Circulating mortalin in blood and activation of the alternative complement pathway as risk indicators in COVID-19 infection.

Background: Mortalin/GRP75 is a ubiquitous mitochondrial chaperone related to the cytosolic heat shock protein 70. It protects cells from various types of damages and from senescence. Our goal was to determine whether COVID-19 patients have circulating mortalin in their blood and to assess its prognostic value in anticipating disease severity. Methods: Mortalin was determined by ELISA in the sera of 83 COVID-19 patients enrolled in the study. Patients were categorized into 4 groups: critical patients who died (FATAL) or required intensive care and survived (ICU), patients of mild severity (hospitalized but not critical) who required nasal oxygen support (HOSP+O2), and patients who did not need oxygen therapy (HOSP). Results: The mortalin concentration in the serum of all COVID-19 patients in the cohort was 194-2324 pg/mL. A comparison of the mortalin levels by peak severity among the various patient groups showed a highly significant difference between the HOSP and FATAL groups and a significant difference between the HOSP and the ICU groups. COVID-19 patients who eventually failed to survive had at hospitalization a markedly higher level of mortalin in their sera. Cox regression analysis revealed a high mortality hazard (HR=3.96, p<0.01) in patients with high mortalin circulating levels (above the median, ≥651 pg/mL). This was confirmed in survival curve analysis (Kaplan-Meier; p=0.0032, log-rank test). Mortalin remained an independent predictor of mortality even after adjusting for age and sex or various complement activation products. Complement activation data collected in an earlier study in the same cohort was compared regarding the mortalin levels. Patients with higher circulating mortalin levels also had higher levels of complement C3a but reduced levels of properdin. Discussion: This is the first report on circulating mortalin in COVID-19 patients. Higher mortalin levels were associated with more severe illnesses and a higher risk of death. We claim that quantifying the blood levels of mortalin and activated complement proteins will provide important information on the prognosis of COVID-19 patients and will serve as a useful tool for guiding their clinical management and treatment.

External quality assurance program for diagnostic complement laboratories: evaluation of the results of the past seven years.

Introduction: The complement external quality assurance (EQA) program was first organized in 2010 by a group of researchers working in diagnostic complement laboratories. Starting in 2016, INSTAND e.V., a German, non-profit interdisciplinary scientific medical society dedicated to providing expert EQA programs for medical laboratories, started organizing the EQAs for complement diagnostic laboratories together with the same group of experienced scientists and doctors who also work as EQA experts. The aim of the current work is to provide descriptive analysis of the past seven years' complement EQA results and evaluate timeline changes in proficiency testing. Methods: Each year, in March and October, blinded samples (normal, pathological) were sent to the participating diagnostic laboratories, where complement parameters were evaluated exactly as in daily routine samples. Since no reference method/target values exist for these parameters, and participants used different units for measurement, the reported results were compared to the stable mean (Algorithm A) of the participants using the same method/measurement units. A reported result was qualified as "passed" if it fell into the 30-50% evaluation/target range around the mean of reported results (depending on the given parameter). Results: While the number of participating laboratories has increased in the past years (from around 120 to 347), the number of complement laboratories providing multiple determinations remained mostly unchanged (around 30 worldwide). C3, C4, C1-inhibitor antigen and activity determinations provided the best proficiency results, with >90% passing quotas in the past years, independent of the applied method. Determination of the functional activity of the three activation pathways was good in general, but results showed large variance, especially with the pathological samples. Complement factor C1q and regulators FH and FI are determined by only a few laboratories, with variable outcomes (in general in the 85-90% pass range). Activation products sC5b-9 and Bb were determined in 30 and 10 laboratories, respectively, with typical passing quotas in the 70-90% range, without a clear tendency over the past years. Conclusion: With these accumulated data from the past seven years, it is now possible to assess sample-, method-, and evaluation related aspects to further improve proficiency testing and protocolize diagnostic complement determinations.

Natural Autoantibodies in Biologic-Treated Rheumatoid Arthritis and Ankylosing Spondylitis Patients: Associations with Vascular Pathophysiology.

