sMR and PTX3 levels associate with COVID-19 outcome and survival but not with Long COVID.

Biomarkers for monitoring COVID-19 disease course are lacking. Study aim was to identify biomarkers associated with disease severity, survival, long-term outcome, and Long COVID. As excessive macrophages activation is a hallmark of COVID-19 and complement activation is key in this, we selected the following proteins involved in these processes: PTX3, C1q, C1-INH, C1s/C1-INH, and sMR. EDTA-plasma concentrations were measured in 215 patients and 47 controls using ELISA. PTX3, sMR, C1-INH, and C1s/C1-INH levels were associated with disease severity. PTX3 and sMR were also associated with survival and long-term immune recovery. Lastly, sMR levels associate with ICU admittance. sMR (AUC 0.85) and PTX3 (AUC 0.78) are good markers for disease severity, especially when used in combination (AUC 0.88). No association between biomarker levels and Long COVID was observed. sMR has not previously been associated with COVID-19 disease severity, ICU admittance or survival and may serve as marker for disease course.

Detrimental interactions of hypoxia and complement MASP-1 in endothelial cells as a model for atherosclerosis-related diseases.

Both hypoxia and the complement lectin pathway (CLP) are involved in atherosclerosis and atherosclerosis-related stroke and acute myocardial infarction (AMI). We have previously shown that mannose-binding lectin-associated serine protease-1 (MASP-1), the most abundant enzyme of CLP, induces an inflammatory phenotype of endothelial cells (ECs) by cleaving protease activated receptors (PARs). In the absence of data, we aimed to investigate whether hypoxia and MASP-1 interact at the level of ECs, to better understand their role in atherosclerosis-related diseases. Hypoxia attenuated the wound healing ability of ECs, increased ICAM-1 and decreased ICAM-2 expression and upregulated PAR2 gene expression. Hypoxia and MASP-1 increased GROα and IL-8 production, and endothelial permeability without potentiating each other's effects, whereas they cooperatively disrupted vascular network integrity, activated the Ca2+, CREB and NFκB signaling pathways, and upregulated the expression of E-selectin, a crucial adhesion molecule in neutrophil homing. VCAM-1 expression was not influenced either by hypoxia, or by MASP-1. In summary, hypoxia potentiates the effect of MASP-1 on ECs, at least partially by increasing PAR expression, resulting in interaction at several levels, which may altogether exacerbate stroke and AMI progression. Our findings suggest that MASP-1 is a potential drug target in the acute phase of atherosclerosis-related diseases.

Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) as a compensatory protease inhibitor in hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is a genetic disorder that manifests as recurrent angioedema attacks, most frequently due to absent or reduced C1 inhibitor (C1INH) activity. C1INH is a crucial regulator of enzymatic cascades in the complement, fibrinolytic, and contact systems. Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) is an abundant plasma protease inhibitor that can inhibit enzymes in the proteolytic pathways associated with HAE. Nothing is known about its role in HAE. OBJECTIVE: We investigated ITIH4 activation in HAE, establishing it as a potential biomarker, and explored its involvement in HAE-associated proteolytic pathways. METHODS: Specific immunoassays for noncleaved ITIH4 (intact ITIH4) and an assay detecting both intact and cleaved ITIH4 (total ITIH4) were developed. We initially tested serum samples from HAE patients (n = 20), angiotensin-converting enzyme inhibitor-induced edema patients (ACEI) (n = 20), and patients with HAE of unknown cause (HAE-UNK) (n = 20). Validation involved an extended cohort of 80 HAE patients (60 with HAE-C1INH type 1, 20 with HAE-C1INH type 2), including samples taken during attack and quiescent disease periods, as well as samples from 100 healthy controls. RESULTS: In 63% of HAE patients, intact ITIH4 assay showed lower signals than total ITIH4 assay. This difference was not observed in ACEI and HAE-UNK patients. Western blot analysis confirmed cleaved ITIH4 with low intact ITIH4 samples. In serum samples lacking intact endogenous ITIH4, we observed immediate cleavage of added recombinant ITIH4, suggesting continuous enzymatic activity in the serum. Confirmatory HAE cohort analysis revealed significantly lower intact ITIH4 levels in both type 1 and type 2 HAE patients compared to controls, with consistently low intact/total ITIH4 ratios during clinical HAE attacks. CONCLUSION: The disease-specific low intact ITIH4 levels highlight its unique nature in HAE. ITIH4 may exhibit compensatory mechanisms in HAE, suggesting its utility as a diagnostic and prognostic biomarker. The variations during quiescent and active disease periods raise intriguing questions about the dynamics of proteolytic pathways in HAE.

