A survey of ficolin-3 activity in Systemic Lupus Erythematosus reveals a link to hematological disease manifestations and autoantibody profile.

The complement system plays a central role in the pathogenesis of Systemic Lupus Erythematosus (SLE), but most studies have focused on the classical pathway. Ficolin-3 is the main initiator of the lectin pathway of complement in humans, but its role in systemic autoimmune disease has not been conclusively determined. Here, we combined biochemical and genetic approaches to assess the contribution of ficolin-3 to SLE risk and disease manifestations. Ficolin-3 activity was measured by a functional assay in serum or plasma samples from Swedish SLE patients (n = 786) and controls matched for age and sex (n = 566). Genetic variants in an extended 300 kb genomic region spanning the FCN3 locus were analyzed for their association with ficolin-3 activity and SLE manifestations in a Swedish multicenter cohort (n = 985). Patients with ficolin-3 activity in the highest tertile showed a strong enrichment in an SLE cluster defined by anti-Sm/DNA/nucleosome antibodies (OR 3.0, p < 0.001) and had increased rates of hematological disease (OR 1.4, p = 0.078) and lymphopenia (OR = 1.6, p = 0.039). Genetic variants associated with low ficolin-3 activity mapped to an extended haplotype in high linkage disequilibrium upstream of the FCN3 gene. Patients carrying the lead genetic variant associated with low ficolin-3 activity had a lower frequency of hematological disease (OR 0.67, p = 0.018) and lymphopenia (OR 0.63, p = 0.031) and fewer autoantibodies (p = 0.0019). Loss-of-function variants in the FCN3 gene were not associated with SLE, but four (0.5 %) SLE patients developed acquired ficolin-3 deficiency where ficolin-3 activity in serum was depleted following diagnosis of SLE. Taken together, our results provide genetic and biochemical evidence that implicate the lectin pathway in hematological SLE manifestations. We also identify lectin pathway activation through ficolin-3 as a factor that contributes to the autoantibody response in SLE.

Prompt Thrombo-Inflammatory Response to Ischemia-Reperfusion Injury and Kidney Transplant Outcomes.

Introduction: In kidney transplantation (KT), the role of the intravascular innate immune system (IIIS) in response to ischemia-reperfusion injury (IRI) is not well-understood. Here, we studied parallel changes in the generation of key activation products of the proteolytic cascade systems of the IIIS following living donor (LD) and deceased donor (DD) transplantation and evaluated potential associations with clinical outcomes. Methods: In a cohort study, 63 patients undergoing LD (n = 26) and DD (n = 37) transplantation were prospectively included. Fifteen DD kidneys were preserved with hypothermic machine perfusion (HMP), and the remaining were cold stored. Activation products of the kallikrein-kinin, coagulation, and complement systems were measured in blood samples obtained systemically at baseline and locally from the transplant renal vein at 1, 10, and 30 minutes after reperfusion. Results: DD kidneys exhibited a prompt and interlinked activation of all 3 cascade systems of IIIS postreperfusion, indicating a robust and local thrombo-inflammatory response to IRI. In this initial response, the complement activation product sC5b-9 exhibited a robust correlation with other IIIS activation markers and displayed a strong association with short-term and mid-term (24-month) graft dysfunction. In contrast, LD kidneys did not exhibit this thrombo-inflammatory response. The use of HMP was associated with reduced thromboinflammation and preserved mid-term kidney function. Conclusion: Kidneys from DD are vulnerable to a prompt thrombo-inflammatory response to IRI, which adversely affects both short-term and long-term allograft function. Strategies aimed at minimizing graft immunogenicity prior to reperfusion are crucial to mitigate the intricate inflammatory response to IRI.

Regulation of the innate immune system by fragmented heparin-conjugated lipids on lipid bilayered membranes in vitro.

