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Severe cases of coronavirus disease 2019 (COVID-19) are regularly complicated by respiratory failure. Although it has been suggested that elevated levels of blood neutrophils associate with worsening oxygenation in COVID-19, it is unknown whether neutrophils are drivers of the thrombo-inflammatory storm or simple bystanders. To better understand the potential role of neutrophils in COVID-19, we measured levels of the neutrophil activation marker S100A8/A9 (calprotectin) in hospitalized patients and determined its relationship to severity of illness and respiratory status. Patients with COVID-19 (n = 172) had markedly elevated levels of calprotectin in their blood. Calprotectin tracked with other acute phase reactants including C-reactive protein, ferritin, lactate dehydrogenase, and absolute neutrophil count, but was superior in identifying patients requiring mechanical ventilation. In longitudinal samples, calprotectin rose as oxygenation worsened. When tested on day 1 or 2 of hospitalization (n = 94 patients), calprotectin levels were significantly higher in patients who progressed to severe COVID-19 requiring mechanical ventilation (8039 ± 7031 ng/ml, n = 32) as compared to those who remained free of intubation (3365 ± 3146, P < 0.0001). In summary, serum calprotectin levels track closely with current and future COVID-19 severity, implicating neutrophils as potential perpetuators of inflammation and respiratory compromise in COVID-19.

Purpose of review Antiphospholipid syndrome (APS), more common than once believed, is an autoimmune disease best known for its high risk of incident and recurrent thrombotic events. The approach to treatment potentially differs from treatment of thrombosis in the general population, and this article endeavors to review the latest updates on this topic. Recent findings The epidemiology of APS is being increasingly elucidated by large population-based studies, with APS perhaps affecting as many as 1 in 2000 individuals. Vitamin K antagonists, aspirin, and heparinoids continue to have obvious roles in the management of patients with APS. There has recently been intensive study of direct oral anticoagulants in APS, with the most recent randomized studies raising concerns about their inferiority to vitamin K antagonists, at least in some subgroups. Other approaches to treating APS beyond anticoagulants and antiaggregants are also receiving increased attention in mechanistic and preclinical studies with an eye toward future roles in patients with refractory and/or microvascular disease. Pediatric APS is identified as an area in desperate need of additional prospective research. Summary Progress continues to be made in pursuit of improving the lives of individuals afflicted with APS. The most important future directions would seem to involve leveraging modern molecular technologies in order to improve subphenotyping of antiphospholipid antibody-positive individuals. This will help personalize risk profiles and ideally define the optimal approach to therapy based on future risk, rather than past morbid events.

Cutaneous inflammation can signal disease in juvenile dermatomyositis (DM) and childhood-onset systemic lupus erythematosus (cSLE), but we do not fully understand cellular mechanisms of cutaneous inflammation. In this study, we used imaging mass cytometry to characterize cutaneous inflammatory cell populations and cell-cell interactions in juvenile DM as compared to cSLE.We performed imaging mass cytometry analysis on skin biopsy samples from juvenile DM patients (n = 6) and cSLE patients (n = 4). Tissue slides were processed and incubated with metal-tagged antibodies for CD14, CD15, CD16, CD56, CD68, CD11c, HLA-DR, blood dendritic cell antigen 2, CD20, CD27, CD138, CD4, CD8, E-cadherin, CD31, pan-keratin, and type I collagen. Stained tissue was ablated, and raw data were acquired using the Hyperion imaging system. We utilized the Phenograph unsupervised clustering algorithm to determine cell marker expression and permutation test by histoCAT to perform neighborhood analysis.We identified 14 cell populations in juvenile DM and cSLE skin, including CD14+ and CD68+ macrophages, myeloid and plasmacytoid dendritic cells (pDCs), CD4+ and CD8+ T cells, and B cells. Overall, cSLE skin had a higher inflammatory cell infiltrate, with increased CD14+ macrophages, pDCs, and CD8+ T cells and immune cell-immune cell interactions. Juvenile DM skin displayed a stronger innate immune signature, with a higher overall percentage of CD14+ macrophages and prominent endothelial cell-immune cell interaction.Our findings identify immune cell population differences, including CD14+ macrophages, pDCs, and CD8+ T cells, in juvenile DM skin compared to cSLE skin, and highlight a predominant innate immune signature and endothelial cell-immune cell interaction in juvenile DM, providing insight into candidate cell populations and interactions to better understand disease-specific pathophysiology.

