Germline rare variants of lectin pathway genes predispose to asymptomatic SARS-CoV-2 infection in elderly individuals

Background/Objectives: Emerging evidence suggests that complement system infection-dependent hyperactivation may worsen COVID-19 outcome. We investigated the role of predicted high impact variants -referred as Qualifying Variants (QVs) -of complement system genes in predisposing asymptomatic COVID-19 in elderly individuals, known to be more susceptible to severe disease. Method(s): Exploiting Whole-Exome Sequencing (WES) data and 56 complement system genes, we performed a gene-based collapsing test between 164 asymptomatic subjects (age >= 60 y.o.) and 56,885 European individuals from the gnomAD database. We replicated this test comparing the same asymptomatic individuals with 147 hospitalized COVID-19 patients. Result(s): We found an enrichment of QVs in three genes (MASP1, COLEC10 and COLEC11), which belong to the lectin pathway, in the asymptomatic cohort. Moreover, individuals with QVs showed lower serum levels of Masp1 and of prothrombin activity compared to controls while no differences were observed for CH50 and AH50 levels that measure the activity of classical and alternative complement pathways, respectively. Finally, integrative analyses of genome-wide association study and expression quantitative loci traits data showed a correlation between polymorphisms associated with asymptomatic COVID-19 and decreased expression of MASP1, COLEC11 and COLEC10 genes in lung tissue. Conclusion(s): This study suggests that rare genetic variants can protect from severe COVID-19 by mitigating the activation of lectin pathway and prothrombin activity.

Acute angioedena attacks after COVID-19 disease or COVID-19 vaccination in a population afflicted with hereditary angioedema

Background: Few studies demonstrating the involvement of the complement system in COVID-19 pathogenesis have been published, suggesting its role in pulmonary symptoms and endothelial permeability, which is known to be crucial in the origin of Hereditary Angioedema (HAE).1 Post-morten tissue of COVID-19 patients reported depots of complement, activated by the lectin pathway, in type I and II alveolar epithelial cells.2 After this evidence and the link that infectious processes have as triggers of angioedema episodes, in patients with HAE, we propose to study the implication of both the infection and de doses of the COVID vaccine, in the appearance of episodes of angioedema in our population with a diagnos is of HAE. Method(s): Telemedicine interventions (telephone consultations) were carried out by trained Allergists from Hospital Universitario de Canarias, reaching out patients with a confirmed diagnosis of HEA by Skin Allergy Unit (SAU) within the local health district. Result(s): A total of 17 (11 females) were finally screened, and 2 (11.76%) passed a confirmed COVID-19 disease in January 2022 associating no acute attacks or need for rescue medication. Both subjects were fully vaccinated (3 doses-schedule) prior to the infection and suffered from a COVID-19 mild disease only. Only an individual dose of COVID-19 vaccination (Vaxzevria, Astra-Zeneca) -out of 40 overall given doses in 15 subjects and 3 different brands-was associated to an acute episode of abdominal swelling demanding immediate self-administered rescue therapy (icatibant) thus, preventing the patient from rushing to the Emergency Department. The subsequent 2 doses of the COVID-19 vaccination were safely scheduled in the same patient. Conclusion(s): In accordance with former reports4, only mild COVID-19 symptoms were associated in subjects with a confirmed diagnosis of HAE.

Application of urinary proteomics for biomarker discovery in respiratory diseases

