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1.
Crit Rev Microbiol ; 46(6): 689-702, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-1730391

ABSTRACT

Intensive worldwide efforts are underway to determine both the pathogenesis of SARS-CoV-2 infection and the immune responses in COVID-19 patients in order to develop effective therapeutics and vaccines. One type of cell that may contribute to these immune responses is the γδ T lymphocyte, which plays a key role in immunosurveillance of the mucosal and epithelial barriers by rapidly responding to pathogens. Although found in low numbers in blood, γδ T cells consist the majority of tissue-resident T cells and participate in the front line of the host immune defense. Previous studies have demonstrated the critical protective role of γδ T cells in immune responses to other respiratory viruses, including SARS-CoV-1. However, no studies have profoundly investigated these cells in COVID-19 patients to date. γδ T cells can be safely expanded in vivo using existing inexpensive FDA-approved drugs such as bisphosphonate, in order to test its protective immune response to SARS-CoV-2. To support this line of research, we review insights gained from previous coronavirus research, along with recent findings, discussing the potential role of γδ T cells in controlling SARS-CoV-2. We conclude by proposing several strategies to enhance γδ T cell's antiviral function, which may be used in developing therapies for COVID-19.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/immunology , Pneumonia, Viral/immunology , T-Lymphocyte Subsets/immunology , Animals , Betacoronavirus/genetics , COVID-19 , Coronavirus Infections/virology , Humans , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Virus Replication
2.
Clin Infect Dis ; 74(2): 199-209, 2022 01 29.
Article in English | MEDLINE | ID: covidwho-1662119

ABSTRACT

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten public health globally. Patients with severe COVID-19 disease progress to acute respiratory distress syndrome, with respiratory and multiple organ failure. It is believed that dysregulated production of proinflammatory cytokines and endothelial dysfunction contribute to the pathogenesis of severe diseases. However, the mechanisms of SARS-CoV-2 pathogenesis and the role of endothelial cells are poorly understood. METHODS: Well-differentiated human airway epithelial cells were used to explore cytokine and chemokine production after SARS-CoV-2 infection. We measured the susceptibility to infection, immune response, and expression of adhesion molecules in human pulmonary microvascular endothelial cells (HPMVECs) exposed to conditioned medium from infected epithelial cells. The effect of imatinib on HPMVECs exposed to conditioned medium was evaluated. RESULTS: We demonstrated the production of interleukin-6, interferon gamma-induced protein-10, and monocyte chemoattractant protein-1 from the infected human airway cells after infection with SARS-CoV-2. Although HPMVECs did not support productive replication of SARS-CoV-2, treatment of HPMVECs with conditioned medium collected from infected airway cells induced an upregulation of proinflammatory cytokines, chemokines, and vascular adhesion molecules. Imatinib inhibited the upregulation of these cytokines, chemokines, and adhesion molecules in HPMVECs treated with conditioned medium. CONCLUSIONS: We evaluated the role of endothelial cells in the development of clinical disease caused by SARS-CoV-2 and the importance of endothelial cell-epithelial cell interaction in the pathogenesis of human COVID-19 diseases.


Subject(s)
COVID-19 , SARS-CoV-2 , Cell Communication , Endothelial Cells , Epithelial Cells , Humans
3.
Theranostics ; 11(14): 7005-7017, 2021.
Article in English | MEDLINE | ID: covidwho-1524524

ABSTRACT

The tumor suppressor protein p53 remains in a wild type but inactive form in ~50% of all human cancers. Thus, activating it becomes an attractive approach for targeted cancer therapies. In this regard, our lab has previously discovered a small molecule, Inauhzin (INZ), as a potent p53 activator with no genotoxicity. Method: To improve its efficacy and bioavailability, here we employed nanoparticle encapsulation, making INZ-C, an analog of INZ, to nanoparticle-encapsulated INZ-C (n-INZ-C). Results: This approach significantly improved p53 activation and inhibition of lung and colorectal cancer cell growth by n-INZ-C in vitro and in vivo while it displayed a minimal effect on normal human Wi38 and mouse MEF cells. The improved activity was further corroborated with the enhanced cellular uptake observed in cancer cells and minimal cellular uptake observed in normal cells. In vivo pharmacokinetic evaluation of these nanoparticles showed that the nanoparticle encapsulation prolongates the half-life of INZ-C from 2.5 h to 5 h in mice. Conclusions: These results demonstrate that we have established a nanoparticle system that could enhance the bioavailability and efficacy of INZ-C as a potential anti-cancer therapeutic.


