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1.
Clin Epigenetics ; 13(1): 187, 2021 10 11.
Article in English | MEDLINE | ID: covidwho-1526657

ABSTRACT

BACKGROUND: SARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP1) receptors for entry into cells, and the serine protease TMPRSS2 for S protein priming. Inhibition of protease activity or the engagement with ACE2 and NRP1 receptors has been shown to be an effective strategy for blocking infectivity and viral spreading. Valproic acid (VPA; 2-propylpentanoic acid) is an epigenetic drug approved for clinical use. It produces potent antiviral and anti-inflammatory effects through its function as a histone deacetylase (HDAC) inhibitor. Here, we propose VPA as a potential candidate to tackle COVID-19, in which rapid viral spread and replication, and hyperinflammation are crucial elements. RESULTS: We used diverse cell lines (HK-2, Huh-7, HUVEC, Caco-2, and BEAS-2B) to analyze the effect of VPA and other HDAC inhibitors on the expression of the ACE-2 and NRP-1 receptors and their ability to inhibit infectivity, viral production, and the inflammatory response. Treatment with VPA significantly reduced expression of the ACE2 and NRP1 host proteins in all cell lines through a mechanism mediated by its HDAC inhibitory activity. The effect is maintained after SARS-CoV-2 infection. Consequently, the treatment of cells with VPA before infection impairs production of SARS-CoV-2 infectious viruses, but not that of other ACE2- and NRP1-independent viruses (VSV and HCoV-229E). Moreover, the addition of VPA 1 h post-infection with SARS-CoV-2 reduces the production of infectious viruses in a dose-dependent manner without significantly modifying the genomic and subgenomic messenger RNAs (gRNA and sg mRNAs) or protein levels of N protein. The production of inflammatory cytokines (TNF-α and IL-6) induced by TNF-α and SARS-CoV-2 infection is diminished in the presence of VPA. CONCLUSIONS: Our data showed that VPA blocks three essential processes determining the severity of COVID-19. It downregulates the expression of ACE2 and NRP1, reducing the infectivity of SARS-CoV-2; it decreases viral yields, probably because it affects virus budding or virions stability; and it dampens the triggered inflammatory response. Thus, administering VPA could be considered a safe treatment for COVID-19 patients until vaccines have been rolled out across the world.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/prevention & control , Epigenesis, Genetic/physiology , Neuropilin-1/genetics , Receptors, Virus/drug effects , Valproic Acid/pharmacology , Angiotensin-Converting Enzyme 2/drug effects , Antiviral Agents/pharmacology , Cells, Cultured , Enzyme Inhibitors/pharmacology , Epigenesis, Genetic/genetics , Humans , Neuropilin-1/drug effects , SARS-CoV-2
2.
J Infect Dis ; 224(3): 415-419, 2021 08 02.
Article in English | MEDLINE | ID: covidwho-1526165

ABSTRACT

Mutagenic ribonucleosides can act as broad-based antiviral agents. They are metabolized to the active ribonucleoside triphosphate form and concentrate in genomes of RNA viruses during viral replication. ß-d-N4-hydroxycytidine (NHC, initial metabolite of molnupiravir) is >100-fold more active than ribavirin or favipiravir against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with antiviral activity correlated to the level of mutagenesis in virion RNA. However, NHC also displays host mutational activity in an animal cell culture assay, consistent with RNA and DNA precursors sharing a common intermediate of a ribonucleoside diphosphate. These results indicate highly active mutagenic ribonucleosides may hold risk for the host.


Subject(s)
Antiviral Agents/pharmacology , Cytidine/analogs & derivatives , Mutagens/pharmacology , SARS-CoV-2/drug effects , Animals , Antiviral Agents/adverse effects , CHO Cells/drug effects , Cells, Cultured , Cricetulus , Cytidine/adverse effects , Cytidine/pharmacology , Dose-Response Relationship, Drug , Mutagenesis/drug effects , Mutagens/adverse effects , SARS-CoV-2/genetics , Virus Replication/drug effects
3.
Nat Med ; 27(9): 1600-1606, 2021 09.
Article in English | MEDLINE | ID: covidwho-1526089

ABSTRACT

Clinical evidence suggests the central nervous system is frequently impacted by SARS-CoV-2 infection, either directly or indirectly, although the mechanisms are unclear. Pericytes are perivascular cells within the brain that are proposed as SARS-CoV-2 infection points. Here we show that pericyte-like cells (PLCs), when integrated into a cortical organoid, are capable of infection with authentic SARS-CoV-2. Before infection, PLCs elicited astrocytic maturation and production of basement membrane components, features attributed to pericyte functions in vivo. While traditional cortical organoids showed little evidence of infection, PLCs within cortical organoids served as viral 'replication hubs', with virus spreading to astrocytes and mediating inflammatory type I interferon transcriptional responses. Therefore, PLC-containing cortical organoids (PCCOs) represent a new 'assembloid' model that supports astrocytic maturation as well as SARS-CoV-2 entry and replication in neural tissue; thus, PCCOs serve as an experimental model for neural infection.


