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BACKGROUND: Severe COVID-19 carries a high morbidity and mortality. Previous studies have shown an association between COVID-19 severity and SARS-CoV-2 viral load (VL). We sought to measure VL in multiple compartments (urine, plasma, lower respiratory tract) in patients admitted to the intensive care unit (ICU) with severe COVID-19 pneumonia and correlate with clinical outcomes. METHODS: Plasma, urine, and endotracheal aspirate (ETA) samples were obtained on days 1, 3, 7, 14, and 21 from subjects admitted to the ICU with severe COVID-19. VL was measured via reverse transcriptase polymerase chain reaction. Clinical data was collected from the electronic health record. Grouped comparisons were performed using Student's t-test or 1-way ANOVA. Linear regression was used to correlate VL from different compartments collected at the same time. Logistic regression was performed to model ventilator-freedom at 28 days as a function of peak plasma VL. RESULTS: We enrolled 57 subjects with severe COVID-19 and measured VL in plasma (n = 57), urine (n = 25), and ETA (n = 34). Ventilator-associated pneumonia developed in 63% of subjects. 49% of subjects were viremic on study day 1. VL in plasma and ETA both significantly decreased by day 14 (P < 0.05), and the two were weakly correlated on study day 1 (P = 0.0037, r2 = 0.2343) and on all study days (P < 0.001, r2 = 0.2211). VL were not detected in urine. While no associations were observed with peak ETA VL, subjects with higher peak plasma VL experienced a greater number of respiratory complications, including ventilator-associated pneumonia and fewer ventilator-free and hospital-free days. There was no association between VL in either plasma or ETA and mortality. In viremic patients, plasma VL was significantly lower in subjects that were ICU-free and ventilator-free (P < 0.05), with trends noted for hospital-freedom, ventilator-associated pneumonia, and survival to discharge (P < 0.1). By logistic regression, plasma VL was inversely associated with ventilator-freedom at 28 days (odds ratio 0.14, 95% confidence interval 0.02-0.50). CONCLUSIONS: Elevated SARS-CoV-2 VL in the plasma but not in the lower respiratory tract is a novel biomarker in severe COVID-19 for respiratory complications.
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COVID-19 , Unidades de Cuidados Intensivos , SARS-CoV-2 , Carga Viral , Viremia , Humanos , COVID-19/complicaciones , Masculino , Femenino , Persona de Mediana Edad , Anciano , Índice de Severidad de la Enfermedad , Neumonía Asociada al Ventilador/epidemiología , Neumonía Asociada al Ventilador/virología , AdultoRESUMEN
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.
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Trastornos de la Coagulación Sanguínea , COVID-19 , Síndrome de Dificultad Respiratoria , Trombofilia , Trombosis , Trastornos de la Coagulación Sanguínea/complicaciones , COVID-19/complicaciones , Enfermedad Crítica , Fibrinólisis , Humanos , Hipoxia/complicaciones , Microcirculación , Oxígeno , Inhibidor 1 de Activador Plasminogénico , Estudios Prospectivos , Estudios Retrospectivos , Trombofilia/complicaciones , TromboplastinaRESUMEN
Bacterial pneumonia is a major cause of morbidity and mortality worldwide despite the use of antibiotics, and novel therapies are urgently needed. Building on previous work, we aimed to 1) develop a baboon model of severe pneumococcal pneumonia and sepsis with organ dysfunction and 2) test the safety and efficacy of a novel extracorporeal blood filter to remove proinflammatory molecules and improve organ function. After a dose-finding pilot study, 12 animals were inoculated with Streptococcus pneumoniae [5 × 109 colony-forming units (CFU)], given ceftriaxone at 24 h after inoculation, and randomized to extracorporeal blood purification using a filter coated with surface-immobilized heparin sulfate (n = 6) or sham treatment (n = 6) for 4 h at 30 h after inoculation. For safety analysis, four uninfected animals also underwent purification. At 48 h, necropsy was performed. Inoculated animals developed severe pneumonia and septic shock. Compared with sham-treated animals, septic animals treated with purification displayed significantly less kidney injury, metabolic acidosis, hypoglycemia, and shock (P < 0.05). Purification blocked the rise in peripheral blood S. pneumoniae DNA, attenuated bronchoalveolar lavage (BAL) CCL4, CCL2, and IL-18 levels, and reduced renal oxidative injury and classical NLRP3 inflammasome activation. Purification was safe in both uninfected and infected animals and produced no adverse effects. We demonstrate that heparin-based blood purification significantly attenuates levels of circulating S. pneumoniae DNA and BAL cytokines and is renal protective in baboons with severe pneumococcal pneumonia and septic shock. Purification was associated with less severe acute kidney injury, metabolic derangements, and shock. These results support future clinical studies in critically ill septic patients.
