High Circulating Levels of Neutrophil Extracellular Traps Parameters Predicting Poor Outcome in COVID-19.

OBJECTIVE: Exploration of biomarkers to predict the severity of COVID-19 is important to reduce mortality. Upon COVID-19 infection, neutrophil extracellular traps (NET) are formed, which leads to a cytokine storm and host damage. Hence, the extent of NET formation may reflect disease progression and predict mortality in COVID-19. METHODS: We measured 4 NET parameters - cell-free double stranded DNA (cell-free dsDNA), neutrophil elastase, citrullinated histone H3 (Cit-H3), and histone - DNA complex - in 188 COVID-19 patients and 20 healthy controls. Survivors (n=166) were hospitalized with or without oxygen supplementation, while non-survivors (n=22) expired during in-hospital treatment. RESULTS: Cell-free dsDNA was significantly elevated in non-survivors in comparison with survivors and controls. The survival rate of patients with high levels of cell-free dsDNA, neutrophil elastase, and Cit-H3 was significantly lower than that of patients with low levels. These three markers significantly correlated with inflammatory markers (absolute neutrophil count and C-reactive protein). CONCLUSION: Since the increase in NET parameters indicates the unfavourable course of COVID-19 infection, patients predisposed to poor outcome can be rapidly managed through risk stratification by using these NET parameters.

A Method for Evaluation of the Level of Circulating Mitochondrial DNA by ND1 and ND2 Genes.

The measurement of the level of mitochondrial DNA (mtDNA) in the blood is a difficult problem due to high variability of mitochondrial genes, deletions in the mitochondrial genome in some pathological conditions, different sources of mtDNA into the bloodstream (mtDNA from tissues, from blood cells, etc.). We designed primers and TaqMan probes for highly conserved regions of the ND1 and ND2 genes outside the mitochondrial deletions "hot zones". For standardizing the technique, the true concentration of low-molecular-weight mtDNA was determined by real-time PCR for two targets: a fragment of the ND2 gene (122 bp) and the ND1 and ND2 genes (1198 bp). The sensitivity and specificity of the developed approach were verified on a DNA pool isolated from the blood plasma of healthy donors of various nationalities. The concentration of low-molecular-weight mtDNA in the blood plasma of two patients with COVID-19 was monitored over two weeks of inpatient treatment. A significant increase in the content of low-molecular-weight mtDNA was observed during the first 5 days after hospitalization, followed by a drop to the level of healthy donors. The developed technique makes it possible to assess the blood level of low-molecular-weight mtDNA regardless of the quality of sampling and makes it possible to standardize this biological marker in a wide range of infectious and non-infectious pathologies.

Cell-Free DNA: Potential Application in COVID-19 Diagnostics and Management.

WHO has declared COVID-19 as a worldwide, public health emergency. The elderly, pregnant women, and people with associated co-morbidities, including pulmonary disease, heart failure, diabetes, and cancer are the most predisposed population groups to infection. Cell-free DNA is a very commonly applied marker, which is elevated in various pathological conditions. However, it has a much higher sensitivity than standard biochemical markers. cfDNA appears to be an effective marker of COVID-19 complications, and also serves as a marker of certain underlying health conditions and risk factors of severe illness during COVID-19 infection. We aimed to present the possible mechanisms and sources of cfDNA released during moderate and severe infections. Moreover, we attempt to verify how efficiently cfDNA increase could be applied in COVID-19 risk assessment and how it corresponds with epidemiological data.

Plasma cell-free RNA characteristics in COVID-19 patients.

The pathogenesis of COVID-19 is still elusive, which impedes disease progression prediction, differential diagnosis, and targeted therapy. Plasma cell-free RNAs (cfRNAs) carry unique information from human tissue and thus could point to resourceful solutions for pathogenesis and host-pathogen interactions. Here, we performed a comparative analysis of cfRNA profiles between COVID-19 patients and healthy donors using serial plasma. Analyses of the cfRNA landscape, potential gene regulatory mechanisms, dynamic changes in tRNA pools upon infection, and microbial communities were performed. A total of 380 cfRNA molecules were up-regulated in all COVID-19 patients, of which seven could serve as potential biomarkers (AUC > 0.85) with great sensitivity and specificity. Antiviral (NFKB1A, IFITM3, and IFI27) and neutrophil activation (S100A8, CD68, and CD63)-related genes exhibited decreased expression levels during treatment in COVID-19 patients, which is in accordance with the dynamically enhanced inflammatory response in COVID-19 patients. Noncoding RNAs, including some microRNAs (let 7 family) and long noncoding RNAs (GJA9-MYCBP) targeting interleukin (IL6/IL6R), were differentially expressed between COVID-19 patients and healthy donors, which accounts for the potential core mechanism of cytokine storm syndromes; the tRNA pools change significantly between the COVID-19 and healthy group, leading to the accumulation of SARS-CoV-2 biased codons, which facilitate SARS-CoV-2 replication. Finally, several pneumonia-related microorganisms were detected in the plasma of COVID-19 patients, raising the possibility of simultaneously monitoring immune response regulation and microbial communities using cfRNA analysis. This study fills the knowledge gap in the plasma cfRNA landscape of COVID-19 patients and offers insight into the potential mechanisms of cfRNAs to explain COVID-19 pathogenesis.

