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Since human angiotensin-converting enzyme 2 (ACE2) serves as a primary receptor for SARS-CoV-2, characterizing ACE2 regions that allow SARS-CoV-2 to enter human cells is essential for designing peptide-based antiviral blockers and elucidating the pathogenesis of the virus. We identified and synthesized a 25-mer mimetic peptide (encompassing positions 22-46 of the ACE2 alpha-helix α1) implicated in the S1 receptor-binding domain (RBD)-ACE2 interface. The mimetic (wild-type, WT) ACE2 peptide significantly inhibited SARS-CoV-2 infection of human pulmonary Calu-3 cells in vitro. In silico protein modeling predicted that residues F28, K31, F32, F40, and Y41 of the ACE2 alpha-helix α1 are critical for the original, Delta, and Omicron strains of SARS-CoV-2 to establish the Spike RBD-ACE2 interface. Substituting these residues with alanine (A) or aspartic acid (D) abrogated the antiviral protective effect of the peptides, indicating that these positions are critical for viral entry into pulmonary cells. WT ACE2 peptide, but not the A or D mutated peptides, exhibited significant interaction with the SARS-CoV-2 S1 RBD, as shown through molecular dynamics simulations. Through identifying the critical amino acid residues of the ACE2 alpha-helix α1, which is necessary for the Spike RBD-ACE2 interface and mobilized during the in vitro viral infection of cells, we demonstrated that the WT ACE2 peptide protects susceptible K18-hACE2 mice against in vivo SARS-CoV-2 infection and is effective for the treatment of COVID-19.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Péptidos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Humanos , Animales , SARS-CoV-2/efectos de los fármacos , COVID-19/virología , Ratones , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Péptidos/farmacología , Péptidos/química , Péptidos/uso terapéutico , Tratamiento Farmacológico de COVID-19 , Antivirales/farmacología , Antivirales/química , Línea Celular , Neumonía/tratamiento farmacológico , Neumonía/virología , Neumonía/prevención & control , Pulmón/virología , Pulmón/patología , FemeninoRESUMEN
BackgroundCOVID-19 (coronavirus disease 2019) is a disease caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), affecting millions of people worldwide, with a high rate of deaths. The present study aims to evaluate ultrasound (US) as a physical method for virus inactivation. Materials and methodsThe US-transductor was exposed to the SARS-CoV-2 viral solution for 30 minutes. Vero-E6 cells were infected with medium exposure or not with the US, using 3-12, 5-10, or 6-18MHz as frequencies applied. We performed confocal microscopy to determine virus infection and replicative process. Moreover, we detected the virus particles with a titration assay. ResultsWe observed an effective infection of SARS-CoV-2 Wuhan, Delta, and Gamma strains in comparison with mock, an uninfected experimental group. The US treatment was able to inhibit the Wuhan strain in all applied frequencies. Interestingly, 3-12 and 6-18MHz did not inhibit SARS-CoV-2 delta and gamma variants infection, on the other hand, 5-10MHz was able to abrogate infection and replication in all experimental conditions. ConclusionsThese results show that SARS-CoV-2 is susceptible to US exposure at a specific frequency 5-10MHz and could be a novel tool for reducing the incidence of SARS-CoV-2 infection.
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Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS) that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that complement component 5a (C5a), through its cellular receptor C5aR1, has potent proinflammatory actions, and plays immunopathological roles in inflammatory diseases, we investigated whether C5a/C5aR1 pathway could be involved in COVID-19 pathophysiology. C5a/C5aR1 signaling increased locally in the lung, especially in neutrophils of critically ill COVID-19 patients compared to patients with influenza infection, as well as in the lung tissue of K18-hACE2 Tg mice (Tg mice) infected with SARS-CoV-2. Genetic and pharmacological inhibition of C5aR1 signaling ameliorated lung immunopathology in Tg-infected mice. Mechanistically, we found that C5aR1 signaling drives neutrophil extracellular trap (NET)s-dependent immunopathology. These data confirm the immunopathological role of C5a/C5aR1 signaling in COVID-19 and indicate that antagonist of C5aR1 could be useful for COVID-19 treatment.