Background: To search for molecular biomarkers of pulmonary pathologies using non-invasive samples, such as urine, is of high clinical relevance. However, there are almost no proteomic studies using urine applied to respiratory diseases. Aim(s): To develop a biomarker discovery strategy using non-targeted proteomics in urine with applicability to different pulmonary diseases. Method(s): Urine samples were centrifuged and DTT treated to decrease uromodulin (THP). Low-THP samples were concentrated (ultrafiltration), ultracentrifugated, and exosome free urine was analysed using LC-MS/MS. GO terms/Pathway analyses were performed using STRING database. Result(s): Urine proteome (765 proteins) was enriched (FDR < 0.05) in proteins from different tissues, including respiratory system (N = 124), lung (N = 107), and immune system (N = 88). We detected an enrichment of relevant pathways for respiratory diseases, including several innate (e.g., TLR and NFkB pathways, complement system), and adaptive (e.g., interleukin signalling) immune system pathways. Some of these proteins have been previously studied in respiratory system disease (e.g., MPO, NAPSA, CHL1, FREM2, PLG), lower respiratory tract disease (e.g., NCAM1, MTOR, SERPINA1), viral infectious disease (e.g., ITIH4, CD209, CLEC4M, CD55), or specific pathologies such as coronavirus infection (e.g., ACE2, TMPRSS2), bronchiectasis (e.g., SAA1, SAA2, ELANE) or asthma (e.g., IGFALS, IGFBP7, HSPG2, DPP4, CD44, IL6R, MASP1). Conclusion(s): We have developed a protocol for the detection of proteomic biomarkers in urine. This proteome is enriched in proteins from the immune and respiratory systems, with a potential clinical and translational relevance.

Immune system disturbances after a new coronavirus infection COVID-19.

During the pandemic, a large number of works devoted to COVID infection have appeared, which have made it possible to understand the pathogenetic features of the disease and to accumulate significant clinical experience. However, the question remains about the degree of participation of humoral and cellular (primarily T-cell) immunity in the mechanisms of immune defense and resistance to COVID-19, the individual features of the immune response in different subjects. Post-COVID syndrome is currently a separate diagnosis included in the ICD-10 International Classification of Diseases, but the long-term effects of the SARS-CoV-2 on the immune system are not yet well established. At the same time, a long-term increased activity of the immune system can contribute to the development of autoimmune reactions. The review of the literature presents the results of studies, mainly devoted to immune system disorders after COVID infection. The changes in subpopulations of T-lymphocytes, B-lymphocytes, their functional properties, the complement system and other factors of humoral immunity, as well as the production of a number of cytokines are described. Data on immune disorders in post-COVID syndrome and during the convalescence period are presented in detail. Since COVID-19 is an infection that has a significant impact on the hematopoietic system and hemostasis, special attention is paid to the category of subjects with an increased risk of severe complications. Among the latter are elderly patients, persons suffering from diabetes mellitus, oncological and oncohematological patients, in particular, with hematopoietic and lymphoid tissue neoplasia, such as chronic lymphocytic leukemia, lymphoma, multiple myeloma. The review pays special attention to the peculiarities of the course of COVID-19 and the response of the immune system to vaccination in patients with oncohematological diseases. Deciphering the significance of individual links of cellular and humoral immunity in patients who have undergone COVID-19 is an important issue in creating effective vaccines and improving therapeutic methods.Copyright © 2022 Interregional public organization Association of infectious disease specialists of Saint-Petersburg and Leningrad region (IPO AIDSSPbR). All rights reserved.

Mechanisms of kidney injury in COVID-19 and diagnostic methods.

The COVID-19 pandemic is a global healthcare crisis. The frequency of acute kidney injury (AKI) in patients with COVID-19 and the features of its diagnostics indicate the relevance of the topic. Objective of the review. To analyze mechanisms of AKI development in patients with COVID-19 and provide support for methodological approaches to ensure its timely diagnosis. Material and methods. The methodological approaches used in the review are based on a sufficient number of literature sources (more than 150 sources), of which 34 articles are included in the review: 15 original studies, 12 reviews, 2 meta-analyses, 5 re-ports, and letters to the editor. Results. The mechanisms of AKI development and progression, including the direct cytotoxic effect of the SARS-CoV-2 virus, dis-ruption of metabolic pathways of renal blood flow regulation, and the complement system, are considered. We also analyzed AKI risk factors in patients with acute respiratory distress: diabetes mellitus, chronic kidney injury, arterial hypertension with im-paired NOx production, and eNOS expression as significant factors of vasodilation in renal microcirculatory vessels. The analy-sis showed the most perspective directions in the diagnostics of AKI functional stages. These include molecular test methods (pro-teome and metabolome) in blood and urine;they helped define damage markers to proximal tubules and the glomerular system, thus improving the diagnostics accuracy and validity, therapy efficiency, and end points of disease prognosis. Conclusion. The Coronado study aims to assess the phenotypic features of patients with diabetes mellitus and COVID-19. More specific markers of the acute kidney injury functional stage were determined;these markers will improve the diagnostics accuracy and validity, therapy efficiency, and end points of disease prognosis.Copyright © 2023, Media Sphera Publishing Group. All rights reserved.