Human Recombinant DNase I (Pulmozyme®) Inhibits Lung Metastases in Murine Metastatic B16 Melanoma Model That Correlates with Restoration of the DNase Activity and the Decrease SINE/LINE and c-Myc Fragments in Blood Cell-Free DNA.

Tumor-associated cell-free DNAs (cfDNA) play an important role in the promotion of metastases. Previous studies proved the high antimetastatic potential of bovine pancreatic DNase I and identified short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs)and fragments of oncogenes in cfDNA as the main molecular targets of enzyme in the bloodstream. Here, recombinant human DNase I (commercial name Pulmozyme®), which is used for the treatment of cystic fibrosis in humans, was repurposed for the inhibition of lung metastases in the B16 melanoma model in mice. We found that Pulmozyme® strongly reduced migration and induced apoptosis of B16 cells in vitro and effectively inhibited metastases in lungs and liver in vivo. Pulmozyme® was shown to be two times more effective when administered intranasally (i.n.) than bovine DNase I, but intramuscular (i.m.) administration forced it to exhibit as high an antimetastatic activity as bovine DNase I. Both DNases administered to mice either i.m. or i.n. enhanced the DNase activity of blood serum to the level of healthy animals, significantly decreased cfDNA concentrations, efficiently degraded SINE and LINE repeats and c-Myc fragments in the bloodstream and induced apoptosis and disintegration of neutrophil extracellular traps in metastatic foci; as a result, this manifested as the inhibition of metastases spread. Thus, Pulmozyme®, which is already an approved drug, can be recommended for use in the treatment of lung metastases.

Detection of SARS-CoV-2 Derived Small RNAs and Changes in Circulating Small RNAs Associated with COVID-19.

Cleavage of double-stranded RNA is described as an evolutionary conserved host defense mechanism against viral infection. Small RNAs are the product and triggers of post transcriptional gene silencing events. Up until now, the relevance of this mechanism for SARS-CoV-2-directed immune responses remains elusive. Herein, we used high throughput sequencing to profile the plasma of active and convalescent COVID-19 patients for the presence of small circulating RNAs. The existence of SARS-CoV-2 derived small RNAs in plasma samples of mild and severe COVID-19 cases is described. Clusters of high siRNA abundance were discovered, homologous to the nsp2 3'-end and nsp4 virus sequence. Four virus-derived small RNA sequences have the size of human miRNAs, and a target search revealed candidate genes associated with ageusia and long COVID symptoms. These virus-derived small RNAs were detectable also after recovery from the disease. The additional analysis of circulating human miRNAs revealed differentially abundant miRNAs, discriminating mild from severe cases. A total of 29 miRNAs were reduced or absent in severe cases. Several of these are associated with JAK-STAT response and cytokine storm.

Evolution of NETosis markers and DAMPs have prognostic value in critically ill COVID-19 patients.

Coronavirus disease 19 (COVID-19) presents with disease severities of varying degree. In its most severe form, infection may lead to respiratory failure and multi-organ dysfunction. Here we study the levels and evolution of the damage associated molecular patterns (DAMPS) cell free DNA (cfDNA), extracellular histone H3 (H3) and neutrophil elastase (NE), and the immune modulators GAS6 and AXL in relation to clinical parameters, ICU scoring systems and mortality in patients (n = 100) with severe COVID-19. cfDNA, H3, NE, GAS6 and AXL were increased in COVID-19 patients compared to controls. These measures associated with occurrence of clinical events and intensive care unit acquired weakness (ICUAW). cfDNA and GAS6 decreased in time in patients surviving to 30 days post ICU admission. A decrease of 27.2 ng/mL cfDNA during ICU stay associated with patient survival, whereas levels of GAS6 decreasing more than 4.0 ng/mL associated with survival. The presence of H3 in plasma was a common feature of COVID-19 patients, detected in 38% of the patients at ICU admission. NETosis markers cfDNA, H3 and NE correlated well with parameters of tissue damage and neutrophil counts. Furthermore, cfDNA correlated with lowest p/f ratio and a lowering in cfDNA was observed in patients with ventilator-free days.