Subject(s)
Antineoplastic Agents/pharmacology , Colorectal Neoplasms/drug therapy , Indoles/pharmacology , Lung Neoplasms/drug therapy , Nanoparticles/chemistry , Phenothiazines/pharmacology , Tumor Suppressor Protein p53/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Biological Availability , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Humans , Indoles/chemistry , Indoles/pharmacokinetics , Indoles/therapeutic use , Mice , Mice, Inbred C57BL , Microscopy, Electron, Transmission , Nanoparticles/toxicity , Nanoparticles/ultrastructure , Phenothiazines/chemistry , Phenothiazines/pharmacokinetics , Phenothiazines/therapeutic use , Spectroscopy, Fourier Transform Infrared , Tumor Suppressor Protein p53/genetics , Xenograft Model Antitumor Assays
4.
Mater Sci Eng C Mater Biol Appl ; 116: 111260, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-1452344

ABSTRACT

Polymeric nanoparticulate systems allow the encapsulation of bio-active substances, giving them protection against external agents and increasing the drug's bioavailability. The use of biocompatible and biodegradable polymers usually guarantees the harmless character of the formulation, and a controlled drug release is also assured. A relatively easy procedure to obtain polymeric formulations of bioactive agents is ionotropic gelation, which allows the synthesis of chitosan (CS) - sodium tri-polyphosphate nanoparticles (NPs) loading encapsulated proteins. In this work, Bovine serum albumin (BSA) model protein and a recombinant porcine alpha interferon variant were used to obtain nanoparticulate formulations. The internalization of the encapsulated material by cells was studied using a BSA-fluorescein system; the fluorescent conjugate was observable inside the cells after 20 h of incubation. The therapeutic CS-alpha interferon formulation showed a maximum of protein released in vitro at around 90 h. This system was found to be safe in a cytotoxicity assay, while biological activity experiments in vitro showed antiviral protection of cells in the presence of encapsulated porcine alpha interferon. In vivo experiments in pigs revealed a significant and sustained antiviral response through overexpression of the antiviral markers OAS2 and PKR. This proves the preservation of porcine alpha interferon biological activity, and also that a lasting response was obtained. This procedure is an effective and safe method to formulate drugs in nanoparticulate systems, representing a significant contribution to the search for more effective drug delivery strategies.


Subject(s)
Chitosan , Nanoparticles , Pharmaceutical Preparations , Animals , Antiviral Agents/pharmacology , Biological Availability , Cattle , Drug Carriers , Drug Delivery Systems , Interferon-alpha , Particle Size , Polymers , Swine
5.
J Am Soc Nephrol ; 32(1): 86-97, 2021 01.
Article in English | MEDLINE | ID: covidwho-1496654

ABSTRACT

BACKGROUND: Cultured cell lines are widely used for research in the physiology, pathophysiology, toxicology, and pharmacology of the renal proximal tubule. The lines that are most appropriate for a given use depend upon the genes expressed. New tools for transcriptomic profiling using RNA sequencing (RNA-Seq) make it possible to catalog expressed genes in each cell line. METHODS: Fourteen different proximal tubule cell lines, representing six species, were grown on permeable supports under conditions specific for the respective lines. RNA-Seq followed standard procedures. RESULTS: Transcripts expressed in cell lines variably matched transcripts selectively expressed in native proximal tubule. Opossum kidney (OK) cells displayed the highest percentage match (45% of proximal marker genes [TPM threshold =15]), with pig kidney cells (LLC-PK1) close behind (39%). Lower-percentage matches were seen for various human lines, including HK-2 (26%), and lines from rodent kidneys, such as NRK-52E (23%). Nominally, identical OK cells from different sources differed substantially in expression of proximal tubule markers. Mapping cell line transcriptomes to gene sets for various proximal tubule functions (sodium and water transport, protein transport, metabolic functions, endocrine functions) showed that different lines may be optimal for experimentally modeling each function. An online resource (https://esbl.nhlbi.nih.gov/JBrowse/KCT/) has been created to interrogate cell line transcriptome data. Proteomic analysis of NRK-52E cells confirmed low expression of many proximal tubule marker proteins. CONCLUSIONS: No cell line fully matched the transcriptome of native proximal tubule cells. However, some of the lines tested are suitable for the study of particular metabolic and transport processes seen in the proximal tubule.