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Hemofiltración , Heparina/química , Neumonía Neumocócica/terapia , Choque Séptico/terapia , Streptococcus pneumoniae/metabolismo , Animales , Citocinas/metabolismo , Masculino , Papio , Proyectos Piloto , Neumonía Neumocócica/sangre , Choque Séptico/sangreRESUMEN
Investigations on ion channels in muscle tissues have mainly focused on physiological muscle function and related disorders, but emerging evidence supports a critical role of ion channels and transporters in developmental processes, such as controlling the myogenic commitment of stem cells. In this review, we provide an overview of ion channels and transporters that influence skeletal muscle myoblast differentiation, cardiac differentiation from pluripotent stem cells, as well as vascular smooth muscle cell differentiation. We highlight examples of model organisms or patients with mutations in ion channels. Furthermore, a potential underlying molecular mechanism involving hyperpolarization of the resting membrane potential and a series of calcium signaling is discussed.
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Proteínas de Transporte de Membrana/metabolismo , Desarrollo de Músculos , Mioblastos/citología , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Animales , Diferenciación Celular , Humanos , Canales Iónicos/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Mioblastos/metabolismo , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/metabolismoRESUMEN
Skeletal muscle myoblast differentiation involves elaborate signaling networks, including the activity of various ion channels and transporters. Several K+ and Ca2+ channels have been shown to affect myogenesis, but little is known about roles of Cl- channels in the associated processes. Here, we report that the leucine-rich repeat containing family 8 (LRRC8)/volume-regulated anion channel (VRAC) promotes mouse myoblast differentiation. All LRRC8 subunits of heteromeric VRAC were expressed during myotube formation of murine C2C12 myoblasts. Pharmacological VRAC inhibitors, siRNA-mediated knockdown of the essential VRAC subunit LRRC8A, or VRAC activity-suppressing overexpression of LRRC8A effectively reduced the expression of the myogenic transcription factor myogenin and suppressed myoblast fusion while not affecting myoblast proliferation. We found that inhibiting VRAC impairs plasma membrane hyperpolarization early during differentiation. At later times (more than 6 h after inducing differentiation), VRAC inhibition no longer suppressed myoblast differentiation, suggesting that VRAC acts upstream of K+ channel activation. Consequently, VRAC inhibition prevented the increase of intracellular steady-state Ca2+ levels that normally occurs during myogenesis. Our results may explain the mechanism for the thinning of skeletal muscle bundles observed in LRRC8A-deficient mice and highlight the importance of the LRRC8/VRAC anion channel in cell differentiation.
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Diferenciación Celular , Potenciales de la Membrana , Proteínas de la Membrana/metabolismo , Mioblastos/metabolismo , Animales , Línea Celular , Proteínas de la Membrana/genética , Ratones , Mioblastos/citología , Mioblastos/fisiología , Miogenina/genética , Miogenina/metabolismoRESUMEN
Leucine-rich repeat containing family 8 (LRRC8) proteins form the volume-regulated anion channel (VRAC). Recently, they were shown to be required for normal differentiation and fusion of C2C12 myoblasts, by promoting membrane hyperpolarization and intracellular Ca2+ signals. However, the mechanism by which they are involved remained obscure. Here, using a FRET-based sensor for VRAC activity, we show temporary activation of VRAC within the first 2 h of myogenic differentiation. During this period, we also observed a significant decrease in the intracellular Cl- concentration that was abolished by the VRAC inhibitor carbenoxolone. However, lowering the intracellular Cl- concentration by extracellular Cl- depletion did not promote differentiation as judged by the percentage of myogenin-positive nuclei or total myogenin levels in C2C12 cells. Instead, it inhibited myosin expression and myotube formation. Together, these data suggest that VRAC is activated and mediates Cl- efflux early on during myogenic differentiation, and a moderate intracellular Cl- concentration is necessary for myoblast fusion.