Complement MASP-1 Modifies Endothelial Wound Healing.

Endothelial wound-healing processes are fundamental for the maintenance and restoration of the circulatory system and are greatly affected by the factors present in the blood. We have previously shown that the complement protein mannan-binding lectin-associated serine protease-1 (MASP-1) induces the proinflammatory activation of endothelial cells and is able to cooperate with other proinflammatory activators. Our aim was to investigate the combined effect of mechanical wounding and MASP-1 on endothelial cells. Transcriptomic analysis showed that MASP-1 alters the expression of wound-healing-related and angiogenesis-related genes. Both wounding and MASP-1 induced Ca2+ mobilization when applied individually. However, MASP-1-induced Ca2+ mobilization was inhibited when the treatment was preceded by wounding. Mechanical wounding promoted CREB phosphorylation, and the presence of MASP-1 enhanced this effect. Wounding induced ICAM-1 and VCAM-1 expression on endothelial cells, and MASP-1 pretreatment further increased VCAM-1 levels. MASP-1 played a role in the subsequent stages of angiogenesis, facilitating the breakdown of the endothelial capillary network on Matrigel®. Our findings extend our general understanding of endothelial wound healing and highlight the importance of complement MASP-1 activation in wound-healing processes.

The Lectin Pathway of the Complement System-Activation, Regulation, Disease Connections and Interplay with Other (Proteolytic) Systems.

The complement system is the other major proteolytic cascade in the blood of vertebrates besides the coagulation-fibrinolytic system. Among the three main activation routes of complement, the lectin pathway (LP) has been discovered the latest, and it is still the subject of intense research. Mannose-binding lectin (MBL), other collectins, and ficolins are collectively termed as the pattern recognition molecules (PRMs) of the LP, and they are responsible for targeting LP activation to molecular patterns, e.g., on bacteria. MBL-associated serine proteases (MASPs) are the effectors, while MBL-associated proteins (MAps) have regulatory functions. Two serine protease components, MASP-1 and MASP-2, trigger the LP activation, while the third component, MASP-3, is involved in the function of the alternative pathway (AP) of complement. Besides their functions within the complement system, certain LP components have secondary ("moonlighting") functions, e.g., in embryonic development. They also contribute to blood coagulation, and some might have tumor suppressing roles. Uncontrolled complement activation can contribute to the progression of many diseases (e.g., stroke, kidney diseases, thrombotic complications, and COVID-19). In most cases, the lectin pathway has also been implicated. In this review, we summarize the history of the lectin pathway, introduce their components, describe its activation and regulation, its roles within the complement cascade, its connections to blood coagulation, and its direct cellular effects. Special emphasis is placed on disease connections and the non-canonical functions of LP components.

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.

Decreased adhesion to endothelium leads to elevated neutrophil granulocyte count in hereditary angioedema patients.