Cardiovascular (CV) morbidity and mortality have been associated with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Natural autoantibodies (nAAb) are involved in innate immunity, as well as autoimmunity, inflammation, and atherosclerosis. There have not been any studies assessing the effects of biologics on nAAbs in RA and AS, also in relation to vascular pathophysiology. Fifty-three anti-TNF-treated RA and AS patients were included in a 12-month follow-up study. Anti-citrate synthase (CS) and anti-topoisomerase I fragment 4 (TOPO-F4) IgM and IgG levels were determined by ELISA. Ultrasonography was performed to assess brachial artery flow-mediated vasodilation (FMD), common carotid intima-media thickness (ccIMT), and arterial pulse-wave velocity (PWV). Other variables were also evaluated at baseline and 6 and 12 months after treatment initiation. Anti-TNF therapy improved FMD in RA and PWV in AS and stabilized ccIMT. TNF inhibition increased anti-CS IgM and IgG, and possibly also anti-TOPO-F4 IgG levels. Various correlation analyses revealed that nAAbs might be independently involved in autoimmunity as well as changes in inflammation and vascular pathology over time in biologic-treated patients (p < 0.05). We also found associations between anti-TOPO-F4 IgG and anti-Hsp60 IgG (p < 0.05). Baseline nAAb levels or nAAb level changes might determine changes in CRP, disease activity, FMD, PWV, and ccIMT over time (p < 0.05). The interplay between arthritis and inflammatory atherosclerosis, as well as the effects of anti-TNF biologics on these pathologies, might independently involve nAAbs.

Complement C7 and clusterin form a complex in circulation.

Introduction: The complement system is part of innate immunity and is comprised of an intricate network of proteins that are vital for host defense and host homeostasis. A distinct mechanism by which complement defends against invading pathogens is through the membrane attack complex (MAC), a lytic structure that forms on target surfaces. The MAC is made up of several complement components, and one indispensable component of the MAC is C7. The role of C7 in MAC assembly is well documented, however, inherent characteristics of C7 are yet to be investigated. Methods: To shed light on the molecular characteristics of C7, we examined the properties of serum-purified C7 acquired using polyclonal and novel monoclonal antibodies. The properties of serum­purified C7 were investigated through a series of proteolytic analyses, encompassing Western blot and mass spectrometry. The nature of C7 protein-protein interactions were further examined by a novel enzyme-linked immunosorbent assay (ELISA), as well as size­exclusion chromatography. Results: Protein analyses showcased an association between C7 and clusterin, an inhibitory complement regulator. The distinct association between C7 and clusterin was also demonstrated in serum-purified clusterin. Further assessment revealed that a complex between C7 and clusterin (C7-CLU) was detected. The C7-CLU complex was also identified in healthy serum and plasma donors, highlighting the presence of the complex in circulation. Discussion: Clusterin is known to dissociate the MAC structure by binding to polymerized C9, nevertheless, here we show clusterin binding to the native form of a terminal complement protein in vivo. The presented data reveal that C7 exhibits characteristics beyond that of MAC assembly, instigating further investigation of the effector role that the C7-CLU complex plays in the complement cascade.

Storage of Transfusion Platelet Concentrates Is Associated with Complement Activation and Reduced Ability of Platelets to Respond to Protease-Activated Receptor-1 and Thromboxane A2 Receptor.

Platelet activation and the complement system are mutually dependent. Here, we investigated the effects of storage time on complement activation and platelet function in routinely produced platelet concentrates. The platelet concentrates (n = 10) were stored at 22 °C for seven days and assessed daily for complement and platelet activation markers. Additionally, platelet function was analyzed in terms of their responsiveness to protease-activated receptor-1 (PAR-1) and thromboxane A2 receptor (TXA2R) activation and their capacity to adhere to collagen. Complement activation increased over the storage period for all analyzed markers, including the C1rs/C1-INH complex (fold change (FC) = 1.9; p < 0.001), MASP-1/C1-INH complex (FC = 2.0; p < 0.001), C4c (FC = 1.8, p < 0.001), C3bc (FC = 4.0; p < 0.01), and soluble C5b-9 (FC = 1.7, p < 0.001). Furthermore, the levels of soluble platelet activation markers increased in the concentrates over the seven-day period, including neutrophil-activating peptide-2 (FC = 2.5; p < 0.0001), transforming growth factor beta 1 (FC = 1.9; p < 0.001) and platelet factor 4 (FC = 2.1; p < 0.0001). The ability of platelets to respond to activation, as measured by surface expression of CD62P and CD63, decreased by 19% and 24% (p < 0.05) for PAR-1 and 69-72% (p < 0.05) for TXA2R activation, respectively, on Day 7 compared to Day 1. The extent of platelet binding to collagen was not significantly impaired during storage. In conclusion, we demonstrated that complement activation increased during the storage of platelets, and this correlated with increased platelet activation and a reduced ability of the platelets to respond to, primarily, TXA2R activation.