Glomerulonephritis associated with SARS-CoV-2 infection

Implication for health policy/practice/research/medical education: Post-infectious glomerulonephritis (PIGN) can develop secondary to infections associated with bacterial, viral, fungal, protozoal, and helminthic parasites. Recently, there is a serious concern regarding the occurrence of kidney dysfunctions and subsequent acute kidney injury (AKI) among COVID-19 patients. The outcome data of COVID-19 in neonates and children demonstrated that the fatality rate is significantly higher in patients with AKI than in patients without AKI. In the current COVID-19 pandemic, few instances of glomerulonephritis (GN) in patients affected by SARS-CoV-2 have been reported. In this review, we investigated the PIGN concentrating on the COVID19-nephropathy, as well as its prevention and diagnosis strategies.Copyright © 2021 The Author(s).

Complement Inhibition and COVID-19: The Story so Far.

Acute respiratory distress syndrome (ARDS) is the most severe complication of COVID-19, a disease caused by severe acute respiratory syndrome coronavirus (SARS CoV) 2. The mechanisms underlying the progression from asymptomatic disease to pneumonia and ARDS are complex and by far unelucidated. As for bacterial sepsis, the release of damage associated molecular patterns and pathogen associated molecular patterns triggers activation of the complement cascade. Subsequently, overexpressed anaphylatoxins recruit inflammatory cells in the lung and other organs and contribute initiating and amplifying a vicious circle of thromboinflammation causing organs damage and eventually death. Preclinical and observational studies in patients with COVID-19 provided evidence that complement inhibition effectively may attenuate lung and systemic inflammation, restore the coagulation/fibrinolysis balance, improve organs function and eventually may save life. Ongoing Phase 2/3 trials should elucidate the benefit to risk profile of complement inhibitors and may clarify the optimal targets in the complement cascade.

Longitudinal Modulation of Loco-Regional Immunity in Ovarian Cancer Patients Receiving Intraperitoneal Chemotherapy.

The immune tumor microenvironment (TME) of epithelial ovarian cancer (EOC) carries both effector and suppressive functions. To define immune correlates of chemotherapy-induced tumor involution, we performed longitudinal evaluation of biomarker expression on serial biological specimens collected during intraperitoneal (IP) platinum-based chemotherapy. Serial biological samples were collected at several time points during IP chemotherapy. RNA from IP fluid cells and tumor tissue was analyzed via NanoString. Meso Scale Discovery (MSD) multiplex assay and ELISA for MUC1 antibodies were performed on plasma and IP fluid. Differentially expressed genes in IP fluid demonstrate an upregulation of B cell function and activation of Th2 immune response along with dampening of Th1 immunity during chemotherapy. MSD analysis of IP fluid and gene expression analysis of tumor tissue revealed activation of Th2 immunity and the complement system. Anti-MUC1 antibodies were detected in IP fluid samples. IP fluid analysis in a secondary cohort also identified chemotherapy-induced B cell function genes. This study shows that serial IP fluid sampling is an effective method to capture changes in the immune TME during chemotherapy and reveals treatment induced changes in B cell function and Th2 immunity.

Anti-Spike Antibodies Present in the Milk of SARS-CoV-2 Vaccinated Mothers Are Complement-Activating.

