T-cell count and T-cell telomere length in patients with severe COVID-19.

Lymphocyte telomere length (TL) is highly variable and shortens with age. Short telomeres may impede TL-dependent T-cell clonal expansion with viral infection. As SARS-CoV-2 infection can induce prolonged and severe T-cell lymphopenia, infected adults, and particularly older adults with short telomeres, may display severe T-cell lymphopenia. To examine the relationship between T-cell TL parameters and T-cell counts, we studied 40 patients hospitalized with severe COVID-19. T-cells were isolated from lymphocytes, counted using flow cytometry, and their TL parameters were measured using the Telomere Shortest Length Assay. The cohort (median age = 62 years, 27% female) was racially and ethnically diverse (33% White, 35% Black, and 33% Other). On intensive care unit study day 1, T-cell count (mean=1.03 x109/L) was inversely related to age (p=0.007) and higher in females than males (p=0.025). Mean TL was 3.88 kilobases (kb), and 45.3% of telomeres were shorter than 3 kb. Using multiple regression analysis and adjusting for age and sex, T-cell count decreased with increased proportion of T-cell telomeres shorter than 3 kb (p=0.033) and increased with mean TL (p=0.052). Our findings suggest an association between the buildup of short telomeres within T-cells and explain in part reduced peripheral blood T-cell counts in patients with severe COVID-19. Shortened T-cell telomeres may be a risk factor for COVID-19-associated T-cell lymphopenia.

Advanced Respiratory Support Days as a Novel Marker of Mortality in COVID-19 Acute Respiratory Distress Syndrome Requiring Extracorporeal Membrane Oxygenation.

Emerging evidence suggests prolonged use of noninvasive respiratory support may increase mortality of patients with coronavirus disease 2019 (COVID-19)-associated acute respiratory distress syndrome who require extracorporeal membrane oxygenation (ECMO). Using a database of adults receiving ECMO for COVID-19, we calculated survival curves and multivariable Cox regression to determine the risk of death associated with pre-ECMO use of high-flow nasal oxygen (HFNO), noninvasive ventilation (NIV), and invasive mechanical ventilation (IMV) days. We investigated the performance of a novel variable, advanced respiratory support days (composite of HFNO, NIV, and IMV days), on Respiratory ECMO Survival Prediction (RESP) score. Subjects (N = 146) with increasing advanced respiratory support days (<5, 5-9, and ≥10) had a stepwise increase in 90 day mortality (32.2%, 57.7%, and 75.4%, respectively; p = 0.002). Ninety-day mortality was significantly higher in subjects (N = 121) receiving NIV >4 days (81.8% vs. 52.4%, p < 0.001). Each additional pre-ECMO advanced respiratory support day increased the odds of right ventricular failure (odds ratio [OR]: 1.066, 95% confidence interval [CI]: 1.002-1.135) and in-hospital mortality (1.17, 95% CI: 1.08-1.27). Substituting advanced respiratory support days for IMV days improved RESP score mortality prediction (area under the curve (AUC) or: 0.64 vs. 0.71). Pre-ECMO advanced respiratory support days were associated with increased 90 day mortality compared with IMV days alone. Adjusting the RESP score for advanced respiratory support days improved mortality prediction.

U-Shaped Association Between Carboxyhemoglobin and Mortality in Patients With Acute Respiratory Distress Syndrome on Venovenous Extracorporeal Membrane Oxygenation.