Subject(s)
Astrocytes/virology , Brain/virology , COVID-19/pathology , Pericytes/virology , Viral Tropism/physiology , Astrocytes/cytology , Brain/pathology , Cell Differentiation/physiology , Cells, Cultured , Humans , Interferon Type I/immunology , SARS-CoV-2 , Virus Replication/physiology
4.
Elife ; 102021 04 27.
Article in English | MEDLINE | ID: covidwho-1513055

ABSTRACT

Dendritic cells (DCs) regulate processes ranging from antitumor and antiviral immunity to host-microbe communication at mucosal surfaces. It remains difficult, however, to genetically manipulate human DCs, limiting our ability to probe how DCs elicit specific immune responses. Here, we develop a CRISPR-Cas9 genome editing method for human monocyte-derived DCs (moDCs) that mediates knockouts with a median efficiency of >94% across >300 genes. Using this method, we perform genetic screens in moDCs, identifying mechanisms by which DCs tune responses to lipopolysaccharides from the human microbiome. In addition, we reveal donor-specific responses to lipopolysaccharides, underscoring the importance of assessing immune phenotypes in donor-derived cells, and identify candidate genes that control this specificity, highlighting the potential of our method to pinpoint determinants of inter-individual variation in immunity. Our work sets the stage for a systematic dissection of the immune signaling at the host-microbiome interface and for targeted engineering of DCs for neoantigen vaccination.


Subject(s)
CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Dendritic Cells/immunology , Gene Editing , Genomics , Immunity, Innate/genetics , Bacteroides thetaiotaomicron/immunology , CRISPR-Associated Protein 9/metabolism , Cells, Cultured , Cytokines/genetics , Cytokines/metabolism , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Gene Expression Regulation , Humans , Immunity, Innate/drug effects , Lipopolysaccharides/pharmacology , Phenotype , Signal Transduction , Toll-Like Receptor 4/agonists , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism
5.
Cell Transplant ; 30: 9636897211054481, 2021.
Article in English | MEDLINE | ID: covidwho-1511642

ABSTRACT

Biological and cellular interleukin-6 (IL-6)-related therapies have been used to treat severe COVID-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure, which prompted further exploration of the role of IL-6 in human umbilical cord mesenchymal stem cell (hUCMSC) therapy. Peripheral blood mononuclear cells (PBMCs) were responders cocultured with hUCMSCs or exogenous IL-6. A PBMC suppression assay was used to analyze the anti-inflammatory effects via MTT assay. The IL-6 concentration in the supernatant was measured using ELISA. The correlation between the anti-inflammatory effect of hUCMSCs and IL-6 levels and the relevant roles of IL-6 and IL-6 mRNA expression was analyzed using the MetaCore functional network constructed from gene microarray data. The location of IL-6 and IL-6 receptor (IL-6R) expression was further evaluated. We reported that hUCMSCs did not initially exert any inhibitory effect on PHA-stimulated proliferation; however, a potent inhibitory effect on PHA-stimulated proliferation was observed, and the IL-6 concentration reached approximately 1000 ng/mL after 72 hours. Exogenous 1000 ng/mL IL-6 inhibited PHA-stimulated inflammation but less so than hUCMSCs. The inhibitory effects of hUCMSCs on PHA-stimulated PBMCs disappeared after adding an IL-6 neutralizing antibody or pretreatment with tocilizumab (TCZ), an IL-6R antagonist. hUCMSCs exert excellent anti-inflammatory effects by inducing higher IL-6 levels, which is different from TCZ. High concentration of IL-6 cytokine secretion plays an important role in the anti-inflammatory effect of hUCMSC therapy. Initial hUCMSC therapy, followed by TCZ, seems to optimize the therapeutic potential to treat COVID-19-related acute respiratory distress syndrome (ARDS).


Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , COVID-19/complications , Interleukin-6/biosynthesis , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Respiratory Distress Syndrome/therapy , SARS-CoV-2 , Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Neutralizing/immunology , Cells, Cultured , Coculture Techniques , Combined Modality Therapy , DNA, Complementary/genetics , Gene Expression Regulation/drug effects , Humans , Inflammation , Interleukin-6/genetics , Interleukin-6/pharmacology , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Lymphocyte Activation/drug effects , Phytohemagglutinins/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, Interleukin-6/antagonists & inhibitors , Receptors, Interleukin-6/biosynthesis , Receptors, Interleukin-6/genetics , Respiratory Distress Syndrome/drug therapy , Respiratory Distress Syndrome/etiology , Umbilical Cord/cytology
6.
J Immunol ; 207(7): 1776-1784, 2021 10 01.
Article in English | MEDLINE | ID: covidwho-1497460

ABSTRACT

Acquired neutrophil dysfunction frequently develops during critical illness, independently increasing the risk for intensive care unit-acquired infection. PI3Kδ is implicated in driving neutrophil dysfunction and can potentially be targeted pharmacologically. The aims of this study were to determine whether PI3Kδ inhibition reverses dysfunction in neutrophils from critically ill patients and to describe potential mechanisms. Neutrophils were isolated from blood taken from critically ill patients requiring intubation and mechanical ventilation, renal support, or blood pressure support. In separate validation experiments, neutrophil dysfunction was induced pharmacologically in neutrophils from healthy volunteers. Phagocytosis and bacterial killing assays were performed, and activity of RhoA and protein kinase A (PKA) was assessed. Inhibitors of PI3Kδ, 3-phosphoinositide-dependent protein kinase-1 (PDK1), and PKA were used to determine mechanisms of neutrophil dysfunction. Sixty-six patients were recruited. In the 27 patients (40.9%) with impaired neutrophil function, PI3Kδ inhibition consistently improved function and significantly increased bacterial killing. These findings were validated in neutrophils from healthy volunteers with salbutamol-induced dysfunction and extended to demonstrate that PI3Kδ inhibition restored killing of clinical isolates of nine pathogens commonly associated with intensive care unit-acquired infection. PI3Kδ activation was associated with PDK1 activation, which in turn phosphorylated PKA, which drove phosphorylation and inhibition of the key regulator of neutrophil phagocytosis, RhoA. These data indicate that, in a significant proportion of critically ill patients, PI3Kδ inhibition can improve neutrophil function through PDK1- and PKA-dependent processes, suggesting that therapeutic use of PI3Kδ inhibitors warrants investigation in this setting.


Subject(s)
COVID-19/immunology , Class I Phosphatidylinositol 3-Kinases/metabolism , Critical Illness , Neutrophils/immunology , Pneumonia/immunology , SARS-CoV-2/physiology , Sepsis/immunology , 3-Phosphoinositide-Dependent Protein Kinases/pharmacology , Adult , Aged , Aged, 80 and over , Bacterial Load , Bacteriolysis , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/metabolism , Female , Humans , Male , Middle Aged , Phagocytosis , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Respiratory Insufficiency , Risk
7.
Methods Mol Biol ; 2311: 185-193, 2021.
Article in English | MEDLINE | ID: covidwho-1482181

ABSTRACT

Studies of blood-brain barrier (BBB) require developing of a novel and convenient in vitro endothelial cell model. We isolated primary human and rodent brain microvascular endothelial cells and developed methods for culturing, characterization, and high-efficiency transfection of endothelial cells. Here, we describe the improved methods to obtain in vitro human and rodent BBB models to study expression of endogenous and exogenous genes of interest.


Subject(s)
Blood-Brain Barrier/physiology , Brain/blood supply , Cell Separation , Endothelial Cells/physiology , Microvessels/cytology , Transfection , Animals , Blood-Brain Barrier/metabolism , Cell Culture Techniques , Cell Differentiation , Cell Proliferation , Cells, Cultured , Endothelial Cells/metabolism , Fetus , Gestational Age , Humans , Mice , Rats
8.
Pharmacol Res Perspect ; 9(1): e00712, 2021 02.
Article in English | MEDLINE | ID: covidwho-1482163