Early prediction of COVID-19 severity using extracellular vesicle COPB2.

The clinical manifestations of COVID-19 vary broadly, ranging from asymptomatic infection to acute respiratory failure and death. But the predictive biomarkers for characterizing the variability are still lacking. Since emerging evidence indicates that extracellular vesicles (EVs) and extracellular RNAs (exRNAs) are functionally involved in a number of pathological processes, we hypothesize that these extracellular components may be key determinants and/or predictors of COVID-19 severity. To test our hypothesis, we collected serum samples from 31 patients with mild COVID-19 symptoms at the time of their admission for discovery cohort. After symptomatic treatment without corticosteroids, 9 of the 31 patients developed severe/critical COVID-19 symptoms. We analyzed EV protein and exRNA profiles to look for correlations between these profiles and COVID-19 severity. Strikingly, we identified three distinct groups of markers (antiviral response-related EV proteins, coagulation-related markers, and liver damage-related exRNAs) with the potential to serve as early predictive biomarkers for COVID-19 severity. As the best predictive marker, EV COPB2 protein, a subunit of the Golgi coatomer complex, exhibited significantly higher abundance in patients remained mild than developed severe/critical COVID-19 and healthy controls in discovery cohort (AUC 1.00 (95% CI: 1.00-1.00)). The validation set included 40 COVID-19 patients and 39 healthy controls, and showed exactly the same trend between the three groups with excellent predictive value (AUC 0.85 (95% CI: 0.73-0.97)). These findings highlight the potential of EV COPB2 expression for patient stratification and for making early clinical decisions about strategies for COVID-19 therapy.

ICODE: the international COVID-19 thrombosis biomarkers colloquium-novel soluble biomarkers: circulating cell-free nucleic acids and other molecules.

This article summarizes the evidence derived from clinical (observational) studies describing novel soluble biomarkers in COVID-19. Our goal was to stimulate further research (preclinical as well as clinical studies) and therefore we discuss potential prognostic value, but also technical details, such as sample preparation. A table provides an overview of the described biomarkers measured in plasma, serum or other (namely bronchoalveolar) fluids.

Circulating mitochondrial DNA is an early indicator of severe illness and mortality from COVID-19.

BackgroundMitochondrial DNA (MT-DNA) are intrinsically inflammatory nucleic acids released by damaged solid organs. Whether circulating cell-free MT-DNA quantitation could be used to predict the risk of poor COVID-19 outcomes remains undetermined.MethodsWe measured circulating MT-DNA levels in prospectively collected, cell-free plasma samples from 97 subjects with COVID-19 at hospital presentation. Our primary outcome was mortality. Intensive care unit (ICU) admission, intubation, vasopressor, and renal replacement therapy requirements were secondary outcomes. Multivariate regression analysis determined whether MT-DNA levels were independent of other reported COVID-19 risk factors. Receiver operating characteristic and area under the curve assessments were used to compare MT-DNA levels with established and emerging inflammatory markers of COVID-19.ResultsCirculating MT-DNA levels were highly elevated in patients who eventually died or required ICU admission, intubation, vasopressor use, or renal replacement therapy. Multivariate regression revealed that high circulating MT-DNA was an independent risk factor for these outcomes after adjusting for age, sex, and comorbidities. We also found that circulating MT-DNA levels had a similar or superior area under the curve when compared against clinically established measures of inflammation and emerging markers currently of interest as investigational targets for COVID-19 therapy.ConclusionThese results show that high circulating MT-DNA levels are a potential early indicator for poor COVID-19 outcomes.FundingWashington University Institute of Clinical Translational Sciences COVID-19 Research Program and Washington University Institute of Clinical Translational Sciences (ICTS) NIH grant UL1TR002345.

Cell-free DNA maps COVID-19 tissue injury and risk of death and can cause tissue injury.

