Molecular imaging of chemokine-like receptor 1 (CMKLR1) in experimental acute lung injury.

The lack of techniques for noninvasive imaging of inflammation has challenged precision medicine management of acute respiratory distress syndrome (ARDS). Here, we determined the potential of positron emission tomography (PET) of chemokine-like receptor-1 (CMKLR1) to monitor lung inflammation in a murine model of lipopolysaccharide-induced injury. Lung uptake of a CMKLR1-targeting radiotracer, [64Cu]NODAGA-CG34, was significantly increased in lipopolysaccharide-induced injury, correlated with the expression of multiple inflammatory markers, and reduced by dexamethasone treatment. Monocyte-derived macrophages, followed by interstitial macrophages and monocytes were the major CMKLR1-expressing leukocytes contributing to the increased tracer uptake throughout the first week of lipopolysaccharide-induced injury. The clinical relevance of CMKLR1 as a biomarker of lung inflammation in ARDS was confirmed using single-nuclei RNA-sequencing datasets which showed significant increases in CMKLR1 expression among transcriptionally distinct subsets of lung monocytes and macrophages in COVID-19 patients vs. controls. CMKLR1-targeted PET is a promising strategy to monitor the dynamics of lung inflammation and response to anti-inflammatory treatment in ARDS.

Inter-rater reliability and prognostic value of baseline Radiographic Assessment of Lung Edema (RALE) scores in observational cohort studies of inpatients with COVID-19.

OBJECTIVES: To reliably quantify the radiographic severity of COVID-19 pneumonia with the Radiographic Assessment of Lung Edema (RALE) score on clinical chest X-rays among inpatients and examine the prognostic value of baseline RALE scores on COVID-19 clinical outcomes. SETTING: Hospitalised patients with COVID-19 in dedicated wards and intensive care units from two different hospital systems. PARTICIPANTS: 425 patients with COVID-19 in a discovery data set and 415 patients in a validation data set. PRIMARY AND SECONDARY OUTCOMES: We measured inter-rater reliability for RALE score annotations by different reviewers and examined for associations of consensus RALE scores with the level of respiratory support, demographics, physiologic variables, applied therapies, plasma host-response biomarkers, SARS-CoV-2 RNA load and clinical outcomes. RESULTS: Inter-rater agreement for RALE scores improved from fair to excellent following reviewer training and feedback (intraclass correlation coefficient of 0.85 vs 0.93, respectively). In the discovery cohort, the required level of respiratory support at the time of CXR acquisition (supplemental oxygen or non-invasive ventilation (n=178); invasive-mechanical ventilation (n=234), extracorporeal membrane oxygenation (n=13)) was significantly associated with RALE scores (median (IQR): 20.0 (14.1-26.7), 26.0 (20.5-34.0) and 44.5 (34.5-48.0), respectively, p<0.0001). Among invasively ventilated patients, RALE scores were significantly associated with worse respiratory mechanics (plateau and driving pressure) and gas exchange metrics (PaO2/FiO2 and ventilatory ratio), as well as higher plasma levels of IL-6, soluble receptor of advanced glycation end-products and soluble tumour necrosis factor receptor 1 (p<0.05). RALE scores were independently associated with 90-day survival in a multivariate Cox proportional hazards model (adjusted HR 1.04 (1.02-1.07), p=0.002). We replicated the significant associations of RALE scores with baseline disease severity and mortality in the independent validation data set. CONCLUSIONS: With a reproducible method to measure radiographic severity in COVID-19, we found significant associations with clinical and physiologic severity, host inflammation and clinical outcomes. The incorporation of radiographic severity assessments in clinical decision-making may provide important guidance for prognostication and treatment allocation in COVID-19.

Disease-Specific Factors Associated with Readmissions or Mortality After Hospital Discharge in COVID-19 Patients: a Retrospective Cohort Study.

