Early IFNß secretion determines variable downstream IL-12p70 responses upon TLR4 activation.

The interleukin-12 (IL-12) family comprises the only heterodimeric cytokines mediating diverse functional effects. We previously reported a striking bimodal IL-12p70 response to lipopolysaccharide (LPS) stimulation in healthy donors. Herein, we demonstrate that interferon ß (IFNß) is a major upstream determinant of IL-12p70 production, which is also associated with numbers and activation of circulating monocytes. Integrative modeling of proteomic, genetic, epigenomic, and cellular data confirms IFNß as key for LPS-induced IL-12p70 and allowed us to compare the relative effects of each of these parameters on variable cytokine responses. Clinical relevance of our findings is supported by reduced IFNß-IL-12p70 responses in patients hospitalized with acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or chronically infected with hepatitis C (HCV). Importantly, these responses are resolved after viral clearance. Our systems immunology approach defines a better understanding of IL-12p70 and IFNß in healthy and infected persons, providing insights into how common genetic and epigenetic variation may impact immune responses to bacterial infection.

Neutrophils in COVID-19: Not Innocent Bystanders.

Unusually for a viral infection, the immunological phenotype of severe COVID-19 is characterised by a depleted lymphocyte and elevated neutrophil count, with the neutrophil-to-lymphocyte ratio correlating with disease severity. Neutrophils are the most abundant immune cell in the bloodstream and comprise different subpopulations with pleiotropic actions that are vital for host immunity. Unique neutrophil subpopulations vary in their capacity to mount antimicrobial responses, including NETosis (the generation of neutrophil extracellular traps), degranulation and de novo production of cytokines and chemokines. These processes play a role in antiviral immunity, but may also contribute to the local and systemic tissue damage seen in acute SARS-CoV-2 infection. Neutrophils also contribute to complications of COVID-19 such as thrombosis, acute respiratory distress syndrome and multisystem inflammatory disease in children. In this Progress review, we discuss the anti-viral and pathological roles of neutrophils in SARS-CoV-2 infection, and potential therapeutic strategies for COVID-19 that target neutrophil-mediated inflammatory responses.

Study protocol for the St James's Hospital, Tallaght University Hospital, Trinity College Dublin Allied Researchers' (STTAR) Bioresource for COVID-19.

Background: The current coronavirus disease 2019 (COVID-19) pandemic began in Ireland with the first confirmed positive case in March 2020. In the early stages of the pandemic clinicians and researchers in two affiliated Dublin hospitals identified the need for a COVID-19 biobanking initiative to support and enhance research into the disease. Through large scale analysis of clinical, regional, and genetic characteristics of COVID-19 patients, biobanks have helped identify, and so protect, at risk patient groups The STTAR Bioresource has been created to collect and store data and linked biological samples from patients with SARS-CoV-2 infection and healthy and disease controls. Aim: The primary objective of this study is to build a biobank, to understand the clinical characteristics and natural history of COVID-19 infection with the long-term goal of research into improved disease understanding, diagnostic tests and treatments. Methods: This is a prospective dual-site cohort study across two tertiary acute university teaching hospitals. Patients are recruited from inpatient wards or outpatient clinics. Patients with confirmed COVID-19 infection as well as healthy and specific disease control groups are recruited.  Biological samples are collected and a case report form detailing demographic and medical background is entered into the bespoke secure online Dendrite database. Impact: The results of this study will be used to inform national and international strategy on health service provision and disease management related to COVID-19. In common with other biobanks, study end points  evolve over time as new research questions emerge. They currently include patient survival, occurrence of severe complications of the disease or its therapy, occurrence of persistent symptoms following recovery from the acute illness and vaccine responses.

Persistent endotheliopathy in the pathogenesis of long COVID syndrome.