ABSTRACT

Mass drug administration of ivermectin has been proposed as a possible malaria elimination tool. Ivermectin exhibits a mosquito-lethal effect well beyond its biological half-life, suggesting the presence of active slowly eliminated metabolites. Human liver microsomes, primary human hepatocytes, and whole blood from healthy volunteers given oral ivermectin were used to identify ivermectin metabolites by ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry. The molecular structures of metabolites were determined by mass spectrometry and verified by nuclear magnetic resonance. Pure cytochrome P450 enzyme isoforms were used to elucidate the metabolic pathways. Thirteen different metabolites (M1-M13) were identified after incubation of ivermectin with human liver microsomes. Three (M1, M3, and M6) were the major metabolites found in microsomes, hepatocytes, and blood from volunteers after oral ivermectin administration. The chemical structure, defined by LC-MS/MS and NMR, indicated that M1 is 3″-O-demethyl ivermectin, M3 is 4-hydroxymethyl ivermectin, and M6 is 3″-O-demethyl, 4-hydroxymethyl ivermectin. Metabolic pathway evaluations with characterized cytochrome P450 enzymes showed that M1, M3, and M6 were produced primarily by CYP3A4, and that M1 was also produced to a small extent by CYP3A5. Demethylated (M1) and hydroxylated (M3) ivermectin were the main human in vivo metabolites. Further studies are needed to characterize the pharmacokinetic properties and mosquito-lethal activity of these metabolites.


Subject(s)
Antiparasitic Agents/pharmacokinetics , Ivermectin/pharmacokinetics , Administration, Oral , Antiparasitic Agents/blood , Antiparasitic Agents/pharmacology , Cells, Cultured , Cytochrome P-450 Enzyme System/metabolism , Demethylation , Hepatocytes/metabolism , Humans , Hydroxylation , Ivermectin/blood , Ivermectin/pharmacology , Metabolic Networks and Pathways , Microsomes, Liver/metabolism
9.
Front Immunol ; 12: 741502, 2021.
Article in English | MEDLINE | ID: covidwho-1477825

ABSTRACT

Host innate immune response follows severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and it is the driver of the acute respiratory distress syndrome (ARDS) amongst other inflammatory end-organ morbidities. Such life-threatening coronavirus disease 2019 (COVID-19) is heralded by virus-induced activation of mononuclear phagocytes (MPs; monocytes, macrophages, and dendritic cells). MPs play substantial roles in aberrant immune secretory activities affecting profound systemic inflammation and end-organ malfunctions. All follow the presence of persistent viral components and virions without evidence of viral replication. To elucidate SARS-CoV-2-MP interactions we investigated transcriptomic and proteomic profiles of human monocyte-derived macrophages. While expression of the SARS-CoV-2 receptor, the angiotensin-converting enzyme 2, paralleled monocyte-macrophage differentiation, it failed to affect productive viral infection. In contrast, simple macrophage viral exposure led to robust pro-inflammatory cytokine and chemokine expression but attenuated type I interferon (IFN) activity. Both paralleled dysregulation of innate immune signaling pathways, specifically those linked to IFN. We conclude that the SARS-CoV-2-infected host mounts a robust innate immune response characterized by a pro-inflammatory storm heralding end-organ tissue damage.


Subject(s)
COVID-19/virology , Immunity, Innate , Macrophages/virology , SARS-CoV-2/pathogenicity , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/immunology , COVID-19/metabolism , Cells, Cultured , Cytokines/genetics , Cytokines/metabolism , Gene Expression Profiling , Gene Regulatory Networks , Host-Pathogen Interactions , Humans , Immunity, Innate/genetics , Inflammation Mediators/metabolism , Macrophages/immunology , Macrophages/metabolism , Proteome , Proteomics , Receptors, Virus/genetics , Receptors, Virus/metabolism , SARS-CoV-2/immunology , Signal Transduction , Transcriptome
10.
J Immunol ; 207(10): 2405-2410, 2021 11 15.
Article in English | MEDLINE | ID: covidwho-1471046

ABSTRACT

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) causes severe acute respiratory syndrome. mRNA vaccines directed at the SARS-CoV-2 spike protein resulted in development of Abs and protective immunity. To determine the mechanism, we analyzed the kinetics of induction of circulating exosomes with SARS-CoV-2 spike protein and Ab following vaccination of healthy individuals. Results demonstrated induction of circulating exosomes expressing spike protein on day 14 after vaccination followed by Abs 14 d after the second dose. Exosomes with spike protein, Abs to SARS-CoV-2 spike, and T cells secreting IFN-γ and TNF-α increased following the booster dose. Transmission electron microscopy of exosomes also demonstrated spike protein Ags on their surface. Exosomes with spike protein and Abs decreased in parallel after four months. These results demonstrate an important role of circulating exosomes with spike protein for effective immunization following mRNA-based vaccination. This is further documented by induction of humoral and cellular immune responses in mice immunized with exosomes carrying spike protein.