INTRODUCTIONThe clinical course of coronavirus 2019 (COVID-19) is heterogeneous, ranging from mild to severe multiorgan failure and death. In this study, we analyzed cell-free DNA (cfDNA) as a biomarker of injury to define the sources of tissue injury that contribute to such different trajectories.METHODSWe conducted a multicenter prospective cohort study to enroll patients with COVID-19 and collect plasma samples. Plasma cfDNA was subject to bisulfite sequencing. A library of tissue-specific DNA methylation signatures was used to analyze sequence reads to quantitate cfDNA from different tissue types. We then determined the correlation of tissue-specific cfDNA measures to COVID-19 outcomes. Similar analyses were performed for healthy controls and a comparator group of patients with respiratory syncytial virus and influenza.RESULTSWe found markedly elevated levels and divergent tissue sources of cfDNA in COVID-19 patients compared with patients who had influenza and/or respiratory syncytial virus and with healthy controls. The major sources of cfDNA in COVID-19 were hematopoietic cells, vascular endothelium, hepatocytes, adipocytes, kidney, heart, and lung. cfDNA levels positively correlated with COVID-19 disease severity, C-reactive protein, and D-dimer. cfDNA profile at admission identified patients who subsequently required intensive care or died during hospitalization. Furthermore, the increased cfDNA in COVID-19 patients generated excessive mitochondrial ROS (mtROS) in renal tubular cells in a concentration-dependent manner. This mtROS production was inhibited by a TLR9-specific antagonist.CONCLUSIONcfDNA maps tissue injury that predicts COVID-19 outcomes and may mechanistically propagate COVID-19-induced tissue injury.FUNDINGIntramural Targeted Anti-COVID-19 grant, NIH.

Sensitive tracking of circulating viral RNA through all stages of SARS-CoV-2 infection.

BACKGROUNDCirculating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA may represent a more reliable indicator of infection than nasal RNA, but quantitative reverse transcription PCR (RT-qPCR) lacks diagnostic sensitivity for blood samples.METHODSA CRISPR-augmented RT-PCR assay that sensitively detects SARS-CoV-2 RNA was employed to analyze viral RNA kinetics in longitudinal plasma samples from nonhuman primates (NHPs) after virus exposure; to evaluate the utility of blood SARS-CoV-2 RNA detection for coronavirus disease 2019 (COVID-19) diagnosis in adults cases confirmed by nasal/nasopharyngeal swab RT-PCR results; and to identify suspected COVID-19 cases in pediatric and at-risk adult populations with negative nasal swab RT-qPCR results. All blood samples were analyzed by RT-qPCR to allow direct comparisons.RESULTSCRISPR-augmented RT-PCR consistently detected SARS-CoV-2 RNA in the plasma of experimentally infected NHPs from 1 to 28 days after infection, and these increases preceded and correlated with rectal swab viral RNA increases. In a patient cohort (n = 159), this blood-based assay demonstrated 91.2% diagnostic sensitivity and 99.2% diagnostic specificity versus a comparator RT-qPCR nasal/nasopharyngeal test, whereas RT-qPCR exhibited 44.1% diagnostic sensitivity and 100% specificity for the same blood samples. This CRISPR-augmented RT-PCR assay also accurately identified patients with COVID-19 using one or more negative nasal swab RT-qPCR results.CONCLUSIONResults of this study indicate that sensitive detection of SARS-CoV-2 RNA in blood by CRISPR-augmented RT-PCR permits accurate COVID-19 diagnosis, and can detect COVID-19 cases with transient or negative nasal swab RT-qPCR results, suggesting that this approach could improve COVID-19 diagnosis and the evaluation of SARS-CoV-2 infection clearance, and predict the severity of infection.TRIAL REGISTRATIONClinicalTrials.gov. NCT04358211.FUNDINGDepartment of Defense, National Institute of Allergy and Infectious Diseases, National Institute of Child Health and Human Development, and the National Center for Research Resources.

Chronic Active Antibody-Mediated Rejection Following COVID-19 Infection in a Kidney Transplant Recipient: A Case Report.

Kidney transplant recipients who develop coronavirus disease 2019 (COVID-19) are at increased risk of life-threatening illness, which often requires reducing immunosuppression despite the potential risk of causing an allograft rejection. Herein, we describe the clinical presentation and course of a kidney transplant recipient who acquired COVID-19 and was hospitalized with severe symptoms and hypoxemia. Upon admission, the patient was found to have elevated de novo donor-specific antibodies (DSA) yielding a positive cytotoxicity crossmatch and concurrent elevated plasma donor-derived cell-free DNA (dd-cfDNA) level, indicating a possible ongoing rejection despite improvement in his serum creatinine. Because of persistent positive COVID-19 tests and stable serum creatinine, a kidney allograft biopsy was initially deferred and his dd-cfDNA and DSA were monitored closely postdischarge. Three months later, because of persistent elevated dd-cfDNA and positive DSA, a kidney allograft biopsy was performed, which showed chronic active antibody-mediated rejection. Accordingly, the patient was treated with intravenous immunoglobulin and his maintenance immunosuppressive regimen was increased.