Surface modification with heparin is a powerful biomaterial coating strategy that protects against innate immunity activation since heparin is a part of the proteoglycan heparan sulfate on cell surfaces in the body. We studied the heparinization of cellular and material surfaces via lipid conjugation to a heparin-binding peptide. In the present study, we synthesized fragmented heparin (fHep)-conjugated phospholipids and studied their regulation of the innate immune system on a lipid bilayered surface using liposomes. Liposomes have versatile applications, such as drug-delivery systems, due to their ability to carry a wide range of molecules. Owing to their morphological similarity to cell membranes, they can also be used to mimic a simple cell-membrane to study protein-lipid interactions. We investigated the interaction of complement-regulators, factor H and C4b-binding protein (C4BP), as well as the coagulation inhibitor antithrombin (AT), with fHep-lipids on the liposomal surface. Herein, we studied the ability of fHep-lipids to recruit factor H, C4BP, and AT using a quartz crystal microbalance with dissipation monitoring. With dynamic light scattering, we demonstrated that liposomes could be modified with fHep-lipids and were stable up to 60 days at 4 °C. Using a capillary western blot-based method (Wes), we showed that fHep-liposomes could recruit factor H in a model system using purified proteins and assist in the degradation of the active complement protein C3b to iC3b. Furthermore, we found that fHep-liposomes could recruit factor H and AT from human plasma. Therefore, the use of fHep-lipids could be a potential coating for liposomes and cell surfaces to regulate the immune system on the lipid surface.

Citrullination of C1-inhibitor as a mechanism of impaired complement regulation in rheumatoid arthritis.

Background: Dysregulated complement activation, increased protein citrullination, and production of autoantibodies against citrullinated proteins are hallmarks of rheumatoid arthritis (RA). Citrullination is induced by immune cell-derived peptidyl-Arg deiminases (PADs), which are overactivated in the inflamed synovium. We characterized the effect of PAD2- and PAD4-induced citrullination on the ability of the plasma-derived serpin C1-inhibitor (C1-INH) to inhibit complement and contact system activation. Methods: Citrullination of the C1-INH was confirmed by ELISA and Western blotting using a biotinylated phenylglyoxal probe. C1-INH-mediated inhibition of complement activation was analyzed by C1-esterase activity assay. Downstream inhibition of complement was studied by C4b deposition on heat-aggregated IgGs by ELISA, using pooled normal human serum as a complement source. Inhibition of the contact system was investigated by chromogenic activity assays for factor XIIa, plasma kallikrein, and factor XIa. In addition, autoantibody reactivity to native and citrullinated C1-INH was measured by ELISA in 101 RA patient samples. Results: C1-INH was efficiently citrullinated by PAD2 and PAD4. Citrullinated C1-INH was not able to bind the serine protease C1s and inhibit its activity. Citrullination of the C1-INH abrogated its ability to dissociate the C1-complex and thus inhibit complement activation. Consequently, citrullinated C1-INH had a decreased capacity to inhibit C4b deposition via the classical and lectin pathways. The inhibitory effect of C1-INH on the contact system components factor XIIa, plasma kallikrein, and factor XIa was also strongly reduced by citrullination. In RA patient samples, autoantibody binding to PAD2- and PAD4-citrullinated C1-INH was detected. Significantly more binding was observed in anti-citrullinated protein antibody (ACPA)-positive than in ACPA-negative samples. Conclusion: Citrullination of the C1-INH by recombinant human PAD2 and PAD4 enzymes impaired its ability to inhibit the complement and contact systems in vitro. Citrullination seems to render C1-INH more immunogenic, and citrullinated C1-INH might thus be an additional target of the autoantibody response observed in RA patients.

Activation of the Complement and Coagulation Systems in the Small Airways in Asthma.