The release of neutrophil extracellular traps (NETs) by hyperactive neutrophils is recognized to play an important role in the thromboinflammatory milieu inherent to severe presentations of COVID-19. At the same time, a variety of functional autoantibodies have been observed in individuals with severe COVID-19 where they likely contribute to immunopathology. Here, we aimed to determine the extent to which autoantibodies might target NETs in COVID-19 and, if detected, to elucidate their potential functions and clinical associations. We measured anti-NET antibodies in 328 individuals hospitalized with COVID-19 alongside 48 healthy controls. We found high anti-NET activity in the IgG and IgM fractions of 27% and 60% of patients, respectively. There was a strong correlation between anti-NET IgG and anti-NET IgM (r=0.4, p

Objectives Patients with coronavirus disease 19 ( COVID-19 ) are at high risk for fibrin-based occlusion of vascular beds of all sizes. Considering endothelial cell activation has regularly been described as part of the COVID-19 thrombo-inflammatory storm, we aimed to find upstream mediators of this activation. Methods Cultured endothelial cells were exposed to sera or plasma from 244 patients hospitalized with COVID-19 or plasma from 100 patients in the intensive care unit with sepsis. Cell adhesion molecules E-selectin, VCAM-1, and ICAM-1 were detected by in-cell ELISA. Soluble E-selectin was measured in serum. Results As compared with healthy controls, sera and plasma from patients with COVID-19, and to a lesser extent plasma from patients with sepsis, increased expression of E-selectin, VCAM-1, and ICAM-1 on cultured endothelial cells. We found modest correlations between serum neutrophil extracellular trap (NET) remnants and upregulation of cell adhesion molecules on endothelial cells. A stronger marker of the ability of COVID-19 serum to activate endothelial cells was the presence of circulating antiphospholipid antibodies, specifically anticardiolipin IgG and IgM and anti-phosphatidlyserine/prothrombin (anti-PS/PT) IgG and IgM. Depletion of total IgG from anticardiolipin-positive and anti-PS/PT-positive samples markedly restrained upregulation of E-selectin, VCAM-1, and ICAM-1. At the same time, supplementation of control serum with patient IgG was sufficient to trigger endothelial cell activation. Conclusions These data are the first to suggest that some patients with COVID-19 have potentially diverse antibodies that drive endothelial cell activation in COVID-19. The data also add important context regarding thrombo-inflammatory effects of autoantibodies in severe COVID-19. KEY MESSAGES What is already known about this subject?: Patients with COVID-19 are at high risk for fibrin-based occlusion of vascular beds of all sizes.Endothelial cell activation has regularly been described as part of the COVID-19 thrombo-inflammatory storm.What does this study add?: The presence of circulating antiphospholipid antibodies may be a predictor of the ability of a patient’s total antibody profile to activate endothelial cells.Purified COVID-19 IgG with high levels of anticardiolipin and anti-PS/PT activity trigger a pro-adhesive phenotype in endothelial cells.How might this impact on clinical practice or future developments?: Patients might be screened for antiphospholipid antibodies to evaluate their risk of having an antibody profile likely to activate endothelial cells.Patients with high antiphospholipid antibody titers might benefit from treatments used in traditional cases of severe APS such as therapeutic anticoagulation, corticosteroids, and plasmapheresis.

Background: Early studies of patients with COVID-19 have demonstrated markedly dysregulated coagulation and a high risk of morbid arterial and venous thrombotic events. While elevated levels of blood neutrophils and neutrophil extracellular traps (NETs) have been described in patients with COVID-19, their potential role in COVID-19-associated thrombosis remains unknown. Objectives: To elucidate the potential role of hyperactive neutrophils and NET release in COVID-19-associated thrombosis. Patients/Methods: This is a retrospective, case-control study of patients hospitalized with COVID-19 who developed thrombosis (n=11), as compared with gender- and age-matched COVID-19 patients without clinical thrombosis (n=33). In addition to capturing clinical data, we measured remnants of NETs (cell-free DNA, myeloperoxidase-DNA complexes, and citrullinated histone H3) and neutrophil-derived S100A8/A9 (calprotectin) in patient sera. Results: The majority of patients (9/11) were receiving at least prophylactic doses of heparinoids at the time thrombosis was diagnosed. As compared with controls, patients with COVID-19-associated thrombosis had significantly higher blood levels of markers of NETs (cell-free DNA, myeloperoxidase-DNA complexes, citrullinated histone H3) and neutrophil activation (calprotectin). The thrombosis group also had higher levels of D-dimer, CRP, ferritin, and platelets, but not troponin or neutrophils. Finally, there were strong associations between markers of hyperactive neutrophils (calprotectin and cell-free DNA) and D-dimer. Conclusion: Elevated levels of neutrophil activation and NET formation in patients hospitalized with COVID-19 are associated with higher risk of morbid thrombotic complications. These observations underscore the need for urgent investigation into the potential relationship between NETs and unrelenting thrombosis in COVID-19.