Subject(s)
Cell Culture Techniques/methods , Kidney Tubules, Proximal/metabolism , Transcriptome , Animals , Biological Transport , Cell Line , Chromatography, Liquid , Gene Expression Profiling , Humans , Internet , Mice , Opossums , Proteomics , RNA-Seq , Rats , Sequence Analysis, RNA , Species Specificity , Swine , Tandem Mass Spectrometry
6.
Jpn J Infect Dis ; 74(5): 421-423, 2021 Sep 22.
Article in English | MEDLINE | ID: covidwho-1436359

ABSTRACT

Green tea extracts effectively inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro in a dose-dependent manner. Ten-fold serially diluted solutions of catechin mixture reagent from green tea were mixed with the viral culture fluid at a volume ratio of 9:1, respectively, and incubated at room temperature for 5 min. The solution of 10 mg/mL catechin reagent reduced the viral titer by 4.2 log and 1.0 mg/mL solution by one log. Pre-infection treatment of cells with the reagent alone did not affect viral growth. In addition, cells treated with only the reagent were assayed for host cell viability using the WST-8 system, and almost no host cell damage by the treatment was observed. These findings suggested that the direct treatment of virus with the reagent before inoculation decreased the viral activity and that catechins might have the potential to suppress SARSCoV-2 infection.


Subject(s)
Antiviral Agents/pharmacology , Catechin/pharmacology , SARS-CoV-2/drug effects , Tea/chemistry , Animals , COVID-19/virology , Cell Survival/drug effects , Chlorocebus aethiops , Dose-Response Relationship, Drug , Humans , Vero Cells , Viral Load/drug effects
7.
Viruses ; 12(5)2020 05 06.
Article in English | MEDLINE | ID: covidwho-1389513

ABSTRACT

SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.


Subject(s)
Antibodies, Viral/immunology , Neutralization Tests/methods , Spike Glycoprotein, Coronavirus/analysis , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Containment of Biohazards , HEK293 Cells , Humans , Lentivirus , Peptidyl-Dipeptidase A/metabolism , Plasma/immunology
8.
mBio ; 11(6)2020 12 11.
Article in English | MEDLINE | ID: covidwho-1388458

