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1.
Trop Anim Health Prod ; 53(2): 265, 2021 Apr 17.
Article in English | MEDLINE | ID: covidwho-1549502

ABSTRACT

BACKGROUND: Bronchial-associated lymphoid tissue (BALT) is responsible for the local immune response of the lung against airborne infections. The structure of this tissue varies according to species and age. AIM: The aim of this study was to describe the possible age-related structural variation of the BALT of the one humped camel. MATERIAL AND METHODS: Fresh specimens from both lungs of 15 clinically healthy male camels (10 months-12 years) were studied with light and electron microscopes. RESULTS: The BALT in the camel was variable from few lymphocytes to well-organized lymphoid tissue with a clear germinal center. The BALT of the bronchi is a constant lymphoid tissue in young and adult camels which may be of the large size with clear germinal center in response to repeated immune reaction and involutes in old age. The BALT of the bronchioles may be induced and develops mainly due to an immune reaction and showed great morphological variations and observed in different ages. High endothelial venules were associated with BALT in the bronchi but not with that of the bronchioles. The BALT-associated epithelium was tall pseudostratified columnar ciliated epithelium with goblet cells in the extrapulmonary bronchi changed to pseudostratified columnar ciliated epithelium mucous secreting cells in the intrapulmonary bronchi and simple columnar ciliated to simple cuboidal epithelium with Clara cells without goblet cells or mucous secreting cells in the bronchioles. CONCLUSIONS: The BALT of the bronchi is a constant lymphoid tissue in young and adult camels and involutes in old age. The BALT of the bronchioles may be induced and develops mainly due to an immune reaction and observed in different ages.


Subject(s)
Bronchi , Camelus , Animals , Epithelium , Lung , Lymphoid Tissue , Male
2.
Front Pharmacol ; 11: 579330, 2020.
Article in English | MEDLINE | ID: covidwho-1389228

ABSTRACT

The Syrian golden hamster (Mesocricetus auratus) has recently been demonstrated as a clinically relevant animal model for SARS-CoV-2 infection. However, lack of knowledge about the tissue-specific expression pattern of various proteins in these animals and the unavailability of reagents like antibodies against this species hampers these models' optimal use. The major objective of our current study was to analyze the tissue-specific expression pattern of angiotensin-converting enzyme 2, a proven functional receptor for SARS-CoV-2 in different organs of the hamster. Using two different antibodies (MA5-32307 and AF933), we have conducted immunoblotting, immunohistochemistry, and immunofluorescence analysis to evaluate the ACE2 expression in different tissues of the hamster. Further, at the mRNA level, the expression of Ace2 in tissues was evaluated through RT-qPCR analysis. Both the antibodies detected expression of ACE2 in kidney, small intestine, tongue, and liver. Epithelium of proximal tubules of kidney and surface epithelium of ileum expresses a very high amount of this protein. Surprisingly, analysis of stained tissue sections showed no detectable expression of ACE2 in the lung or tracheal epithelial cells. Similarly, all parts of the large intestine were negative for ACE2 expression. Analysis of tissues from different age groups and sex didn't show any obvious difference in ACE2 expression pattern or level. Together, our findings corroborate some of the earlier reports related to ACE2 expression patterns in human tissues and contradict others. We believe that this study's findings have provided evidence that demands further investigation to understand the predominant respiratory pathology of SARS-CoV-2 infection and disease.