Background: Carbon monoxide (CO) is an endogenous signaling molecule that activates cytoprotective programs implicated in the resolution of acute respiratory distress syndrome (ARDS) and survival of critical illness. Because CO levels can be measured in blood as carboxyhemoglobin, we hypothesized that carboxyhemoglobin percent (COHb%) may associate with mortality. OBJECTIVES: To examine the relationship between COHb% and outcomes in patients with ARDS requiring venovenous extracorporeal membrane oxygenation (ECMO), a condition where elevated COHb% is commonly observed. DESIGN: Retrospective cohort study. SETTING: Academic medical center ICU. PATIENTS: Patients were included that had ARDS on venovenous ECMO. MEASUREMENTS AND MAIN RESULTS: We examined the association between COHb% and mortality using a Cox proportional hazards model. Secondary outcomes including ECMO duration, ventilator weaning, and hospital and ICU length of stay were examined using both subdistribution and causal-specific hazard models for competing risks. We identified 109 consecutive patients for analysis. Mortality significantly decreased per 1 U increase in COHb% below 3.25% (hazard ratio [HR], 0.35; 95% CI, 0.15-0.80; p = 0.013) and increased per 1 U increase above 3.25% (HR, 4.7; 95% CI, 1.5-14.7; p = 0.007) reflecting a nonlinear association (p = 0.006). Each unit increase in COHb% was associated with reduced likelihood of liberation from ECMO and mechanical ventilation, and increased time to hospital and ICU discharge (all p < 0.05). COHb% was significantly associated with hemolysis but not with initiation of hemodialysis or blood transfusions. CONCLUSIONS: In patients with ARDS on venovenous ECMO, COHb% is a novel biomarker for mortality exhibiting a U-shaped pattern. Our findings suggest that too little CO (perhaps due to impaired host signaling) or excess CO (perhaps due to hemolysis) is associated with higher mortality. Patients with low COHb% may exhibit the most benefit from future therapies targeting anti-oxidant and anti-inflammatory pathways such as low-dose inhaled CO gas.

Management of severe acute respiratory distress syndrome: a primer.

This narrative review explores the physiology and evidence-based management of patients with severe acute respiratory distress syndrome (ARDS) and refractory hypoxemia, with a focus on mechanical ventilation, adjunctive therapies, and veno-venous extracorporeal membrane oxygenation (V-V ECMO). Severe ARDS cases increased dramatically worldwide during the Covid-19 pandemic and carry a high mortality. The mainstay of treatment to improve survival and ventilator-free days is proning, conservative fluid management, and lung protective ventilation. Ventilator settings should be individualized when possible to improve patient-ventilator synchrony and reduce ventilator-induced lung injury (VILI). Positive end-expiratory pressure can be individualized by titrating to best respiratory system compliance, or by using advanced methods, such as electrical impedance tomography or esophageal manometry. Adjustments to mitigate high driving pressure and mechanical power, two possible drivers of VILI, may be further beneficial. In patients with refractory hypoxemia, salvage modes of ventilation such as high frequency oscillatory ventilation and airway pressure release ventilation are additional options that may be appropriate in select patients. Adjunctive therapies also may be applied judiciously, such as recruitment maneuvers, inhaled pulmonary vasodilators, neuromuscular blockers, or glucocorticoids, and may improve oxygenation, but do not clearly reduce mortality. In select, refractory cases, the addition of V-V ECMO improves gas exchange and modestly improves survival by allowing for lung rest. In addition to VILI, patients with severe ARDS are at risk for complications including acute cor pulmonale, physical debility, and neurocognitive deficits. Even among the most severe cases, ARDS is a heterogeneous disease, and future studies are needed to identify ARDS subgroups to individualize therapies and advance care.

Alveolar Mitochondrial Quality Control During Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a leading cause of respiratory failure and death in patients in the intensive care unit. Experimentally, acute lung injury resolution depends on the repair of mitochondrial oxidant damage by the mitochondrial quality control (MQC) pathways, mitochondrial biogenesis, and mitophagy, but nothing is known about this in the human lung. In a case-control autopsy study, we compared the lungs of subjects dying of ARDS (n = 8; cases) and age-/gender-matched subjects dying of nonpulmonary causes (n = 7; controls). Slides were examined by light microscopy and immunofluorescence confocal microscopy, randomly probing for co-localization of citrate synthase with markers of oxidant stress, mitochondrial DNA damage, mitophagy, and mitochondrial biogenesis. ARDS lungs showed diffuse alveolar damage with edema, hyaline membranes, and neutrophils. Compared with controls, a high degree of mitochondrial oxidant damage was seen in type 2 epithelial (AT2) cells and alveolar macrophages by 8-hydroxydeoxyguanosine and malondialdehyde co-staining with citrate synthase. In ARDS, antioxidant protein heme oxygenase-1 and DNA repair enzyme N-glycosylase/DNA lyase (Ogg1) were found in alveolar macrophages but not in AT2 cells. Moreover, MAP1 light chain-3 (LC3) and serine/threonine-protein kinase (Pink1) staining were absent in AT2 cells, suggesting a mitophagy failure. Nuclear respiratory factor-1 staining was missing in the alveolar region, suggesting impaired mitochondrial biogenesis. Widespread hyperproliferation of AT2 cells in ARDS could suggest defective differentiation into type 1 cells. ARDS lungs show profuse mitochondrial oxidant DNA damage but little evidence of MQC activity in AT2 epithelium. Because these pathways are important for acute lung injury resolution, our findings support MQC as a novel pharmacologic target for ARDS resolution.