ABSTRACT

SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as the primary receptor to enter host cells and initiate the infection. The critical binding region of ACE2 is an ∼30-amino-acid (aa)-long helix. Here, we report the design of four stapled peptides based on the ACE2 helix, which is expected to bind to SARS-CoV-2 and prevent the binding of the virus to the ACE2 receptor and disrupt the infection. All stapled peptides showed high helical contents (50 to 94% helicity). In contrast, the linear control peptide NYBSP-C showed no helicity (19%). We have evaluated the peptides in a pseudovirus-based single-cycle assay in HT1080/ACE2 cells and human lung cell line A549/ACE2, overexpressing ACE2. Three of the four stapled peptides showed potent antiviral activity in HT1080/ACE2 (50% inhibitory concentration [IC50]: 1.9 to 4.1 µM) and A549/ACE2 (IC50: 2.2 to 2.8 µM) cells. The linear peptide NYBSP-C and the double-stapled peptide StRIP16, used as controls, showed no antiviral activity. Most significantly, none of the stapled peptides show any cytotoxicity at the highest dose tested. We also evaluated the antiviral activity of the peptides by infecting Vero E6 cells with the replication-competent authentic SARS-CoV-2 (US_WA-1/2020). NYBSP-1 was the most efficient, preventing the complete formation of cytopathic effects (CPEs) at an IC100 of 17.2 µM. NYBSP-2 and NYBSP-4 also prevented the formation of the virus-induced CPE with an IC100 of about 33 µM. We determined the proteolytic stability of one of the most active stapled peptides, NYBSP-4, in human plasma, which showed a half-life (T 1/2) of >289 min.IMPORTANCE SARS-CoV-2 is a novel virus with many unknowns. No vaccine or specific therapy is available yet to prevent and treat this deadly virus. Therefore, there is an urgent need to develop novel therapeutics. Structural studies revealed critical interactions between the binding site helix of the ACE2 receptor and SARS-CoV-2 receptor-binding domain (RBD). Therefore, targeting the entry pathway of SARS-CoV-2 is ideal for both prevention and treatment as it blocks the first step of the viral life cycle. We report the design of four double-stapled peptides, three of which showed potent antiviral activity in HT1080/ACE2 cells and human lung carcinoma cells, A549/ACE2. Most significantly, the active stapled peptides with antiviral activity against SARS-CoV-2 showed high α-helicity (60 to 94%). The most active stapled peptide, NYBSP-4, showed substantial resistance to degradation by proteolytic enzymes in human plasma. The lead stapled peptides are expected to pave the way for further optimization of a clinical candidate.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Peptides/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Virus Attachment/drug effects , A549 Cells , Animals , Binding Sites , Chlorocebus aethiops , Humans , Inhibitory Concentration 50 , Peptides/chemical synthesis , Protein Binding , Vero Cells
9.
Med Hypotheses ; 144: 109976, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-1386300

ABSTRACT

Several attempts to control the dreadfulness of SARS-CoV-2 are still underway. Based on the literature evidences we have speculated a prospective contemporary remedy, which was categorized into Specificity, Remedy, and a Conveyor. In which, pros and cons were discussed and inferred the possible alternatives. (a) Specificity: Implicit to express the ACE2 receptors in conveyor cells to deceive SARS-CoV-2 frompreponetargets. (b) Remedy: As depletion of pulmonary surfactants causes strong acute respiratory distress syndrome, we propose an entity of a cost-effective artificialsurfactantsystem as a remedy to pulmonary complications. (c) Conveyor: We propose red blood cells (RBCs) as a conveyor with embedded artificial surfactant and protruding ACE2 receptors for the target-specific delivery. Overall we postulate focused insights by employing a combinational contemporary strategy to steer towards a prospective direction on combating SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/therapeutic use , COVID-19/virology , Erythrocytes , Pulmonary Surfactants/therapeutic use , Receptors, Virus/therapeutic use , SARS-CoV-2/physiology , Viral Tropism , Angiotensin-Converting Enzyme 2/administration & dosage , COVID-19/complications , COVID-19/prevention & control , Drug Costs , Drug Delivery Systems , Humans , Pulmonary Alveoli/drug effects , Pulmonary Alveoli/virology , Pulmonary Surfactants/administration & dosage , Pulmonary Surfactants/chemical synthesis , Pulmonary Surfactants/economics , Receptors, Virus/administration & dosage , Respiratory Distress Syndrome/prevention & control
11.
Invest Ophthalmol Vis Sci ; 62(7): 6, 2021 06 01.
Article in English | MEDLINE | ID: covidwho-1388618