3.
Front Immunol ; 11: 607314, 2020.
Article in English | MEDLINE | ID: covidwho-1389171

ABSTRACT

Acute lung injury (ALI) is an important cause of morbidity and mortality after viral infections, including influenza A virus H1N1, SARS-CoV, MERS-CoV, and SARS-CoV-2. The angiotensin I converting enzyme 2 (ACE2) is a key host membrane-bound protein that modulates ALI induced by viral infection, pulmonary acid aspiration, and sepsis. However, the contributions of ACE2 sequence variants to individual differences in disease risk and severity after viral infection are not understood. In this study, we quantified H1N1 influenza-infected lung transcriptomes across a family of 41 BXD recombinant inbred strains of mice and both parents-C57BL/6J and DBA/2J. In response to infection Ace2 mRNA levels decreased significantly for both parental strains and the expression levels was associated with disease severity (body weight loss) and viral load (expression levels of viral NA segment) across the BXD family members. Pulmonary RNA-seq for 43 lines was analyzed using weighted gene co-expression network analysis (WGCNA) and Bayesian network approaches. Ace2 not only participated in virus-induced ALI by interacting with TNF, MAPK, and NOTCH signaling pathways, but was also linked with high confidence to gene products that have important functions in the pulmonary epithelium, including Rnf128, Muc5b, and Tmprss2. Comparable sets of transcripts were also highlighted in parallel studies of human SARS-CoV-infected primary human airway epithelial cells. Using conventional mapping methods, we determined that weight loss at two and three days after viral infection maps to chromosome X-the location of Ace2. This finding motivated the hierarchical Bayesian network analysis, which defined molecular endophenotypes of lung infection linked to Ace2 expression and to a key disease outcome. Core members of this Bayesian network include Ace2, Atf4, Csf2, Cxcl2, Lif, Maml3, Muc5b, Reg3g, Ripk3, and Traf3. Collectively, these findings define a causally-rooted Ace2 modulatory network relevant to host response to viral infection and identify potential therapeutic targets for virus-induced respiratory diseases, including those caused by influenza and coronaviruses.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Lung/virology , Virus Diseases/genetics , Animals , Bayes Theorem , Epithelial Cells/virology , Female , Humans , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Respiratory Mucosa/virology , Signal Transduction/genetics
4.
JCI Insight ; 6(7)2021 04 08.
Article in English | MEDLINE | ID: covidwho-1383578

ABSTRACT

Proline-glycine-proline (PGP) and its acetylated form (Ac-PGP) are neutrophil chemoattractants generated by collagen degradation, and they have been shown to play a role in chronic inflammatory disease. However, the mechanism for matrikine regulation in acute inflammation has not been well established. Here, we show that these peptides are actively transported from the lung by the oligopeptide transporter, PEPT2. Following intratracheal instillation of Ac-PGP in a mouse model, there was a rapid decline in concentration of the labeled peptide in the bronchoalveolar lavage (BAL) over time and redistribution to extrapulmonary sites. In vitro knockdown of the PEPT2 transporter in airway epithelia or use of a competitive inhibitor of PEPT2, cefadroxil, significantly reduced uptake of Ac-PGP. Animals that received intratracheal Ac-PGP plus cefadroxil had higher levels of Ac-PGP in BAL and lung tissue. Utilizing an acute LPS-induced lung injury model, we demonstrate that PEPT2 blockade enhanced pulmonary Ac-PGP levels and lung inflammation. We further validated this effect using clinical samples from patients with acute lung injury in coculture with airway epithelia. This is the first study to our knowledge to determine the in vitro and in vivo significance of active matrikine transport as a mechanism of modulating acute inflammation and to demonstrate that it may serve as a potential therapeutic target.


Subject(s)
Acute Lung Injury/immunology , COVID-19 , Cefadroxil/pharmacology , Inflammation/metabolism , Oligopeptides , Proline/analogs & derivatives , Symporters , Animals , Anti-Bacterial Agents/pharmacology , Biological Transport, Active/immunology , COVID-19/immunology , COVID-19/metabolism , Cells, Cultured , Chemotactic Factors/immunology , Chemotactic Factors/pharmacology , Chemotaxis, Leukocyte/immunology , Disease Models, Animal , Extracellular Matrix , Extracellular Matrix Proteins/metabolism , Humans , Mice , Oligopeptides/immunology , Oligopeptides/pharmacology , Proline/immunology , Proline/pharmacology , Symporters/antagonists & inhibitors , Symporters/metabolism
5.
Int J Mol Sci ; 22(5)2021 Mar 04.
Article in English | MEDLINE | ID: covidwho-1389392