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Cloruros/metabolismo , Proteínas de la Membrana/metabolismo , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Animales , Carbenoxolona/farmacología , Diferenciación Celular/fisiología , Fusión Celular , Línea Celular , Citosol/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Transporte Iónico/efectos de los fármacos , Ratones , Desarrollo de Músculos/fisiología , Mioblastos Esqueléticos/efectos de los fármacosRESUMEN
The volume-regulated anion channel (VRAC) is a key player in the volume regulation of vertebrate cells. This ubiquitously expressed channel opens upon osmotic cell swelling and potentially other cues and releases chloride and organic osmolytes, which contributes to regulatory volume decrease (RVD). A plethora of studies have proposed a wide range of physiological roles for VRAC beyond volume regulation including cell proliferation, differentiation and migration, apoptosis, intercellular communication by direct release of signaling molecules and by supporting the exocytosis of insulin. VRAC was additionally implicated in pathological states such as cancer therapy resistance and excitotoxicity under ischemic conditions. Following extensive investigations, 5 years ago leucine-rich repeat-containing family 8 (LRRC8) heteromers containing LRRC8A were identified as the pore-forming components of VRAC. Since then, molecular biological approaches have allowed further insight into the biophysical properties and structure of VRAC. Heterologous expression, siRNA-mediated downregulation and genome editing in cells, as well as the use of animal models have enabled the assessment of the proposed physiological roles, together with the identification of new functions including spermatogenesis and the uptake of antibiotics and platinum-based cancer drugs. This review discusses the recent molecular biological insights into the physiology of VRAC in relation to its previously proposed roles.
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Proteínas de la Membrana/metabolismo , Humanos , Canales Iónicos/metabolismoRESUMEN
OBJECTIVES: Metabolic derangements in sepsis stem from mitochondrial injury and contribute significantly to organ failure and mortality; however, little is known about mitochondrial recovery in human sepsis. We sought to test markers of mitochondrial injury and recovery (mitochondrial biogenesis) noninvasively in peripheral blood mononuclear cells from patients with sepsis and correlate serial measurements with clinical outcomes. DESIGN: Prospective case-control study. SETTING: Academic Medical Center and Veterans Affairs Hospital. PATIENTS: Uninfected control patients (n = 20) and septic ICU patients (n = 37). INTERVENTIONS: Blood samples were collected once from control patients and serially with clinical data on days 1, 3, and 5 from septic patients. Gene products for HMOX1, NRF1, PPARGC1A, and TFAM, and mitochondrial DNA ND1 and D-loop were measured by quantitative reverse transcriptase-polymerase chain reaction. Proinflammatory cytokines were measured in plasma and neutrophil lysates. MEASUREMENTS AND MAIN RESULTS: Median (interquartile range) Acute Physiology and Chronic Health Evaluation II and Sequential Organ Failure Assessment scores were 21 (8) and 10 (4), respectively, and 90-day mortality was 19%. Transcript levels of all four genes in peripheral blood mononuclear cells were significantly reduced in septic patients on day 1 (p < 0.05), whereas mitochondrial DNA copy number fell and plasma D-loop increased (both p < 0.05), indicative of mitochondrial damage. D-loop content was directly proportional to tumor necrosis factor-α and high-mobility group protein B1 cytokine expression. By day 5, we observed transcriptional activation of mitochondrial biogenesis and restoration of mitochondrial DNA copy number (p < 0.05). Patients with early activation of mitochondrial biogenesis were ICU-free by 1 week. CONCLUSIONS: Our findings support data that sepsis-induced mitochondrial damage is reversed by activation of mitochondrial biogenesis and that gene transcripts measured noninvasively in peripheral blood mononuclear cells can serve as novel biomarkers of sepsis recovery.
