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1.
J Biol Chem ; 295(36): 12686-12696, 2020 09 04.
Article in English | MEDLINE | ID: covidwho-1387615

ABSTRACT

Type II transmembrane serine proteases (TTSPs) are a group of enzymes participating in diverse biological processes. Some members of the TTSP family are implicated in viral infection. TMPRSS11A is a TTSP expressed on the surface of airway epithelial cells, which has been shown to cleave and activate spike proteins of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome coronaviruses (CoVs). In this study, we examined the mechanism underlying the activation cleavage of TMPRSS11A that converts the one-chain zymogen to a two-chain enzyme. By expression in human embryonic kidney 293, esophageal EC9706, and lung epithelial A549 and 16HBE cells, Western blotting, and site-directed mutagenesis, we found that the activation cleavage of human TMPRSS11A was mediated by autocatalysis. Moreover, we found that TMPRSS11A activation cleavage occurred before the protein reached the cell surface, as indicated by studies with trypsin digestion to remove cell surface proteins, treatment with cell organelle-disturbing agents to block intracellular protein trafficking, and analysis of a soluble form of TMPRSS11A without the transmembrane domain. We also showed that TMPRSS11A was able to cleave the SARS-CoV-2 spike protein. These results reveal an intracellular autocleavage mechanism in TMPRSS11A zymogen activation, which differs from the extracellular zymogen activation reported in other TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation.


Subject(s)
Epithelial Cells/metabolism , Membrane Proteins/metabolism , Proteolysis , Serine Proteases/metabolism , A549 Cells , Cells, Cultured , HEK293 Cells , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mutation , Protein Domains , Protein Transport , Respiratory Mucosa/cytology , Serine Proteases/chemistry , Serine Proteases/genetics , Spike Glycoprotein, Coronavirus/metabolism , Trypsin/metabolism
2.
Lancet Microbe ; 1(7): e290-e299, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-1087376

ABSTRACT

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets multiple organs and causes severe coagulopathy. Histopathological organ changes might not only be attributable to a direct virus-induced effect, but also the immune response. The aims of this study were to assess the duration of viral presence, identify the extent of inflammatory response, and investigate the underlying cause of coagulopathy. Methods: This prospective autopsy cohort study was done at Amsterdam University Medical Centers (UMC), the Netherlands. With informed consent from relatives, full body autopsy was done on 21 patients with COVID-19 for whom autopsy was requested between March 9 and May 18, 2020. In addition to histopathological evaluation of organ damage, the presence of SARS-CoV-2 nucleocapsid protein and the composition of the immune infiltrate and thrombi were assessed, and all were linked to disease course. Findings: Our cohort (n=21) included 16 (76%) men, and median age was 68 years (range 41-78). Median disease course (time from onset of symptoms to death) was 22 days (range 5-44 days). In 11 patients tested for SARS-CoV-2 tropism, SARS-CoV-2 infected cells were present in multiple organs, most abundantly in the lungs, but presence in the lungs became sporadic with increased disease course. Other SARS-CoV-2-positive organs included the upper respiratory tract, heart, kidneys, and gastrointestinal tract. In histological analyses of organs (sampled from nine to 21 patients per organ), an extensive inflammatory response was present in the lungs, heart, liver, kidneys, and brain. In the brain, extensive inflammation was seen in the olfactory bulbs and medulla oblongata. Thrombi and neutrophilic plugs were present in the lungs, heart, kidneys, liver, spleen, and brain and were most frequently observed late in the disease course (15 patients with thrombi, median disease course 22 days [5-44]; ten patients with neutrophilic plugs, 21 days [5-44]). Neutrophilic plugs were observed in two forms: solely composed of neutrophils with neutrophil extracellular traps (NETs), or as aggregates of NETs and platelets.. Interpretation: In patients with lethal COVID-19, an extensive systemic inflammatory response was present, with a continued presence of neutrophils and NETs. However, SARS-CoV-2-infected cells were only sporadically present at late stages of COVID-19. This suggests a maladaptive immune response and substantiates the evidence for immunomodulation as a target in the treatment of severe COVID-19. Funding: Amsterdam UMC Corona Research Fund.