Patients with COVID-19 are at high risk for thrombotic arterial and venous occlusions. Lung histopathology often reveals fibrin-based blockages in the small blood vessels of patients who succumb to the disease. Antiphospholipid syndrome is an acquired and potentially life-threatening thrombophilia in which patients develop pathogenic autoantibodies targeting phospholipids and phospholipid-binding proteins (aPL antibodies). Case series have recently detected aPL antibodies in patients with COVID-19. Here, we measured eight types of aPL antibodies in serum samples from 172 patients hospitalized with COVID-19. These aPL antibodies included anticardiolipin IgG, IgM, and IgA; anti–β2 glycoprotein I IgG, IgM, and IgA; and anti-phosphatidylserine/prothrombin (aPS/PT) IgG and IgM. We detected aPS/PT IgG in 24% of serum samples, anticardiolipin IgM in 23% of samples, and aPS/PT IgM in 18% of samples. Antiphospholipid autoantibodies were present in 52% of serum samples using the manufacturer’s threshold and in 30% using a more stringent cutoff (≥40 ELISA-specific units). Higher titers of aPL antibodies were associated with neutrophil hyperactivity, including the release of neutrophil extracellular traps (NETs), higher platelet counts, more severe respiratory disease, and lower clinical estimated glomerular filtration rate. Similar to IgG from patients with antiphospholipid syndrome, IgG fractions isolated from patients with COVID-19 promoted NET release from neutrophils isolated from healthy individuals. Furthermore, injection of IgG purified from COVID-19 patient serum into mice accelerated venous thrombosis in two mouse models. These findings suggest that half of patients hospitalized with COVID-19 become at least transiently positive for aPL antibodies and that these autoantibodies are potentially pathogenic.

Objective The release of neutrophil extracellular traps (NETs) by hyperactive neutrophils has recently been recognized to play an important role in antiphospholipid syndrome (APS). This study was undertaken to evaluate autoantibodies targeting NETs in patients with primary APS, and to determine their potential functions and clinical associations. Methods We measured global anti-NET activity in 76 patients with primary APS, 23 patients with systemic lupus erythematosus without antiphospholipid antibodies (aPL), 11 patients with a history of unprovoked venous thrombosis without aPL, and 44 healthy controls. The ability of APS sera to degrade NETs was also assessed. Results We found markedly elevated levels of anti-NET IgG and IgM in patients with primary APS compared with healthy controls (for IgG, mean ± SD optical density 0.55 ± 0.34 versus 0.33 ± 0.17; for IgM, mean ± SD optical density 0.76 ± 0.51 versus 0.26 ± 0.23). This anti-NET activity did not correlate with levels of traditional aPL and was relatively stable over time. Mechanistically, anti-NET antibodies (especially of the IgG isotype) impaired the ability of patient sera to degrade NETs (r = 0.4, P = 0.003). Levels of anti-NET IgM inversely correlated with complement C4 (r = 0.4, P = 0.019). Clinically, anti-NET antibodies associated with certain APS clinical manifestations, and in particular recurrent venous thrombosis (odds ratio 4.3; P = 0.002). Interestingly, anti-NET antibody levels also appeared to be associated with unprovoked venous thrombosis in the general population (for IgM, mean ± SD optical density 0.67 ± 0.34 versus 0.26 ± 0.23). Conclusion Our data indicate high levels of anti-NET antibodies in patients with primary APS, which may impair NET clearance and activate the complement cascade. These findings may ultimately enable more effective risk stratification.