Subject(s)
Antibodies, Viral/metabolism , COVID-19 Vaccines/immunology , COVID-19/immunology , Exosomes/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , T-Lymphocytes/metabolism , Animals , Blood Circulation , Cells, Cultured , Exosomes/immunology , Healthy Volunteers , Humans , Immunization , Interferon-gamma/metabolism , Mice , Mice, Inbred C57BL , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes/immunology , Tumor Necrosis Factor-alpha/metabolism , Vaccination
11.
Int J Mol Sci ; 22(19)2021 Oct 05.
Article in English | MEDLINE | ID: covidwho-1457746

ABSTRACT

Various pathogens, such as Ebola virus, Marburg virus, Nipah virus, Hendra virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and SARS-CoV-2, are threatening human health worldwide. The natural hosts of these pathogens are thought to be bats. The rousette bat, a megabat, is thought to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Additionally, the rousette bat showed a transient infection in the experimental inoculation of SARS-CoV-2. In the current study, we established and characterized intestinal organoids from Leschenault's rousette, Rousettus leschenaultii. The established organoids successfully recapitulated the characteristics of intestinal epithelial structure and morphology, and the appropriate supplements necessary for long-term stable culture were identified. The organoid showed susceptibility to Pteropine orthoreovirus (PRV) but not to SARS-CoV-2 in experimental inoculation. This is the first report of the establishment of an expandable organoid culture system of the rousette bat intestinal organoid and its sensitivity to bat-associated viruses, PRV and SARS-CoV-2. This organoid is a useful tool for the elucidation of tolerance mechanisms of the emerging rousette bat-associated viruses such as Ebola and Marburg virus.


Subject(s)
COVID-19/virology , Chiroptera/virology , Organoids/virology , Orthoreovirus/physiology , Reoviridae Infections/virology , SARS-CoV-2/physiology , Animals , COVID-19/veterinary , Cell Culture Techniques , Cells, Cultured , Chiroptera/physiology , Humans , Intestines/cytology , Intestines/virology , Organoids/cytology , Reoviridae Infections/veterinary
12.
Immunity ; 54(9): 2159-2166.e6, 2021 09 14.
Article in English | MEDLINE | ID: covidwho-1454205

ABSTRACT

The emergence of SARS-CoV-2 antigenic variants with increased transmissibility is a public health threat. Some variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies. Here, we analyzed receptor binding domain-binding monoclonal antibodies derived from SARS-CoV-2 mRNA vaccine-elicited germinal center B cells for neutralizing activity against the WA1/2020 D614G SARS-CoV-2 strain and variants of concern. Of five monoclonal antibodies that potently neutralized the WA1/2020 D614G strain, all retained neutralizing capacity against the B.1.617.2 variant, four also neutralized the B.1.1.7 variant, and only one, 2C08, also neutralized the B.1.351 and B.1.1.28 variants. 2C08 reduced lung viral load and morbidity in hamsters challenged with the WA1/2020 D614G, B.1.351, or B.1.617.2 strains. Clonal analysis identified 2C08-like public clonotypes among B cells responding to SARS-CoV-2 infection or vaccination in 41 out of 181 individuals. Thus, 2C08-like antibodies can be induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern.


Subject(s)
Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , B-Lymphocytes/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , Germinal Center/immunology , Lung/virology , SARS-CoV-2/physiology , Animals , Cells, Cultured , Clone Cells , Cricetinae , Disease Models, Animal , Humans , Neutralization Tests , Spike Glycoprotein, Coronavirus/immunology , Vaccination , Viral Load
13.
J Immunol ; 207(10): 2399-2404, 2021 11 15.
Article in English | MEDLINE | ID: covidwho-1450887