As many aspects of hereditary angioedema (HAE) due to C1-inhibitor (C1-INH) deficiency (C1-INH-HAE) cannot be explained with elevated bradykinin level alone, it has recently become clear that other factors also play an important role in the pathogenesis. One of these factors could be elevated neutrophil granulocyte (NG) counts, which are associated with increased NG activation in C1-INH-HAE patients; however, their origin has not been elucidated so far. Here, we aimed to investigate whether the excess of NGs is due to disturbed maturation, biased circulating/marginated pool equilibrium or decreased elimination. We enrolled 20 attack-free C1-INH-HAE patients together with 21 healthy controls and collected blood samples. We compared cell surface maturation markers, adhesion molecules, cytokine receptors, and Ca2+-mobilization of NG by flow cytometry, activation markers by ELISA, and NG/endothelial cell adhesion by automated pipetting system. Cell-surface markers showed normal maturation of NGs in C1-INH-HAE patients. Adhesion of NGs to endothelial cells pretreated with lipopolysaccharide or phorbol 12-myristate 13-acetate was significantly weaker in samples from C1-INH-HAE patients and bradykinin had no effect on the adhesion. NGs from C1-INH-HAE patients were in an activated state when assessed by soluble activation markers without any stimulation. Our data support that the maturation of NGs in C1-INH-HAE patients is normal, whereas adhesion properties of patient-derived NGs to the endothelium are reduced compared to those from healthy controls, indicating a bias between the circulating and marginated pools of NGs in patients. Bradykinin may not be responsible for reduced adhesion properties of NGs.

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.

Cooperation of Complement MASP-1 with Other Proinflammatory Factors to Enhance the Activation of Endothelial Cells.

Endothelial cells play an important role in sensing danger signals and regulating inflammation. Several factors are capable of inducing a proinflammatory response (e.g., LPS, histamine, IFNγ, and bradykinin), and these factors act simultaneously during the natural course of the inflammatory reaction. We have previously shown that the complement protein mannan-binding lectin-associated serine protease-1 (MASP-1) also induces a proinflammatory activation of the endothelial cells. Our aim was to investigate the possible cooperation between MASP-1 and other proinflammatory mediators when they are present in low doses. We used HUVECs and measured Ca2+ mobilization, IL-8, E-selectin, VCAM-1 expression, endothelial permeability, and mRNA levels of specific receptors. LPS pretreatment increased the expression of PAR2, a MASP-1 receptor, and furthermore, MASP-1 and LPS enhanced each other's effects in regulating IL-8, E-selectin, Ca2+ mobilization, and changes in permeability in a variety of ways. Cotreatment of MASP-1 and IFNγ increased the IL-8 expression of HUVECs. MASP-1 induced bradykinin and histamine receptor expression, and consequently, increased Ca2+ mobilization was found. Pretreatment with IFNγ enhanced MASP-1-induced Ca2+ mobilization. Our findings highlight that well-known proinflammatory mediators and MASP-1, even at low effective doses, can strongly synergize to enhance the inflammatory response of endothelial cells.

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.

C1-inhibitor/C1-inhibitor antibody complexes in acquired angioedema due to C1-inhibitor deficiency.

BACKGROUND: Autoantibodies against C1-inhibitor (C1-INH-Ab) have a diagnostic value in acquired angioedema due to C1-inhibitor deficiency (C1-INH-AAE), even though antibodies can circulate in complexes, which can be undetectable by proven methods. Our aim was to measure C1-INH/C1-INH-Ab complexes (CAC) and investigate their connection to C1-INH-Ab and the changes in their titer over time. RESULTS: 19 patients were diagnosed with C1-INH-AAE in the Hungarian Angioedema Center of Reference and Excellence; 79% of them had an underlying disease. Samples were examined with a newly developed in-house complex ELISA method. Patients with high C1-INH-Ab titer had a CAC titer which did not exceed the normal level and the ones with high CAC titer had a C1-INH-Ab titer which did not exceed the normal level. In case of those patients who had C1-INH-Ab and CAC of the same type of immunoglobulin, the increasing titer of C1-INH-Ab went together with the decreasing level of CAC and vice versa. CAC titer was already increased before the diagnosis of the underlying disease. CONCLUSIONS: Free circulating and complex antibodies are in a dynamically changing equilibrium. CAC measurements can help to predict the development of an underlying disease. The efficiency of the treatment for underlying disease can be monitored by the decreasing CAC titers. Our results show that the CAC can be of important additional information besides the complement panel examination in case of C1-INH-AAE. Measurement of CAC is recommended to be done parallelly with C1-INH-Ab, so as to detect both free and bound antibodies.