In severe cases of coronavirus disease 2019 (COVID-19), viral pneumonia progresses to respiratory failure. Neutrophil extracellular traps (NETs) are extracellular webs of chromatin, microbicidal proteins, and oxidant enzymes that are released by neutrophils to contain infections. However, when not properly regulated, NETs have potential to propagate inflammation and microvascular thrombosis, including in the lungs of patients with acute respiratory distress syndrome. While elevated levels of blood neutrophils predict worse outcomes in COVID-19, the role of NETs has not been investigated. We now report that sera from patients with COVID-19 (n=50 patients, n=84 samples) have elevated levels of cell-free DNA, myeloperoxidase(MPO)-DNA, and citrullinated histone H3 (Cit-H3); the latter two are highly specific markers of NETs. Highlighting the potential clinical relevance of these findings, cell-free DNA strongly correlated with acute phase reactants including C-reactive protein, D-dimer, and lactate dehydrogenase, as well as absolute neutrophil count. MPO-DNA associated with both cell-free DNA and absolute neutrophil count, while Cit-H3 correlated with platelet levels. Importantly, both cell-free DNA and MPO-DNA were higher in hospitalized patients receiving mechanical ventilation as compared with hospitalized patients breathing room air. Finally, sera from individuals with COVID-19 triggered NET release from control neutrophils in vitro. In summary, these data reveal high levels of NETs in many patients with COVID-19, where they may contribute to cytokine release and respiratory failure. Future studies should investigate the predictive power of circulating NETs in longitudinal cohorts, and determine the extent to which NETs may be novel therapeutic targets in severe COVID-19.

In severe cases of coronavirus disease 2019 (COVID-19), viral pneumonia progresses to respiratory failure. Neutrophil extracellular traps (NETs) are extracellular webs of chromatin, microbicidal proteins, and oxidant enzymes that are released by neutrophils to contain infections. However, when not properly regulated, NETs have potential to propagate inflammation and microvascular thrombosis—including in the lungs of patients with acute respiratory distress syndrome. While elevated levels of blood neutrophils predict worse outcomes in COVID-19, the role of NETs has not been investigated. We now report that sera from patients with COVID-19 (n=50 patients, n=84 samples) have elevated levels of cell-free DNA, myeloperoxidase(MPO)-DNA, and citrullinated histone H3 (Cit-H3); the latter two are highly specific markers of NETs. Highlighting the potential clinical relevance of these findings, cell-free DNA strongly correlated with acute phase reactants including C-reactive protein, D-dimer, and lactate dehydrogenase, as well as absolute neutrophil count. MPO-DNA associated with both cell-free DNA and absolute neutrophil count, while Cit-H3 correlated with platelet levels. Importantly, both cell-free DNA and MPO-DNA were higher in hospitalized patients receiving mechanical ventilation as compared with hospitalized patients breathing room air. Finally, sera from individuals with COVID-19 triggered NET release from control neutrophils in vitro. In summary, these data reveal high levels of NETs in many patients with COVID-19, where they may contribute to cytokine release and respiratory failure. Future studies should investigate the predictive power of circulating NETs in longitudinal cohorts, and determine the extent to which NETs may be novel therapeutic targets in severe COVID-19.

Antiphospholipid syndrome (APS), more common than once believed, is an autoimmune disease best known for its high risk of incident and recurrent thrombotic events. The approach to treatment potentially differs from treatment of thrombosis in the general population, and this article endeavors to review the latest updates on this topic.The epidemiology of APS is being increasingly elucidated by large population-based studies, with APS perhaps affecting as many as 1 in 2000 individuals. Vitamin K antagonists, aspirin, and heparinoids continue to have obvious roles in the management of patients with APS. There has recently been intensive study of direct oral anticoagulants in APS, with the most recent randomized studies raising concerns about their inferiority to vitamin K antagonists, at least in some subgroups. Other approaches to treating APS beyond anticoagulants and antiaggregants are also receiving increased attention in mechanistic and preclinical studies with an eye toward future roles in patients with refractory and/or microvascular disease. Pediatric APS is identified as an area in desperate need of additional prospective research.Progress continues to be made in pursuit of improving the lives of individuals afflicted with APS. The most important future directions would seem to involve leveraging modern molecular technologies in order to improve subphenotyping of antiphospholipid antibody-positive individuals. This will help personalize risk profiles and ideally define the optimal approach to therapy based on future risk, rather than past morbid events.

Despite its potential to cause significant morbidity in children, pediatric antiphospholipid syndrome (APS) is an understudied condition. In this review, we will cover what is known about pediatric APS epidemiology and how the clinician might approach the diagnosis of pediatric APS. We will highlight similarities and differences with the adult disease, both for primary APS and in the context of lupus. Clinical manifestations beyond thrombosis, especially neurologic and hematologic in nature, will be discussed. We will also consider what unique implications antiphospholipid antibody-positivity may have for children with lupus and for neonates born to mothers with APS. The approach to treatment will be covered, including the unique impact of APS medications on children as compared with adults. Finally, the importance of future mechanistic research is emphasized as physicians endeavor to provide the personalized care that children with APS clearly deserve.