Feasibility Study of Cord Tissue Derived Mesenchymal Stromal Cells in COVID-19-Related Acute Respiratory Distress Syndrome.

BACKGROUND: Treatment options for patients with COVID-19-related acute respiratory distress syndrome (ARDS) are desperately needed. Allogeneic human umbilical cord derived mesenchymal stromal cells (hCT-MSCs) have potential therapeutic benefits in these critically ill patients, but feasibility and safety data are lacking. MATERIALS AND METHODS: In this phase I multisite study, 10 patients with COVID-19-related ARDS were treated with 3 daily intravenous infusions of hCT-MSCs (1 million cells/kg, maximum dose 100 million cells). The primary endpoint assessed safety. RESULTS: Ten patients (7 females, 3 males; median age 62 years (range 39-79)) were enrolled at 2 sites and received a total of 30 doses of study product. The average cell dose was 0.93 cells/kg (range 0.56-1.45 cells/kg and total dose range 55-117 million cells) with 5/30 (17%) of doses lower than intended dose. Average cell viability was 85% (range 63%-99%) with all but one meeting the >70% release criteria. There were no infusion-related reactions or study-related adverse events, 28 non-serious adverse events in 3 unique patients, and 2 serious adverse events in 2 unique patients, which were expected and unrelated to the study product. Five patients died: 3 by day 28 and 5 by day 90 of the study (median 27 days, range 7-76 days). All deaths were determined to be unrelated to the hCT-MSCs. CONCLUSION: We were able to collect relevant safety outcomes for the use of hCT-MSCs in patients with COVID-19-related ARDS. Future studies to explore their safety and efficacy are warranted.

Timing of Intubation in COVID-19: When It Is Too Early and When It Is Too Late.

The timing of initiating mechanical ventilation in patients with acute respiratory distress syndrome due to COVID-19 remains controversial. At the outset of the pandemic, "very early" intubation was recommended in patients requiring oxygen flows above 6 L per minute but was followed closely thereafter by avoidance of invasive mechanical ventilation (IMV) due to a perceived (yet over-estimated) risk of mortality after intubation. While the use of noninvasive methods of oxygen delivery, such as high-flow nasal oxygen (HFNO) or noninvasive positive pressure ventilation (NIV), can avert the need for mechanical ventilation in some, accumulating evidence suggests delayed intubation is also associated with an increased mortality in a subset of COVID-19 patients. Close monitoring is necessary in COVID-19 patients on HFNO or NIV to identify signs of noninvasive failure and ensure appropriate provision of IMV.

Use of Inhaled Epoprostenol in Patients With COVID-19 Receiving Humidified, High-Flow Nasal Oxygen Is Associated With Progressive Respiratory Failure.

BACKGROUND: The clinical benefit of using inhaled epoprostenol (iEpo) through a humidified high-flow nasal cannula (HHFNC) remains unknown for patients with COVID-19. RESEARCH QUESTION: Can iEpo prevent respiratory deterioration for patients with positive SARS-CoV-2 findings receiving HHFNC? STUDY DESIGN AND METHODS: This multicenter retrospective cohort analysis included patients aged 18 years or older with COVID-19 pneumonia who required HHFNC treatment. Patients who received iEpo were propensity score matched to patients who did not receive iEpo. The primary outcome was time to mechanical ventilation or death without mechanical ventilation and was assessed using Kaplan-Meier curves and Cox proportional hazard ratios. The effects of residual confounding were assessed using a multilevel analysis, and a secondary analysis adjusted for outcome propensity also was performed in a multivariable model that included the entire (unmatched) patient cohort. RESULTS: Among 954 patients with positive SARS-CoV-2 findings receiving HHFNC therapy, 133 patients (13.9%) received iEpo. After propensity score matching, the median number of days until the composite outcome was similar between treatment groups (iEpo: 5.0 days [interquartile range, 2.0-10.0 days] vs no-iEpo: 6.5 days [interquartile range, 2.0-11.0 days]; P = .26), but patients who received iEpo were more likely to meet the composite outcome in the propensity score-matched, multilevel, and multivariable unmatched analyses (hazard ratio, 2.08 [95% CI, 1.73-2.50]; OR, 4.72 [95% CI, 3.01-7.41]; and OR, 1.35 [95% CI, 1.23-1.49]; respectively). INTERPRETATION: In patients with COVID-19 receiving HHFNC therapy, use of iEpo was associated with the need for invasive mechanical ventilation.