ABSTRACT

Immunity to pulmonary infection typically requires elicitation of lung-resident T cells that subsequently confer protection against secondary infection. The presence of tissue-resident T cells in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) convalescent patients is unknown. Using a sublethal mouse model of coronavirus disease 2019, we determined if SARS-CoV-2 infection potentiated Ag-specific pulmonary resident CD4+ and CD8+ T cell responses and if these cells mediated protection against secondary infection. S protein-specific T cells were present in resident and circulating populations. However, M and N protein-specific T cells were detected only in the resident T cell pool. Using an adoptive transfer strategy, we found that T cells from SARS-CoV-2 immune animals did not protect naive mice. These data indicate that resident T cells are elicited by SARS-CoV-2 infection but are not sufficient for protective immunity.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Lung/immunology , SARS-CoV-2/physiology , Adoptive Transfer , Angiotensin-Converting Enzyme 2/genetics , Animals , Cells, Cultured , Disease Models, Animal , Disease Resistance , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Spike Glycoprotein, Coronavirus/immunology , T-Cell Antigen Receptor Specificity
14.
Int J Mol Sci ; 22(19)2021 Sep 30.
Article in English | MEDLINE | ID: covidwho-1444232

ABSTRACT

Natural or experimental infection of domestic cats and virus transmission from humans to captive predatory cats suggest that felids are highly susceptible to SARS-CoV-2 infection. However, it is unclear which cells and compartments of the respiratory tract are infected. To address this question, primary cell cultures derived from the nose, trachea, and lungs of cat and lion were inoculated with SARS-CoV-2. Strong viral replication was observed for nasal mucosa explants and tracheal air-liquid interface cultures, whereas replication in lung slices was less efficient. Infection was mainly restricted to epithelial cells and did not cause major pathological changes. Detection of high ACE2 levels in the nose and trachea but not lung further suggests that susceptibility of feline tissues to SARS-CoV-2 correlates with ACE2 expression. Collectively, this study demonstrates that SARS-CoV-2 can efficiently replicate in the feline upper respiratory tract ex vivo and thus highlights the risk of SARS-CoV-2 spillover from humans to felids.


Subject(s)
COVID-19/veterinary , Cats/virology , Lions/virology , Angiotensin-Converting Enzyme 2/analysis , Animals , COVID-19/transmission , COVID-19/virology , Cat Diseases/transmission , Cat Diseases/virology , Cells, Cultured , Disease Susceptibility , Humans , Lung/cytology , Lung/virology , Nose/cytology , Nose/virology , SARS-CoV-2/isolation & purification , Trachea/cytology , Trachea/virology
15.
Int J Mol Sci ; 22(19)2021 Sep 28.
Article in English | MEDLINE | ID: covidwho-1444228

ABSTRACT

For a yet unknown reason, a substantial share of patients suffering from COVID-19 develop long-lasting neuropsychiatric symptoms ranging from cognitive deficits to mood disorders and/or an extreme fatigue. We previously reported that in non-neural cells, angiotensin-1 converting enzyme 2 (ACE2), the gene coding for the SARS-CoV2 host receptor, harbors tight co-expression links with dopa-decarboxylase (DDC), an enzyme involved in the metabolism of dopamine. Here, we mined and integrated data from distinct human expression atlases and found that, among a wide range of tissues and cells, enterocytes of the small intestine express the highest expression levels of ACE2, DDC and several key genes supporting the metabolism of neurotransmitters. Based on these results, we performed co-expression analyses on a recently published set of RNA-seq data obtained from SARS-CoV2-infected human intestinal organoids. We observed that in SARS-CoV2-infected enterocytes, ACE2 co-regulates not only with DDC but also with a specific group of genes involved in (i) the dopamine/trace amines metabolic pathway, (ii) the absorption of microbiota-derived L-DOPA and (iii) the absorption of neutral amino acids serving as precursors to neurotransmitters. We conclude that in patients with long COVID, a chronic infection and inflammation of small intestine enterocytes might be indirectly responsible for prolonged brain alterations.


Subject(s)
Brain/pathology , COVID-19/complications , Gene Expression Regulation , Intestine, Small/pathology , Angiotensin-Converting Enzyme 2/genetics , Aromatic-L-Amino-Acid Decarboxylases/genetics , Brain/metabolism , COVID-19/genetics , COVID-19/pathology , Cells, Cultured , Enterocytes/metabolism , Enterocytes/pathology , Humans , Intestine, Small/metabolism , SARS-CoV-2/isolation & purification
16.
Molecules ; 26(17)2021 Aug 27.
Article in English | MEDLINE | ID: covidwho-1403854

ABSTRACT

This paper presents the results of the first part of testing a novel electrospun fiber mat based on a unique macromolecule: polyisobutylene (PIB). A PIB-based compound containing zinc oxide (ZnO) was electrospun into self-supporting mats of 203.75 and 295.5 g/m2 that were investigated using a variety of techniques. The results show that the hydrophobic mats are not cytotoxic, resist fibroblast cell adhesion and biofilm formation and are comfortable and easy to breathe through for use as a mask. The mats show great promise for personal protective equipment and other applications.