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Infection with SARS-CoV-2 induces COVID-19, an inflammatory disease that is usually self-limited, but depending on patient conditions may culminate with critical illness and patient death. The virus triggers activation of intracellular sensors, such as the NLRP3 inflammasome, which promotes inflammation and aggravates the disease. Thus, identification of host components associated with NLRP3 inflammasome is key for understanding the physiopathology of the disease. Here, we reported that SARS-CoV-2 induces upregulation and activation of human Caspase-4/CASP4 (mouse Caspase-11/CASP11) and this process contributes to inflammasome activation in response to SARS-CoV-2. CASP4 was expressed in lung autopsy of lethal cases of COVID-19 and CASP4 expression correlates with expression of inflammasome components and inflammatory mediators such as CASP1, IL1B, IL18 and IL6. In vivo infections performed in transgenic hACE2 humanized mouse, deficient or sufficient for Casp11, indicate that hACE2 Casp11-/- mice were protected from disease development, with reduced body weight loss, reduced temperature variation, increased pulmonary parenchymal area, reduced clinical score of the disease and reduced mortality. Collectively, our data establishes that CASP4/11 contributes to disease pathology and contributes for future immunomodulatory therapeutic interventions to COVID-19.
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Inflammasome activation is associated with disease severity in patients who are infected with SARS-CoV-2 and influenza viruses, but the specific cell types involved in inflammasome activation, as well as the balance of inflammasome activation versus viral replication in COVID-19 exacerbation and the induction of patient death, are unknown. In this study, we assessed lung autopsies of 47 COVID-19 and 12 influenza fatal cases and examined the inflammatory profiles and inflammasome activation; additionally, we correlated these factors with clinical and histopathological patient conditions. We observed an overall stronger inflammasome activation in lethal cases of SARS-CoV-2 compared to influenza and found a different profile of inflammasome-activating cells during these diseases. In COVID-19 patients, inflammasome activation is mostly mediated by macrophages and endothelial cells, whereas in influenza, type I and type II pneumocytes contribute more significantly. An analysis of gene expression allowed for the classification of COVID-19 patients into two different clusters. Cluster 1 (n=16 patients) died with higher viral loads and exhibited a reduced inflammatory profile than Cluster 2 (n=31 patients). Illness time, mechanical ventilation time, pulmonary fibrosis, respiratory functions, histopathological status, thrombosis, and inflammasome activation significantly differed between the two clusters. Our data demonstrated two distinct profiles in lethal cases of COVID-19, thus indicating that the balance of viral replication and inflammasome-mediated pulmonary inflammation may lead to different clinical conditions, yet both lead to patient death. An understanding of this process is critical for decisions between immune-mediated or antiviral-mediated therapies for the treatment of critical cases of COVID-19.
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COVID-19 is characterized by severe acute lung injury, which is associated with neutrophils infiltration and release of neutrophil extracellular traps (NETs). COVID-19 treatment options are scarce. Previous work has shown an increase in NETs release in the lung and plasma of COVID-19 patients suggesting that drugs that prevent NETs formation or release could be potential therapeutic approaches for COVID-19 treatment. Here, we report the efficacy of NET-degrading DNase I treatment in a murine model of COVID-19. DNase I decreased detectable levels of NETs, improved clinical disease, and reduced lung, heart, and kidney injuries in SARS-CoV-2-infected K18-hACE2 mice. Furthermore, our findings indicate a potential deleterious role for NETs lung tissue in vivo and lung epithelial (A549) cells in vitro, which might explain part of the pathophysiology of severe COVID-19. This deleterious effect was diminished by the treatment with DNase I. Together, our results support the role of NETs in COVID-19 immunopathology and highlight NETs disruption pharmacological approaches as a potential strategy to ameliorate COVID-19 clinical outcomes.
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The release of neutrophil extracellular traps (NETs) is associated with inflammation, coagulopathy, and organ damage found in severe cases of COVID-19. However, the molecular mechanisms underlying the release of NETs in COVID-19 remain unclear. Using a single-cell transcriptome analysis we observed that the expression of GSDMD and inflammasome-related genes were increased in neutrophils from COVID-19 patients. Furthermore, high expression of GSDMD was found associated with NETs structures in the lung tissue of COVID-19 patients. The activation of GSDMD in neutrophils requires live SARS-CoV-2 and occurs after neutrophil infection via ACE2 receptors and serine protease TMPRSS2. In a mouse model of SARS-CoV-2 infection, the treatment with GSDMD inhibitor (disulfiram) reduced NETs release and organ damage. These results demonstrated that GSDMD-dependent NETosis plays a critical role in COVID-19 immunopathology, and suggests that GSDMD inhibitors, can be useful to COVID-19 treatment. In BriefHere, we showed that the activation of the Gasdermin-D (GSDMD) pathway in neutrophils controls NET release during COVID-19. The inhibition of GSDMD with disulfiram, abrogated NET formation reducing lung inflammation and tissue damage. These findings suggest GSDMD as a target for improving the COVID-19 therapy.