Although only 0.8-1% of SARS-CoV-2 infections are in the 0-9 age-group, pneumonia is still the leading cause of infant mortality globally. Antibodies specifically directed against SARS-CoV-2 spike protein (S) are produced during severe COVID-19 manifestations. Following vaccination, specific antibodies are also detected in the milk of breastfeeding mothers. Since antibody binding to viral antigens can trigger activation of the complement classical - pathway, we investigated antibody-dependent complement activation by anti-S immunoglobulins (Igs) present in breast milk following SARS-CoV-2 vaccination. This was in view of the fact that complement could play a fundamentally protective role against SARS-CoV-2 infection in newborns. Thus, 22 vaccinated, lactating healthcare and school workers were enrolled, and a sample of serum and milk was collected from each woman. We first tested for the presence of anti-S IgG and IgA in serum and milk of breastfeeding women by ELISA. We then measured the concentration of the first subcomponents of the three complement pathways (i.e., C1q, MBL, and C3) and the ability of anti-S Igs detected in milk to activate the complement in vitro. The current study demonstrated that vaccinated mothers have anti-S IgG in serum as well as in breast milk, which is capable of activating complement and may confer a protective benefit to breastfed newborns.

Mechanisms of kidney injury in COVID-19 and diagnostic methods

The COVID-19 pandemic is a global healthcare crisis. The frequency of acute kidney injury (AKI) in patients with COVID-19 and the features of its diagnostics indicate the relevance of the topic. Objective of the review. To analyze mechanisms of AKI development in patients with COVID-19 and provide support for methodological approaches to ensure its timely diagnosis. Material and methods. The methodological approaches used in the review are based on a sufficient number of literature sources (more than 150 sources), of which 34 articles are included in the review: 15 original studies, 12 reviews, 2 meta-analyses, 5 re-ports, and letters to the editor. Results. The mechanisms of AKI development and progression, including the direct cytotoxic effect of the SARS-CoV-2 virus, dis-ruption of metabolic pathways of renal blood flow regulation, and the complement system, are considered. We also analyzed AKI risk factors in patients with acute respiratory distress: diabetes mellitus, chronic kidney injury, arterial hypertension with im-paired NOx production, and eNOS expression as significant factors of vasodilation in renal microcirculatory vessels. The analy-sis showed the most perspective directions in the diagnostics of AKI functional stages. These include molecular test methods (pro-teome and metabolome) in blood and urine;they helped define damage markers to proximal tubules and the glomerular system, thus improving the diagnostics accuracy and validity, therapy efficiency, and end points of disease prognosis. Conclusion. The Coronado study aims to assess the phenotypic features of patients with diabetes mellitus and COVID-19. More specific markers of the acute kidney injury functional stage were determined;these markers will improve the diagnostics accuracy and validity, therapy efficiency, and end points of disease prognosis.

Associations between Serum Levels of C3, C4, and Total Classical Complement Activity in Covid-19 Patients at the Time of Admission and Clinical Outcome

In the present study, we investigated the association between complement system status at the time of admission and clinical outcomes in COVID-19 patients. This single-center study was carried out with sixty-one adult patients with COVID-19 who were hospitalized at Imam Hassan Hospital of North Khorasan University of Medical Sciences (Bojnurd, Iran) with less than three days passage since onset of COVID-19 symptoms. Twenty-three healthy volunteers with demographic features similar to the patient group (matched by age and gender) were included in the study as a control group. Patient information including demographic information, demographic data, clinical characteristics, and clinical outcomes were obtained from electronic medical records. Of 61 hospitalized patients with COVID-19, 28 (47.54%) were female, and the average age was 48.7+/-8.8 years. The healthy control group included 23 cases (11 (47.8%) female, 12 (52.1%) males, mean age 46.4+/-4.4 years). Twenty-one of the 61 patients (34.4%) were admitted to the ICU, and sixteen of them (26.2%) died. Thirty-three (54.10%) patients with COVID-19 were hospitalized for less than 7 days, and 28 (45.90%) of them were hospitalized for >= 7 days. Our results show that length of hospital stay in the no-ICU group was significantly lower than the ICU admission or death groups (6.49+/-0.24 vs. 8.85+/-1.59 and 10.53+/-1.80, p = 0.0002). The levels of C3, C4, and CH50 were determined through the immunoturbidimetric method and single-radial-haemolysis plates, respectively, on serum samples obtained from patients at the time of admission or those in the control group. Our results indicate that C3, C4 and CH50 levels were markedly lower in COVID-19 patients than in the control group. We also found that complement parameter levels in COVID-19 patients who died or were admitted to ICU were significantly lower than in non-ICU COVID-19 patients. In general, it seems that serum level of C3, C4, and CH50 at admission may predict disease progression or adverse clinical outcome in COVID-19 patients. Copyright © 2022 Saint Petersburg Pasteur Institute. All rights reserved.