ABSTRACT

Purpose: To investigate the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 in human retina. Methods: Human post-mortem eyes from 13 non-diabetic control cases and 11 diabetic retinopathy cases were analyzed for the expression of ACE2. To compare the vascular ACE2 expression between different organs that involve in diabetes, the expression of ACE2 was investigated in renal specimens from nondiabetic and diabetic nephropathy patients. Expression of TMPRSS2, a cell-surface protease that facilitates SARS-CoV-2 entry, was also investigated in human nondiabetic retinas. Primary human retinal endothelial cells (HRECs) and primary human retinal pericytes (HRPCs) were further used to confirm the vascular ACE2 expression in human retina. Results: We found that ACE2 was expressed in multiple nonvascular neuroretinal cells, including the retinal ganglion cell layer, inner plexiform layer, inner nuclear layer, and photoreceptor outer segments in both nondiabetic and diabetic retinopathy specimens. Strikingly, we observed significantly more ACE2 positive vessels in the diabetic retinopathy specimens. By contrast, in another end-stage organ affected by diabetes, the kidney, ACE2 in nondiabetic and diabetic nephropathy showed apical expression of ACE2 tubular epithelial cells, but no endothelial expression in glomerular or peritubular capillaries. Western blot analysis of protein lysates from HRECs and HRPCs confirmed expression of ACE2. TMPRSS2 expression was present in multiple retinal neuronal cells, vascular and perivascular cells, and Müller glia. Conclusions: Together, these results indicate that retina expresses ACE2 and TMPRSS2. Moreover, there are increased vascular ACE2 expression in diabetic retinopathy retinas.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Diabetic Retinopathy/enzymology , Receptors, Virus/metabolism , Retina/enzymology , SARS-CoV-2/physiology , Adult , Aged , Aged, 80 and over , Binding Sites , Blotting, Western , Cells, Cultured , Diabetic Nephropathies/enzymology , Diabetic Nephropathies/pathology , Diabetic Nephropathies/virology , Diabetic Retinopathy/pathology , Diabetic Retinopathy/virology , Endothelium, Vascular/enzymology , Endothelium, Vascular/virology , Female , Fluorescent Antibody Technique, Indirect , Humans , Immunohistochemistry , Male , Middle Aged , Pericytes/enzymology , Pericytes/virology , Retinal Vessels/enzymology , Retinal Vessels/pathology , Retinal Vessels/virology , Serine Endopeptidases/metabolism
12.
Bioessays ; 43(2): e2000232, 2021 02.
Article in English | MEDLINE | ID: covidwho-1372696

ABSTRACT

Immunity against SARS-CoV-2 that is acquired by convalescent COVID-19 patients is examined in reference to (A) the Th17 cell generation system in psoriatic epidermis and (B) a recently discovered phenomenon in which Th17 cells are converted into tissue-resident memory T (TRM ) cells with Th1 phenotype. Neutrophils that are attracted to the site of infection secrete IL-17A, which stimulates lung epithelial cells to express CCL20. Natural Th17 (nTh17) cells are recruited to the infection site by CCL20 and expand in the presence of IL-23. These nTh17 cells are converted to TRM cells upon encounter with SARS-CoV-2 and continue to exist as ex-Th17 cells, which exert Th1-like immunity during a memory response. G-CSF can induce nTh17 cell accumulation at the infection site because it promotes neutrophil egress from the bone marrow. Hence, G-CSF may be effective against COVID-19. Administration of G-CSF to patients infected with SARS-CoV-2 is worth a clinical trial.


Subject(s)
Granulocyte Colony-Stimulating Factor/therapeutic use , Neutrophils/immunology , SARS-CoV-2/immunology , Th1 Cells/immunology , Th17 Cells/immunology , COVID-19/drug therapy , COVID-19/immunology , Chemokine CCL20/metabolism , Humans , Immunologic Memory/immunology , Interleukin-17/metabolism , Interleukin-23 Subunit p19/immunology , Neutrophils/drug effects , Th17 Cells/drug effects
13.
Int J Mol Sci ; 21(21)2020 Nov 03.
Article in English | MEDLINE | ID: covidwho-1344351

ABSTRACT

Progressive respiratory failure is seen as a major cause of death in severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection. Relatively little is known about the associated morphologic and molecular changes in the circulation of these patients. In particular, platelet and erythrocyte pathology might result in severe vascular issues, and the manifestations may include thrombotic complications. These thrombotic pathologies may be both extrapulmonary and intrapulmonary and may be central to respiratory failure. Previously, we reported the presence of amyloid microclots in the circulation of patients with coronavirus disease 2019 (COVID-19). Here, we investigate the presence of related circulating biomarkers, including C-reactive protein (CRP), serum ferritin, and P-selectin. These biomarkers are well-known to interact with, and cause pathology to, platelets and erythrocytes. We also study the structure of platelets and erythrocytes using fluorescence microscopy (using the markers PAC-1 and CD62PE) and scanning electron microscopy. Thromboelastography and viscometry were also used to study coagulation parameters and plasma viscosity. We conclude that structural pathologies found in platelets and erythrocytes, together with spontaneously formed amyloid microclots, may be central to vascular changes observed during COVID-19 progression, including thrombotic microangiopathy, diffuse intravascular coagulation, and large-vessel thrombosis, as well as ground-glass opacities in the lungs. Consequently, this clinical snapshot of COVID-19 strongly suggests that it is also a true vascular disease and considering it as such should form an essential part of a clinical treatment regime.