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ADN Mitocondrial/sangre , Leucocitos Mononucleares/metabolismo , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Sepsis/metabolismo , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/genética , Enfermedades Mitocondriales/sangre , Enfermedades Mitocondriales/genética , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la Polimerasa , Sepsis/sangre , Sepsis/genéticaRESUMEN
Mitochondrial damage is often overlooked in acute lung injury (ALI), yet most of the lung's physiological processes, such as airway tone, mucociliary clearance, ventilation-perfusion (Va/Q) matching, and immune surveillance require aerobic energy provision. Because the cell's mitochondrial quality control (QC) process regulates the elimination and replacement of damaged mitochondria to maintain cell survival, we serially evaluated mitochondrial biogenesis and mitophagy in the alveolar regions of mice in a validated Staphylococcus aureus pneumonia model. We report that apart from cell lysis by direct contact with microbes, modest epithelial cell death was detected despite significant mitochondrial damage. Cell death by TdT-mediated dUTP nick-end labeling staining occurred on days 1 and 2 postinoculation: apoptosis shown by caspase-3 cleavage was present on days 1 and 2, while necroptosis shown by increased levels of phospho- mixed lineage kinase domain-like protein (MLKL) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1) was present on day 1 Cell death in alveolar type I (AT1) cells assessed by bronchoalveolar lavage fluid receptor for advanced glycation end points (RAGE) levels was high, yet AT2 cell death was limited while both mitochondrial biogenesis and mitophagy were induced. These mitochondrial QC mechanisms were evaluated mainly in AT2 cells by localizing increases in citrate synthase content, increases in nuclear mitochondrial biogenesis regulators nuclear respiratory factor-1 (NRF-1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and increases in light chain 3B protein (LC3-I)/LC3II ratios. Concomitant changes in p62, Pink 1, and Parkin protein levels indicated activation of mitophagy. By confocal microscopy, mitochondrial biogenesis and mitophagy were often observed on day 1 within the same AT2 cells. These findings imply that mitochondrial QC activation in pneumonia-damaged AT2 cells promotes cell survival in support of alveolar function.
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Células Epiteliales Alveolares/patología , Mitocondrias/patología , Neumonía Estafilocócica/etiología , Neumonía Estafilocócica/patología , Infecciones Estafilocócicas/complicaciones , Staphylococcus aureus/patogenicidad , Células Epiteliales Alveolares/metabolismo , Animales , Apoptosis , Biomarcadores/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Neumonía Estafilocócica/metabolismo , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patologíaRESUMEN
Acute respiratory failure is a common clinical finding caused by insufficient oxygenation (hypoxemia) or ventilation (hypocapnia). Understanding the pathophysiology of acute respiratory failure can help to facilitate recognition, diagnosis, and treatment. The cause of acute respiratory failure can be identified through utilization of physical examination findings, laboratory analysis, and chest imaging.
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Síndrome de Dificultad Respiratoria , Insuficiencia Respiratoria , Humanos , Síndrome de Dificultad Respiratoria/diagnóstico , Síndrome de Dificultad Respiratoria/epidemiología , Síndrome de Dificultad Respiratoria/terapia , Insuficiencia Respiratoria/diagnóstico , Insuficiencia Respiratoria/epidemiología , Insuficiencia Respiratoria/etiologíaRESUMEN
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.
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COVID-19 , Linfopenia , Masculino , Humanos , Femenino , Anciano , Persona de Mediana Edad , Linfocitos T , SARS-CoV-2 , Recuento de Linfocitos , TelómeroRESUMEN
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.
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COVID-19 , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Síndrome de Dificultad Respiratoria , Masculino , Femenino , Humanos , Adulto , Persona de Mediana Edad , Anciano , COVID-19/terapia , COVID-19/etiología , Estudios de Factibilidad , Trasplante de Células Madre Mesenquimatosas/efectos adversos , Síndrome de Dificultad Respiratoria/etiología , Síndrome de Dificultad Respiratoria/terapiaRESUMEN
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.
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Aptámeros de Nucleótidos , COVID-19 , Anticoagulantes/farmacología , Anticoagulantes/uso terapéutico , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/farmacología , Aptámeros de Nucleótidos/uso terapéutico , Puente Cardiopulmonar/efectos adversos , Factor X , Heparina , Humanos , TrombinaRESUMEN
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.