Efficacy and safety of the investigational complement C5 inhibitor zilucoplan in patients hospitalized with COVID-19: an open-label randomized controlled trial.

BACKGROUND: The efficacy and safety of complement inhibition in COVID-19 patients is unclear. METHODS: A multicenter randomized controlled, open-label trial. Hospitalized COVID-19 patients with signs of systemic inflammation and hypoxemia (PaO2/FiO2 below 350 mmHg) were randomized (2:1 ratio) to receive standard of care with or without the C5 inhibitor zilucoplan daily for 14 days, under antibiotic prophylaxis. The primary outcome was improvement in oxygenation at day 6 and 15. RESULTS: 81 patients were randomly assigned to zilucoplan (n = 55) or the control group (n = 26). 78 patients were included in the safety and primary analysis. Most were men (87%) and the median age was 63 years. The mean improvement in PaO2/FiO2 from baseline to day 6 was 56.4 mmHg in the zilucoplan group and 20.6 mmHg in the control group (mean difference + 35.8; 95% confidence interval (CI) - 9.4 to 80.9; p = 0.12), an effect also observed at day 15. Day 28 mortality was 9% in the zilucoplan and 21% in the control group (odds ratio 0.4; 95% CI 0.1 to 1.5). At long-term follow up, the distance walked in a 6-min test was 539.7 m in zilucoplan and 490.6 m in the control group (p = 0.18). Zilucoplan lowered serum C5b-9 (p < 0.001) and interleukin-8 (p = 0.03) concentration compared with control. No relevant safety differences between the zilucoplan and control group were identified. CONCLUSION: Administration of zilucoplan to COVID-19 patients in this proof-of-concept randomized trial was well tolerated under antibiotic prophylaxis. While not reaching statistical significance, indicators of respiratory function (PaO2/FiO2) and clinical outcome (mortality and 6-min walk test) suggest that C5 inhibition might be beneficial, although this requires further research in larger randomized studies.

Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Anti-C5a Antibody BDB-001 for Severe COVID-19: A Randomized, Double-Blind, Placebo-Controlled Phase 1 Clinical Trial in Healthy Chinese Adults.

INTRODUCTION: Severe Coronavirus Disease 2019 (COVID-19) progresses with inflammation and coagulation, due to an overactive complement system. Complement component 5a (C5a) plays a key role in the complement system to trigger a powerful "cytokine and chemokine storm" in viral infection. BDB-001, a recombinant human immunoglobulin G4 (IgG4) that specially binds to C5a, has the potential to inhibit the C5a-triggered cytokine storm in treating COVID-19 patients and other inflammation diseases. Here, we have explored its safety, tolerability, pharmacokinetics, and pharmacodynamics in healthy adults. This trial is registered with http://www.chinadrugtrials.org.cn/(CTR20200429 ). METHODS: Thirty-two enrolled participants were randomized into three single-dose cohorts (2, 4, and 8 mg/kg) and 1 multi-dose cohort (4 mg/kg), and received either BDB-001 or placebo (3:1) double-blindly. The safety and tolerability after administration were evaluated for 21 days for single-dose cohorts and 28 days for the multi-dose cohort. The pharmacokinetics of BDB-001 in plasma and pharmacodynamics as free C5a in plasma were analyzed. RESULTS: The incidence of drug-related adverse events (AEs) was low, and all AEs were mild or moderate: neither AEs ≥ 3 (NCI-Common Terminology Criteria For Adverse Events, CTCAE 5.0) nor serious adverse events (SAEs) were found. The area under the concentration-time curve from time zero to 480 h (AUC0-480h), that from time zero to infinity (AUCinf), and peak plasma concentration ©max) increased dose-dependently from 2 to 8 mg/kg in the single-dose cohorts and were characterized by a nonlinear pharmacokinetics of target-mediated drug disposal (TMDD). The accumulation index by AUC0-tau after five administrations (4 mg/kg) from the multi-dose cohort was 6.42, suggesting an accumulation effect. Furthermore, inhibition of C5a at the plasma level was observed. CONCLUSION: The results of this phase I study supported that BDB-001 is a potent anti-C5a inhibitor with safety, tolerability, and no immunogenicity. TRIAL REGISTRATION NUMBER: CTR20200429.