Age and Comorbidities Predict COVID-19 Outcome, Regardless of Innate Immune Response Severity: A Single Institutional Cohort Study.

The COVID-19 pandemic has claimed over eight hundred thousand lives in the United States alone, with older individuals and those with comorbidities being at higher risk of severe disease and death. Although severe acute respiratory syndrome coronavirus 2-induced hyperinflammation is one of the mechanisms underlying the high mortality, the association between age and innate immune responses in COVID-19 mortality remains unclear. DESIGN: Flow cytometry of fresh blood and multiplexed inflammatory chemokine measurements of sera were performed on samples collected longitudinally from our cohort. Aggregate impact of comorbid conditions was calculated with the Charlson Comorbidity Index, and association between patient factors and outcomes was calculated via Cox proportional hazard analysis and repeated measures analysis of variance. SETTING: A cohort of severely ill COVID-19 patients requiring ICU admission was followed prospectively. PATIENTS: In total, 67 patients (46 male, age 59 ± 14 yr) were included in the study. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Mortality in our cohort was 41.8%. We identified older age (hazard ratio [HR] 1.09 [95% CI 1.07-1.11]; p = 0.001), higher comorbidity index (HR 1.24 [95% CI 1.14-1.35]; p = 0.039), and hyponatremia (HR 0.90 [95% CI 0.82-0.99]; p = 0.026) to each independently increase risk for death in COVID-19. We also found that neutrophilia (R = 0.2; p = 0.017), chemokine C-C motif ligand (CCL) 2 (R = 0.3; p = 0.043), and C-X-C motif chemokine ligand 9 (CXCL9) (R = 0.3; p = 0.050) were weakly but significantly correlated with mortality. Older age was associated with lower monocyte (R = -0.2; p = 0.006) and cluster of differentiation (CD) 16+ cell counts (R = -0.2; p = 0.002) and increased CCL11 concentration (R = 0.3; p = 0.050). Similarly, younger patients (< 65 yr) demonstrated a rise in CD4 (b-coefficient = 0.02; p = 0.036) and CD8 (0.01; p = 0.001) counts, as well as CCL20 (b-coefficient = 6.8; p = 0.036) during their ICU stay. This CD8 count rise was also associated with survival (b-coefficient = 0.01; p = 0.023). CONCLUSIONS: Age, comorbidities, and hyponatremia independently predict mortality in severe COVID-19. Neutrophilia and higher CCL2 and CXCL9 levels are also associated with higher mortality, while independent of age.

Effects of high-intensity interval training with hyperbaric oxygen.