3.
Autoimmun Rev ; 20(4): 102792, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-1086776

ABSTRACT

Autoimmunity may be generated by a variety of factors by creating a hyper-stimulated state of the immune system. It had been established long ago that viruses are a substantial component of environmental factors that contribute to the production of autoimmune antibodies, as well as autoimmune diseases. Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human immunodeficiency virus (HIV) are viruses that withhold these autoimmune abilities. In a similar manner, SARS-CoV-2 may be counted to similar manifestations, as numerous records demonstrating the likelihood of COVID-19 patients to develop multiple types of autoantibodies and autoimmune diseases. In this review, we focused on the association between COVID-19 and the immune system concerning the tendency of patients to develop over 15 separate types of autoantibodies and above 10 distinct autoimmune diseases. An additional autoimmunity manifestation may be one of the common initial symptoms in COVID-19 patients, anosmia, the complete loss of the ability to sense smell, and other olfactory alterations. We summarize current knowledge on principal mechanisms that may contribute to the development of autoimmunity in the disease: the ability of SARS-CoV-2 to hyper-stimulate the immune system, induce excessive neutrophil extracellular traps formation with neutrophil-associated cytokine responses and the molecular resemblance between self-components of the host and the virus. Additionally, we will examine COVID-19 potential risk on the new-onsets of autoimmune diseases, such as antiphospholipid syndrome, Guillain-Barré syndrome, Kawasaki disease and numerous others. It is of great importance to recognize those autoimmune manifestations of COVID-19 in order to properly cope with their outcomes in the ongoing pandemic and the long-term post-pandemic period. Lastly, an effective vaccine against SARS-CoV-2 may be the best solution in dealing with the ongoing pandemic. We will discuss the new messenger RNA vaccination strategy with an emphasis on autoimmunity implications.


Subject(s)
Autoimmune Diseases , COVID-19 , Epstein-Barr Virus Infections , Autoimmunity , COVID-19 Vaccines , Herpesvirus 4, Human , Humans , SARS-CoV-2
4.
Nephrologe ; : 1-6, 2020 Dec 11.
Article in German | MEDLINE | ID: covidwho-986660

ABSTRACT

The aim of this article is to explain the clinical benefits of the growing knowledge about severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to the lungs, SARS-CoV­2 can invade multiple cell types in other organs, such as the kidneys and replicate there. Important damaging pathways of the virus, such as vascular endotheliitis, thrombotic events and systemic cytokine release are still incompletely understood. Coronavirus disease 2019 (COVID-19) is a systemic disease that necessitates intensive medical care and in particular, internal medicine involvement and represents a major challenge for all disciplines of internal medicine. Among these, nephrology in particular is involved in the fight against COVID-19 in a variety of ways: urine investigations can provide indications of multiple organ involvement, endotheliitis, microthrombi and microcirculation damage, etc. Experience with low serum albumin levels and antithrombin III activity in nephrotic patients helps to point out the decreasing effects of loop diuretics and heparin to other specialist disciplines. Nephrological knowledge of the complications of hypoalbuminemia and "resistance" to diuretics must lead to an early implementation of renal replacement procedures in order to be able to prevent mechanical ventilation in COVID-19 intensive care patients with increased extracellular lung fluid. The kidneys can be used as a seismograph for severe courses of COVID-19 and nephrological knowledge can be brought to use to optimize the intensive medical care for critically ill patients. Both together have the potential to considerably reduce morbidity and mortality further.

5.
Cells ; 9(12)2020 12 12.
Article in English | MEDLINE | ID: covidwho-971834

ABSTRACT

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.


Subject(s)
Antibodies, Viral/blood , COVID-19/immunology , Extracellular Traps/immunology , Immunoglobulin A/blood , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Aged, 80 and over , Biomarkers/blood , C-Reactive Protein/immunology , COVID-19/epidemiology , Case-Control Studies , Cell-Free Nucleic Acids/blood , Female , Humans , Male , Middle Aged , SARS-CoV-2 , Severity of Illness Index , Young Adult
6.
J Clin Med ; 9(9)2020 Sep 11.
Article in English | MEDLINE | ID: covidwho-892446