BACKGROUND: Persistent symptoms including breathlessness, fatigue, and decreased exercise tolerance have been reported in patients after acute SARS-CoV-2 infection. The biological mechanisms underlying this "long COVID" syndrome remain unknown. However, autopsy studies have highlighted the key roles played by pulmonary endotheliopathy and microvascular immunothrombosis in acute COVID-19. OBJECTIVES: To assess whether endothelial cell activation may be sustained in convalescent COVID-19 patients and contribute to long COVID pathogenesis. PATIENTS AND METHODS: Fifty patients were reviewed at a median of 68 days following SARS-CoV-2 infection. In addition to clinical workup, acute phase markers, endothelial cell (EC) activation and NETosis parameters and thrombin generation were assessed. RESULTS: Thrombin generation assays revealed significantly shorter lag times (p < .0001, 95% CI -2.57 to -1.02 min), increased endogenous thrombin potential (p = .04, 95% CI 15-416 nM/min), and peak thrombin (p < .0001, 95% CI 39-93 nM) in convalescent COVID-19 patients. These prothrombotic changes were independent of ongoing acute phase response or active NETosis. Importantly, EC biomarkers including von Willebrand factor antigen (VWF:Ag), VWF propeptide (VWFpp), and factor VIII were significantly elevated in convalescent COVID-19 compared with controls (p = .004, 95% CI 0.09-0.57 IU/ml; p = .009, 95% CI 0.06-0.5 IU/ml; p = .04, 95% CI 0.03-0.44 IU/ml, respectively). In addition, plasma soluble thrombomodulin levels were significantly elevated in convalescent COVID-19 (p = .02, 95% CI 0.01-2.7 ng/ml). Sustained endotheliopathy was more frequent in older, comorbid patients, and those requiring hospitalization. Finally, both plasma VWF:Ag and VWFpp levels correlated inversely with 6-min walk tests. CONCLUSIONS: Collectively, our findings demonstrate that sustained endotheliopathy is common in convalescent COVID-19 and raise the intriguing possibility that this may contribute to long COVID pathogenesis.

Investigating the Relationship between Vitamin D and Persistent Symptoms Following SARS-CoV-2 Infection.

The emergence of persistent symptoms following SARS-CoV-2 infection, known as long COVID, is providing a new challenge to healthcare systems. The cardinal features are fatigue and reduced exercise tolerance. Vitamin D is known to have pleotropic effects far beyond bone health and is associated with immune modulation and autoimmunity. We hypothesize that vitamin D levels are associated with persistent symptoms following COVID-19. Herein, we investigate the relationship between vitamin D and fatigue and reduced exercise tolerance, assessed by the Chalder Fatigue Score, six-minute walk test and modified Borg scale. Multivariable linear and logistic regression models were used to evaluate the relationships. A total of 149 patients were recruited at a median of 79 days after COVID-19 illness. The median vitamin D level was 62 nmol/L, with n = 36 (24%) having levels 30-49 nmol/L and n = 14 (9%) with levels <30 nmol/L. Fatigue was common, with n = 86 (58%) meeting the case definition. The median Borg score was 3, while the median distance covered for the walk test was 450 m. No relationship between vitamin D and the measures of ongoing ill-health assessed in the study was found following multivariable regression analysis. These results suggest that persistent fatigue and reduced exercise tolerance following COVID-19 are independent of vitamin D.

Persistent Poor Health after COVID-19 Is Not Associated with Respiratory Complications or Initial Disease Severity.

Rationale: Much is known about the acute infective process of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of the coronavirus disease (COVID-19) pandemic. The marked inflammatory response and coagulopathic state in acute SARS-CoV-2 infection may promote pulmonary fibrosis. However, little is known about the incidence and seriousness of post-COVID-19 pulmonary pathology. Objectives: To describe the respiratory recovery and self-reported health after infection at the time of outpatient attendance. Methods: Infection severity was graded into three groups: 1) not requiring admission, 2) requiring hospital admission, and 3) requiring intensive care unit care. Participants underwent chest radiography and a 6-minute walk test (6MWT). Fatigue and subjective return to health were assessed, and concentrations of CRP (C-reactive protein), IL-6 (interleukin-6), sCD25 (soluble CD25), and D-dimer were measured. The associations between initial illness and abnormal chest X-ray findings, 6MWT distance, and perception of maximal exertion were investigated. Results: A total of 487 patients were offered an outpatient appointment, of whom 153 (31%) attended for assessment at a median of 75 days after diagnosis. A total of 74 (48%) had required hospital admission during acute infection. Persistently abnormal chest X-ray findings were seen in 4%. The median 6MWT distance covered was 460 m. A reduced distance covered was associated with frailty and length of inpatient stay. A total of 95 (62%) patients believed that they had not returned to full health, whereas 47% met the case definition for fatigue. Ongoing ill health and fatigue were associated with an increased perception of exertion. None of the measures of persistent respiratory disease were associated with initial disease severity. Conclusions: This study highlights the rates of objective respiratory disease and subjective respiratory symptoms after COVID-19 and the complex multifactorial nature of post-COVID-19 ill health.

ADAMTS13 regulation of VWF multimer distribution in severe COVID-19.