Open ADAMTS-13 conformation index predicts earlier relapse in immune-mediated thrombotic thrombocytopenic purpura.

BACKGROUND: ADAMTS-13 adopts an open conformation in patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) in acute phase while being closed in healthy donors. We reported that a substantial number of patients with iTTP in remission with restored ADAMTS-13 activity (>50%) still had an open ADAMTS-13 conformation, although a closed conformation is expected given the extent of remission. OBJECTIVES: To investigate whether open ADAMTS-13, represented by a conformation index >0.5, is associated with a risk of earlier ADAMTS-13 and/or clinical relapse. METHODS: We collected follow-up data (ADAMTS-13 parameters, ADAMTS-13 and clinical relapse, and treatment) from 81 patients with iTTP in remission with ADAMTS-13 activity >50%. RESULTS: During follow-up, 19 ADAMTS-13 and 10 clinical relapses were reported (median follow-up period, 20 months). First, open or closed ADAMTS-13 conformation was dichotomized based on the 0.5 conformation index cutoff. Open ADAMTS-13 (conformation index, >0.5) was not identified as a risk factor for ADAMTS-13 and clinical relapse (log-rank test and Cox regression model). In contrast, by identifying the optimal conformation index cutoff for relapse prediction, using classification and regression tree analysis, a conformation index >0.645 and >0.835 was shown to be a risk factor for ADAMTS-13 relapse (hazard ratio, 3.3; 95% CI, 1.3-8.3; P = .01) and clinical relapse (hazard ratio, 4.4; 95% CI, 1.3-15.3; P = .02), respectively. CONCLUSION: Patients with open ADAMTS-13 with a conformation index >0.645 and >0.835 have a >3- and >4-fold higher risk of earlier ADAMTS-13 and clinical relapse, respectively. Hence, ADAMTS-13 conformation index could be used to complement ADAMTS-13 activity monitoring to timely notice ADAMTS-13 relapse and prevent clinical relapse.

MAP-2:CD55 chimeric construct effectively modulates complement activation.

The complement system is a complex, tightly regulated protein cascade involved in pathogen defense and the pathogenesis of several diseases. Thus, the development of complement modulators has risen as a potential treatment for complement-driven inflammatory pathologies. The enzymatically inactive MAP-2 has been reported to inhibit the lectin pathway by competing with its homologous serine protease MASP-2. The membrane-bound complement inhibitor CD55 acts on the C3/C5 convertase level. Here, we fused MAP-2 to the four N-terminal domains of CD55 generating a targeted chimeric inhibitor to modulate complement activation at two different levels of the complement cascade. Its biological properties were compared in vitro with the parent molecules. While MAP-2 and CD55 alone showed a minor inhibition of the three complement pathways when co-incubated with serum (IC50MAP-2+CD55 1-4 = 60.98, 36.10, and 97.01 nM on the classical, lectin, and alternative pathways, respectively), MAP-2:CD551-4 demonstrated a potent inhibitory activity (IC50MAP-2:CD55 1-4 = 2.94, 1.76, and 12.86 nM, respectively). This inhibitory activity was substantially enhanced when pre-complexes were formed with the lectin pathway recognition molecule mannose-binding lectin (IC50MAP-2:CD55 1-4 = 0.14 nM). MAP-2:CD551-4 was also effective at protecting sensitized sheep erythrocytes in a classical hemolytic assay (CH50 = 13.35 nM). Finally, the chimeric inhibitor reduced neutrophil activation in full blood after stimulation with Aspergillus fumigatus conidia, as well as phagocytosis of conidia by isolated activated neutrophils. Our results demonstrate that MAP-2:CD551-4 is a potent complement inhibitor reinforcing the idea that engineered fusion proteins are a promising design strategy for identifying and developing drug candidates to treat complement-mediated diseases.