Hyperbaric Oxygen (HBO2) has been proposed as a pre-conditioning method to enhance exercise performance. Most prior studies testing this effect have been limited by inadequate methodologies. Its potential efficacy and mechanism of action remain unknown. We hypothesized that HBO2 could enhance aerobic capacity by inducing mitochondrial biogenesis via redox signaling in skeletal muscle. HBO2 was administered in combination with high-intensity interval training (HIIT), a potent redox stimulus known to induce mitochondrial biogenesis. Aerobic capacity was tested during acute hypobaric hypoxia seeking to shift the limiting site of whole body V̇O2 from convection to diffusion, more closely isolating any effect of improved oxidative capacity. Healthy volunteers were screened with sea-level (SL) V̇O2peak testing. Seventeen subjects were enrolled (10 men, 7 women, ages 26.5±1.3 years, BMI 24.6±0.6 kg m-2, V̇O2peak SL = 43.4±2.1). Each completed 6 HIIT sessions over 2 weeks randomized to breathing normobaric air, "HIIT+Air" (PiO2 = 0.21 ATM) or HBO2 (PiO2 = 1.4 ATM) during training, "HIIT+HBO2" group. Training workloads were individualized based on V̇O2peak SL test. Vastus Lateralis (VL) muscle biopsies were performed before and after HIIT in both groups. Baseline and post-training V̇O2peak tests were conducted in a hypobaric chamber at PiO2 = 0.12 ATM. HIIT significantly increased V̇O2peak in both groups: HIIT+HBO2 31.4±1.5 to 35.2±1.2 ml kg-1·min-1 and HIIT+Air 29.0±3.1 to 33.2±2.5 ml kg-1·min-1 (p = 0.005) without an additional effect of HBO2 (p = 0.9 for interaction of HIIT x HBO2). Subjects randomized to HIIT+HBO2 displayed higher skeletal muscle mRNA levels of PPARGC1A, a regulator of mitochondrial biogenesis, and HK2 and SLC2A4, regulators of glucose utilization and storage. All other tested markers of mitochondrial biogenesis showed no additional effect of HBO2 to HIIT. When combined with HIIT, short-term modest HBO2 (1.4 ATA) has does not increase whole-body V̇O2peak during acute hypobaric hypoxia. (ClinicalTrials.gov Identifier: NCT02356900; https://clinicaltrials.gov/ct2/show/NCT02356900).

It's about the patients: Practical antibiotic stewardship in outpatient settings in the United States.

Antibiotic-resistant pathogens cause over 35,000 preventable deaths in the United States every year, and multiple strategies could decrease morbidity and mortality. As antibiotic stewardship requirements are being deployed for the outpatient setting, community providers are facing systematic challenges in implementing stewardship programs. Given that the vast majority of antibiotics are prescribed in the outpatient setting, there are endless opportunities to make a smart and informed choice when prescribing and to move the needle on antibiotic stewardship. Antibiotic stewardship in the community, or "smart prescribing" as we suggest, should factor in antibiotic efficacy, safety, local resistance rates, and overall cost, in addition to patient-specific factors and disease presentation, to arrive at an appropriate therapy. Here, we discuss some of the challenges, such as patient/parent pressure to prescribe, lack of data or resources for implementation, and a disconnect between guidelines and real-world practice, among others. We have assembled an easy-to-use best practice guide for providers in the outpatient setting who lack the time or resources to develop a plan or consult lengthy guidelines. We provide specific suggestions for antibiotic prescribing that align real-world clinical practice with best practices for antibiotic stewardship for two of the most common bacterial infections seen in the outpatient setting: community-acquired pneumonia and skin and soft-tissue infection. In addition, we discuss many ways that community providers, payors, and regulatory bodies can make antibiotic stewardship easier to implement and more streamlined in the outpatient setting.

Differential chromatin accessibility in peripheral blood mononuclear cells underlies COVID-19 disease severity prior to seroconversion.

SARS-CoV-2 infection triggers profound and variable immune responses in human hosts. Chromatin remodeling has been observed in individuals severely ill or convalescing with COVID-19, but chromatin remodeling early in disease prior to anti-spike protein IgG seroconversion has not been defined. We performed the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and RNA-seq on peripheral blood mononuclear cells (PBMCs) from outpatients with mild or moderate symptom severity at different stages of clinical illness. Early in the disease course prior to IgG seroconversion, modifications in chromatin accessibility associated with mild or moderate symptoms were already robust and included severity-associated changes in accessibility of genes in interleukin signaling, regulation of cell differentiation and cell morphology. Furthermore, single-cell analyses revealed evolution of the chromatin accessibility landscape and transcription factor motif accessibility for individual PBMC cell types over time. The most extensive remodeling occurred in CD14+ monocytes, where sub-populations with distinct chromatin accessibility profiles were observed prior to seroconversion. Mild symptom severity was marked by upregulation of classical antiviral pathways, including those regulating IRF1 and IRF7, whereas in moderate disease, these classical antiviral signals diminished, suggesting dysregulated and less effective responses. Together, these observations offer novel insight into the epigenome of early mild SARS-CoV-2 infection and suggest that detection of chromatin remodeling in early disease may offer promise for a new class of diagnostic tools for COVID-19.