Subject(s)
Polyenes/chemistry , Polymers/chemistry , Biofilms/drug effects , Cell Adhesion/drug effects , Cells, Cultured , Fibroblasts/drug effects , Humans , Materials Testing/methods , Nanofibers/chemistry , Zinc Oxide/chemistry
17.
Cells ; 10(9)2021 09 04.
Article in English | MEDLINE | ID: covidwho-1403545

ABSTRACT

Stroke is the third leading cause of mortality in women and it kills twice as many women as breast cancer. A key role in the pathophysiology of stroke plays the disruption of the blood-brain barrier (BBB) within the neurovascular unit. While estrogen induces vascular protective actions, its influence on stroke remains unclear. Moreover, experiments assessing its impact on endothelial cells to induce barrier integrity are non-conclusive. Since pericytes play an active role in regulating BBB integrity and function, we hypothesize that estradiol may influence BBB by regulating their activity. In this study using human brain vascular pericytes (HBVPs) we investigated the impact of estradiol on key pericyte functions known to influence BBB integrity. HBVPs expressed estrogen receptors (ER-α, ER-ß and GPER) and treatment with estradiol (10 nM) inhibited basal cell migration but not proliferation. Since pericyte migration is a hallmark for BBB disruption following injury, infection and inflammation, we investigated the effects of estradiol on TNFα-induced PC migration. Importantly, estradiol prevented TNFα-induced pericyte migration and this effect was mimicked by PPT (ER-α agonist) and DPN (ER-ß agonist), but not by G1 (GPR30 agonist). The modulatory effects of estradiol were abrogated by MPP and PHTPP, selective ER-α and ER-ß antagonists, respectively, confirming the role of ER-α and ER-ß in mediating the anti-migratory actions of estrogen. To delineate the intracellular mechanisms mediating the inhibitory actions of estradiol on PC migration, we investigated the role of AKT and MAPK activation. While estradiol consistently reduced the TNFα-induced MAPK and Akt phosphorylation, only the inhibition of MAPK, but not Akt, significantly abrogated the migratory actions of TNFα. In transendothelial electrical resistance measurements, estradiol induced barrier function (TEER) in human brain microvascular endothelial cells co-cultured with pericytes, but not in HBMECs cultured alone. Importantly, transcriptomics analysis of genes modulated by estradiol in pericytes showed downregulation of genes known to increase cell migration and upregulation of genes known to inhibit cell migration. Taken together, our findings provide the first evidence that estradiol modulates pericyte activity and thereby improves endothelial integrity.


Subject(s)
Brain/blood supply , Cell Movement/drug effects , Estradiol/pharmacology , Gene Expression Profiling , Pericytes/cytology , Cell Movement/genetics , Cell Proliferation/drug effects , Cells, Cultured , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Gene Expression Regulation/drug effects , Humans , Mitogen-Activated Protein Kinases/metabolism , Pericytes/drug effects , Pericytes/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Estrogen/metabolism , Tumor Necrosis Factor-alpha/metabolism
18.
Clin Immunol ; 231: 108850, 2021 10.
Article in English | MEDLINE | ID: covidwho-1401322

ABSTRACT

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has resulted in considerable morbidity and mortality in humans. Little is known regarding the development of immunological memory following SARS-CoV-2 infection or whether immunological memory can provide long-lasting protection against reinfection. Urgent need for vaccines is a considerable issue for all governments worldwide. METHODS: A total of 39 patients were recruited in this study. Tonsillar mononuclear cells (MNCs) were co-cultured in RPMI medium and stimulated with the full-length SARS-CoV-2 spike protein in the presence and absence of a CpG-DNA adjuvant. An enzyme-linked immunosorbent assay (ELISA) was utilised to measure the specific antibody response to the spike protein in the cell culture supernatants. RESULTS: The SARS-CoV-2 spike protein primed a potent memory B cell-mediated immune response in nasal-associated lymphoid tissue (NALT) from patients previously infected with the virus. Additionally, spike protein combined with the CpG-DNA adjuvant induced a significantly increased level of specific anti-spike protein IgG antibody compared with the spike protein alone (p < 0.0001, n = 24). We also showed a strong positive correlation between the specific anti-spike protein IgG antibody level in a serum samples and that produced by MNCs derived from the same COVID-19-recovered patients following stimulation (r = 0.76, p = 0.0002, n = 24). CONCLUSION: Individuals with serological evidence of previous SARS-CoV-2 exposure showed a significant anti-spike protein-specific memory humoral immune response to the viral spike protein upon stimulation. Additionally, our results demonstrated the functional response of NALT-derived MNCs to the viral spike protein. CpG-DNA adjuvant combined with spike protein induced significantly stronger humoral immune responses than the spike protein alone. These data indicate that the S protein antigen combined with CpG-DNA adjuvant could be used as a future vaccine candidate.