ABSTRACT

Alveolar type II (ATII) cells are a key structure of the distal lung epithelium, where they exert their innate immune response and serve as progenitors of alveolar type I (ATI) cells, contributing to alveolar epithelial repair and regeneration. In the healthy lung, ATII cells coordinate the host defense mechanisms, not only generating a restrictive alveolar epithelial barrier, but also orchestrating host defense mechanisms and secreting surfactant proteins, which are important in lung protection against pathogen exposure. Moreover, surfactant proteins help to maintain homeostasis in the distal lung and reduce surface tension at the pulmonary air-liquid interface, thereby preventing atelectasis and reducing the work of breathing. ATII cells may also contribute to the fibroproliferative reaction by secreting growth factors and proinflammatory molecules after damage. Indeed, various acute and chronic diseases are associated with intensive inflammation. These include oedema, acute respiratory distress syndrome, fibrosis and numerous interstitial lung diseases, and are characterized by hyperplastic ATII cells which are considered an essential part of the epithelialization process and, consequently, wound healing. The aim of this review is that of revising the physiologic and pathologic role ATII cells play in pulmonary diseases, as, despite what has been learnt in the last few decades of research, the origin, phenotypic regulation and crosstalk of these cells still remain, in part, a mystery.


Subject(s)
Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/physiology , Lung Diseases/physiopathology , Lung/physiology , Alveolar Epithelial Cells/cytology , Animals , COVID-19/physiopathology , Humans , Immunity, Innate , Ions/metabolism , Lung/anatomy & histology , Lung Diseases/etiology , Lung Diseases/pathology , Pulmonary Surfactant-Associated Proteins/metabolism , Regeneration
6.
Am J Physiol Lung Cell Mol Physiol ; 320(6): L1183-L1185, 2021 06 01.
Article in English | MEDLINE | ID: covidwho-1388546
7.
mBio ; 12(2)2021 04 13.
Article in English | MEDLINE | ID: covidwho-1388457

ABSTRACT

Mammalian cells detect microbial molecules known as pathogen-associated molecular patterns (PAMPs) as indicators of potential infection. Upon PAMP detection, diverse defensive responses are induced by the host, including those that promote inflammation and cell-intrinsic antimicrobial activities. Host-encoded molecules released from dying or damaged cells, known as damage-associated molecular patterns (DAMPs), also induce defensive responses. Both DAMPs and PAMPs are recognized for their inflammatory potential, but only the latter are well established to stimulate cell-intrinsic host defense. Here, we report a class of DAMPs that engender an antiviral state in human epithelial cells. These DAMPs include oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine), PGPC (1-palmitoyl-2-glutaryl phosphatidylcholine), and POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphatidylcholine], oxidized lipids that are naturally released from dead or dying cells. Exposing cells to these DAMPs prior to vesicular stomatitis virus (VSV) infection limits viral replication. Mechanistically, these DAMPs prevent viral entry, thereby limiting the percentage of cells that are productively infected and consequently restricting viral load. We found that the antiviral actions of oxidized lipids are distinct from those mediated by the PAMP Poly I:C, in that the former induces a more rapid antiviral response without the induction of the interferon response. These data support a model whereby interferon-independent defensive activities can be induced by DAMPs, which may limit viral replication before PAMP-mediated interferon responses are induced. This antiviral activity may impact viruses that disrupt interferon responses in the oxygenated environment of the lung, such as influenza virus and SARS-CoV-2.IMPORTANCE In this work, we explored how a class of oxidized lipids, spontaneously created during tissue damage and unprogrammed cell lysis, block the earliest events in RNA virus infection in the human epithelium. This gives us novel insight into the ways that we view infection models, unveiling a built-in mechanism to slow viral growth that neither engages the interferon response nor is subject to known viral antagonism. These oxidized phospholipids act prior to infection, allowing time for other, better-known innate immune mechanisms to take effect. This discovery broadens our understanding of host defenses, introducing a soluble factor that alters the cellular environment to protect from RNA virus infection.


Subject(s)
Alarmins/pharmacology , Antiviral Agents/pharmacology , RNA Viruses/drug effects , Virus Internalization/drug effects , Virus Replication/drug effects , A549 Cells , Cell Death/drug effects , Humans , Immunity, Innate , Interferons/genetics , Interferons/metabolism , Kinetics , Pathogen-Associated Molecular Pattern Molecules/pharmacology , Phosphatidylcholines/pharmacology , RNA Viruses/physiology , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Vesiculovirus/drug effects , Vesiculovirus/physiology , Viral Load
8.
Sci Rep ; 11(1): 6621, 2021 03 23.
Article in English | MEDLINE | ID: covidwho-1387468