BACKGROUND: Several studies have shown the importance of the complement and coagulation systems in the pathogenesis of asthma. OBJECTIVES: We explored whether we could detect differentially abundant complement and coagulation proteins in the samples obtained from the small airway lining fluid by collection of exhaled particles in patients with asthma and whether these proteins are associated with small airway dysfunction and asthma control. METHOD: Exhaled particles were obtained from 20 subjects with asthma and 10 healthy controls (HC) with the PExA method and analysed with the SOMAscan proteomics platform. Lung function was assessed by nitrogen multiple breath washout test and spirometry. RESULTS: 53 proteins associated with the complement and coagulation systems were included in the analysis. Nine of those proteins were differentially abundant in subjects with asthma as compared to HC, and C3 was significantly higher in inadequately controlled asthma as compared to well-controlled asthma. Several proteins were associated with physiological tests assessing small airways. CONCLUSIONS: The study highlights the role of the local activation of the complement and coagulation systems in the small airway lining fluid in asthma and their association with both asthma control and small airway dysfunction. The findings highlight the potential of complement factors as biomarkers to identify different sub-groups among patients with asthma that could potentially benefit from a therapeutic approach targeting the complement system.

Complement and platelets: prothrombotic cell activation requires membrane attack complex-induced release of danger signals.

Complement activation in the diseases paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) results in cytolysis and fatal thrombotic events, which are largely refractory to anticoagulation and/or antiplatelet therapy. Anticomplement therapy, however, efficiently prevents thrombotic events in PNH and aHUS, but the underlying mechanisms remain unresolved. We show that complement-mediated hemolysis in whole blood induces platelet activation similarly to activation by adenosine 5'-diphosphate (ADP). Blockage of C3 or C5 abolished platelet activation. We found that human platelets failed to respond functionally to the anaphylatoxins C3a and C5a. Instead, complement activation did lead to prothrombotic cell activation in the whole blood when membrane attack complex (MAC)-mediated cytolysis occurred. Consequently, we demonstrate that ADP receptor antagonists efficiently inhibited platelet activation, although full complement activation, which causes hemolysis, occurred. By using an established model of mismatched erythrocyte transfusions in rats, we crossvalidated these findings in vivo using the complement inhibitor OmCI and cobra venom factor. Consumptive complement activation in this animal model only led to a thrombotic phenotype when MAC-mediated cytolysis occurred. In conclusion, complement activation only induces substantial prothrombotic cell activation if terminal pathway activation culminates in MAC-mediated release of intracellular ADP. These results explain why anticomplement therapy efficiently prevents thromboembolisms without interfering negatively with hemostasis.

Combined Heterozygous Genetic Variations in Complement C2 and C8B: An Explanation for Multidimensional Immune Imbalance?

The complement system plays a crucial role in host defense, homeostasis, and tissue regeneration and bridges the innate and the adaptive immune systems. Although the genetic variants in complement C2 (c.839_849+17del; p.(Met280Asnfs*5)) and C8B (c.1625C>T; p.(Thr542Ile)) are known individually, here, we report on a patient carrying their combination in a heterozygous form. The patient presented with a reduced general condition and suffers from a wide variety of autoimmune diseases. While no autoimmune disease-specific autoantibodies could be detected, genetic analysis revealed abnormalities in the two complement genes C2 and C8B. Therefore, we performed a comprehensive investigation of the innate immune system on a cellular and humoral level to define the functional consequences. We found slightly impaired functionality of neutrophils and monocytes regarding phagocytosis and reactive oxygen species generation and a diminished expression of the C5aR1. An extensive complement analysis revealed a declined activation potential for the alternative and classical pathway. Reconstitution with purified C2 and C8 into patient serum failed to normalize the dysfunction, whereas the addition of C3 improved the hemolytic activity. In clinical transfer, in vitro supplementation of the patient's plasma with FFP as a complement source could fully restore full complement functionality. This study describes for the first time a combined heterozygous genetic variation in complement C2 and C8B which, however, cannot fully explain the overall dysfunctions and calls for further complement deficiency research and corresponding therapies.

Everyday navigation between adaptation and resistance: How young people negotiate their well-being in relation to assigned migrant positions in school.

