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1.
American Journal of Respiratory and Critical Care Medicine ; 205(1), 2022.
Article in English | EMBASE | ID: covidwho-1927800

ABSTRACT

Rationale Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterised by an IL-6 driven cytokinemia, associated with a rapidly developing acute respiratory distress syndrome (ARDS). A blunted AAT response to IL-6 in SARS-CoV-2 has been associated with increased morbidity and mortality. One of the main functions of IL-6 is regulation of acute-phase proteins such as alpha-1 antitrypsin (AAT), a key lung anti-protease. We investigated the proteaseanti- protease balance in the circulation and pulmonary compartments in SARS-CoV-2 acute respiratory distress syndrome (ARDS). In addition, we investigated the effect of anti-IL-6 therapy on anti-protease defence. Methods Levels and activity of AAT and neutrophil elastase (NE) were measured in plasma (n=20), airway tissue (n=8) and tracheal secretions (n=13) of people with severe SARS-CoV-2 infection. AAT and IL-6 levels were also evaluated over time in people with moderate SARS-CoV-2 infection who received standard of care +/- tocilizumab (n=30). Results AAT plasma levels doubled in severe SARS-CoV-2 ARDS patients (329g/L +/- 08 g/L as compared to baseline levels 174g/L +/- 011 g/L, P<0001). In lung parenchyma AAT levels were increased. Despite no increase in neutrophils, an increased percentage of neutrophils involved in NET formation were observed in the alveoli. A protease-anti-protease imbalance was detected in tracheal aspirates (TA). NE was active and AAT inactivated, reflecting cleavage and complexation with NE. The major airway anti-protease, secretory leukoprotease inhibitor (SLPI) was decreased in SARS-CoV-2-infected lungs and cleaved in TAs. Induction of AAT in SARS-CoV-2 infection occurred mainly through IL-6 signalling. Tocilizumab (IL-6 receptor antagonist) down-regulated AAT during infection (13g/L+/-0225 from 2469 g/L+/-0197, P<00001) while IL-6 remained elevated (NS=0.0998) as reflected by the IL-6/AAT ratio (P=0046). Conclusion This study shows that the AAT response to SARS-CoV-2 infection is compartmentalized with an appropriate increase in plasma and alveoli but an inadequate response in airways. This underlines a significant, but potentially treatable, protease-antiprotease imbalance in SARS-CoV-2 ARDS as well as highlighting IL-6's importance in SARS-CoV-2 pathology not only as a pro-inflammatory cytokine but as an anti-inflammatory regulator. In conclusion there is unopposed NE activity in the airways of people with SARS-CoV-2 ARDS which could be amenable to AAT therapy. Our data suggest caution in the use of IL-6 blocking therapies in SARS-CoV-2-infected individuals.