Complement system as the potential therapeutic target in the management of COVID-19 patients

The emerging COVID-19 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been raised as a global health challenge. Despite the breakthrough in the development of the COVID-19 vac-cine, it still continues as a serious crisis, worldwide. The aberrant immune responses are strongly associated with the severity of the disease and an increased rate of morbidity and mortality among COVID-19 patients. The complement cascade activation is mediated by classical, lectin, and alternative pathways which could induce an inflammatory state during the COVID-19 infection. The growing body of research suggests that complement system activation plays an important role in the immunopathogenesis of SARS-CoV-2. Therefore, the blockade of complement cascades may be an effective approach to prevent the multi-organ complications of COVID-19. In this review, we will highlight the role of the complement system in the immunopathology of COVID-19, emphasizing the potential therapeutical targets to ameliorate COVID-19 infection. © 2022 Gilda Parsamanesh, et al.

The complement system in preeclampsia: a review of its activation and endothelial injury in the triad of COVID 19 infection and HIV-associated preeclampsia.

This review assessed the complement system and its activation with respect to the pathological features of severe acute respiratory syndrome (SARS-CoV-2), human immunodeficiency virus (HIV) infection, and preeclampsia (PE). The complement system is the first defensive response of the host innate immune system to viral pathogens, including SARS-Cov-2. SARS-CoV-2 entry results in the release of proinflammatory cytokines and chemical mediators to create a "cytokine storm". Endothelial cell (EC) dysfunction and cell-mediated injury are also observed. These factors exacerbate inflammation. During HIV infection and PE, various complement components are elevated, causing a hyperinflammatory state. Furthermore, EC dysfunction and cell-mediated injury are also observed. The similarities in the pathological aspects of these three disorders may emanate from excessive complement activation. This review serves as a platform for further research on the complement system, coronavirus disease-2019, HIV, and PE.

Low molecular weight cytotoxic components (DAMPs) form the post-COVID-19 syndrome.

We studied the role of cytotoxic components (DAMPs) formed in the body of patients with COVID-19 in ensuring the long-term preservation of post-COVID-19 manifestations and the possibility of creating an experimental model by transferring DAMPs to rats. In patients with post-COVID-19 syndrome (PCS) 2 months after SARS-CoV-2 infection we determined the presence of cytotoxic components in the blood serum (Terasaki test, Dunaliella viridis test and content of DAMPs). In post-COVID-19 syndrome patients with a high content of serum cytotoxic oligopeptide fraction (selective group, n = 16) we determined the number of leukocytes, lymphocytes, neutrophil granulocytes and monocytes in the blood, the content of C-reactive protein (CRP), the concentration of C3 and C4 complement components and circulating immune complexes, the serum content of IL-6, IL -10, IL-18, TNF-α, phagocytic activity of neutrophils, presence of neutrophil traps and autoantibodies ANA. It has been shown that in patients with PCS, there are components with cytotoxicity in the blood serum, form specific immunopathological patterns, which are characterized by: an increased content of CRP, complement system components C3 and C4 and cytokines (TNF-α, IL-6, IL-10, IL-18) activation, the formation of a wide range of autoantibodies ANA, the low efficiency of endocytosis in oxygen-independent phagocytosis; their phagocytic activity reaches its functional limit, and against this background, activation of neutrophil traps occurs, which can contribute to further induction of DAMPs. This self-sustaining cell-killing activation provided long-term preservation of PCS symptoms. The transfer of blood serum components from selective group patients with PCS to rats was accompanied by the appearance of cytotoxic components in them which induced sensitization and immunopathological reactions. Preventive administration of a biologically active substance with polyfunctional properties MF to experimental animals "corrected" the initial functional state of the body's immune-metabolic system and eliminated or facilitated immuno-inflammatory reactions.