Concerning the so-called "refugee crisis" in 2015 and how it affected the position of young migrants in society, researchers have underscored the value of studies challenging one-sided images of migrant youth. This study examines how migrant positions are constituted, negotiated, and related to young people's well-being. The study was undertaken using an ethnographic approach combined with the theoretical concept of translocational positionality to acknowledge how positions are created through historical and political processes and, at the same time, are context-dependent over time and space and thus contain incongruities. Our findings show how the newly arrived youth used multiple ways to navigate the school's everyday life and ascribed migrant positions to achieve well-being as illustrated through the distancing, adapting, defense, and the contradictory positions. Based on our findings, we understand the negotiations that occur in forming migrant positions within the school as asymmetric. At the same time, the youths' diverse and often contradictory positionality showed in various ways the striving for increased agency and well-being.

Biomimetic polyelectrolyte coating of stem cells suppresses thrombotic activation and enhances its survival and function.

Mesenchymal stem cells (MSCs) therapy is a promising approach for treating inflammatory diseases due to their immunosuppressive and tissue repair characteristics. However, allogenic transplantation of MSCs induces thrombotic complications in some patients which limits its potential for clinical translation. To address this challenge, we have exploited the bioactivity of heparin, a well-known anticoagulant and immunosuppressive polysaccharide that is widely used in clinics. We have developed a smart layer-by-layer (LbL) coating strategy using gelatin and heparin polymers exploiting their overall positive and negative charges that enabled efficient complexation with the MSCs' glycocalyx. The stable coating of MSCs suppressed complement attack and mitigated thrombotic activation as demonstrated in human whole blood. Gratifyingly, the MSC coating retained its immunosuppressive properties and differentiation potential when exposed to inflammatory conditions and differentiation factors. We believe the simple coating procedure of MSCs will increase allogenic tolerance and circumvent the major challenge of MSCs transplantation.

Impact of surface coating and systemic anticoagulants on hemostasis and inflammation in a human whole blood model.

BACKGROUND: Surface compatibility with blood is critical both for scientific investigations on hemostasis and clinical applications. Regarding in vitro and ex vivo investigations, minimal alteration in physiological hemostasis is of particular importance to draw reliable conclusions on the human coagulation system. At the same time, artificial coagulation activation must be avoided, which is relevant for the patient, for example to prevent stent graft occlusion. The aim was to evaluate the advantages and disadvantages of antithrombotic and antifouling surface coatings in the context of their suitability for ex vivo incubation and the study of coagulation properties. METHODS: We investigated the impact of different protocols for surface coating of synthetic material and different anticoagulants on hemostasis and platelet activation in ex vivo human whole blood. Blood samples from healthy donors were incubated in coated microtubes on a rotating wheel at 37°C. Two protocols for surface coating were analyzed for hemostatic parameters and metabolic status, a heparin-based coating (CHC, Corline Heparin Conjugate) without further anticoagulation and a passivating coating (MPC, 2-methacryloyloxethyl phosphorylcholine) with added anticoagulants (enoxaparin, ENOX; or fondaparinux, FPX). Employing the MPC-based coating, the anticoagulants enoxaparin and fondaparinux were compared regarding their differential effects on plasmatic coagulation by thrombelastometry and on platelet activation by flowcytometry and platelet function assays. RESULTS: Using the CHC coating, significant coagulation cascade activation was observed, whereas parameters remained mostly unchanged with MPC-based protocols. Extended incubation caused significantly elevated levels of the soluble membrane attack complex. Neither ENOX nor FPX caused a relevant impairment of platelet function or activation capacity and thrombelastometric parameters remained unchanged with both protocols. For translational purposes, we additionally modeled endotoxemia with the MPC-based protocols by incubating with lipopolysaccharide plus/minus thrombin. While coagulation parameters remained unchanged, elevated Interleukin 8 and Matrix Metalloproteinase 9 demonstrated preserved immune cell responsiveness. CONCLUSIONS: The MPC-based protocols demonstrated better hemocompatibility compared to CHC, and ENOX and FPX proved useful for additional anticoagulation. Furthermore, this simple-to-use whole blood model may be useful for experimental analyses of the early coagulatory and immunological response without decalcification.