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.

BRAF increases endothelial cell stiffness through reorganization of the actin cytoskeleton.

The dynamics of the actin cytoskeleton and its connection to endothelial cell-cell junctions determine the barrier function of endothelial cells. The proper regulation of barrier opening/closing is necessary for the normal function of vessels, and its dysregulation can result in chronic and acute inflammation leading to edema formation. By using atomic force microscopy, we show here that thrombin-induced permeability of human umbilical vein endothelial cells, associated with actin stress fiber formation, stiffens the cell center. The depletion of the MEK/ERK kinase BRAF reduces thrombin-induced permeability prevents stress fiber formation and cell stiffening. The peripheral actin ring becomes stabilized by phosphorylated myosin light chain, while cofilin is excluded from the cell periphery. All these changes can be reverted by the inhibition of ROCK, but not of the MEK/ERK module. We propose that the balance between the binding of cofilin and myosin to F-actin in the cell periphery, which is regulated by the activity of ROCK, determines the local dynamics of actin reorganization, ultimately driving or preventing stress fiber formation.

A novel pathogenetic factor of laryngeal attack in hereditary angioedema? Involvement of protease activated receptor 1.

BACKGROUND: Hereditary angioedema (HAE) is a rare, life-threatening disease. The knowledge about the molecular pathogenesis of HAE has derived mainly from investigating blood samples. However, limited data are available on the role of the molecular mechanisms in the affected tissues during HAE attack. OBJECTIVE: The aim of our study was to explore the histological changes occurring in HAE attacks. METHODS: Post mortem macro-, microscopic and immunohistological assessment of upper airway tissues of a patient with HAE due to C1 inhibitor deficiency (C1-INH-HAE) type 2 who died from laryngeal HAE attack was compared with a non-HAE patient who died from other condition without any signs of angioedema. RESULTS: Compared to the control patient, we demonstrated stronger T cell/monocyte infiltration and a more intense C1-INH staining in the C1-INH-HAE patient. The expression of both bradykinin receptors (B1/B2) was observed with a slightly lower level in the C1-INH-HAE patient than in the control patient. PAR1 expression was strongly reduced in the C1-INH-HAE patient suggesting overactivation of this hyperpermeability inducing receptor. CONCLUSION: Our unique case and novel results correspond to the knowledge about C1-INH and BDKRs observed in plasma; however, it revealed new information about the pathomechanism of HAE attack focusing on the potential involvement of PAR1 in edema formation. This observation, if it is verified by subcutaneous biopsy studies, may designate a new therapeutic target in HAE.

Complement Levels at Admission Reflecting Progression to Severe Acute Kidney Injury (AKI) in Coronavirus Disease 2019 (COVID-19): A Multicenter Prospective Cohort Study.