Complement and endothelial cell activation in COVID-19 patients compared to controls with suspected SARS-CoV-2 infection: A prospective cohort study.

Background: Thromboinflammation may influence disease outcome in COVID-19. We aimed to evaluate complement and endothelial cell activation in patients with confirmed COVID-19 compared to controls with clinically suspected but excluded SARS-CoV-2 infection. Methods: In a prospective, observational, single-center study, patients presenting with clinically suspected COVID-19 were recruited in the emergency department. Blood samples on presentation were obtained for analysis of C5a, sC5b-9, E-selectin, Galectin-3, ICAM-1 and VCAM-1. Results: 153 cases and 166 controls (suffering mainly from non-SARS-CoV-2 respiratory viral infections, non-infectious inflammatory conditions and bacterial pneumonia) were included. Hospital admission occurred in 62% and 45% of cases and controls, respectively. C5a and VCAM-1 concentrations were significantly elevated and E-selectin concentrations decreased in COVID-19 out- and inpatients compared to the respective controls. However, relative differences in outpatients vs. inpatients in most biomarkers were comparable between cases and controls. Elevated concentrations of C5a, Galectin-3, ICAM-1 and VCAM-1 on presentation were associated with the composite outcome of ICU- admission or 30-day mortality in COVID-19 and controls, yet more pronounced in COVID-19. C5a and sC5b-9 concentrations were significantly higher in COVID-19 males vs. females, which was not observed in the control group. Conclusions: Our data indicate an activation of the complement cascade and endothelium in COVID-19 beyond a nonspecific inflammatory trigger as observed in controls (i.e., "over"-activation).

Pitfalls in complement analysis: A systematic literature review of assessing complement activation.

Background: The complement system is an essential component of our innate defense and plays a vital role in the pathogenesis of many diseases. Assessment of complement activation is critical in monitoring both disease progression and response to therapy. Complement analysis requires accurate and standardized sampling and assay procedures, which has proven to be challenging. Objective: We performed a systematic analysis of the current methods used to assess complement components and reviewed whether the identified studies performed their complement measurements according to the recommended practice regarding pre-analytical sample handling and assay technique. Results are supplemented with own data regarding the assessment of key complement biomarkers to illustrate the importance of accurate sampling and measuring of complement components. Methods: A literature search using the Pubmed/MEDLINE database was performed focusing on studies measuring the key complement components C3, C5 and/or their split products and/or the soluble variant of the terminal C5b-9 complement complex (sTCC) in human blood samples that were published between February 2017 and February 2022. The identified studies were reviewed whether they had used the correct sample type and techniques for their analyses. Results: A total of 92 out of 376 studies were selected for full-text analysis. Forty-five studies (49%) were identified as using the correct sample type and techniques for their complement analyses, while 25 studies (27%) did not use the correct sample type or technique. For 22 studies (24%), it was not specified which sample type was used. Conclusion: A substantial part of the reviewed studies did not use the appropriate sample type for assessing complement activation or did not mention which sample type was used. This deviation from the standardized procedure can lead to misinterpretation of complement biomarker levels and hampers proper comparison of complement measurements between studies. Therefore, this study underlines the necessity of general guidelines for accurate and standardized complement analysis.