Subject(s)
COVID-19/immunology , COVID-19/virology , Immunologic Memory/physiology , Lymphoid Tissue/physiology , SARS-CoV-2/immunology , Antibodies, Viral/metabolism , B-Lymphocytes , Cells, Cultured , DNA , Enzyme-Linked Immunosorbent Assay , Humans , Immunity, Cellular , Immunity, Humoral , Immunoglobulin G/metabolism , Lymphoid Tissue/virology , Nose , Oligodeoxyribonucleotides , Spike Glycoprotein, Coronavirus/immunology
19.
Biochem Biophys Res Commun ; 577: 146-151, 2021 11 05.
Article in English | MEDLINE | ID: covidwho-1401239

ABSTRACT

The human lung cell A549 is susceptible to infection with a number of respiratory viruses. However, A549 cells are resistant to Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection in conventional submerged culture, and this would appear to be due to low expression levels of the SARS-CoV-2 entry receptor: angiotensin-converting enzyme-2 (ACE2). Here, we examined SARS-CoV-2 susceptibility to A549 cells after adaptation to air-liquid interface (ALI) culture. A549 cells in ALI culture yielded a layer of mucus on their apical surface, exhibited decreased expression levels of the proliferation marker KI-67 and intriguingly became susceptible to SARS-CoV-2 infection. We found that A549 cells increased the endogenous expression levels of ACE2 and TMPRSS2 following adaptation to ALI culture conditions. Camostat, a TMPRSS2 inhibitor, reduced SARS-CoV-2 infection in ALI-cultured A549 cells. These findings indicate that ALI culture switches the phenotype of A549 cells from resistance to susceptibility to SARS-CoV-2 infection through upregulation of ACE2 and TMPRSS2.


Subject(s)
Alveolar Epithelial Cells/virology , COVID-19/virology , Cell Culture Techniques/methods , SARS-CoV-2/physiology , A549 Cells , Alveolar Epithelial Cells/pathology , Cells, Cultured , Disease Susceptibility , Gene Expression Regulation, Neoplastic , Humans , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Up-Regulation/genetics
20.
Life Sci Alliance ; 4(1)2021 01.
Article in English | MEDLINE | ID: covidwho-1389961

ABSTRACT

Viruses rely on their host for reproduction. Here, we made use of genomic and structural information to create a biomass function capturing the amino and nucleic acid requirements of SARS-CoV-2. Incorporating this biomass function into a stoichiometric metabolic model of the human lung cell and applying metabolic flux balance analysis, we identified host-based metabolic perturbations inhibiting SARS-CoV-2 reproduction. Our results highlight reactions in the central metabolism, as well as amino acid and nucleotide biosynthesis pathways. By incorporating host cellular maintenance into the model based on available protein expression data from human lung cells, we find that only few of these metabolic perturbations are able to selectively inhibit virus reproduction. Some of the catalysing enzymes of such reactions have demonstrated interactions with existing drugs, which can be used for experimental testing of the presented predictions using gene knockouts and RNA interference techniques. In summary, the developed computational approach offers a platform for rapid, experimentally testable generation of drug predictions against existing and emerging viruses based on their biomass requirements.


Subject(s)
Host-Pathogen Interactions , Lung , SARS-CoV-2 , Virus Replication , Antiviral Agents/pharmacology , Biomass , COVID-19/prevention & control , COVID-19/virology , Cells, Cultured , Culture Media/chemistry , Culture Media/metabolism , Glycolysis/physiology , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/physiology , Humans , Lung/cytology , Lung/metabolism , Metabolic Flux Analysis , Models, Biological , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Systems Biology , Virus Replication/drug effects , Virus Replication/physiology
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