ABSTRACT

The human bronchial epithelium is the first line of defense against atmospheric particles, pollutants, and respiratory pathogens such as the novel SARS-CoV-2. The epithelial cells form a tight barrier and secrete proteins that are major components of the mucosal immune response. Functional in vitro models of the human lung are essential for screening the epithelial response and assessing the toxicity and barrier crossing of drugs, inhaled particles, and pollutants. However, there is a lack of models to investigate the effect of chronic exposure without resorting to animal testing. Here, we developed a 3D model of the human bronchial epithelium using Calu-3 cell line and demonstrated its viability and functionality for 21 days without subculturing. We investigated the effect of reduced Fetal Bovine Serum supplementation in the basal medium and defined the minimal supplementation needed to maintain a functional epithelium, so that the amount of exogenous serum proteins could be reduced during drug testing. The long-term evolution of the epithelial cell secretome was fully characterized by quantitative mass spectrometry in two preclinical models using Calu-3 or primary NHBE cells. 408 common secreted proteins were identified while significant differences in protein abundance were observed with time, suggesting that 7-10 days are necessary to establish a mature secretome in the Calu-3 model. The associated Reactome pathways highlight the role of the secreted proteins in the immune response of the bronchial epithelium. We suggest this preclinical 3D model can be used to evaluate the long-term toxicity of drugs or particles on the human bronchial epithelium, and subsequently to investigate their effect on the epithelial cell secretions.


Subject(s)
Epithelial Cells/metabolism , Proteome/analysis , Proteomics/methods , Angiotensin-Converting Enzyme 2/metabolism , Bronchi/cytology , COVID-19/pathology , COVID-19/virology , Cell Culture Techniques , Cell Line , Culture Media/chemistry , Epithelial Cells/cytology , Humans , Mass Spectrometry , Models, Biological , Principal Component Analysis , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology
9.
J Infect Dis ; 224(1): 21-30, 2021 07 02.
Article in English | MEDLINE | ID: covidwho-1379462

ABSTRACT

The differentiation between influenza and coronavirus disease 2019 (COVID-19) could constitute a diagnostic challenge during the ongoing winter owing to their clinical similitude. Thus, novel biomarkers are required to enable making this distinction. Here, we evaluated whether the surfactant protein D (SP-D), a collectin produced at the alveolar epithelium with known immune properties, was useful to differentiate pandemic influenza A(H1N1) from COVID-19 in critically ill patients. Our results revealed high serum SP-D levels in patients with severe pandemic influenza but not those with COVID-19. This finding was validated in a separate cohort of mechanically ventilated patients with COVID-19 who also showed low plasma SP-D levels. However, plasma SP-D levels did not distinguish seasonal influenza from COVID-19 in mild-to-moderate disease. Finally, we found that high serum SP-D levels were associated with death and renal failure among severe pandemic influenza cases. Thus, our studies have identified SP-D as a unique biomarker expressed during severe pandemic influenza but not COVID-19.


Subject(s)
COVID-19/genetics , Gene Expression , Host-Pathogen Interactions/genetics , Influenza A Virus, H1N1 Subtype , Influenza, Human/genetics , Pulmonary Surfactant-Associated Protein D/genetics , SARS-CoV-2 , Adult , Aged , Biomarkers , COVID-19/blood , COVID-19/diagnosis , COVID-19/virology , Coinfection , Enzyme-Linked Immunosorbent Assay , Female , Humans , Influenza, Human/diagnosis , Influenza, Human/virology , Male , Middle Aged , Prognosis , Pulmonary Surfactant-Associated Protein D/blood , Severity of Illness Index , Symptom Assessment , Young Adult
10.
Jpn J Infect Dis ; 74(4): 285-292, 2021 Jul 21.
Article in English | MEDLINE | ID: covidwho-1323436