The Rationale of Complement Blockade of the MCPggaac Haplotype following Atypical Hemolytic Uremic Syndrome of Three Southeastern European Countries with a Literature Review.

We present eight cases of the homozygous MCPggaac haplotype, which is considered to increase the likelihood and severity of atypical hemolytic uremic syndrome (aHUS), especially in combination with additional risk aHUS mutations. Complement blockade (CBT) was applied at a median age of 92 months (IQR 36-252 months). The median number of relapses before CBT initiation (Eculizumab) was two. Relapses occurred within an average of 22.16 months (median 17.5, minimum 8 months, and maximum 48 months) from the first subsequent onset of the disease (6/8 patients). All cases were treated with PI/PEX, and rarely with renal replacement therapy (RRT). When complement blockade was applied, children had no further disease relapses. Children with MCPggaac haplotype with/without additional gene mutations can achieve remission through renal replacement therapy without an immediate need for complement blockade. If relapse of aHUS occurs soon after disease onset or relapses are repeated frequently, a permanent complement blockade is required. However, the duration of such a blockade remains uncertain. If complement inhibition is not applied within 4-5 relapses, proteinuria and chronic renal failure will eventually occur.

Immunosuppressive Therapy of Antibody-Mediated aHUS and TTP.

The recent classification of pediatric thrombotic microangiopathies (TMA) takes into consideration mechanisms of disease for guidance to targeted therapies. We present our experience with seven patients with antibody mediated atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura (TTP). Five children had aHUS with antibodies against complement factor H (CFH-ab) and two with TTP with antibodies against metalloproteinase ADAMTS13. In the aHUS cases diagnosed and treated before the eculizumab era, CFH-ab was detected using the ELISA assay. Mutational analysis of selected complement genes was performed. TTP was diagnosed if, in addition to microangiopathic hemolytic anemia and thrombocytopenia, ischemic organ involvement and severe deficiency in ADAMTS13 activity were present. Treatment protocol consisted of plasma exchanges (PE) and steroid pulses, followed by the combination of cyclophosphamide and rituximab to achieve long-term immunosuppression. Four patients with CFH-ab and the TTP patients with ADAMTS13 antibodies came into sustained remission. After a median follow-up of 11.7 (range 7.7-12.9) years without maintenance therapy, no disease recurrence was observed; nevertheless, six patients, two had hypertension and two had proteinuria as a late consequence. One patient, with late diagnosis of CFH-ab and additional genetic risk factors who was treated only with PE and plasma substitution, reached end-stage renal disease and was later successfully transplanted using eculizumab prophylaxis. In the cases of antibody-mediated TMAs, PE and early immunosuppressive treatment may result in sustained remission with preserved kidney function. Further data are needed to establish optimal treatment of anti-FH antibody-associated HUS.

Class switch towards spike protein-specific IgG4 antibodies after SARS-CoV-2 mRNA vaccination depends on prior infection history.

Vaccinations against SARS-CoV-2 reduce the risk of developing serious COVID-19 disease. Monitoring spike-specific IgG subclass levels after vaccinations may provide additional information on SARS-CoV-2 specific humoral immune response. Here, we examined the presence and levels of spike-specific IgG antibody subclasses in health-care coworkers vaccinated with vector- (Sputnik, AstraZeneca) or mRNA-based (Pfizer-BioNTech, Moderna) vaccines against SARS-CoV-2 and in unvaccinated COVID-19 patients. We found that vector-based vaccines elicited lower total spike-specific IgG levels than mRNA vaccines. The pattern of spike-specific IgG subclasses in individuals infected before mRNA vaccinations resembled that of vector-vaccinated subjects or unvaccinated COVID-19 patients. However, the pattern of mRNA-vaccinated individuals without SARS-CoV-2 preinfection showed a markedly different pattern. In addition to IgG1 and IgG3 subclasses presented in all groups, a switch towards distal IgG subclasses (spike-specific IgG4 and IgG2) appeared almost exclusively in individuals who received only mRNA vaccines or were infected after mRNA vaccinations. In these subjects, the magnitude of the spike-specific IgG4 response was comparable to that of the spike-specific IgG1 response. These data suggest that the priming of the immune system either by natural SARS-CoV-2 infection or by vector- or mRNA-based vaccinations has an important impact on the characteristics of the developed specific humoral immunity.