BACKGROUND: Consistent with fulminant endothelial cell activation, elevated plasma von Willebrand factor (VWF) antigen levels have been reported in patients with COVID-19. The multimeric size and function of VWF are normally regulated through A Disintegrin And Metalloprotease with ThrombSpondin Motif type 1 motif, member 13 (ADAMTS-13)--mediated proteolysis. OBJECTIVES: This study investigated the hypothesis that ADAMTS-13 regulation of VWF multimer distribution may be impaired in severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection contributing to the observed microvascular thrombosis. PATIENTS AND METHODS: Patients with COVID-19 (n = 23) were recruited from the Beaumont Hospital Intensive Care Unit (ICU) in Dublin. Plasma VWF antigen, multimer distribution, ADAMTS-13 activity, and known inhibitors thereof were assessed. RESULTS: We observed markedly increased VWF collagen-binding activity in patients with severe COVID-19 compared to controls (median 509.1 versus 94.3 IU/dl). Conversely, plasma ADAMTS-13 activity was significantly reduced (median 68.2 IU/dl). In keeping with an increase in VWF:ADAMTS-13 ratio, abnormalities in VWF multimer distribution were common in patients with COVID-19, with reductions in high molecular weight VWF multimers. Terminal sialylation regulates VWF susceptibility to proteolysis by ADAMTS-13 and other proteases. We observed that both N- and O-linked sialylation were altered in severe COVID-19. Furthermore, plasma levels of the ADAMTS-13 inhibitors interleukin-6, thrombospondin-1, and platelet factor 4 were significantly elevated. CONCLUSIONS: These findings support the hypothesis that SARS-CoV-2 is associated with profound quantitative and qualitative increases in plasma VWF levels, and a multifactorial down-regulation in ADAMTS-13 function. Further studies will be required to determine whether therapeutic interventions to correct ADAMTS-13-VWF multimer dysfunction may be useful in COVID-microvascular thrombosis and angiopathy.

Prolonged elevation of D-dimer levels in convalescent COVID-19 patients is independent of the acute phase response.

BACKGROUND: Persistent fatigue, breathlessness, and reduced exercise tolerance have been reported following acute COVID-19 infection. Although immuno-thrombosis has been implicated in acute COVID-19 pathogenesis, the biological mechanisms underpinning long COVID remain unknown. We hypothesized that pulmonary microvascular immuno-thrombosis may be important in this context. METHODS: One hundred fifty COVID-19 patients were reviewed at St James's Hospital Dublin between May and September 2020 at a median of 80.5 (range 44-155) days after initial diagnosis. These included patients hospitalized during initial illness (n = 69) and others managed entirely as out-patients (n = 81). Clinical examination, chest x-ray, and 6-min walk tests were performed. In addition, a range of coagulation and inflammatory markers were assessed. RESULTS: Increased D-dimer levels (>500 ng/ml) were observed in 25.3% patients up to 4 months post-SARS-CoV-2 infection. On univariate analysis, elevated convalescent D-dimers were more common in COVID-19 patients who had required hospital admission and in patients aged more than 50 years (p < .001). Interestingly, we observed that 29% (n = 11) of patients with elevated convalescent D-dimers had been managed exclusively as out-patients during their illness. In contrast, other coagulation (prothrombin time, activated partial thromboplastin time, fibrinogen, platelet count) and inflammation (C-reactive protein, interleukin-6, and sCD25) markers had returned to normal in >90% of convalescent patients. CONCLUSIONS: Elucidating the biological mechanisms responsible for sustained D-dimer increases may be of relevance in long COVID pathogenesis and has implications for clinical management of these patients.

Making healthcare accessible for single adults with complex needs experiencing long-term homelessness: A realist evaluation protocol.

Background: Over the last several years, homelessness has increased in Ireland and across Europe. Rates have recently declined since the coronavirus disease 2019 (COVID-19) pandemic, but it is unclear whether emergency housing measures will remain in place permanently. Populations experiencing long-term homelessness face a higher burden of multi-morbidity at an earlier age than housed populations and have poorer health outcomes. However, this population also has more difficulty accessing appropriate health services. A realist review by the authors found that important health system contexts which impact access are resourcing, training, funding cycles, health system fragmentation, health system goals, how care is organised, culture, leadership and flexibility of care delivery. Using a realist evaluation approach, this research will explore and refine key system-level factors, highlighted in our realist review, in a local health care system. Aim: The aim of this study is to understand how funding procedures and health system performance management impact service settings, staff, providers and their ability to make services accessible to populations experiencing homelessness. Methods: A realist evaluation will be undertaken to explain how funding and health system performance management impact healthcare accessibility for populations experiencing homelessness. Data will be collected using qualitative and realist interview techniques and focus group methodology. Secondary data such as policy documents and budgets will utilised. The analysis will follow Pawson and Tilley's iterative phases starting with building an Initial programme theory, then data collection, data analysis, synthesis and finally building a refined programme theory. Conclusion: Building on a realist review conducted by the same research team, this study will further test and refine findings that explain how health system factors impact healthcare accessibility for populations experiencing homelessness. The study has the potential to inform policy makers, health planners and managers of contextual factors that can be modified to increase healthcare accessibility.