ABSTRACT

Isolation of seasonal coronaviruses, which include human coronavirus (HCoV) OC43, HCoV-HKU1, and HCoV-NL63, from primary cultures is difficult because it requires experienced handling, an exception being HCoV-229E, which can be isolated using cell lines such as RD-18S and HeLa-ACE2-TMPRSS2. We aimed to isolate seasonal CoVs in Yamagata, Japan to obtain infective virions useful for further research and to accelerate fundamental studies on HCoVs and SARS-CoV-2. Using modified air-liquid interface (ALI) culture of the normal human airway epithelium from earlier studies, we isolated 29 HCoVs (80.6%: 16, 6, 6, and 1 isolates of HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E, respectively) from 36 cryopreserved nasopharyngeal specimens. In ALI cultures of HCoV-OC43 and HCoV-NL63, the harvested medium contained more than 1 × 104 genome copies/µL at every tested time point during the more than 100 days of culture. Four isolates of HCoV-NL63 were further subcultured and successfully propagated in an LLC-MK2 cell line. Our results suggest that ALI culture is useful for isolating seasonal CoVs and sustainably obtaining HCoV-OC43 and HCoV-NL63 virions. Furthermore, the LLC-MK2 cell line in combination with ALI cultures can be used for the large-scale culturing of HCoV-NL63. Further investigations are necessary to develop methods for culturing difficult-to-culture seasonal CoVs in cell lines.


Subject(s)
Coronavirus/isolation & purification , Epithelium/virology , Respiratory System/virology , Respiratory Tract Infections/virology , Coronavirus/genetics , Genome, Viral/genetics , Humans , Japan
11.
Clin Infect Dis ; 73(2): e503-e512, 2021 07 15.
Article in English | MEDLINE | ID: covidwho-1315661

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) is primarily an acute respiratory tract infection. Distinctively, a substantial proportion of COVID-19 patients develop olfactory dysfunction. Especially in young patients, loss of smell can be the first or only symptom. The roles of inflammatory obstruction of the olfactory clefts, inflammatory cytokines affecting olfactory neuronal function, destruction of olfactory neurons or their supporting cells, and direct invasion of olfactory bulbs in causing olfactory dysfunction are uncertain. METHODS: We investigated the location for the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from the olfactory epithelium (OE) to the olfactory bulb in golden Syrian hamsters. RESULTS: After intranasal inoculation with SARS-CoV-2, inflammatory cell infiltration and proinflammatory cytokine/chemokine responses were detected in the nasal turbinate tissues. The responses peaked between 2 and 4 days postinfection, with the highest viral load detected at day 2 postinfection. In addition to the pseudo-columnar ciliated respiratory epithelial cells, SARS-CoV-2 viral antigens were also detected in the mature olfactory sensory neurons labeled by olfactory marker protein, in the less mature olfactory neurons labeled by neuron-specific class III ß-tubulin at the more basal position, and in the sustentacular cells, resulting in apoptosis and severe destruction of the OE. During the entire course of infection, SARS-CoV-2 viral antigens were not detected in the olfactory bulb. CONCLUSIONS: In addition to acute inflammation at the OE, infection of mature and immature olfactory neurons and the supporting sustentacular cells by SARS-CoV-2 may contribute to the unique olfactory dysfunction related to COVID-19, which is not reported with SARS-CoV-2.


Subject(s)
COVID-19 , Olfactory Receptor Neurons , Animals , Cricetinae , Humans , Mesocricetus , Olfactory Mucosa , SARS-CoV-2
12.
J Infect Dis ; 224(1): 31-38, 2021 07 02.
Article in English | MEDLINE | ID: covidwho-1294729

ABSTRACT

Virus-virus interactions influence the epidemiology of respiratory infections. However, the impact of viruses causing upper respiratory infections on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication and transmission is currently unknown. Human rhinoviruses cause the common cold and are the most prevalent respiratory viruses of humans. Interactions between rhinoviruses and cocirculating respiratory viruses have been shown to shape virus epidemiology at the individual host and population level. Here, we examined the replication kinetics of SARS-CoV-2 in the human respiratory epithelium in the presence or absence of rhinovirus. We show that human rhinovirus triggers an interferon response that blocks SARS-CoV-2 replication. Mathematical simulations show that this virus-virus interaction is likely to have a population-wide effect as an increasing prevalence of rhinovirus will reduce the number of new coronavirus disease 2019 cases.