Differentiating between activation via the lectin or the classical complement pathway in patients with systemic lupus erythematosus.

Complement activation is a hallmark of systemic lupus erythematosus (SLE) and can proceed through the classical (CP), lectin (LP), or alternative pathway (AP). When managing SLE patients, pathway-specific complement activation is rarely monitored as clinical assays are unavailable. In this study, we aim to differentiate between CP- or LP-mediated complement activation in SLE patients by quantifying pathway-specific protein complexes, namely C1s/C1-inhibitor (C1-INH) (CP-specific activation) and MASP-1/C1-INH (LP-specific activation). Levels for both complexes were assessed in 156 SLE patients and 50 controls using two newly developed ELISAs. We investigated whether pathway-specific complement activation was associated with disease activity and lupus nephritis (LN). Disease activity stratification was performed using SLEDAI scores assessed at inclusion. C1s/C1-INH concentrations were significantly increased in active SLE patients (SLEDAI ≥6) when compared with SLE patients with low disease activity (SLEDAI <6, P < 0.01) and correlated with SLEDAI score (r = .29, P < 0.01). In active LN, MASP-1/C1-INH plasma concentrations were significantly increased compared with nonactive LN (P = 0.02). No differences in MASP-1/C1-INH plasma concentrations were observed between active SLE patients and patients with low disease activity (P = 0.11) nor did we observe a significant correlation with disease activity (r = 0.12, P = 0.15). Our data suggest that the CP and the LP are activated in SLE. The CP is activated in active SLE disease, whereas activation of the LP might be more specific to disease manifestations like LN. Our results warrant further research into specific complement pathway activation in SLE patients to potentially improve specific-targeted and tailored-treatment approaches.

Pregnancy in Complement-Mediated Thrombotic Microangiopathy: Maternal and Neonatal Outcomes.

Rationale & Objective: Pregnancy, delivery, and neonatal outcomes in women with complement-mediated thrombotic microangiopathy (cTMA) have not been well described. A better understanding of these outcomes is necessary to provide women with competent pregnancy counseling. Study Design: Cohort study. Setting and Participants: Women with a history of cTMA and pregnancies enrolled into the Vienna thrombotic microangiopathy cohort. Exposure: New onset or relapses of cTMA. Outcomes: Pregnancy, delivery, and neonatal outcomes of pregnancies in women (a) before cTMA manifestation, (b) complicated by pregnancy-associated cTMA (P-cTMA), and (c) after first manifestation of cTMA or P-cTMA. Analytical Approach: Mixed models were used to adjust the comparison of pregnancy, delivery, and neonatal outcomes between conditions (before, with, and after cTMA) for repeated pregnancies using the mother's ID as random factor. In addition, the fixed factors, mother's age and neonate's sex, were used for adjustment. For (sex-adjusted and age-adjusted) centile outcomes, only the mother's age was used. Adjusted odds ratios were derived from a generalized linear mixed model with live birth as the outcome. Least squares means and pairwise differences between them were derived from the linear mixed models for the remaining outcomes. Results: 28 women reported 74 pregnancies. Despite higher rates of fetal loss before the diagnosis of P-cTMA and preterm births with P-cTMA, most of the women were able to conceive successfully. Neonatal development in all 3 conditions of pregnancies was excellent. Pregnancy and neonatal outcomes were better in women with a pregnancy after the diagnosis of cTMA. Limitations: Although our data set comprises a considerable number of 74 pregnancies, the effective sample size is lower because only 28 mothers with multiple pregnancies were observed. The statistical power for detecting clinically relevant effects was probably low. A recall bias for miscarriages cannot be ruled out. Conclusions: Prepregnancy counseling of women with a history of cTMA can be supportive of their desire to become pregnant.