Subject(s)
Blood Platelets/pathology , Cardiovascular Diseases/virology , Coronavirus Infections/blood , Coronavirus Infections/pathology , Erythrocytes/pathology , Ferritins/blood , P-Selectin/blood , Pneumonia, Viral/blood , Pneumonia, Viral/pathology , Betacoronavirus/isolation & purification , Blood Coagulation/physiology , Blood Platelets/virology , COVID-19 , Cardiovascular Diseases/blood , Cardiovascular Diseases/pathology , Coronavirus Infections/virology , Erythrocytes/virology , Female , Humans , Male , Middle Aged , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Thrombosis/pathology , Thrombosis/virology
14.
Pharmacol Res ; 158: 104850, 2020 08.
Article in English | MEDLINE | ID: covidwho-1318927

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread worldwide through person-to-person contact, causing a public health emergency of international concern. At present, there is no specific antiviral treatment recommended for SARS-CoV-2 infection. Liu Shen capsule (LS), a traditional Chinese medicine, has been proven to have a wide spectrum of pharmacological properties, such as anti-inflammatory, antiviral and immunomodulatory activities. However, little is known about the antiviral effect of LS against SARS-CoV-2. Herein, the study was designed to investigate the antiviral activity of SARS-CoV-2 and its potential effect in regulating the host's immune response. The inhibitory effect of LS against SARS-CoV-2 replication in Vero E6 cells was evaluated by using the cytopathic effect (CPE) and plaque reduction assay. The number of virions of SARS-CoV-2 was observed under transmission electron microscope after treatment with LS. Proinflammatory cytokine expression levels upon SARS-CoV-2 infection in Huh-7 cells were measured by real-time quantitative PCR assays. The results showed that LS could significantly inhibit SARS-CoV-2 replication in Vero E6 cells, and reduce the number of virus particles and it could markedly reduce pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, IL-8, CCL-2/MCP-1 and CXCL-10/IP-10) production at the mRNA levels. Moreover, the expression of the key proteins in the NF-κB/MAPK signaling pathway was detected by western blot and it was found that LS could inhibit the expression of p-NF-κB p65, p-IκBα and p-p38 MAPK, while increasing the expression of IκBα. These findings indicate that LS could inhibit SARS-CoV-2 virus infection via downregulating the expression of inflammatory cytokines induced virus and regulating the activity of NF-κB/MAPK signaling pathway in vitro, making its promising candidate treatment for controlling COVID-19 disease.


Subject(s)
Betacoronavirus/drug effects , Complex Mixtures/pharmacology , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Signal Transduction/drug effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Anti-Inflammatory Agents/pharmacology , Antiviral Agents/pharmacology , COVID-19 , Cell Proliferation/drug effects , Cells, Cultured , Chlorocebus aethiops , Coronavirus Infections/virology , Humans , Inflammation Mediators/metabolism , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Virion/drug effects
16.
Commun Biol ; 4(1): 654, 2021 06 02.
Article in English | MEDLINE | ID: covidwho-1253994