Surface modulation of extracellular vesicles with cell-penetrating peptide-conjugated lipids for improvement of intracellular delivery to endothelial cells.

Exosomes (diameter 30-200 nm) are a subtype of extracellular vesicles secreted by cells containing DNA, microRNA (miRNA), and proteins. Exosomes are expected to be valuable as a means of delivering drugs or functional miRNAs in treatment of diseases. However, the delivery of exosomes is not sufficiently effective, even though exosomes have intrinsic delivery functions. Cell-penetrating peptides (CPPs) are short peptide families that facilitate cellular intake of molecules and vesicles. We previously reported that the modification of cells, and liposomes with CPP-conjugated-lipids, CPPs conjugated with poly (ethylene glycol)-conjugated phospholipids (PEG-lipid), that induce adhesion by CPPs, can be useful for cell-based assays and harvesting liposomes. In this study, we aimed to modulate the exosome surface using Tat peptide (YGRKKRRQRRR)-PEG-lipids to improve intracellular delivery to endothelial cells. We isolated and characterized exosomes from the medium of HEK 293 T cell cultures. Tat conjugated PEG-lipids with different spacer molecular weights and lipid types were incorporated into exosomes using fluorescein isothiocyanate labeling to optimize the number of Tat-PEG-lipids immobilized on the exosome surface. The exosomes modified with Tat-PEG-lipids were incubated with human umbilical vein endothelial cells (HUVECs) to study the interaction. Tat conjugated with 5 kDa PEG and C16 lipids incorporated on the exosome surface were highly detected inside HUVECs by flow cytometry. Fluorescence was negligible in HUVECs for control groups. Thus, Tat-PEG-lipids can be modified on the exosome surface, improving the intracellular delivery of exosomes.

Therapeutic regulation of complement activation in extracorporeal circuits and intravascular treatments with special reference to the alternative pathway amplification loop.

A number of clinical treatment modalities involve contact between blood and biomaterials: these include extracorporeal circuits such as hemodialysis, cardiopulmonary bypass, plasmapheresis, and intravascular treatments. Common side effects arising from these treatments are caused by activation of the cascade systems of the blood. Many of these side effects are mediated via the complement system, including thromboinflammatory reactions and rejection of implants. Depending on the composition of the materials, complement activation is triggered via all the activation pathways but is by far mostly driven by the alternative pathway amplification loop. On biomaterial surfaces the alternative pathway amplification is totally unregulated and leads under optimal conditions to deposition of complement fragments, mostly C3b, on the surface leading to a total masking of the underlying surface. In this review, we discuss the mechanism of the complement activation, clinical consequences of the activation, and potential strategies for therapeutic regulation of the activation, using hemodialysis as demonstrator.

Impact of spontaneous liposome modification with phospholipid polymer-lipid conjugates on protein interactions.

Liposome surface coating has been studied to avoid the immunological responses caused by the complement system, and alternative materials to poly(ethylene glycol) (PEG) have been explored recently since the production of anti-PEG IgM antibodies has been found in humans. We previously reported a liposome coating with poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC))-conjugated lipids (PMPC-lipids) and demonstrated its protective effect on blood protein interactions. Here, we attempted to modify the liposome surface by exogenously adding PMPC-lipids, which were spontaneously incorporated into the outer membrane via hydrophobic interactions. The polymerization degree of the PMPC segment was regulated from 10 to 100. The incorporated ratio of PMPC-lipid increased with a decrease in the degree of PMPC polymerization. Due to surface modification with PMPC-lipids, increase in the length of the PMPC-chain increased the size of the liposomes. The modified liposomes were kept stable for 14 d in terms of their size, polydispersity, and surface properties, where approximately 70% of PMPC-lipids were incorporated into the liposome surface. We demonstrated that liposome surface modification with PMPC-lipids can inhibit protein adsorption when exposed to serum, regardless of the degree of polymerization of PMPC. In addition, the PMPC-lipid modified surface was not recognized by the anti-PEG IgM antibody, whereas PEG-lipid was recognized by the antibody. Thus, we successfully fabricated an inert liposome surface via spontaneous modification with PMPC-lipids, where only the outer bilayer surface was modified. This technique can be available for full loading of water-soluble active pharmaceutical ingredient inside the modified liposome.