Complement-Mediated Thrombotic Microangiopathy Related to COVID-19 or SARS-CoV-2 Vaccination.

Introduction: Infectious diseases and vaccinations are trigger factors for thrombotic microangiopathy. Consequently, the COVID-19 pandemic could have an effect on disease manifestation or relapse in patients with atypical hemolytic syndrome/complement-mediated thrombotic microangiopathy (aHUS/cTMA). Methods: We employed the Vienna TMA cohort database to examine the incidence of COVID-19 related and of SARS-CoV-2 vaccination-related relapse of aHUS/cTMA among patients previously diagnosed with aHUS/cTMA during the first 2.5 years of the COVID-19 pandemic. We calculated incidence rates, including respective confidence intervals (CIs) and used Cox proportional hazard models for comparison of aHUS/cTMA episodes following infection or vaccination. Results: Among 27 patients with aHUS/cTMA, 13 infections triggered 3 (23%) TMA episodes, whereas 70 vaccinations triggered 1 TMA episode (1%; odds ratio 0.04; 95% CI 0.003-0.37, P = 0.01). In total, the incidence of TMA after COVID-19 or SARS-CoV-2 vaccination was 6 cases per 100 patient years (95% CI 0.017-0.164) (4.5/100 patient years for COVID-19 and 1.5/100 patient years for SARS-CoV-2 vaccination). The mean follow-up time was 2.31 ± 0.26 years (total amount: 22,118 days; 62.5 years) to either the end of the follow-up or TMA relapse (outcome). Between 2012 and 2022 we did not find a significant increase in the incidence of aHUS/cTMA. Conclusion: COVID-19 is associated with a higher risk for aHUS/cTMA recurrence when compared to SARS-CoV-2 vaccination. Overall, the incidence of aHUS/cTMA after COVID-19 infection or SARS-CoV-2 vaccination is low and comparable to that described in the literature.

[Reducing the risk of infections in hereditary and acquired complement deficiencies.] / Infekciók kockázatának csökkentése veleszületett és szerzett komplementdefektusokban.

Hereditary complement deficiencies are relatively rare worldwide, they account for about 1-10% of primary immunodeficiencies. Acquired complement deficiencies are more prevalent and with the more frequent use of complement inhibitor therapy, the incidence of patients with iatrogenic complement deficiency is increasing. Alike in the inherited forms, patients have a high risk of severe and life-threatening infections caused by encapsulated bacteria (sepsis, meningitis). The most frequent pathogens are Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae. C5 and C3 complement inhibitor therapies are available in Hungary, which are mostly indicated in the treatment of paroxysmal nocturnal hemoglobinuria, myasthenia gravis, neuromyelitis optica and atypical haemolytic uremic syndrome. It is of utmost importance to prevent severe, potentially life-threatening bacterial infections in this group of patients. Nevertheless, there is no Hungarian guidance to decrease the risk of infections, preventive measures are incomplete and not standardized posing potential risk of infections for these patients, so far. In this review, we aim to summarize the international clinical practices and guidance on the infection prevention in complement deficient patients. This recommendation might be a source of an evidence-based Hungarian guideline regarding vaccination and antibiotic prophylaxis in this specifically vulnerable group of patients. Orv Hetil. 2023; 164(25): 971-980.

Complement lectin pathway activation is associated with COVID-19 disease severity, independent of MBL2 genotype subgroups.

Introduction: While complement is a contributor to disease severity in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, all three complement pathways might be activated by the virus. Lectin pathway activation occurs through different pattern recognition molecules, including mannan binding lectin (MBL), a protein shown to interact with SARS-CoV-2 proteins. However, the exact role of lectin pathway activation and its key pattern recognition molecule MBL in COVID-19 is still not fully understood. Methods: We therefore investigated activation of the lectin pathway in two independent cohorts of SARS-CoV-2 infected patients, while also analysing MBL protein levels and potential effects of the six major single nucleotide polymorphisms (SNPs) found in the MBL2 gene on COVID-19 severity and outcome. Results: We show that the lectin pathway is activated in acute COVID-19, indicated by the correlation between complement activation product levels of the MASP-1/C1-INH complex (p=0.0011) and C4d (p<0.0001) and COVID-19 severity. Despite this, genetic variations in MBL2 are not associated with susceptibility to SARS-CoV-2 infection or disease outcomes such as mortality and the development of Long COVID. Conclusion: In conclusion, activation of the MBL-LP only plays a minor role in COVID-19 pathogenesis, since no clinically meaningful, consistent associations with disease outcomes were noted.