BACKGROUND: Understanding the implications of disease-specific factors beyond baseline patient characteristics for coronavirus disease 2019 (COVID-19) may allow for identification of indicators for safe hospital discharge. OBJECTIVE: Assess whether disease-specific factors are associated with adverse events post-discharge using a data-driven approach. DESIGN: Retrospective cohort study. SETTING: Fifteen medical centers within Kaiser Permanente Southern California. PARTICIPANTS: Adult patients (n=3508) discharged alive following hospitalization for COVID-19 between 05/01/2020 and 09/30/2020. INTERVENTIONS: None. MAIN MEASURES: Adverse events defined as all-cause readmission or mortality within 14 days of discharge. Least absolute shrinkage and selection operator (LASSO) was used for variable selection and logistic regression was performed to estimate odds ratio (OR) and 95% confidence interval (CI). KEY RESULTS: Four variables including age, Elixhauser index, treatment with remdesivir, and symptom duration at discharge were selected by LASSO. Treatment with remdesivir was inversely associated with adverse events (OR: 0.46 [95%CI: 0.36-0.61]), while symptom duration ≤ 10 days was associated with adverse events (OR: 2.27 [95%CI: 1.79-2.87]) in addition to age (OR: 1.02 [95%CI: 1.01-1.03]) and Elixhauser index (OR: 1.15 [95%CI: 1.11-1.20]). A significant interaction between remdesivir and symptom duration was further observed (p=0.01). The association of remdesivir was stronger among those with symptom duration ≤10 days vs >10 days at discharge (OR: 0.30 [95%CI: 0.19-0.47] vs 0.62 [95%CI: 0.44-0.87]), while the association of symptom duration ≤ 10 days at discharge was weaker among those treated with remdesivir vs those not treated (OR: 1.31 [95%CI: 0.79-2.17] vs 2.71 [95%CI 2.05-3.59]). CONCLUSIONS: Disease-specific factors including treatment with remdesivir, symptom duration, and their interplay may help guide clinical decision making at time of discharge.

Antibodies targeting conserved non-canonical antigens and endemic coronaviruses associate with favorable outcomes in severe COVID-19.

While there have been extensive analyses characterizing cellular and humoral responses across the severity spectrum in COVID-19, outcome predictors within severe COVID-19 remain less comprehensively elucidated. Furthermore, properties of antibodies (Abs) directed against viral antigens beyond spike and their associations with disease outcomes remain poorly defined. We perform deep molecular profiling of Abs directed against a wide range of antigenic specificities in severe COVID-19 patients. The profiles included canonical (spike [S], receptor-binding domain [RBD], and nucleocapsid [N]) and non-canonical (orf3a, orf8, nsp3, nsp13, and membrane [M]) antigenic specificities. Notably, multivariate Ab profiles directed against canonical or non-canonical antigens are equally discriminative of survival in severe COVID-19. Intriguingly, pre-pandemic healthy controls have cross-reactive Abs directed against nsp13, a protein conserved across coronaviruses. Consistent with these findings, a model built on Ab profiles for endemic coronavirus antigens also predicts COVID-19 outcome. Our results suggest the importance of studying Abs targeting non-canonical severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and endemic coronavirus antigens in COVID-19.

Stability of SARS-CoV-2-Encoded Proteins and Their Antibody Levels Correlate with Interleukin 6 in COVID-19 Patients.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has become a severe global public health crisis. Therefore, understanding the molecular details of SARS-CoV-2 will be critical for fighting the virus's spread and preventing future pandemics. In this study, we globally profiled the stability of SARS-CoV-2-encoded proteins, studied their degradation pathways, and determined their correlation with the antibody responses in patient plasma. We identified 18 proteins with unstable half-lives and 6 relatively stable proteins with longer half-lives. The labile SARS-CoV-2 proteins were degraded mainly by the ubiquitin-proteasome pathway. We also observed a significant correlation between antibody levels and protein half-lives, which indicated that a stable antigen of SARS-CoV-2 could be more effective for eliciting antibody responses. In addition, levels of antiviral antibodies targeting NSP10 were found to be negatively correlated with systemic levels of interleukin 6 (IL-6) in patients. These findings may facilitate the development of novel therapeutic or diagnostic approaches. IMPORTANCE SARS-CoV-2, the etiological cause of COVID-19, carries 29 genes in its genome. However, our knowledge of the viral proteins in biological and biochemical aspects is limited. In this study, we globally profiled the stability of the viral proteins in living lung epithelial cells. Importantly, the labile SARS-CoV-2-encoded proteins were mainly degraded through the ubiquitin-proteasome pathway. Stable proteins, including spike and nucleocapsid, of SARS-CoV-2 were more effective in eliciting antibody production. The levels of antiviral antibodies targeting NSP10 were negatively correlated with systemic levels of IL-6 in COVID-19 patients.