Modulation of Neutrophil Activity by Soluble Complement Cleavage Products-An In-Depth Analysis.

The cellular and fluid phase-innate immune responses of many diseases predominantly involve activated neutrophil granulocytes and complement factors. However, a comparative systematic analysis of the early impact of key soluble complement cleavage products, including anaphylatoxins, on neutrophil granulocyte function is lacking. Neutrophil activity was monitored by flow cytometry regarding cellular (electro-)physiology, cellular activity, and changes in the surface expression of activation markers. The study revealed no major effects induced by C3a or C4a on neutrophil functions. By contrast, exposure to C5a or C5a des-Arg stimulated neutrophil activity as reflected in changes in membrane potential, intracellular pH, glucose uptake, and cellular size. Similarly, C5a and C5a des-Arg but no other monitored complement cleavage product enhanced phagocytosis and reactive oxygen species generation. C5a and C5a des-Arg also altered the neutrophil surface expression of several complement receptors and neutrophil activation markers, including C5aR1, CD62L, CD10, and CD11b, among others. In addition, a detailed characterization of the C5a-induced effects was performed with a time resolution of seconds. The multiparametric response of neutrophils was further analyzed by a principal component analysis, revealing CD11b, CD10, and CD16 to be key surrogates of the C5a-induced effects. Overall, we provide a comprehensive insight into the very early interactions of neutrophil granulocytes with activated complement split products and the resulting neutrophil activity. The results provide a basis for a better and, importantly, time-resolved and multiparametric understanding of neutrophil-related (patho-)physiologies.

Complement C3 inhibition in severe COVID-19 using compstatin AMY-101.

Complement C3 activation contributes to COVID-19 pathology, and C3 targeting has emerged as a promising therapeutic strategy. We provide interim data from ITHACA, the first randomized trial evaluating a C3 inhibitor, AMY-101, in severe COVID-19 (PaO2/FiO2 ≤ 300 mmHg). Patients received AMY-101 (n = 16) or placebo (n = 15) in addition to standard of care. AMY-101 was safe and well tolerated. Compared to placebo (8 of 15, 53.3%), a higher, albeit nonsignificant, proportion of AMY-101-treated patients (13 of 16, 81.3%) were free of supplemental oxygen at day 14. Three nonresponders and two placebo-treated patients succumbed to disease-related complications. AMY-101 significantly reduced CRP and ferritin and restrained thrombin and NET generation. Complete and sustained C3 inhibition was observed in all responders. Residual C3 activity in the three nonresponders suggested the presence of a convertase-independent C3 activation pathway overriding the drug's inhibitory activity. These findings support the design of larger trials exploring the potential of C3-based inhibition in COVID-19 or other complement-mediated diseases.

Complement C3 activation in the ICU: Disease and therapy as Bonnie and Clyde.

Patients in the intensive care unit (ICU) often straddle the divide between life and death. Understanding the complex underlying pathomechanisms relevant to such situations may help intensivists select broadly acting treatment options that can improve the outcome for these patients. As one of the most important defense mechanisms of the innate immune system, the complement system plays a crucial role in a diverse spectrum of diseases that can necessitate ICU admission. Among others, myocardial infarction, acute lung injury/acute respiratory distress syndrome (ARDS), organ failure, and sepsis are characterized by an inadequate complement response, which can potentially be addressed via promising intervention options. Often, ICU monitoring and existing treatment options rely on massive intervention strategies to maintain the function of vital organs, and these approaches can further contribute to an unbalanced complement response. Artificial surfaces of extracorporeal organ support devices, transfusion of blood products, and the application of anticoagulants can all trigger or amplify undesired complement activation. It is, therefore, worth pursuing the evaluation of complement inhibition strategies in the setting of ICU treatment. Recently, clinical studies in COVID-19-related ARDS have shown promising effects of central inhibition at the level of C3 and paved the way for prospective investigation of this approach. In this review, we highlight the fundamental and often neglected role of complement in the ICU, with a special focus on targeted complement inhibition. We will also consider complement substitution therapies to temporarily counteract a disease/treatment-related complement consumption.