3.
Blood ; 138:1, 2021.
Article in English | EMBASE | ID: covidwho-1582278

ABSTRACT

Severe SARS-CoV-2 infection is complicated by dysregulation of the blood coagulation system and high rates of thrombosis, but virus-intrinsic mechanisms underlying this phenomenon are poorly understood. Increased intracellular calcium concentrations promote externalization of phosphatidylserine (PS), the membrane anionic phospholipid required for assembly and activation of the tenase and prothrombinase complexes to drive blood coagulation. TMEM16F is a ubiquitous phospholipid scramblase that mediates externalization of PS in a calcium-dependent manner. As SARS-CoV-2 ORF3a encodes a presumed cation channel with the ability to transport calcium, we hypothesized that ORF3a expression by infected host cells perturbs the cellular calcium rheostat, driving TMEM16F-dependent externalization of PS and enhancing procoagulant activity. Using a doxycycline-inducible system, synchronized expression of ORF3a in A549 pulmonary epithelial cells resulted in a time-dependent augmentation of tissue factor (TF) procoagulant activity exceeding 9-fold by 48 hours (p < 0.0001), with no change in TF cell-surface expression. This enhancement was dependent upon PS as determined by inhibition with the PS-binding protein lactadherin. Over 2-fold enhancement of prothrombinase activity (p < 0.0001) was also observed by 48 hours. ORF3a increased intracellular calcium levels by 18-fold at 48 hours (p < 0.0001), as determined by the intracellular calcium indicator fluo-4. After 16 hours of ORF3a expression, more than 60% of cells had externalized PS (p < 0.001) without increased cell death, as quantified by flow cytometry following annexin V binding. Immunofluorescence microscopy staining for ORF3a, annexin V, and nuclei confirmed ORF3a expression within internal and cell surface membranes and increased PS externalization. PS externalization was insensitive to the pan-caspase inhibitor z-VAD-FMK, and there was no evidence of apoptotic activation as determined by caspase-3 cleavage. By contrast, ORF3a expression did not augment coagulation in cells deficient in the calcium-dependent phospholipid scramblase TMEM16F. Similarly, ORF3a-enhanced TF procoagulant activity (p < 0.01) and prothrombinase activity (p<0.05) was completely abrogated using TMEM16 inhibitors, including the uricosuric agent benzbromarone that has been registered for human use in over 20 countries. Live SARS-CoV-2 infection of A549-ACE2 cells increased cell surface factor Xa generation at MOI 0.1 (p < 0.01) but not MOI 0.01 or following heat inactivation of the virus, and RNA sequencing confirmed ORF3a induction without increased F3 expression. RNA sequencing of human SARS-CoV-2 infected lung autopsy and control tissue (n= 53) confirmed these findings in vivo. Immunofluorescence staining for ORF3a and KRT8/18 and CD31 in SARS-CoV-2 infected human lung autopsy specimens demonstrated ORF3a expression in pulmonary epithelium and endothelial cells, highlighting the potential pathologic relevance of this mechanism. Here we demonstrate that expression of the SARS-CoV-2 accessory protein ORF3a increases the intracellular calcium concentration and TMEM16F-dependent PS scrambling to augment procoagulant activity of the tenase and prothrombinase complexes. Our studies of human cells and tissues infected with SARS-CoV-2 support the pathologic relevance of this mechanism. We highlight the therapeutic potential to target the ORF3a-TMEM16F axis as with benzbromarone to mitigate dysregulation of coagulation and thrombosis during severe SARS-CoV-2 infection. Disclosures: Schwartz: Miromatrix Inc: Membership on an entity's Board of Directors or advisory committees;Alnylam Inc.: Consultancy, Speakers Bureau. Schulman: CSL Behring: Consultancy, Research Funding.

4.
PubMed; 2021.
Preprint in English | PubMed | ID: ppcovidwho-296897

ABSTRACT

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has infected over 115 million people and caused over 2.5 million deaths worldwide. Yet, the molecular mechanisms underlying the clinical manifestations of COVID-19, as well as what distinguishes them from common seasonal influenza virus and other lung injury states such as Acute Respiratory Distress Syndrome (ARDS), remains poorly understood. To address these challenges, we combined transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues, matched with spatial protein and expression profiling (GeoMx) across 357 tissue sections. These results define both body-wide and tissue-specific (heart, liver, lung, kidney, and lymph nodes) damage wrought by the SARS-CoV-2 infection, evident as a function of varying viral load (high vs. low) during the course of infection and specific, transcriptional dysregulation in splicing isoforms, T cell receptor expression, and cellular expression states. In particular, cardiac and lung tissues revealed the largest degree of splicing isoform switching and cell expression state loss. Overall, these findings reveal a systemic disruption of cellular and transcriptional pathways from COVID-19 across all tissues, which can inform subsequent studies to combat the mortality of COVID-19, as well to better understand the molecular dynamics of lethal SARS-CoV-2 infection and other viruses.

5.
Research and Practice in Thrombosis and Haemostasis ; 5(SUPPL 2), 2021.
Article in English | EMBASE | ID: covidwho-1508970

ABSTRACT

Background : Continuous renal replacement therapy (CRRT) is an important modality in critical care that utilizes a closed blood circuit with a hollow fiber membrane hemofilter (Figure 1A). CRRT circuit failure is a costly complication with an unclear mechanism, prominently observed during the coronavirus disease 2019 (COVID-19) pandemic. Aims : In this retrospective case series, we study the basis of circuit failure, particularly in the setting of COVID-19, via histopathologic examination of hemofilters after use in CRRT circuits. Methods : Study patients were identified from critically ill patients, admitted to Weill Cornell Medical Center between April and June 2020, who had acute kidney injury requiring CRRT. Circuit failure was defined as the inability of the circuit to provide the prescribed continuous blood flow rate to the patient, necessitating a hemofilter change in advance of its 72-hour lifespan. Hemofilters from failed and functional circuits were formalin-fixed, sectioned and evaluated with histopathologic stains and immunohistochemistry (IHC). Chart review was completed in accordance with IRB-approved protocols. Results : Circuit failure occurred in 4 out of 6 study patients. Two patients with severe COVID-19 experienced circuit failure despite anticoagulation with argatroban or regional citrate (Table 1). All hemofilters displayed the accumulation of dense, platelet-rich aggregates at the arterial ends (Figure 1B-C), with a greater volume found in failed hemofilters. The hollow fiber membranes in hemofilters used for patients with severe COVID-19 displayed fibrinogen localized along the blood-facing lumens by IHC (Figure 1D). While hemofilters used for patients with COVID-19 exhibited such luminal fibrinogen staining regardless of circuit function, for patients without COVID-19 this was seen only in failed hemofilters. Conclusions : Fibrinogen adsorption on biocompatible hemofilter surfaces exposed to patient blood appears to be an initial event preceding CRRT circuit failure. With COVID-19, this adsorption occurs despite intensified anticoagulation. Further mechanistic study of hemofilter failure may facilitate direct therapeutic strategies to prevent this complication.