Suppression of Fibrinolysis and Hypercoagulability, Severity of Hypoxemia, and Mortality in COVID-19 Patients: A Retrospective Cohort Study.

BACKGROUND: COVID-19 causes hypercoagulability, but the association between coagulopathy and hypoxemia in critically ill patients has not been thoroughly explored. This study hypothesized that severity of coagulopathy would be associated with acute respiratory distress syndrome severity, major thrombotic events, and mortality in patients requiring intensive care unit-level care. METHODS: Viscoelastic testing by rotational thromboelastometry and coagulation factor biomarker analyses were performed in this prospective observational cohort study of critically ill COVID-19 patients from April 2020 to October 2020. Statistical analyses were performed to identify significant coagulopathic biomarkers such as fibrinolysis-inhibiting plasminogen activator inhibitor 1 and their associations with clinical outcomes such as mortality, extracorporeal membrane oxygenation requirement, occurrence of major thrombotic events, and severity of hypoxemia (arterial partial pressure of oxygen/fraction of inspired oxygen categorized into mild, moderate, and severe per the Berlin criteria). RESULTS: In total, 53 of 55 (96%) of the cohort required mechanical ventilation and 9 of 55 (16%) required extracorporeal membrane oxygenation. Extracorporeal membrane oxygenation-naïve patients demonstrated lysis indices at 30 min indicative of fibrinolytic suppression on rotational thromboelastometry. Survivors demonstrated fewer procoagulate acute phase reactants, such as microparticle-bound tissue factor levels (odds ratio, 0.14 [0.02, 0.99]; P = 0.049). Those who did not experience significant bleeding events had smaller changes in ADAMTS13 levels compared to those who did (odds ratio, 0.05 [0, 0.7]; P = 0.026). Elevations in plasminogen activator inhibitor 1 (odds ratio, 1.95 [1.21, 3.14]; P = 0.006), d-dimer (odds ratio, 3.52 [0.99, 12.48]; P = 0.05), and factor VIII (no clot, 1.15 ± 0.28 vs. clot, 1.42 ± 0.31; P = 0.003) were also demonstrated in extracorporeal membrane oxygenation-naïve patients who experienced major thrombotic events. Plasminogen activator inhibitor 1 levels were significantly elevated during periods of severe compared to mild and moderate acute respiratory distress syndrome (severe, 44.2 ± 14.9 ng/ml vs. mild, 31.8 ± 14.7 ng/ml and moderate, 33.1 ± 15.9 ng/ml; P = 0.029 and 0.039, respectively). CONCLUSIONS: Increased inflammatory and procoagulant markers such as plasminogen activator inhibitor 1, microparticle-bound tissue factor, and von Willebrand factor levels are associated with severe hypoxemia and major thrombotic events, implicating fibrinolytic suppression in the microcirculatory system and subsequent micro- and macrovascular thrombosis in severe COVID-19.

Differential chromatin accessibility in peripheral blood mononuclear cells underlies COVID-19 disease severity prior to seroconversion.

SARS-CoV-2 infection triggers profound and variable immune responses in human hosts. Chromatin remodeling has been observed in individuals severely ill or convalescing with COVID-19, but chromatin remodeling early in disease prior to anti-spike protein IgG seroconversion has not been defined. We performed the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and RNA-seq on peripheral blood mononuclear cells (PBMCs) from outpatients with mild or moderate symptom severity at different stages of clinical illness. Early in the disease course prior to IgG seroconversion, modifications in chromatin accessibility associate with mild or moderate symptoms are already robust and include severity-associated changes in accessibility of genes in interleukin signaling, regulation of cell differentiation and cell morphology. Furthermore, single-cell analyses revealed evolution of the chromatin accessibility landscape and transcription factor motif accessibility for individual PBMC cell types over time. The most extensive remodeling occurred in CD14+ monocytes, where sub-populations with distinct chromatin accessibility profiles were observed prior to seroconversion. Mild symptom severity is marked by upregulation classical antiviral pathways including those regulating IRF1 and IRF7, whereas in moderate disease these classical antiviral signals diminish suggesting dysregulated and less effective responses. Together, these observations offer novel insight into the epigenome of early mild SARS-CoV-2 infection and suggest that detection of chromatin remodeling in early disease may offer promise for a new class of diagnostic tools for COVID-19.