Variation of Complement Protein Levels in Maternal Plasma and Umbilical Cord Blood during Normal Pregnancy: An Observational Study.

The complement system constitutes a crucial part of the innate immunity, mediating opsonization, lysis, inflammation, and elimination of potential pathogens. In general, there is an increased activity of the complement system during pregnancy, which is essential for maintaining the host's defense and fetal survival. Unbalanced or excessive activation of the complement system in the placenta is associated with pregnancy complications, such as miscarriage, preeclampsia, and premature birth. Nonetheless, the actual clinical value of monitoring the activation of the complement system during pregnancy remains to be investigated. Unfortunately, normal reference values specifically for pregnant women are missing, and for umbilical cord blood (UCB), data on complement protein levels are scarce. Herein, complement protein analyses (C1q, C3, C4, C3d levels, and C3d/C3 ratio) were performed in plasma samples from 100 healthy, non-medicated and non-smoking pregnant women, collected during different trimesters and at the time of delivery. In addition, UCB was collected at all deliveries. Maternal plasma C1q and C3d/C3 ratio showed the highest mean values during the first trimester, whereas C3, C4, and C3d had rising values until delivery. We observed low levels of C1q and C4 as well as increased C3d and C3d/C3 ratio, particularly during the first trimester, as a sign of complement activation in some women. However, the reference limits of complement analyses applied for the general population appeared appropriate for the majority of the samples. As expected, the mean complement concentrations in UCB were much lower than in maternal plasma, due to the immature complement system in neonates.

Angiopoietin-2 Inhibition of Thrombomodulin-Mediated Anticoagulation-A Novel Mechanism That May Contribute to Hypercoagulation in Critically Ill COVID-19 Patients.

Hypercoagulation and endothelial dysfunction play central roles in severe forms of COVID-19 infections, but the molecular mechanisms involved are unclear. Increased plasma levels of the inflammatory cytokine and TIE2 receptor antagonist Angiopoietin-2 were reported in severely ill COVID-19 patients. In vitro experiments suggest that Angiopoietin-2 bind and inhibits thrombomodulin. Thrombomodulin is expressed on the luminal surface of endothelial cells where it is an important member of the intrinsic anticoagulant pathway through activation of protein C. Using clinical data, mouse models, and in vitro assays, we tested if Angiopoietin-2 plays a causal role in COVID-19-associated hypercoagulation through direct inhibition of thrombin/thrombomodulin-mediated physiological anticoagulation. Angiopoietin-2 was measured in 61 patients at admission, and after 10 days in the 40 patients remaining in the ICU. We found that Angiopoietin-2 levels were increased in COVID-19 patients in correlation with disease severity, hypercoagulation, and mortality. In support of a direct effect of Angiopoietin-2 on coagulation, we found that injected Angiopoietin-2 in mice associated to thrombomodulin and resulted in a shortened tail bleeding time, decreased circulating levels of activated protein C, and increased plasma thrombin/antithrombin complexes. Conversely, bleeding time was increased in endothelial-specific Angiopoietin-2 knockout mice, while knockout of Tie2 had no effect on tail bleeding. Using in vitro assays, we found that Angiopoietin-2 inhibited thrombomodulin-mediated anticoagulation and protein C activation in human donor plasma. Our data suggest a novel in vivo mechanism for Angiopoietin-2 in COVID-19-associated hypercoagulation, implicating that Angiopoietin-2 inhibitors may be effective in the treatment of hypercoagulation in severe COVID-19 infection.

Impaired Antibody Response Is Associated with Histone-Release, Organ Dysfunction and Mortality in Critically Ill COVID-19 Patients.