ABSTRACT

Understanding of the pathogenesis of the coronavirus disease-2019 (COVID-19) remains incomplete, particularly in respect to the multi-organ dysfunction it may cause. We were the first to report the analogous biological and physiological features of COVID-19 pathogenesis and the harmful amplification loop between inflammation and tissue damage induced by the dysregulation of neutrophil extracellular traps (NETs) formation. Given the rapid evolution of this disease, the nature of its symptoms, and its potential lethality, we hypothesize that COVID-19 progresses under just such an amplifier loop, leading to a massive, uncontrolled inflammation process. Here, we describe in-depth the correlations of COVID-19 symptoms and biological features with those where uncontrolled NET formation is implicated in various sterile or infectious diseases. General clinical conditions, as well as numerous pathological and biological features, are analogous with NETs deleterious effects. Among NETs by-products implicated in COVID-19 pathogenesis, one of the most significant appears to be elastase, in accelerating virus entry and inducing hypertension, thrombosis and vasculitis. We postulate that severe acute respiratory syndrome-coronavirus 2 (SARS-CoV2) may evade innate immune response, causing uncontrolled NETs formation and multi-organ failure. In addition, we point to indicators that NETS-associated diseases are COVID-19 risk factors. Acknowledging that neutrophils are the principal origin of extracellular and circulating DNA release, we nonetheless, explain why targeting NETs rather than neutrophils themselves may in practice be a better strategy. This paper also offers an in-depth review of NET formation, function and pathogenic dysregulation, as well as of current and prospective future therapies to control NETopathies. As such, it enables us also to suggest new therapeutic strategies to fight COVID-19. In combination with or independent of the latest tested approaches, we propose the evaluation, in the short term, of treatments with DNase-1, with the anti-diabetic Metformin, or with drugs targeting elastase (i.e., Silvelestat). With a longer perspective, we also advocate a significant increase in research on the development of toll-like receptors (TLR) and C-type lectin-like receptors (CLEC) inhibitors, NET-inhibitory peptides, and on anti-IL-26 therapies.

7.
Microb Risk Anal ; 16: 100140, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-779468

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) infect the human respiratory tract. A prototype thermodynamic equilibrium model is presented here for the probability of the virions getting through the mucus barrier and infecting epithelial cells based on the binding affinity (Kmucin) of the virions to mucin molecules in the mucus and parameters for binding and infection of the epithelial cell. Both MERS-CoV and SARS-CoV-2 bind strongly to their cellular receptors, DDP4 and ACE2, respectively, and infect very efficiently both bronchus and lung ex vivo cell cultures which are not protected by a mucus barrier. According to the model, mucin binding could reduce the infectivity for MERS-CoV compared to SARS-CoV-2 by at least 100-fold depending on the magnitude of Kmucin. Specifically Kmucin values up to 106 M-1 have little protective effect and thus the mucus barrier would not remove SARS-CoV-2 which does not bind to sialic acids (SA) and hence would have a very low Kmucin. Depending on the viability of individual virions, the ID50 for SARS-CoV-2 is estimated to be ~500 virions (viral RNA genomic copies) representing 1 to 2 pfu. In contrast MERS-CoV binds both SA and human mucin and a Kmucin of 5 × 109 M-1 as reported for lectins would mop up 99.83% of the virus according to the model with the ID50 for MERS-CoV estimated to be ~295,000 virions (viral RNA genomic copies) representing 819 pfu. This could in part explain why MERS-CoV is poorly transmitted from human to human compared to SARS-CoV-2. Some coronaviruses use an esterase to escape the mucin, although MERS-CoV does not. Instead, it is shown here that "clustering" of virions into single aerosol particles as recently reported for rotavirus in extracellular vesicles could provide a co-operative mechanism whereby MERS-CoV could theoretically overcome the mucin barrier locally and a small proportion of 10 µm diameter aerosol particles could contain ~70 virions based on reported maximum levels in saliva. Although recent evidence suggests SARS-CoV-2 initiates infection in the nasal epithelium, the thermodynamic equilibrium models presented here could complement published approaches for modelling the physical entry of pathogens to the lung based on the fate and transport of the pathogen particles (as for anthrax spores) to develop a dose-response model for aerosol exposure to respiratory viruses. This would enable the infectivity through aerosols to be defined based on molecular parameters as well as physical parameters. The role of the spike proteins of MERS-CoV and SARS-CoV-2 binding to SA and heparan sulphate, respectively, may be to aid non-specific attachment to the host cell. It is proposed that a high Kmucin is the cost for subsequent binding of MERS-CoV to SAs on the cell surface to partially overcome the unfavourable entropy of immobilisation as the virus adopts the correct orientation for spike protein interactions with its protein cellular receptor DPP4.