Plasma SARS-CoV-2 RNA levels as a biomarker of lower respiratory tract SARS-CoV-2 infection in critically ill patients with COVID-19.

Plasma SARS-CoV-2 viral RNA (vRNA) levels are predictive of COVID-19 outcomes in hospitalized patients, but whether plasma vRNA reflects lower respiratory tract (LRT) vRNA levels is unclear. We compared plasma and LRT vRNA levels in serially collected samples from mechanically ventilated patients with COVID-19. LRT and plasma vRNA levels were strongly correlated at first sampling (n=33, r=0.83, p<10-9) and then declined in parallel in available serial samples except in non-survivors who exhibited delayed vRNA clearance in LRT samples. Plasma vRNA measurement may offer a practical surrogate of LRT vRNA burden in critically ill patients, especially early after ICU admission.

Severe Acute Respiratory Syndrome Coronavirus 2 Viremia Is Associated With Coronavirus Disease 2019 Severity and Predicts Clinical Outcomes.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA (vRNA) is detected in the bloodstream of some patients with coronavirus disease 2019 (COVID-19), but it is not clear whether this RNAemia reflects viremia (ie, virus particles) and how it relates to host immune responses and outcomes. METHODS: SARS-CoV-2 vRNA was quantified in plasma samples from observational cohorts of 51 COVID-19 patients including 9 outpatients, 19 hospitalized (non-intensive care unit [ICU]), and 23 ICU patients. vRNA levels were compared with cross-sectional indices of COVID-19 severity and prospective clinical outcomes. We used multiple imaging methods to visualize virions in plasma. RESULTS: SARS-CoV-2 vRNA was detected in plasma of 100%, 52.6%, and 11.1% of ICU, non-ICU, and outpatients, respectively. Virions were detected in plasma pellets using electron tomography and immunostaining. Plasma vRNA levels were significantly higher in ICU > non-ICU > outpatients (P < .0001); for inpatients, plasma vRNA levels were strongly associated with higher World Health Organization (WHO) score at admission (P = .01), maximum WHO score (P = .002), and discharge disposition (P = .004). A plasma vRNA level >6000 copies/mL was strongly associated with mortality (hazard ratio, 10.7). Levels of vRNA were significantly associated with several inflammatory biomarkers (P < .01) but not with plasma neutralizing antibody titers (P = .8). CONCLUSIONS: Visualization of virus particles in plasma indicates that SARS-CoV-2 RNAemia is due, at least in part, to viremia. The levels of SARS-CoV-2 RNAemia correlate strongly with disease severity, patient outcome, and specific inflammatory biomarkers but not with neutralizing antibody titers.

People critically ill with COVID-19 exhibit peripheral immune profiles predictive of mortality and reflective of SARS-CoV-2 lung viral burden.

Despite extensive analyses, there remains an urgent need to delineate immune cell states that contribute to mortality in people critically ill with COVID-19. Here, we present high-dimensional profiling of blood and respiratory samples from people with severe COVID-19 to examine the association between cell-linked molecular features and mortality outcomes. Peripheral transcriptional profiles by single-cell RNA sequencing (RNA-seq)-based deconvolution of immune states are associated with COVID-19 mortality. Further, persistently high levels of an interferon signaling module in monocytes over time lead to subsequent concerted upregulation of inflammatory cytokines. SARS-CoV-2-infected myeloid cells in the lower respiratory tract upregulate CXCL10, leading to a higher risk of death. Our analysis suggests a pivotal role for viral-infected myeloid cells and protracted interferon signaling in severe COVID-19.