8.
Journal of Molecular Diagnostics ; 22(11):S38-S39, 2020.
Article in English | Web of Science | ID: covidwho-1070113
9.
Journal of the American Society of Nephrology ; 31:299, 2020.
Article in English | EMBASE | ID: covidwho-984783

ABSTRACT

Background: Patients infected with the novel coronavirus 2019 (COVID19) have a wide spectrum of symptoms ranging from asymptomatic carriers to multisystem organ failure and death. While 20-40% of critically ill patients develop acute kidney injury (AKI) during the course of the disease, only few are biopsied. The most severely affected patients, frequently with multiple co-morbidities, provide insight into renal disease at autopsy. Methods: 30 of 34 autopsies performed on COVID patients had kidneys available for routine evaluation. Clinicopathologic features are presented. Results: The 34 patients range in age from 30-100 years (mean 68.5), 24 males and 10 females, 13 Caucasian, 10 Hispanic, 5 African American, 3 Indian, 3 Asian. All cases were positive by RT PCR nasal swab for SARS-CoV-2 except 3 (presumed false negative). All had on average 3.4 comorbidities (range: 0-7, hypertension (HTN), diabetes (DM), obesity, COPD, asthma, stroke, dementia, cancer), frequently HTN (20) and DM (20), 11 required intubation. 18 patients had AKI (53%), 2 previously ESRD, and 5 required renal replacement therapy. Presenting Cr ranged from 0.7-9.6 mg/dl (mean 1.7). Renal pathology included diabetic nephropathy (14, 47%), with tubulointerstitial scarring ranging from <25% (60%), 25-50% (23%), to >50% (17%), and moderate (40%) or severe (40%) chronic vascular sclerosis. Other findings: obesity related glomerulopathy (2), atheroemboli (1), bilateral infarction (1), papillary necrosis (2), and thrombotic microangiopathy (2). No collapsing glomerulopathy was seen. Tubular autolysis prevents complete assessment of ATN. Platelet thrombi were seen by CD61 staining in 43% of cases to involve >20% of glomeruli and peritubular capillaries. C5b-9 staining was strong, 2-3+ arteriolar in 67% and glomeruli in 20%, suggesting localized complement activation. By electron microscopy, viral particles were identified within cells of glomeruli and tubulo-interstitium. Conclusions: Pathology in autopsy kidneys from 30 patients with COVID display pre-existing chronic disease correlating with co-morbidities, presenting with AKI or ESRD (59%). Despite varied tissue autolysis and the absence of significant proteinuria, the majority of AKI is presumed to be acute tubular injury due to ischemia and other causes. The viral particles in the renal glomerular and tubular cells may play a role in renal cytopathic injury.

10.
PubMed; 2020.
Preprint in English | PubMed | ID: ppcovidwho-2670

ABSTRACT

Heart injury has been reported in up to 20% of COVID-19 patients, yet the cause of myocardial histopathology remains unknown. In order to study the cause of myocardial pathology in COVID-19 patients, we used a hamster model to determine whether following infection SARS-CoV-2, the causative agent of COVID-19, can be detected in heart tissues. Here, we clearly demonstrate that viral RNA and nucleocapsid protein is present in cardiomyocytes in the hearts of infected hamsters. Interestingly, functional cardiomyocyte associated gene expression was decreased in infected hamster hearts, corresponding to an increase in reactive oxygen species (ROS). This data using an animal model was further validated using autopsy heart samples of COVID-19 patients. Moreover, we show that both human pluripotent stem cell-derived cardiomyocytes (hPSC-derived CMs) and adult cardiomyocytes (CMs) can be infected by SARS-CoV-2 and that CCL2 is secreted upon SARS-CoV-2 infection, leading to monocyte recruitment. Increased CCL2 expression and macrophage infiltration was also observed in the hearts of infected hamsters. Using single cell RNA-seq, we also show that macrophages are able to decrease SARS-CoV-2 infection of CMs. Overall, our study provides direct evidence that SARS-CoV-2 infects CMs in vivo and proposes a mechanism of immune-cell infiltration and pathology in heart tissue of COVID-19 patients.

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