Subject(s)
Antibiosis , COVID-19/virology , Coinfection , Picornaviridae Infections/virology , Rhinovirus/physiology , SARS-CoV-2/physiology , Virus Replication , COVID-19/epidemiology , Cell Line , Cells, Cultured , Fluorescent Antibody Technique , Humans , Respiratory Mucosa/virology
13.
Curr Opin Virol ; 49: 151-156, 2021 08.
Article in English | MEDLINE | ID: covidwho-1271612

ABSTRACT

Intestinal microbiota have profound effects on viral infections locally and systemically. While they can directly influence enteric virus infections, there is also an increasing appreciation for the role of microbiota-derived metabolites in regulating virus infections. Because metabolites diffuse across the intestinal epithelium and enter circulation, they can influence host response to pathogens at extraintestinal sites. In this review, we summarize the effects of three types of microbiota-derived metabolites on virus infections. While short-chain fatty acids serve to regulate the extent of inflammation associated with viral infections, the flavonoid desaminotyrosine and bile acids generally regulate interferon responses. A common theme that emerges is that microbiota-derived metabolites can have proviral and antiviral effects depending on the virus in question. Understanding the molecular mechanisms by which microbiota-derived metabolites impact viral infections and the highly conditional nature of these responses should pave the way to developing novel rational antivirals.


Subject(s)
Bacteria/metabolism , Gastrointestinal Microbiome/physiology , Virus Diseases/microbiology , Virus Diseases/physiopathology , Bile Acids and Salts/metabolism , Fatty Acids, Volatile/metabolism , Flavonoids/metabolism , Humans , Inflammation , Interferons/metabolism , Virus Diseases/immunology
14.
Mol Med Rep ; 24(2)2021 Aug.
Article in English | MEDLINE | ID: covidwho-1271003

ABSTRACT

Coronavirus disease 2019 (COVID­19), caused by the severe acute respiratory syndrome coronavirus­2 (SARS­CoV­2), led to an outbreak of viral pneumonia in December 2019. The present study aimed to investigate the host inflammatory response signature­caused by SARS­CoV­2 in human corneal epithelial cells (HCECs). The expression level of angiotensin­converting enzyme 2 (ACE2) in the human cornea was determined via immunofluorescence. In vitro experiments were performed in HCECs stimulated with the SARS­CoV­2 spike protein. Moreover, the expression levels of ACE2, IL­8, TNF­α, IL­6, gasdermin D (GSDMD) and IL­1ß in HCECs were detected using reverse transcription­quantitative PCR and/or western blotting. It was identified that ACE2 was expressed in normal human corneal epithelium and HCECs cultured in vitro. Furthermore, the expression levels of IL­8, TNF­α and IL­6 in HCECs were decreased following SARS­CoV­2 spike protein stimulation, while the expression levels of GSDMD and IL­1ß were increased. In conclusion, the present results demonstrated that the SARS­CoV­2 spike protein suppressed the host inflammatory response and induced pyroptosis in HCECs. Therefore, blocking the ACE2 receptor in HCECs may reduce the infection rate of COVID­19.


Subject(s)
Epithelium, Corneal/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Adult , Aged , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Cells, Cultured , Cornea/cytology , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/virology , Epithelium, Corneal/virology , Female , Humans , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Male , Middle Aged , Phosphate-Binding Proteins/genetics , Phosphate-Binding Proteins/metabolism , Pyroptosis , Spike Glycoprotein, Coronavirus/genetics , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Up-Regulation
15.
Arthritis Res Ther ; 23(1): 166, 2021 06 10.
Article in English | MEDLINE | ID: covidwho-1266501

ABSTRACT

BACKGROUND: To investigate whether methotrexate treatment may affect the susceptibility to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: Clinical assessment of symptoms, SARS-CoV-2 RNA, and anti-SARS-CoV-2 IgG in an initial case series of four families and confirmatory case series of seven families, within which one family member developed coronavirus disease 19 (COVID-19) and exposed another family member receiving methotrexate treatment; experimental part with methotrexate treatment of mice and organoids followed by the assessment of mRNA and protein expression of the SARS-CoV-2 receptor angiotensin-converting enzyme (ACE)-2. RESULTS: In the initial case series, three of four women on a joint ski trip developed COVID-19, while the fourth woman, under treatment with methotrexate, remained virus-free. Two of the three diseased women infected their husbands, while the third husband treated with methotrexate remained virus-free. In addition, 7 other families were identified in a follow-up case series, in which one member developed COVID-19, while the other, receiving methotrexate, remained healthy. Experimentally, when mice were treated with methotrexate, ACE2 expression significantly decreased in the lung, in the intestinal epithelium, and in intestinal organoids. CONCLUSION: These clinical and experimental data indicate that methotrexate has certain protective effects on SARS-CoV-2 infection via downregulating ACE2.