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Millions of COVID-19 patients have succumbed to respiratory and systemic inflammation. Hyperstimulation of toll-like receptor (TLR) signaling is a key driver of immunopathology following infection by viruses. We found that severely ill COVID-19 patients in the Intensive Care Unit (ICU) display hallmarks of such hyper-stimulation with abundant agonists of nucleic acid-sensing TLRs present in their blood and lungs. These nucleic acid-containing Damage and Pathogen Associated Molecular Patterns (DAMPs/PAMPs) can be depleted using nucleic acid-binding microfibers to limit the patient samples' ability to hyperactivate such innate immune receptors. Single-cell RNA-sequencing revealed that CD16+ monocytes from deceased but not recovered ICU patients exhibit a TLR-tolerant phenotype and a deficient anti-viral response after ex vivo TLR stimulation. Plasma proteomics confirmed such myeloid hyperactivation and revealed DAMP/PAMP carrier consumption in deceased patients. Treatment of these COVID-19 patient samples with MnO nanoparticles effectively neutralizes TLR activation by the abundant nucleic acid-containing DAMPs/PAMPs present in their lungs and blood. Finally, MnO nanoscavenger treatment limits the ability of DAMPs/PAMPs to induce TLR tolerance in monocytes. Thus, treatment with microfiber- or nanoparticle-based DAMP/PAMP scavengers may prove useful for limiting SARS-CoV-2 induced hyperinflammation, preventing monocytic TLR tolerance, and improving outcomes in severely ill COVID-19 patients.
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COVID-19 , Ácidos Nucleicos , Humanos , Moléculas de Patrón Molecular Asociado a Patógenos , SARS-CoV-2 , Receptores Toll-LikeRESUMEN
Because T cell differentiation leads to an expanded repertoire of chemokine receptors, a subgroup of G protein-coupled receptors, we hypothesized that the repertoire of G proteins might be altered in parallel. We analyzed the abundance of mRNA and/or protein of six G protein α-subunits in human CD4(+) and CD8(+) T cell subsets from blood. Although most G protein α-subunits were similarly expressed in all subsets, the abundance of Gα(o), a protein not previously described in hematopoietic cells, was much higher in memory versus naive cells. Consistent with these data, activation of naive CD4(+) T cells in vitro significantly increased the abundance of Gα(o) in cells stimulated under nonpolarizing or T(H)17 (but not T(H)1 or T(H)2)-polarizing conditions. In functional studies, the use of a chimeric G protein α-subunit, Gα(qo5), demonstrated that chemokine receptors could couple to Gα(o)-containing G proteins. We also found that Gα(i1), another α-subunit not described previously in leukocytes, was expressed in naive T cells but virtually absent from memory subsets. Corresponding to their patterns of expression, siRNA-mediated knockdown of Gα(o) in memory (but not naive) and Gα(i1) in naive (but not memory) CD4(+) T cells inhibited chemokine-dependent migration. Moreover, although even in Gα(o)- and Gα(i1)-expressing cells mRNAs of these α-subunits were much less abundant than Gα(i2) or Gα(i3), knockdown of any of these subunits impaired chemokine receptor-mediated migration similarly. Together, our data reveal a change in the repertoire of Gα(i/o) subunits during T cell differentiation and suggest functional equivalence among Gα(i/o) subunits irrespective of their relative abundance.
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Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Western Blotting , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Células Cultivadas , Quimiocina CXCL12/metabolismo , Quimiotaxis/genética , Femenino , Sangre Fetal/citología , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Recién Nacido , Células Jurkat , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Embarazo , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores CCR6/genética , Receptores CCR6/metabolismo , Receptores CXCR6 , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Receptores Virales/genética , Receptores Virales/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The role of concurrent illness in coronavirus disease 2019 (COVID-19) is unknown. Patients with leukemia may display altered thromboinflammatory responses. We report a 53-year-old man presenting with acute leukemia and COVID-19 who developed thrombotic complications and acute respiratory distress syndrome. Multiple analyses, including rotational thromboelastometry and flow cytometry on blood and bronchoalveolar lavage, are reported to characterize coagulation and immune profiles. The patient developed chemotherapy-induced neutropenia that may have protected his lungs from granulocyte-driven hyperinflammatory acute lung injury. However, neutropenia also alters viral clearing, potentially enabling ongoing viral propagation. This case depicts a precarious equilibrium between leukemia and COVID-19.
