), prothrombin fragment F1+2 and fibrin fragment D-dimer. Forty healthy subjects served as basal controls.At baseline, RA patients showed a moderate-to-high disease activity and median ESR of 51 mm/1(st) hour (interquartile range 25-63). Plasma levels of CRP (p=0.0001), IL-6 (p=0.043), sIL-6R (p=0.003), TNF-alpha (p=0.0001), F1+2 (p=0.0001) and D-dimer (p=0.002) were higher than those of healthy controls. After four weeks we observed reduction of DAS28 (p=0.0001), ESR (p=0.0001), CRP (p=0.014), TNF-alpha (p=0.006), F1+2 (p=0.009) and D-dimer (p=0.04). No differences were observed between naïve and maintenance patients.The reduction of prothrombotic biomarkers parallels the reduction of inflammatory parameters and clinical symptoms in RA patients treated with tocilizumab, both four weeks after the first administration and during maintenance therapy.

Summary Background Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy characterized by hemolysis, platelet consumption, and renal injury. Eculizumab, a mAb that blocks complement activity, has been successfully used in aHUS. Objectives To optimize eculizumab therapy in aHUS patients by monitoring complement functional tests and markers of disease activity. Patients/Methods We studied 18 patients with aHUS (10 males; eight females; age range, 2–40 years) treated with eculizumab to induce and/or maintain disease remission. Patients were followed up for a cumulative observation period of 160 months, during which blood samples were obtained at various time intervals to measure complement activity (Wieslab for the classical, alternative and mannose-binding lectin complement pathways) and the parameters of disease activity (haptoglobin and lactate dehydrogenase serum levels, and platelet count). The intravenous eculizumab doses of 12–33 mg kg−1 were initially administered every week, with the interval between doses being gradually extended to 2 weeks, 3 weeks and 4 weeks on the basis of strict laboratory and clinical control. Results Complement activity was normal before eculizumab treatment, regardless of the state of the disease (activity or remission). It was completely suppressed 1 week, 2 weeks and 3 weeks after the last eculizumab infusion (mean values ± standard deviation: 1% ± 1% to 3% ± 5% for both the classical and alternative pathways; P = 0.0001 vs. baseline), and partially suppressed after 4 weeks (22% ± 26% and 16% ± 27%; P = 0.0001 vs. baseline). The increase in the time interval between eculizumab infusions did not change disease activity markers. Conclusions Monitoring complement tests can allow a safe reduction in the frequency of eculizumab administration in aHUS while keeping the disease in remission.