ABSTRACT

SARS-CoV-2 infection of human airway epithelium activates genetic programs leading to progressive hyperinflammation in COVID-19 patients. Here, we report on transcriptomes activated in primary airway cells by interferons and their suppression by Janus kinase (JAK) inhibitors. Deciphering the regulation of the angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, is paramount for understanding the cell tropism of SARS-CoV-2 infection. ChIP-seq for activating histone marks and Pol II loading identified candidate enhancer elements controlling the ACE2 locus, including the intronic dACE2 promoter. Employing RNA-seq, we demonstrate that interferons activate expression of dACE2 and, to a lesser extent, the genuine ACE2 gene. Interferon-induced gene expression was mitigated by the JAK inhibitors baricitinib and ruxolitinib, used therapeutically in COVID-19 patients. Through integrating RNA-seq and ChIP-seq data we provide an in-depth understanding of genetic programs activated by interferons, and our study highlights JAK inhibitors as suitable tools to suppress these in bronchial cells.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Antiviral Agents/pharmacology , COVID-19/drug therapy , Interferons/pharmacology , Janus Kinase Inhibitors/pharmacology , Transcriptional Activation/drug effects , COVID-19/genetics , Cell Line , Humans , Respiratory Mucosa/cytology , Respiratory Mucosa/drug effects , Respiratory Mucosa/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Transcriptome/drug effects
17.
Cell Death Discov ; 7(1): 114, 2021 May 18.
Article in English | MEDLINE | ID: covidwho-1233706

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) has caused a global health emergency. A key feature of COVID-19 is dysregulated interferon-response. Type-I interferon (IFN-I) is one of the earliest antiviral innate immune responses following viral infection and plays a significant role in the pathogenesis of SARS-CoV-2. In this study, using a proteomics-based approach, we identified that SARS-CoV-2 infection induces delayed and dysregulated IFN-I signaling in Huh7 cells. We demonstrate that SARS-CoV-2 is able to inhibit RIG-I mediated IFN-ß production. Our results also confirm the recent findings that IFN-I pretreatment is able to reduce the susceptibility of Huh7 cells to SARS-CoV-2, but not post-treatment. Moreover, senescent Huh7 cells, in spite of showing accentuated IFN-I response were more susceptible to SARS-CoV-2 infection, and the virus effectively inhibited IFIT1 in these cells. Finally, proteomic comparison between SARS-CoV-2, SARS-CoV, and MERS-CoV revealed a distinct differential regulatory signature of interferon-related proteins emphasizing that therapeutic strategies based on observations in SARS-CoV and MERS-CoV should be used with caution. Our findings provide a better understanding of SARS-CoV-2 regulation of cellular interferon response and a perspective on its use as a treatment. Investigation of different interferon-stimulated genes and their role in the inhibition of SARS-CoV-2 pathogenesis may direct novel antiviral strategies.

18.
Front Microbiol ; 12: 651403, 2021.
Article in English | MEDLINE | ID: covidwho-1231355

ABSTRACT

Coronavirus disease 2019 (COVID-19) has caused serious public health, social, and economic damage worldwide and effective drugs that prevent or cure COVID-19 are urgently needed. Approved drugs including Hydroxychloroquine, Remdesivir or Interferon were reported to inhibit the infection or propagation of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), however, their clinical efficacies have not yet been well demonstrated. To identify drugs with higher antiviral potency, we screened approved anti-parasitic/anti-protozoal drugs and identified an anti-malarial drug, Mefloquine, which showed the highest anti-SARS-CoV-2 activity among the tested compounds. Mefloquine showed higher anti-SARS-CoV-2 activity than Hydroxychloroquine in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 1.28 µM, IC90 = 2.31 µM, and IC99 = 4.39 µM in VeroE6/TMPRSS2 cells. Mefloquine inhibited viral entry after viral attachment to the target cell. Combined treatment with Mefloquine and Nelfinavir, a replication inhibitor, showed synergistic antiviral activity. Our mathematical modeling based on the drug concentration in the lung predicted that Mefloquine administration at a standard treatment dosage could decline viral dynamics in patients, reduce cumulative viral load to 7% and shorten the time until virus elimination by 6.1 days. These data cumulatively underscore Mefloquine as an anti-SARS-CoV-2 entry inhibitor.