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Lesión Pulmonar Aguda/complicaciones , Trastornos de la Coagulación Sanguínea/complicaciones , Trastornos de la Coagulación Sanguínea/patología , COVID-19/complicaciones , COVID-19/patología , Leucemia Mieloide Aguda/complicaciones , Lesión Pulmonar Aguda/diagnóstico , Lesión Pulmonar Aguda/patología , Trastornos de la Coagulación Sanguínea/diagnóstico , Lavado Broncoalveolar , COVID-19/diagnóstico , Citometría de Flujo , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Neutropenia/complicaciones , Neutropenia/diagnóstico , Neutropenia/patología , SARS-CoV-2 , Tromboelastografía , Factores de VirulenciaRESUMEN
Highly sensitive, specific, and point-of-care (POC) serological assays are an essential tool to manage coronavirus disease 2019 (COVID-19). Here, we report on a microfluidic POC test that can profile the antibody response against multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens-spike S1 (S1), nucleocapsid (N), and the receptor binding domain (RBD)-simultaneously from 60 µl of blood, plasma, or serum. We assessed the levels of antibodies in plasma samples from 31 individuals (with longitudinal sampling) with severe COVID-19, 41 healthy individuals, and 18 individuals with seasonal coronavirus infections. This POC assay achieved high sensitivity and specificity, tracked seroconversion, and showed good concordance with a live virus microneutralization assay. We can also detect a prognostic biomarker of severity, IP-10 (interferon-γ-induced protein 10), on the same chip. Because our test requires minimal user intervention and is read by a handheld detector, it can be globally deployed to combat COVID-19.
Asunto(s)
Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , Pruebas en el Punto de Atención , SARS-CoV-2/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , COVID-19/sangre , COVID-19/virología , Prueba Serológica para COVID-19/instrumentación , Humanos , Reproducibilidad de los Resultados , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiología , Sensibilidad y Especificidad , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Highly sensitive, specific, and point-of-care (POC) serological assays are an essential tool to manage the COVID-19 pandemic. Here, we report on a microfluidic, multiplexed POC test that can profile the antibody response against multiple SARS-CoV-2 antigens - Spike S1 (S1), Nucleocapsid (N), and the receptor binding domain (RBD) - simultaneously from a 60 microliter drop of blood, plasma, or serum. We assessed the levels of anti-SARS-CoV-2 antibodies in plasma samples from 19 individuals (at multiple time points) with COVID-19 that required admission to the intensive care unit and from 10 healthy individuals. This POC assay shows good concordance with a live virus microneutralization assay, achieved high sensitivity (100%) and specificity (100%), and successfully tracked the longitudinal evolution of the antibody response in infected individuals. We also demonstrated that we can detect a chemokine, IP-10, on the same chip, which may provide prognostic insight into patient outcomes. Because our test requires minimal user intervention and is read by a handheld detector, it can be globally deployed in the fight against COVID-19 by democratizing access to laboratory quality tests.
RESUMEN
Sepsis involves a disordered host response to systemic infection leading to high morbidity and mortality. Despite intense research, targeted sepsis therapies beyond antibiotics have remained elusive. The cornerstone of sepsis research is the development of animal models to mimic human bacterial infections and test novel pharmacologic targets. Nonhuman primates (NHPs) have served as an attractive, but expensive, animal to model human bacterial infections due to their nearly identical cardiopulmonary anatomy and physiology, as well as host response to infection. Several NHP species have provided substantial insight into sepsis-mediated inflammation, endothelial dysfunction, acute lung injury, and multi-organ failure. The use of NHPs has usually focused on translating therapies from early preclinical models to human clinical trials. However, despite successful sepsis interventions in NHP models, there are still no FDA-approved sepsis therapies. This review highlights major NHP models of bacterial sepsis and their relevance to clinical medicine.