IgA2 Antibodies against SARS-CoV-2 Correlate with NET Formation and Fatal Outcome in Severely Diseased COVID-19 Patients.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to an adaptive immune response in the host and the formation of anti-SARS-CoV-2 specific antibodies. While IgG responses against SARS-CoV-2 have been characterized quite well, less is known about IgA. IgA2 activates immune cells and induces inflammation and neutrophil extracellular trap (NET) formation which may contribute to organ injury and fatal outcome in SARS-CoV-2-infected patients. SARS-CoV-2 spike protein specific antibody levels were measured in plasma samples of 15 noninfected controls and 82 SARS-CoV-2-infected patients with no or mild symptoms, moderate symptoms (hospitalization) or severe disease (intensive care unit, ICU). Antibody levels were compared to levels of C-reactive protein (CRP) and circulating extracellular DNA (ecDNA) as markers for general inflammation and NET formation, respectively. While levels of SARS-CoV-2-specific IgG were similar in all patient groups, IgA2 antibodies were restricted to severe disease and showed the strongest discrimination between nonfatal and fatal outcome in patients with severe SARS-CoV-2 infection. While anti-SARS-CoV-2 IgG and IgA2 levels correlated with CRP levels in severely diseased patients, only anti-SARS-CoV-2 IgA2 correlated with ecDNA. These data suggest that the formation of anti-SARS-CoV-2 IgA2 during SARS-CoV-2 infection is a marker for more severe disease related to NET formation and poor outcome.

Microbial cell-free DNA in plasma of patients with sepsis: a potential diagnostic methodology.

Sepsis is a life-threatening clinical condition demanding accurate and rapid diagnosis of the culprit pathogen, thereby to improve prognosis. Pathogen determination through blood culture is the gold standard for diagnosis but has limitations due to low sensitivity. Recently, circulating DNAs derived from pathogenic organisms were found in the plasma of patients with sepsis and were further proved to be more sensitive biomarkers for the diagnosis of the pathogen origin in sepsis. However, the fundamental molecular characteristics of circulating DNA in patients with sepsis remain unclear. Here, we used specific PCR and Sanger sequencing to verify the microbiology culture results via the corresponding plasma circulating DNA. We analyzed the composition and molecular characteristics of circulating DNA in septic patients using next-generation sequencing technology. We showed the presence of pathogen-derived circulating DNA in the plasma of patients with sepsis. The sizes of circulating DNA fragments derived from pathogenic bacteria showed a skewed unimodal distribution, while those derived from host cells showed a normal unimodal distribution. Lengths of fragments at peak concentration for both origins ranged from 150 bp to 200 bp, and reads mapping to pathogenic bacteria genome distributed uniformly on the reference. Our findings have improved our understanding of microbial circulating DNA in patients with sepsis as a potential methodology for the accurate diagnosis of sepsis, especially in light of an urgent need for such a diagnosis associated with the COVID-19 infection.

Use of DAMPs and SAMPs as Therapeutic Targets or Therapeutics: A Note of Caution.

This opinion article discusses the increasing attention paid to the role of activating damage-associated molecular patterns (DAMPs) in initiation of inflammatory diseases and suppressing/inhibiting DAMPs (SAMPs) in resolution of inflammatory diseases and, consequently, to the future roles of these novel biomarkers as therapeutic targets and therapeutics. Since controlled production of DAMPs and SAMPs is needed to achieve full homeostatic restoration and repair from tissue injury, only their pathological, not their homeostatic, concentrations should be therapeutically tackled. Therefore, distinct caveats are proposed regarding choosing DAMPs and SAMPs for therapeutic purposes. For example, we discuss the need to a priori identify and define a context-dependent "homeostatic DAMP:SAMP ratio" in each case and a "homeostatic window" of DAMP and SAMP concentrations to guarantee a safe treatment modality to patients. Finally, a few clinical examples of how DAMPs and SAMPs might be used as therapeutic targets or therapeutics in the future are discussed, including inhibition of DAMPs in hyperinflammatory processes (e.g., systemic inflammatory response syndrome, as currently observed in Covid-19), administration of SAMPs in chronic inflammatory diseases, inhibition of SAMPs in hyperresolving processes (e.g., compensatory anti-inflammatory response syndrome), and administration/induction of DAMPs in vaccination procedures and anti-cancer therapy.