Influence of complement system genomic variations on the course of COVID-19

Background: The pandemic of SARS-CoV- 2 is a severe worldwide problem increasing morbidity and mortality.1, 2 Severe COVID-19 presents as multiple organ failure caused by systemic inflammation, thrombin generation, and hypofibrinolysis. Diffuse microvascular thrombi and inter-alveolar deposits of complement fragments are observed. The enhanced immunothrombosis is mediated by direct overactivation of complement by virus surface components or damaged cells.3-5 Aims: The study aimed to find whether genetic changes responsible for complement dysregulation known in atypical hemolytic-uremic syndrome (aHUS) can be found in severe COVID-19 patients. Method(s): The study included adult COVID-19 subjects undergoing extracorporeal membrane oxygenation support for severe acute respiratory distress syndrome. Two independent physicians signed informed consent, and the study was approved by a local ethics committee (No. 109/2021) and supported by the University Hospital fund. Next-Generation Sequencing Panel of C3 component, membrane cofactor protein (CD46), complement factor B (CFB), complement factor H (CFH), complement factor H related genes 1-5 (CFHR 1-5), diacylglycerol Kinase Epsilon, thrombomodulin (THBD) and mannose-binding lectin (MBL) genes were performed, with confirmations of positive results by Sanger sequencing. Result(s): Twenty-two patients (13 were male) aged 33 to 65 years were included. No pathogenic gene variants in the C3, CD46, CFB, CFH genes, CFHR 5, CFI, THBD were detected. However, we have shown the presence of modifiers (CFH-H3 haplotype, MCP-GGAAC haplotype, and CFH/CFHR1), which may, together with triggers (infection), increase the severity of the disease (aHUS).6-8 Moreover, we have identified single nucleotide polymorphisms in exon 1 at codon 52 (c.154C>T) and 54 (c.161G>A) of the MBL2 gene promoter associated with low serum levels or dysfunctional MBL and higher incidence of infections. Conclusion(s): We did not detect any complement-related pathogenic gene variants known in aHUS. Thus, It is unlikely that complement dysregulation is the main factor influencing immunothrombosis in a cohort of the most severe COVID-19 patients.

Role of the Complement System and Vascular Endothelium in COVID-19 Pathogenesis

Background: Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 infection has become a global pandemic, presenting with varying degrees of severity from respiratory distress to multi-organ damage. Kidneys are of several organs affected in COVID-19, with acute kidney injury (AKI) being a common consequence, occurring in more than 30% of patients with severe COVID-19. While the underlying mechanisms of COVID-19 pathogenesis remain poorly understood, there is evidence linking complement system overactivation and endothelial injury to organ damage that increases the risk of mortality in COVID-19. Evidence from previous coronavirus epidemics also suggest direct involvement of inflammation, complement dysregulation, and endothelial cell dysfunction. Thus, we hypothesize that vascular endothelial injury resulting from complement overactivation contributes to COVID-19-associated organ injury. Method(s): Clinical information and sera from SARS-CoV-2+ patients with mild (n=7) and severe COVID-19 (n=7) diseases were obtained from the Canadian COVID-19 Prospective Cohort Study (CANCOV). Complement activation on ECs was evaluated via immunofluorescence assays, measuring the deposition of complement products C3b and C5b-9 on Human Umbilical Vein Endothelial Cells exposed to control or patient sera. In addition, a permeability assay using a transwell model was used to measure the integrity of the endothelial monolayer exposed to patient sera. Result(s): Complement was found to be overactivated on ECs treated with SARSCoV-2+ patient sera compared to those treated with normal human serum as evidenced by significantly increased C3b and C5b-9 deposition. While ECs treated with sera from patients with mild COVID-19 seemed to have higher C3b deposition, ECs treated with sera from patients with severe COVID-19 disease were associated with higher C5b-9 deposition. In addition, increased permeability of the monolayer incubated with SARSCoV-2+ patient sera was seen over time regardless of disease severity. However, ECs tretaed with severe COVID-19 patient sera had signficiantly increased vascular leakiness as evidenced by increased permeability of the treated monolayer. Conclusion(s): Thus, we conclude that complement is overactivated in SARS-CoV-2+ patients and use of anti-complement therapies may be an effective strategy in treating COVID-19 associated vascular injury, hyperinflammation, and organ damage.