Hemolytic uremic syndrome in the setting of COVID-19 successfully treated with complement inhibition therapy: An instructive case report of a previously healthy toddler and review of literature.

Introduction: As the global pandemic continues, new complications of COVID-19 in pediatric population have turned up, one of them being hemolytic uremic syndrome (HUS), a complement-mediated thrombotic microangiopathy (CM-TMA) characterized by triad of thrombocytopenia, microangiopathic hemolytic anemia and acute kidney injury (AKI). With both multisystem inflammatory syndrome in children (MIS-C) and HUS sharing complement dysregulation as one of the key factors, the aim of this case report is to highlight differences between these two conditions and also emphasize the importance of complement blockade as a treatment modality. Case report: We describe a 21-month-old toddler who initially presented with fever and confirmed COVID-19. His condition quickly deteriorated and he developed oliguria, accompanied with diarrhea, vomiting and oral intake intolerance. HUS was suspected, supported with compelling laboratory findings, including decreased platelets count and C3 levels, elevated LDH, urea, serum creatinine and sC5b-9 and presence of schistocytes in peripheral blood, negative fecal Shiga toxin and normal ADAMTS13 metalloprotease activity. The patient was given C5 complement blocker Ravulizumab and started to display rapid improvement. Conclusion: Although reports of HUS in the setting of COVID-19 continue to pour in, the questions of exact mechanism and similarities to MIS-C remain. Our case for the first time accentuates the use of complement blockade as a valuable treatment option in this scenario. We sincerely believe that reporting on HUS as a complication of COVID-19 in children will give rise to improved diagnosis and treatment, as well as better understanding of both of these intricating diseases.

Use of eculizumab in children with allogeneic haematopoietic stem cell transplantation associated thrombotic microangiopathy - a multicentre retrospective PDWP and IEWP EBMT study.

Terminal complement blockade by humanised monoclonal antibody eculizumab has been used to treat transplantation-associated thrombotic microangiopathy (TA-TMA) in recent years. This retrospective international study conducted by the Paediatric Diseases (PDWP) and Inborn Error Working Party (IEWP) of the European Society for Blood and Marrow Transplantation (EBMT) describes outcome and response of 82 paediatric patients from 29 centres who developed TA-TMA and were treated with eculizumab between January 2014 and May 2019. The median time from hematopoietic stem cell transplantation (HSCT) to TA-TMA manifestation was 92 days (range: 7-606) and from TA-TMA diagnosis to the start of eculizumab treatment 6 days (range: 0-135). Most patients received eculizumab weekly (72%, n = 55) with a standard weight (kg)-based dose (78%, n = 64). Six months from beginning of eculizumab therapy, the cumulative incidence of TA-TMA resolution was 36.6% (95% CI: 26.2-47) and the overall survival (OS) was 47.1% (95% CI: 35.9-57.5). All 43 patients with unresolved TA-TMA died. The cause of death was HSCT-related in 41 patients. This study also documents poor outcome of patients without aGvHD and their frequent concomitant viral infections. Considering recent publications, intensified eculizumab dosing and complement monitoring could potentially improve upon outcomes observed in this study.

Evidence, detailed characterization and clinical context of complement activation in acute multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) is a rare, life-threatening complication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. MIS-C develops with high fever, marked inflammation and shock-like picture several weeks after exposure to, or mild infection with SARS-CoV-2. Deep immune profiling identified activated macrophages, neutrophils, B-plasmablasts and CD8 + T cells as key determinants of pathogenesis together with multiple inflammatory markers. The disease rapidly responds to intravenous immunoglobulin (IVIG) treatment with clear changes of immune features. Here we present the results of a comprehensive analysis of the complement system in the context of MIS-C activity and describe characteristic changes during IVIG treatment. We show that activation markers of the classical, alternative and terminal pathways are highly elevated, that the activation is largely independent of anti-SARS-CoV-2 humoral immune response, but is strongly associated with markers of macrophage activation. Decrease of complement activation is closely associated with rapid improvement of MIS-C after IVIG treatment.