Interleukin-36 Cytokines in Infectious and Non-Infectious Lung Diseases

The IL-36 family of cytokines were identified in the early 2000’s as a new subfamily of the IL-1 cytokine family, and since then, the role of IL-36 cytokines during various inflammatory processes has been characterized. While most of the research has focused on the role of these cytokines in autoimmune skin diseases such as psoriasis and dermatitis, recent studies have also shown the importance of IL-36 cytokines in the lung inflammatory response during infectious and non-infectious diseases. In this review, we discuss the biology of IL-36 cytokines in terms of how they are produced and activated, as well as their effects on myeloid and lymphoid cells during inflammation. We also discuss the role of these cytokines during lung infectious diseases caused by bacteria and influenza virus, as well as other inflammatory conditions in the lungs such as allergic asthma, lung fibrosis, chronic obstructive pulmonary disease, cystic fibrosis and cancer. Finally, we discuss the current therapeutic advances that target the IL-36 pathway and the possibility to extend these tools to treat lung inflammatory diseases.

Neutrophils and lymphopenia, an unknown axis in severe COVID-19 disease.

The Coronavirus Disease 2019 (COVID-19) is caused by the betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus that can mediate asymptomatic or fatal infections characterized by pneumonia, acute respiratory distress syndrome (ARDS), and multi-organ failure. Several studies have highlighted the importance of B and T lymphocytes, given that neutralizing antibodies and T cell responses are required for an effective immunity. In addition, other reports have described myeloid cells such as macrophages and monocytes play a major role in the immunity against SARS-CoV-2 as well as dysregulated pro-inflammatory signature that characterizes severe COVID-19. During COVID-19, neutrophils have been defined as a heterogeneous group of cells, functionally linked to severe inflammation and thrombosis triggered by degranulation and NETosis, but also to suppressive phenotypes. The physiological role of suppressive neutrophils during COVID-19 and their implications in severe disease have been poorly studied and is not well understood. Here, we discuss the current evidence regarding the role of neutrophils with suppressive properties such as granulocytic myeloid-derived suppressor cells (G-MDSCs) and their possible role in suppressing CD4+ and CD8+ T lymphocytes expansion and giving rise to lymphopenia in severe COVID-19 infection.

COVID-19 versus Non-COVID-19 Acute Respiratory Distress Syndrome: Comparison of Demographics, Physiologic Parameters, Inflammatory Biomarkers, and Clinical Outcomes.

Rationale: There is an urgent need for improved understanding of the mechanisms and clinical characteristics of acute respiratory distress syndrome (ARDS) due to coronavirus disease (COVID-19).Objectives: To compare key demographic and physiologic parameters, biomarkers, and clinical outcomes of COVID-19 ARDS and ARDS secondary to direct lung injury from other etiologies of pneumonia.Methods: We enrolled 27 patients with COVID-19 ARDS in a prospective, observational cohort study and compared them with a historical, pre-COVID-19 cohort of patients with viral ARDS (n = 14), bacterial ARDS (n = 21), and ARDS due to culture-negative pneumonia (n = 30). We recorded clinical demographics; measured respiratory mechanical parameters; collected serial peripheral blood specimens for measurement of plasma interleukin (IL)-6, IL-8, and IL-10; and followed patients prospectively for patient-centered outcomes. We conducted between-group comparisons with nonparametric tests and analyzed time-to-event outcomes with Kaplan-Meier and Cox proportional hazards models.Results: Patients with COVID-19 ARDS had higher body mass index and were more likely to be Black, or residents of skilled nursing facilities, compared with those with non-COVID-19 ARDS (P < 0.05). Patients with COVID-19 had lower delivered minute ventilation compared with bacterial and culture-negative ARDS (post hoc P < 0.01) but not compared with viral ARDS. We found no differences in static compliance, hypoxemic indices, or carbon dioxide clearance between groups. Patients with COVID-19 had lower IL-6 levels compared with bacterial and culture-negative ARDS at early time points after intubation but no differences in IL-6 levels compared with viral ARDS. Patients with COVID-19 had longer duration of mechanical ventilation but similar 60-day mortality in both unadjusted and adjusted analyses.Conclusions: COVID-19 ARDS bears several similarities to viral ARDS but demonstrates lower minute ventilation and lower systemic levels of IL-6 compared with bacterial and culture-negative ARDS. COVID-19 ARDS was associated with longer dependence on mechanical ventilation compared with non-COVID-19 ARDS. Such detectable differences of COVID-19 do not merit deviation from evidence-based management of ARDS but suggest priorities for clinical research to better characterize and treat this new clinical entity.