Combining Heparin and a FX/Xa Aptamer to Reduce Thrombin Generation in Cardiopulmonary Bypass and COVID-19.

Known limitations of unfractionated heparin (UFH) have encouraged the evaluation of anticoagulant aptamers as alternatives to UFH in highly procoagulant settings such as cardiopulmonary bypass (CPB). Despite progress, these efforts have not been totally successful. We take a different approach and explore whether properties of an anticoagulant aptamer can complement UFH, rather than replace it, to address shortcomings with UFH use. Combining RNA aptamer 11F7t, which targets factor X/Xa, with UFH (or low molecular weight heparin) yields a significantly enhanced anticoagulant cocktail effective in normal and COVID-19 patient blood. This aptamer-UFH combination (1) supports continuous circulation of human blood through an ex vivo membrane oxygenation circuit, as is required for patients undergoing CPB and COVID-19 patients requiring extracorporeal membrane oxygenation, (2) allows for a reduced level of UFH to be employed, (3) more effectively limits thrombin generation compared to UFH alone, and (4) is rapidly reversed by the administration of protamine sulfate, the standard treatment for reversing UFH clinically following CPB. Thus, the combination of factor X/Xa aptamer and UFH has significantly improved anticoagulant properties compared to UFH alone and underscores the potential of RNA aptamers to improve medical management of acute care patients requiring potent yet rapidly reversible anticoagulation.

Nuclear respiratory factor-1 negatively regulates TGF-ß1 and attenuates pulmonary fibrosis.

The preclinical model of bleomycin-induced lung fibrosis is useful to study mechanisms related to human pulmonary fibrosis. Using BLM in mice, we find low HO-1 expression. Although a unique Rhenium-CO-releasing molecule (ReCORM) up-regulates HO-1, NRF-1, CCN5, and SMAD7, it reduces TGFß1, TGFßr1, collagen, α-SMA, and phosphorylated Smad2/3 levels in mouse lung and in human lung fibroblasts. ChIP assay studies confirm NRF-1 binding to the promoters of TGFß1 repressors CCN5 and Smad7. ReCORM did not blunt lung fibrosis in Hmox1-deficient alveolar type 2 cell knockout mice, suggesting this gene participates in lung protection. In human lung fibroblasts, TGFß1-dependent production of α-SMA is abolished by ReCORM or by NRF-1 gene transfection. We demonstrate effective HO-1/NRF-1 signaling in lung AT2 cells protects against BLM induced lung injury and fibrosis by maintaining mitochondrial health, function, and suppressing the TGFß1 pathway. Thus, protection of AT2 cell mitochondrial integrity via HO-1/NRF-1 presents an innovative therapeutic target.

Rapid test to assess the escape of SARS-CoV-2 variants of concern.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are concerning in the ongoing coronavirus disease 2019 (COVID-19) pandemic. Here, we developed a rapid test, termed CoVariant-SCAN, that detects neutralizing antibodies (nAbs) capable of blocking interactions between the angiotensin-converting enzyme 2 receptor and the spike protein of wild-type (WT) SARS-CoV-2 and three other variants: B.1.1.7, B.1.351, and P.1. Using CoVariant-SCAN, we assessed neutralization/blocking of monoclonal antibodies and plasma from COVID-19­positive and vaccinated individuals. For several monoclonal antibodies and most plasma samples, neutralization against B.1.351 and P.1 variants is diminished relative to WT, while B.1.1.7 is largely cross-neutralized. We also showed that we can rapidly adapt the platform to detect nAbs against an additional variant­B.1.617.2 (Delta)­without reengineering or reoptimizing the assay. Results using CoVariant-SCAN are consistent with live virus neutralization assays and demonstrate that this easy-to-deploy test could be used to rapidly assess nAb response against multiple SARS-CoV-2 variants.