Background: Dysregulation of complement system is thought to be a major player in development of multi-organ damage and adverse outcomes in patients with coronavirus disease 2019 (COVID-19). This study aimed to examine associations between complement system activity and development of severe acute kidney injury (AKI) among hospitalized COVID-19 patients. Materials and Methods: In this multicenter, international study, complement as well as inflammatory and thrombotic parameters were analyzed in COVID-19 patients requiring hospitalization at one US and two Hungarian centers. The primary endpoint was development of severe AKI defined by KDIGO stage 2+3 criteria, while the secondary endpoint was need for renal replacement therapy (RRT). Complement markers with significant associations with endpoints were then correlated with a panel of inflammatory and thrombotic biomarkers and assessed for independent association with outcome measures using logistic regression. Results: A total of 131 hospitalized COVID-19 patients (median age 66 [IQR, 54-75] years; 54.2% males) were enrolled, 33 from the US, and 98 from Hungary. There was a greater prevalence of complement over-activation and consumption in those who developed severe AKI and need for RRT during hospitalization. C3a/C3 ratio was increased in groups developing severe AKI (3.29 vs. 1.71; p < 0.001) and requiring RRT (3.42 vs. 1.79; p < 0.001) in each cohort. Decrease in alternative and classical pathway activity, and consumption of C4 below reference range, as well as elevation of complement activation marker C3a above the normal was more common in patients progressing to severe AKI. In the Hungarian cohort, each standard deviation increase in C3a (SD = 210.1) was independently associated with 89.7% increased odds of developing severe AKI (95% CI, 7.6-234.5%). Complement was extensively correlated with an array of inflammatory biomarkers and a prothrombotic state. Conclusion: Consumption and dysregulation of complement system is associated with development of severe AKI in COVID-19 patients and could represent a promising therapeutic target for reducing thrombotic microangiopathy in SARS-CoV-2 infection.

Blood-brain barrier dysfunction in L-ornithine induced acute pancreatitis in rats and the direct effect of L-ornithine on cultured brain endothelial cells.

BACKGROUND: In severe acute pancreatitis (AP) the CNS is affected manifesting in neurological symptoms. Earlier research from our laboratory showed blood-brain barrier (BBB) permeability elevation in a taurocholate-induced AP model. Here we aimed to further explore BBB changes in AP using a different, non-invasive in vivo model induced by L-ornithine. Our goal was also to identify whether L-ornithine, a cationic amino acid, has a direct effect on brain endothelial cells in vitro contributing to the observed BBB changes. METHODS: AP was induced in rats by the intraperitoneal administration of L-ornithine-HCl. Vessel permeability and the gene expression of the primary transporter of L-ornithine, cationic amino acid transporter-1 (Cat-1) in the brain cortex, pancreas, liver and lung were determined. Ultrastructural changes were followed by transmission electron microscopy. The direct effect of L-ornithine was tested on primary rat brain endothelial cells and a triple co-culture model of the BBB. Viability and barrier integrity, including permeability and TEER, nitrogen monoxide (NO) and reactive oxygen species (ROS) production and NF-κB translocation were measured. Fluorescent staining for claudin-5, occludin, ZO-1, ß-catenin, cell adhesion molecules Icam-1 and Vcam-1 and mitochondria was performed. Cell surface charge was measured by laser Doppler velocimetry. RESULTS: In the L-ornithine-induced AP model vessel permeability for fluorescein and Cat-1 expression levels were elevated in the brain cortex and pancreas. On the ultrastructural level surface glycocalyx and mitochondrial damage, tight junction and basal membrane alterations, and glial edema were observed. L-ornithine decreased cell impedance and elevated the BBB model permeability in vitro. Discontinuity in the surface glycocalyx labeling and immunostaining of junctional proteins, cytoplasmic redistribution of ZO-1 and ß-catenin, and elevation of Vcam-1 expression were measured. ROS production was increased and mitochondrial network was damaged without NF-κB, NO production or mitochondrial membrane potential alterations. Similar ultrastructural changes were seen in L-ornithine treated brain endothelial cells as in vivo. The basal negative zeta potential of brain endothelial cells became more positive after L-ornithine treatment. CONCLUSION: We demonstrated BBB damage in the L-ornithine-induced rat AP model suggesting a general, AP model independent effect. L-ornithine induced oxidative stress, decreased barrier integrity and altered BBB morphology in a culture BBB model. These data suggest a direct effect of the cationic L-ornithine on brain endothelium. Endothelial surface glycocalyx injury was revealed both in vivo and in vitro, as an additional novel component of the BBB-related pathological changes in AP.