8.
Biochem Biophys Res Commun ; 533(1): 195-200, 2020 11 26.
Article in English | MEDLINE | ID: covidwho-753910

ABSTRACT

The pandemic of COVID-19 is spreading unchecked due to the lack of effective antiviral measures. Silver nanoparticles (AgNP) have been studied to possess antiviral properties and are presumed to inhibit SARS-CoV-2. Due to the need for an effective agent against SARS-CoV-2, we evaluated the antiviral effect of AgNPs. We evaluated a plethora of AgNPs of different sizes and concentration and observed that particles of diameter around 10 nm were effective in inhibiting extracellular SARS-CoV-2 at concentrations ranging between 1 and 10 ppm while cytotoxic effect was observed at concentrations of 20 ppm and above. Luciferase-based pseudovirus entry assay revealed that AgNPs potently inhibited viral entry step via disrupting viral integrity. These results indicate that AgNPs are highly potent microbicides against SARS-CoV-2 but should be used with caution due to their cytotoxic effects and their potential to derange environmental ecosystems when improperly disposed.


Subject(s)
Antiviral Agents/administration & dosage , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Metal Nanoparticles/administration & dosage , Pneumonia, Viral/drug therapy , Silver/administration & dosage , Animals , Antiviral Agents/toxicity , Betacoronavirus/physiology , COVID-19 , Cell Line , Cell Survival/drug effects , Chlorocebus aethiops , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Dose-Response Relationship, Drug , Humans , Metal Nanoparticles/toxicity , Metal Nanoparticles/ultrastructure , Pandemics , Particle Size , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , SARS-CoV-2 , Silver/toxicity , Vero Cells , Virus Internalization/drug effects
9.
Front Mol Biosci ; 7: 197, 2020.
Article in English | MEDLINE | ID: covidwho-732872

ABSTRACT

Here we report our perspective on applying GapmeR technology in combination with recombinant angiotensin-converting enzyme 2 (ACE2) in the treatment of COVID-19 patients. GapmeR is a cell-permeating antisense single-stranded DNA molecule that can be designed to specifically target intracellular severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Once internalized into host cells, such as lung alveolar cells, GapmeR molecules can bind to the viral RNA. This RNA/DNA hybrid will then be degraded by the RNase H enzyme abundantly present in the host cells. GapmeRs can be delivered to COVID-19 patients through inhalation or via nebulization. SARS-CoV-2-targeted GapmeR can also be given to frontline healthcare workers as a prophylactic protection. The recombinant ACE2 protein, the efficacy of which is being evaluated in clinical trials, will bind to the spike (S) glycoprotein of extracellular SARS-CoV-2 and potentially block viral infectivity. We propose that combining inhalable SARS-CoV-2-targeted GapmeRs with recombinant ACE2 could provide a viable and rapidly implementable more effective therapeutic approach for eradicating SARS-CoV-2 and save millions of lives.

10.
Med Hypotheses ; 144: 110030, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-612684

ABSTRACT

Novel Coronavirus (SARS CoV-2), the etiological agent for the highly contagious Corona virus disease-2019 (COVID-19) pandemic has threatened global health and economy infecting around 5.8 million people and causing over 359,200 deaths (as of 28th May 2020, https://www.worldometers.info/coronavirus/). The clinical manifestations of infected patients generally range from asymptomatic or mild to severe illness, or even death. The ability of the virus to evade the host immune response have been major reasons for high morbidity and mortality. One of the important clinical observations under conditions of critical illness show increased risk of developing disseminated intravascular coagulation. Molecular mechanisms of how SARS CoV-2 induces such conditions still remain unclear. This report describes the presence of two unique motifs in the SARS CoV-2 nucleocapsid phosphoprotein (N-protein) that can potentially interact with fibrinogen and possibly prothrombin. This is based on an established function of secretory proteins in Staphylococcus aureus (S. aureus)-coagulase, Efb (Extracellular fibrinogen binding) and vWBP (von Willebrand factor Binding Protein), which are known to regulate the blood clotting cascade and the functions of host immune response. It is hypothesized that having protein interaction motifs that are homologous to these S. aureus proteins, the N-protein of this virus can mimic their functions, which may in turn play a crucial role in formation of blood clots in the host and help the virus evade host immune response. However, this hypothesis needs to be tested in vitro. Considering the overwhelming increase in the incidence of SARS CoV-2 infection globally, this information may be useful for further investigation and could help in deducing new therapeutic strategies to combat advanced stages of this disease.


Subject(s)
Bacterial Proteins/chemistry , COVID-19/virology , Coronavirus Nucleocapsid Proteins/chemistry , Fibrinogen/chemistry , Host-Pathogen Interactions/immunology , SARS-CoV-2 , Amino Acid Motifs , COVID-19/metabolism , Humans , Immune System , Models, Theoretical , Peptides/chemistry , Phosphoproteins/chemistry , Protein Binding , Protein Domains , Staphylococcus aureus/enzymology , von Willebrand Factor/chemistry
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