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This study aimed to evaluate the efficacy and toxicity of tenofovir (TDF) and TDF combined with emtricitabine (TDF/FTC) in patients with mild to moderate COVID-19 infections. We conducted a randomized, double-blind, placebo-controlled clinical trial in patients with clinical suspicion of mild to moderate respiratory infection caused by SARS-CoV-2 who were treated at an outpatient clinic. Patients were randomly recruited to take 10 days of TDF (300 mg/day), TDF (300 mg/day) combined with FTC (200 mg/day) or placebo Vitamin C (500 mg/day). The primary parameter was the score of symptoms and predictive signs of COVID-19, assessed on the seventh day of patient follow-up. From a total of 309 patients with clinical suspicion of SARS-CoV-2, 227 met the inclusion criteria and were randomly distributed into the following groups: (a) 75 (one did not initiate treatment) in the TDF group; (b) 74 in the TDF combined with FTC group; and (c) 77 in the Vitamin C group (placebo). Of the 226 patients, 139 (62%) were positive for SARS-CoV-2. Fever ([≥]37.8{degrees}C), ageusia or dysgeusia, anosmia or dysosmia, and two or more clinical symptoms or signs were significantly associated with SARS-CoV-2 infection. There was no significant change in clinical score based on clinical symptoms and signs between treatment groups. Patients with mild to moderate infection by SARS-CoV-2 had higher concentrations of G-CSF, IL-1{beta}, IL-6 and TNF- compared to patients without infection. Patients with mild to moderate respiratory infection, with fever ([≥]37.8{degrees}C), loss of smell, loss of taste and two or more symptoms, have a better prediction for the diagnosis of COVID-19. Patients with SARS-CoV-2 showed higher and more persistent proinflammatory cytokines profile compared to patients not infected with SARS-CoV-2. Pharmacological intervention with TDF or TDF combined with FTC did not change the clinical signs and symptoms score in mild to moderate respiratory infection in patients with SARS-CoV-2 compared to the Vitamin C group (placebo).
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BackgroundPatients with coronavirus disease-2019 (COVID-19) present varying clinical complications. Different viral load and host response related to genetic and immune background are probably the reasons for these differences. We aimed to sought clinical and pathological correlation that justifies the different clinical outcomes among COVID-19 autopsies cases. MethodsMinimally invasive autopsy was performed on forty-seven confirmed COVID-19 patients from May-July, 2020. Electronic medical record of all patients was collected and a comprehensive histopathological evaluation was performed. Immunohistochemistry, immunofluorescence, special stain, western blotting and post-mortem real-time reverse transcriptase polymerase chain reaction on fresh lung tissue were performed. ResultsWe show that 5/47 (10,6%) patients present a progressive decline in oxygenation index for acute respiratory distress syndrome (PaO2/FiO2 ratio), low compliance levels, interstitial fibrosis, high -SMA+ cells/protein expression, high collagens I/III deposition and NETs(P<0.05), named as fibrotic phenotype (N=5). Conversely, 10/47 (21,2%) patients demonstrated progressive increase in PaO2/FiO2 ratio, high pulmonary compliance levels, preserved elastic framework, increase thrombus formation and high platelets and D-dimer levels at admission (P<0.05), named as thrombotic phenotype. While 32/47 (68,1%) had a mixed phenotypes between both ones. ConclusionsWe believe that categorization of patients based on these two phenotypes can be used to develop prognostic tools and potential therapies since the PaO2/FiO2 ratio variation and D-dimer levels correlate with the underlying fibrotic or thrombotic pathologic process, respectively; which may indicate possible clinical outcome of the patient.