Associations between the von Willebrand Factor-ADAMTS13 Axis, Complement Activation, and COVID-19 Severity and Mortality.

BACKGROUND: Endothelial and complement activation were both associated with immunothrombosis, a key determinant of COVID-19 severity, but their interrelation has not yet been investigated. OBJECTIVES: We aimed to determine von Willebrand factor (VWF) antigen (VWF:Ag) concentration, VWF collagen binding activity (VWF:CBA), a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity (ADAMTS13:Ac), and their ratios in hospitalized COVID-19 patients, and to investigate how these parameters and their constellation with complement activation relate to disease severity and in-hospital mortality in COVID-19. METHODS: Samples of 102 hospitalized patients with polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 positivity were included in our observational cohort study. Patients were stratified according to the peak severity of COVID-19 disease in agreement with the World Health Organization ordinal scale. Twenty-six convalescent plasma donors with previous COVID-19 disease formed the control group. VWF:Ag concentration and VWF:CBA were determined by enzyme-linked immunosorbent assay (ELISA); ADAMTS13:Ac was determined by fluorescence resonance energy transfer. Complement C3 and C3a were measured by turbidimetry and ELISA, respectively. Clinical covariates and markers of inflammation were extracted from hospital records. RESULTS: VWF:Ag and VWF:CBA were elevated in all groups of hospitalized COVID-19 patients and increased in parallel with disease severity. ADAMTS13:Ac was decreased in patients with severe COVID-19, with the lowest values in nonsurvivors. High (> 300%) VWF:Ag concentrations or decreased (< 67%) ADAMTS13:Ac were associated with higher risk of severe COVID-19 disease or in-hospital mortality. The concomitant presence of decreased ADAMTS13:Ac and increased C3a/C3 ratio-indicating complement overactivation and consumption-was a strong independent predictor of in-hospital mortality. CONCLUSION: Our results suggest that an interaction between the VWF-ADAMTS13 axis and complement overactivation and consumption plays an important role in the pathogenesis of COVID-19.

Complement Overactivation and Consumption Predicts In-Hospital Mortality in SARS-CoV-2 Infection.

Objectives: Uncontrolled thromboinflammation plays an important role in the pathogenesis of coronavirus disease (COVID-19) caused by SARS-CoV-2 virus. Complement was implicated as key contributor to this process, therefore we hypothesized that markers of the complement profile, indicative for the activation state of the system, may be related to the severity and mortality of COVID-19. Methods: In this prospective cohort study samples of 102 hospitalized and 26 outpatients with PCR-confirmed COVID-19 were analyzed. Primary outcome was in-hospital, COVID-19 related mortality, and secondary outcome was COVID-19 severity as assessed by the WHO ordinal scale. Complement activity of alternative and classical pathways, its factors, regulators, and activation products were measured by hemolytic titration, turbidimetry, or enzyme-immunoassays. Clinical covariates and markers of inflammation were extracted from hospital records. Results: Increased complement activation was characteristic for hospitalized COVID-19 patients. Complement activation was significantly associated with markers of inflammation, such as interleukin-6, C-reactive protein, and ferritin. Twenty-five patients died during hospital stay due to COVID-19 related illness. Patients with uncontrolled complement activation leading to consumption of C3 and decrease of complement activity were more likely to die, than those who had complement activation without consumption. Cox models identified anaphylatoxin C3a, and C3 overactivation and consumption (ratio of C3a/C3) as predictors of in-hospital mortality [HR of 3.63 (1.55-8.45, 95% CI) and 6.1 (2.1-17.8), respectively]. Conclusion: Increased complement activation is associated with advanced disease severity of COVID-19. Patients with SARS-CoV-2 infection are more likely to die when the disease is accompanied by overactivation and consumption of C3. These results may provide observational evidence and further support to studies on complement inhibitory drugs for the treatment of COVID-19.