19.
mSphere ; 6(3)2021 05 12.
Article in English | MEDLINE | ID: covidwho-1230164

ABSTRACT

Human coronavirus (HCoV)-OC43 rarely shows a cytopathic effect (CPE) after infection of various cell lines, and the indirect immunoperoxidase assay (IPA), a relatively complex procedure, has long been used as an alternative assay. Because HCoV-OC43 uses cell-surface transmembrane protease serine 2 (TMPRSS2) for cell entry, VeroE6 cells expressing TMPRSS2 may show a clear CPE after HCoV-OC43 infection. The aim of this study was to construct a 50% tissue culture infectious dose (TCID50) assay for HCoV-OC43 based on CPE evaluation using VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells showed clear CPEs 3 to 4 days after low-titer HCoV-OC43 infection. Evaluation of viral kinetics indicated that the viral titer in the culture supernatant of VeroE6/TMPRSS2 cells in the early stages of infection was higher than that of other cells. In comparison, between the CPE-based and the IPA-based (i.e., the reference titer) methods, the titer measured with CPE evaluation 4 to 5 days after infection using VeroE6/TMPRSS2 cells showed a much smaller difference from the reference titer than that measured using other cells. Thus, the TCID50 assay using CPE evaluation with VeroE6/TMPRSS2 cells provides the correct titer value and will greatly contribute to future research on HCoV-OC43.IMPORTANCE HCoV-OC43 rarely shows a cytopathic effect (CPE) in infected cell lines, and thus the plaque and TCID50 assays by CPE observation are not applicable for titration; the indirect immunoperoxidase assay (IPA) is used instead. However, the IPA is relatively complex, time-consuming, costly, and not suitable for simultaneous titration of many samples. We developed a TCID50 assay using CPE evaluation with TMPRSS2-expressing VeroE6/TMPRSS2 cells that provides the same accuracy as the conventional IPA-based viral titration and does not require any staining procedures using antibodies or substrates. This titration method will greatly contribute to future research on HCoV-OC43 by allowing simple, low-cost, and accurate titration of this virus.


Subject(s)
Coronavirus OC43, Human/physiology , Cytopathogenic Effect, Viral , Receptors, Virus/metabolism , Serine Endopeptidases/metabolism , Viral Load/methods , Animals , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus OC43, Human/isolation & purification , Humans , Immunoenzyme Techniques , Receptors, Virus/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serine Endopeptidases/genetics , Vero Cells/virology , Virus Cultivation , Virus Internalization , Virus Replication
20.
Oncol Lett ; 21(6): 458, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1225869

ABSTRACT

Cryoablation is an emerging type of treatment for cancer. The sensitization of tumors using cryosensitizing agents prior to treatment enhances ablation efficiency and may improve clinical outcomes. Water efflux, which is regulated by aquaporin channels, contributes to cancer cell damage achieved through cryoablation. An increase in aquaporin (AQP) 3 is cryoprotective, whereas its inhibition augments cryodamage. The present study aimed to investigate aquaporin (AQP1, AQP3 and AQP5) gene expression and cellular localization in response to cryoinjury. Cultured breast cancer cells (MDA-MB-231 and MCF-7) were exposed to freezing to induce cryoinjury. RNA and protein extracts were then analyzed using reverse transcription-quantitative PCR and western blotting, respectively. Localization of aquaporins was studied using immunocytochemistry. Additionally, cells were transfected with small interfering RNA to silence aquaporin gene expression and cell viability was assessed using the Sulforhodamine B assay. Cryoinjury did not influence gene expression of AQPs, except for a 4-fold increase of AQP1 expression in MDA-MD-231 cells. There were no clear differences in AQP protein expression for either cell lines upon exposure to frozen and non-frozen temperatures, with the exception of fainter AQP5 bands for non-frozen MCF-7 cells. The exposure of cancer cells to freezing temperatures altered the localization of AQP1 and AQP3 proteins in both MCF-7 and MDA-MD-231 cells. The silencing of AQP1, AQP3 and AQP5 exacerbated MDA-MD-231 cell damage associated with freezing compared with control siRNA. This was also observed with AQP3 and AQP5 silencing in MCF-7 cells. Inhibition of aquaporins may potentially enhance cryoinjury. This cryosensitizing process may be used as an adjunct to breast cancer cryotherapy, especially in the border area targeted by cryoablation where freezing temperatures are not cold enough to induce cellular damage.

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