PURPOSE: the pathophysiologic mechanisms explaining differences in clinical outcomes following COVID-19 are not completely described. This study aims to investigate antibody responses in critically ill patients with COVID-19 in relation to inflammation, organ failure and 30-day survival. METHODS: All patients with PCR-verified COVID-19 and gave consent, and who were admitted to a tertiary Intensive care unit (ICU) in Sweden during March-September 2020 were included. Demography, repeated blood samples and measures of organ function were collected. Analyses of anti-SARS-CoV-2 antibodies (IgM, IgA and IgG) in plasma were performed and correlated to patient outcome and biomarkers of inflammation and organ failure. RESULTS: A total of 115 patients (median age 62 years, 77% male) were included prospectively. All patients developed severe respiratory dysfunction, and 59% were treated with invasive ventilation. Thirty-day mortality was 22.6% for all included patients. Patients negative for any anti-SARS-CoV-2 antibody in plasma during ICU admission had higher 30-day mortality compared to patients positive for antibodies. Patients positive for IgM had more ICU-, ventilator-, renal replacement therapy- and vasoactive medication-free days. IgA antibody concentrations correlated negatively with both SAPS3 and maximal SOFA-score and IgM-levels correlated negatively with SAPS3. Patients with antibody levels below the detection limit had higher plasma levels of extracellular histones on day 1 and elevated levels of kidney and cardiac biomarkers, but showed no signs of increased inflammation, complement activation or cytokine release. After adjusting for age, positive IgM and IgG antibodies were still associated with increased 30-day survival, with odds ratio (OR) 7.1 (1.5-34.4) and 4.2 (1.1-15.7), respectively. CONCLUSION: In patients with severe COVID-19 requiring intensive care, a poor antibody response is associated with organ failure, systemic histone release and increased 30-day mortality.

Strong Association of Combined Genetic Deficiencies in the Classical Complement Pathway With Risk of Systemic Lupus Erythematosus and Primary Sjögren's Syndrome.

OBJECTIVE: Complete genetic deficiency of the complement component C2 is a strong risk factor for monogenic systemic lupus erythematosus (SLE), but whether heterozygous C2 deficiency adds to the risk of SLE or primary Sjögren's syndrome (SS) has not been studied systematically. This study was undertaken to investigate potential associations of heterozygous C2 deficiency and C4 copy number variation with clinical manifestations in patients with SLE and patients with primary SS. METHODS: The presence of the common 28-bp C2 deletion rs9332736 and C4 copy number variation was examined in Scandinavian patients who had received a diagnosis of SLE (n = 958) or primary SS (n = 911) and in 2,262 healthy controls through the use of DNA sequencing. The concentration of complement proteins in plasma and classical complement function were analyzed in a subgroup of SLE patients. RESULTS: Heterozygous C2 deficiency-when present in combination with a low C4A copy number-substantially increased the risk of SLE (odds ratio [OR] 10.2 [95% confidence interval (95% CI) 3.5-37.0]) and the risk of primary SS (OR 13.0 [95% CI 4.5-48.4]) when compared to individuals with 2 C4A copies and normal C2. For patients heterozygous for rs9332736 with 1 C4A copy, the median age at diagnosis was 7 years earlier in patients with SLE and 12 years earlier in patients with primary SS when compared to patients with normal C2. Reduced C2 levels in plasma (P = 2 × 10-9 ) and impaired function of the classical complement pathway (P = 0.03) were detected in SLE patients with heterozygous C2 deficiency. Finally, in a primary SS patient homozygous for C2 deficiency, we observed low levels of anti-Scl-70, which suggests a risk of developing systemic sclerosis or potential overlap between primary SS and other systemic autoimmune diseases. CONCLUSION: We demonstrate that a genetic pattern involving partial deficiencies of C2 and C4A in the classical complement pathway is a strong risk factor for SLE and for primary SS. Our results emphasize the central role of the complement system in the pathogenesis of both SLE and primary SS.