Distinction of early complement classical and lectin pathway activation via quantification of C1s/C1-INH and MASP-1/C1-INH complexes using novel ELISAs.

The most commonly used markers to assess complement activation are split products that are produced through activation of all three pathways and are located downstream of C3. In contrast, C4d derives from the cleavage of C4 and indicates either classical (CP) or lectin pathway (LP) activation. Although C4d is perfectly able to distinguish between CP/LP and alternative pathway (AP) activation, no well-established markers are available to differentiate between early CP and LP activation. Active enzymes of both pathways (C1s/C1r for the CP, MASP-1/MASP-2 for the LP) are regulated by C1 esterase inhibitor (C1-INH) through the formation of covalent complexes. Aim of this study was to develop validated immunoassays detecting C1s/C1-INH and MASP-1/C1-INH complex levels. Measurement of the complexes reveals information about the involvement of the respective pathways in complement-mediated diseases. Two sandwich ELISAs detecting C1s/C1-INH and MASP-1/C1-INH complex were developed and tested thoroughly, and it was investigated whether C1s/C1-INH and MASP-1/C1-INH complexes could serve as markers for either early CP or LP activation. In addition, a reference range for these complexes in healthy adults was defined, and the assays were clinically validated utilizing samples of 414 COVID-19 patients and 96 healthy controls. The immunoassays can reliably measure C1s/C1-INH and MASP-1/C1-INH complex concentrations in EDTA plasma from healthy and diseased individuals. Both complex levels are increased in serum when activated with zymosan, making them suitable markers for early classical and early lectin pathway activation. Furthermore, measurements of C1-INH complexes in 96 healthy adults showed normally distributed C1s/C1-INH complex levels with a physiological concentration of 1846 ± 1060 ng/mL (mean ± 2SD) and right-skewed distribution of MASP-1/C1-INH complex levels with a median concentration of 36.9 (13.18 - 87.89) ng/mL (2.5-97.5 percentile range), while levels of both complexes were increased in COVID-19 patients (p<0.0001). The newly developed assays measure C1-INH complex levels in an accurate way. C1s/C1-INH and MASP-1/C1-INH complexes are suitable markers to assess early classical and lectin pathway activation. An initial reference range was set and first studies showed that these markers have added value for investigating and unraveling complement activation in human disease.

MCPggaac haplotype is associated with poor graft survival in kidney transplant recipients with de novo thrombotic microangiopathy.

De novo thrombotic microangiopathy (TMA) is associated with poor kidney graft survival, and as we previously described, it is a recipient driven process with suspected genetic background. Direct Sanger sequencing was performed in 90 KTR with de novo TMA and 90 corresponding donors on selected regions in CFH, CD46, C3, and CFB genes that involve variations with a functional effect or confer a risk for aHUS. Additionally, 37 recipients of paired kidneys who did not develop TMA were analyzed for the MCPggaac haplotype. Three-years death-censored graft survival was assessed using Kaplan-Meier and Cox regression models. The distribution of haplotypes in all groups was in the Hardy-Weinberg equilibrium and there was no clustering of haplotypes in any group. In the TMA group, we found that MCPggaac haplotype carriers were at a significantly higher risk of graft loss compared to individuals with the wild-type genotype. Worse 3-year death-censored graft survival was associated with longer cold ischemia time (HR 1.20, 95% CI 1.06, 1.36) and recipients' MCPggaac haplotype (HR 3.83, 95% CI 1.42, 10.4) in the multivariable Cox regression model. There was no association between donor haplotypes and kidney graft survival. Similarly, there was no effect of the MCPggaac haplotype on 3-year graft survival in recipients of paired kidneys without de novo TMA. Kidney transplant recipients carrying the MCPggaac haplotype with de novo TMA are at an increased risk of premature graft loss. These patients might benefit from therapeutic strategies based on complement inhibition.