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COVID-19 is a disease of dysfunctional immune responses, but the mechanisms triggering immunopathogenesis are not established. The functional plasticity of macrophages allows this cell type to promote pathogen elimination and inflammation or suppress inflammation and promote tissue remodeling and injury repair. During an infection, the clearance of dead and dying cells, a process named efferocytosis, can modulate the interplay between these contrasting functions. Here, we show that engulfment of SARS-CoV2-infected apoptotic cells exacerbates inflammatory cytokine production, inhibits the expression of efferocytic receptors, and impairs continual efferocytosis by macrophages. We also provide evidence supporting that lung monocytes and macrophages from severe COVID-19 patients have compromised efferocytic capacity. Our findings reveal that dysfunctional efferocytosis of SARS-CoV-2-infected cell corpses suppress macrophage anti-inflammation and efficient tissue repair programs and provide mechanistic insights for the excessive production of pro-inflammatory cytokines and accumulation of tissue damage associated with COVID-19 immunopathogenesis.
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IntroductionNeutrophilia and high levels of proinflammatory cytokines and other mediators of inflammation are common finds in patients with severe acute respiratory syndrome due to COVID-19. By its action on leukocytes, we propose colchicine as an intervention worthy of being tested. ObjectiveTo evaluate whether the addition of colchicine to standard treatment for COVID-19 results in better outcomes. MethodsWe present the interim analysis of a single-center randomized, double-blinded, placebo controlled clinical trial of colchicine for the treatment of moderate to severe COVID-19, with 38 patients allocated 1:1 from April 11 to July 06, 2020. Colchicine regimen was 0.5 mg thrice daily for 5 days, then 0.5 mg twice daily for 5 days. The first dose was 1.0 mg whether body weight was [≥] 80 kg. EndpointsThe primary endpoints were the need for supplemental oxygen; time of hospitalization; need for admission and length of stay in intensive care units; and death rate and causes of mortality. As secondary endpoints, we assessed: serum C-reactive protein, serum Lactate dehydrogenase and relation neutrophil to lymphocyte of peripheral blood samples from day zero to day 7; the number, type, and severity of adverse events; frequency of interruption of the study protocol due to adverse events; and frequency of QT interval above 450 ms. ResultsThirty-five patients (18 for Placebo and 17 for Colchicine) completed the study. Both groups were comparable in terms of demographic, clinical and laboratory data at baseline. Median (and interquartile range) time of need for supplemental oxygen was 3.0 (1.5-6.5) days for the Colchicine group and 7.0 (3.0-8.5) days for Placebo group (p = 0.02). Median (IQR) time of hospitalization was 6.0 (4.0-8.5) days for the Colchicine group and 8.5 (5.5-11.0) days for Placebo group (p = 0.03). At day 2, 53% vs 83% of patients maintained the need for supplemental oxygen, while at day 7 the values were 6% vs 39%, in the Colchicine and Placebo groups, respectively (log rank; p = 0.01). Hospitalization was maintained for 53% vs 78% of patients at day 5 and 6% vs 17% at day 10, for the Colchicine and Placebo groups, respectively (log rank; p = 0.01). One patient per group needed admission to ICU. No recruited patient died. At day 4, patients of Colchicine group presented significant reduction of serum C-reactive protein compared to baseline (p < 0.001). The majority of adverse events were mild and did not lead to patient withdrawal. Diarrhea was more frequent in the Colchicine group (p = 0.17). Cardiac adverse events were absent. DiscussionThe use of colchicine reduced the length of supplemental oxygen therapy and the length of hospitalization. Clinical improvement was in parallel with a reduction on serum levels of C-reactive protein. The drug was safe and well tolerated. Colchicine may be considered a beneficial and not expensive option for COVID-19 treatment. Clinical trials with larger numbers of patients should be conducted to further evaluate the efficacy and safety of colchicine as an adjunctive therapy for hospitalized patients with moderate to severe COVID-19.
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Severe cases of COVID-19 are characterized by a strong inflammatory process that may ultimately lead to organ failure and patient death. The NLRP3 inflammasome is a molecular platform that promotes inflammation via cleavage and activation of key inflammatory molecules including active caspase-1 (Casp1p20), IL-1{beta} and IL-18. Although the participation of the inflammasome in COVID-19 has been highly speculated, the inflammasome activation and participation in the outcome of the disease is unknown. Here we demonstrate that the NLRP3 inflammasome is activated in response to SARS-CoV-2 infection and it is active in COVID-19, influencing the clinical outcome of the disease. Studying moderate and severe COVID-19 patients, we found active NLRP3 inflammasome in PBMCs and tissues of post-mortem patients upon autopsy. Inflammasome-derived products such as Casp1p20 and IL-18 in the sera correlated with the markers of COVID-19 severity, including IL-6 and LDH. Moreover, higher levels of IL-18 and Casp1p20 are associated with disease severity and poor clinical outcome. Our results suggest that the inflammasome is key in the pathophysiology of the disease, indicating this platform as a marker of disease severity and a potential therapeutic target for COVID-19.