Von Willebrand factor propeptide in severe coronavirus disease 2019 (COVID-19): evidence of acute and sustained endothelial cell activation.

Endothelial cell (EC) activation plays a key role in the pathogenesis of pulmonary microvascular occlusion, which is a hallmark of severe coronavirus disease 2019 (COVID-19). Consistent with EC activation, increased plasma von Willebrand factor antigen (VWF:Ag) levels have been reported in COVID-19. Importantly however, studies in other microangiopathies have shown that plasma VWF propeptide (VWFpp) is a more sensitive and specific measure of acute EC activation. In the present study, we further investigated the nature of EC activation in severe COVID-19. Markedly increased plasma VWF:Ag [median (interquatile range, IQR) 608·8 (531-830)iu/dl] and pro-coagulant factor VIII (FVIII) levels [median (IQR) 261·9 (170-315) iu/dl] were seen in patients with severe severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Sequential testing showed that these elevated VWF-FVIII complex levels remained high for up to 3 weeks. Similarly, plasma VWFpp levels were also markedly elevated [median (IQR) 324·6 (267-524) iu/dl]. Interestingly however, the VWFpp/VWF:Ag ratio was reduced, demonstrating that decreased VWF clearance contributes to the elevated plasma VWF:Ag levels in severe COVID-19. Importantly, plasma VWFpp levels also correlated with clinical severity indices including the Sequential Organ Failure Assessment (SOFA) score, Sepsis-Induced Coagulopathy (SIC) score and the ratio of arterial oxygen partial pressure to fractional inspired oxygen (P/F ratio). Collectively, these findings support the hypothesis that sustained fulminant EC activation is occurring in severe COVID-19, and further suggest that VWFpp may have a role as a biomarker in this setting.

Persistent fatigue following SARS-CoV-2 infection is common and independent of severity of initial infection.

Fatigue is a common symptom in those presenting with symptomatic COVID-19 infection. However, it is unknown if COVID-19 results in persistent fatigue in those recovered from acute infection. We examined the prevalence of fatigue in individuals recovered from the acute phase of COVID-19 illness using the Chalder Fatigue Score (CFQ-11). We further examined potential predictors of fatigue following COVID-19 infection, evaluating indicators of COVID-19 severity, markers of peripheral immune activation and circulating pro-inflammatory cytokines. Of 128 participants (49.5 ± 15 years; 54% female), more than half reported persistent fatigue (67/128; 52.3%) at median of 10 weeks after initial COVID-19 symptoms. There was no association between COVID-19 severity (need for inpatient admission, supplemental oxygen or critical care) and fatigue following COVID-19. Additionally, there was no association between routine laboratory markers of inflammation and cell turnover (leukocyte, neutrophil or lymphocyte counts, neutrophil-to-lymphocyte ratio, lactate dehydrogenase, C-reactive protein) or pro-inflammatory molecules (IL-6 or sCD25) and fatigue post COVID-19. Female gender and those with a pre-existing diagnosis of depression/anxiety were over-represented in those with fatigue. Our findings demonstrate a significant burden of post-viral fatigue in individuals with previous SARS-CoV-2 infection after the acute phase of COVID-19 illness. This study highlights the importance of assessing those recovering from COVID-19 for symptoms of severe fatigue, irrespective of severity of initial illness, and may identify a group worthy of further study and early intervention.

COVID19 coagulopathy in Caucasian patients.

Although the pathophysiology underlying severe COVID19 remains poorly understood, accumulating data suggest that a lung-centric coagulopathy may play an important role. Elevated D-dimer levels which correlated inversely with overall survival were recently reported in Chinese cohort studies. Critically however, ethnicity has major effects on thrombotic risk, with a 3-4-fold lower risk in Chinese compared to Caucasians and a significantly higher risk in African-Americans. In this study, we investigated COVID19 coagulopathy in Caucasian patients. Our findings confirm that severe COVID19 infection is associated with a significant coagulopathy that correlates with disease severity. Importantly however, Caucasian COVID19 patients on low molecular weight heparin thromboprophylaxis rarely develop overt disseminated intravascular coagulation (DIC). In rare COVID19 cases where DIC does develop, it tends to be restricted to late-stage disease. Collectively, these data suggest that the diffuse bilateral pulmonary inflammation observed in COVID19 is associated with a novel pulmonary-specific vasculopathy termed pulmonary intravascular coagulopathy (PIC) as distinct to DIC. Given that thrombotic risk is significantly impacted by race, coupled with the accumulating evidence that coagulopathy is important in COVID19 pathogenesis, our findings raise the intriguing possibility that pulmonary vasculopathy may contribute to the unexplained differences that are beginning to emerge highlighting racial susceptibility to COVID19 mortality.