Characteristics of patients discharged and readmitted after COVID-19 hospitalisation within a large integrated health system in the United States.

BACKGROUND: Limited studies have explored post-discharge outcomes following Coronavirus Disease 2019 (COVID-19) hospitalisation. We sought to characterise patients discharged following a COVID-19 hospitalisation within a large integrated health system in the United States. METHODS: We performed a retrospective study of 2180 COVID-19 patients discharged between 1 April 2020 and 31 July 2020. The primary endpoint was all-cause observation stay or inpatient readmission within 30 days from discharge. Bivariate and multivariable logistic regression analyses were performed to estimate the association between key socio-demographic and clinical characteristics with risk of 30-day readmission. RESULTS: The 30-day readmission rate was 7.6% (n = 166); 30-day mortality rate was 1% (n = 19). Most readmissions were respiratory-related (58%) and occurred at a median time of 5 days post discharge. Adjusted models showed that prior hospitalisations (Odds Ratio = 2.36, [95% Confidence Interval: 1.59-3.50]), chronic pulmonary disease (1.57 [1.09-2.28]), and discharge to home health (1.46 [1.01-2.11]) were significantly associated with 30-day readmission. Longer duration from diagnosis to index admission was borderline associated with lower odds of readmission (0.95 [0.91-1.00]). CONCLUSION: Readmission and mortality rates for COVID-19 following discharge are low. Most readmissions occur early and are due to respiratory causes and may reflect the prolonged acute disease course.

Brief Report: Hydroxychloroquine does not induce hemolytic anemia or organ damage in a "humanized" G6PD A- mouse model.

BACKGROUND: Hydroxychloroquine (HCQ) is widely used in the treatment of malaria, rheumatologic disease such as lupus, and most recently, COVID-19. These uses raise concerns about its safe use in the setting of glucose-6-phosphate dehydrogenase (G6PD) deficiency, especially as 11% of African American men carry the G6PD A- variant. However, limited data exist regarding the safety of HCQ in this population. STUDY DESIGN AND METHODS: Recently, we created a novel "humanized" mouse model containing the G6PD deficiency A- variant (Val68Met) using CRISPR technology. We tested the effects of high-dose HCQ administration over 5 days on hemolysis in our novel G6PD A- mice. In addition to standard hematologic parameters including plasma hemoglobin, erythrocyte methemoglobin, and reticulocytes, hepatic and renal function were assessed after HCQ. RESULTS: Residual erythrocyte G6PD activity in G6PD A- mice was ~6% compared to wild-type (WT) littermates. Importantly, we found no evidence of clinically significant intravascular hemolysis, methemoglobinemia, or organ damage in response to high-dose HCQ. CONCLUSIONS: Though the effects of high doses over prolonged periods was not assessed, this study provides early, novel safety data of the use of HCQ in the setting of G6PD deficiency secondary to G6PD A-. In addition to novel safety data for HCQ, to our knowledge, we are the first to present the creation of a "humanized" murine model of G6PD deficiency.

Practical Guidelines for Collection, Manipulation and Inactivation of SARS-CoV-2 and COVID-19 Clinical Specimens.

SARS-CoV-2 is a novel coronavirus that causes the acute respiratory disease-Coronavirus disease 2019 (COVID-19)-which has led to a global health crisis. Currently, no prophylactics or therapies exist to control virus spread or mitigate the disease. Thus, the risk of infection for physicians and scientists is high, requiring work to be conducted in Biosafety Level-3 (BSL-3) facilities if virus will be isolated or propagated. However, inactivation of the virus can enable safe handling at a reduced biosafety level, making samples accessible to a diverse array of institutions and investigators. Institutions of all types have an immediate need for guidelines that outline safe collection, handling, and inactivation of samples suspected to contain active virus. Here we provide a practical guide for physicians and researchers wishing to work with materials from patients who are COVID-19 positive or suspected positive. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Practical guidelines for the safe collection and handling of specimens collected from COVID-19 and suspected COVID-19 patients Basic Protocol 2: Inactivating SARS-CoV-2.