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Although SARS-CoV-2 severe infection is associated with a hyperinflammatory state, lymphopenia is an immunological hallmark, and correlates with poor prognosis in COVID-19. However, it remains unknown if circulating human lymphocytes and monocytes are susceptible to SARS-CoV-2 infection. In this study, SARS-CoV-2 infection of human peripheral blood mononuclear cells (PBMCs) was investigated both in vitro and in vivo. We found that in vitro infection of whole PBMCs from healthy donors was productive of virus progeny. Results revealed that monocytes, as well as B and T lymphocytes, are susceptible to SARS-CoV-2 active infection and viral replication was indicated by detection of double-stranded RNA. Moreover, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from COVID-19 patients, and less frequently in CD4+T lymphocytes. The rates of SARS-CoV-2-infected monocytes in PBMCs from COVID-19 patients increased over time from symptom onset. Additionally, SARS-CoV-2-positive monocytes and B and CD4+T lymphocytes were detected by immunohistochemistry in post mortem lung tissue. SARS-CoV-2 infection of blood circulating leukocytes in COVID-19 patients may have important implications for disease pathogenesis, immune dysfunction, and virus spread within the host.
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Severe COVID-19 patients develop acute respiratory distress syndrome that may progress to respiratory failure. These patients also develop cytokine storm syndrome, and organ dysfunctions, which is a clinical picture that resembles sepsis. Considering that neutrophil extracellular traps (NETs) have been described as an important factors of tissue damage in sepsis, we investigated whether NETs would be produced in COVID-19 patients and participate in the lung tissue damage. A cohort of 32 hospitalized patients with a confirmed diagnosis of COVID-19 and respective healthy controls were enrolled. NETs concentration was assessed by MPO-DNA PicoGreen assay or by confocal immunofluorescence. The cytotoxic effect of SARS-CoV-2-induced NETs was analyzed in human epithelial lung cells (A549 cells). The concentration of NETs was augmented in plasma and tracheal aspirate from COVID-19 patients and their neutrophils spontaneously released higher levels of NETs. NETs were also found in the lung tissue specimens from autopsies of COVID-19 patients. Notably, viable SARS-CoV-2 can directly induce in vitro release of NETs by healthy neutrophils in a PAD-4-dependent manner. Finally, NETs released by SARS-CoV-2-activated neutrophils promote lung epithelial cell death in vitro. These results unravel a possible detrimental role of NETs in the pathophysiology of COVID-19. Therefore, the inhibition of NETs represent a potential therapeutic target for COVID-19.
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BACKGROUND: Human respiratory syncytial virus (HRSV) is one of the major etiologic agents of respiratory tract infections among children worldwide. METHODOLOGY/PRINCIPAL FINDINGS: Here through a comprehensive analysis of the two major HRSV groups A and B (n=1983) which comprise of several genotypes, we present a complex pattern of population dynamics of HRSV over a time period of 50 years (1956-2006). Circulation pattern of HRSV revealed a series of expansions and fluctuations of co-circulating lineages with a predominance of HRSVA. Positively selected amino acid substitutions of the G glycoprotein occurred upon population growth of GB3 with a 60-nucleotide insertion (GB3 Insert), while other genotypes acquired substitutions upon both population growth and decrease, thus possibly reflecting a role for immune selected epitopes in linkage to the traced substitution sites that may have important relevance for vaccine design. Analysis evidenced the co-circulation and predominance of distinct HRSV genotypes in Brazil and suggested a year-round presence of the virus. In Brazil, GA2 and GA5 were the main culprits of HRSV outbreaks until recently, when the GB3 Insert became highly prevalent. Using Bayesian methods, we determined the dispersal patterns of genotypes through several inferred migratory routes. CONCLUSIONS/SIGNIFICANCE: Genotypes spread across continents and between neighboring areas. Crucially, genotypes also remained at any given region for extended periods, independent of seasonal outbreaks possibly maintained by re-infecting the general population.