Subject(s)
COVID-19 , Animals , Humans , Methotrexate , Mice , RNA, Viral , SARS-CoV-2
16.
PLoS One ; 16(6): e0251955, 2021.
Article in English | MEDLINE | ID: covidwho-1262543

ABSTRACT

Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2-induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway and/or gastrointestinal barrier damage and mitigate virus spread.


Subject(s)
COVID-19/metabolism , COVID-19/virology , Coronavirus Envelope Proteins/metabolism , SARS-CoV-2/metabolism , Zonula Occludens-1 Protein/metabolism , COVID-19/pathology , Host-Pathogen Interactions , Humans , PDZ Domains , Protein Binding , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , SARS-CoV-2/isolation & purification , Tight Junctions/metabolism
17.
bioRxiv ; 2021 Jun 07.
Article in English | MEDLINE | ID: covidwho-1262289

ABSTRACT

Previous studies suggested that the SARS-CoV-2 virus may gain access to the brain by using a route along the olfactory nerve. However, there is a general consensus that the obligatory virus entry receptor, angiotensin converting enzyme 2 (ACE2), is not expressed in olfactory receptor neurons, and the timing of arrival of the virus in brain targets is inconsistent with a neuronal transfer along olfactory projections. We determined whether nervus terminalis neurons and their peripheral and central projections should be considered as a potential alternative route from the nose to the brain. Nervus terminalis neurons in postnatal mice were double-labeled with antibodies against ACE2 and two nervus terminalis markers, gonadotropin-releasing hormone (GnRH) and choline acetyltransferase (CHAT). We show that a small fraction of CHAT-labeled nervus terminalis neurons, and the large majority of GnRH-labeled nervus terminalis neurons with cell bodies in the region between the olfactory epithelium and the olfactory bulb express ACE2 and cathepsins B and L. Nervus terminalis neurons therefore may provide a direct route for the virus from the nasal epithelium, possibly via innervation of Bowman's glands, to brain targets, including the telencephalon and diencephalon. This possibility needs to be examined in suitable animal models and in human tissues.

18.
EMBO J ; 40(15): e107826, 2021 08 02.
Article in English | MEDLINE | ID: covidwho-1261483

ABSTRACT

SARS-CoV-2 infection causes broad-spectrum immunopathological disease, exacerbated by inflammatory co-morbidities. A better understanding of mechanisms underpinning virus-associated inflammation is required to develop effective therapeutics. Here, we discover that SARS-CoV-2 replicates rapidly in lung epithelial cells despite triggering a robust innate immune response through the activation of cytoplasmic RNA sensors RIG-I and MDA5. The inflammatory mediators produced during epithelial cell infection can stimulate primary human macrophages to enhance cytokine production and drive cellular activation. Critically, this can be limited by abrogating RNA sensing or by inhibiting downstream signalling pathways. SARS-CoV-2 further exacerbates the local inflammatory environment when macrophages or epithelial cells are primed with exogenous inflammatory stimuli. We propose that RNA sensing of SARS-CoV-2 in lung epithelium is a key driver of inflammation, the extent of which is influenced by the inflammatory state of the local environment, and that specific inhibition of innate immune pathways may beneficially mitigate inflammation-associated COVID-19.


Subject(s)
COVID-19/immunology , DEAD Box Protein 58/immunology , Epithelial Cells/immunology , Interferon-Induced Helicase, IFIH1/immunology , Macrophages/immunology , RNA, Viral/immunology , Receptors, Immunologic/immunology , SARS-CoV-2 , COVID-19/genetics , COVID-19/virology , Cell Line , Cytokines/genetics , Cytokines/immunology , Epithelial Cells/virology , Host-Pathogen Interactions , Humans , Immunity, Innate , Inflammation/genetics , Inflammation/immunology , Inflammation/virology , Janus Kinases/immunology , Lung/cytology , Lung/immunology , Lung/virology , Macrophage Activation , NF-kappa B/immunology , Respiratory Mucosa/cytology , Respiratory Mucosa/immunology , Respiratory Mucosa/virology , SARS-CoV-2/genetics , SARS-CoV-2/physiology , STAT Transcription Factors/immunology , Virus Replication
19.
Braz J Otorhinolaryngol ; 2021 Jun 05.
Article in English | MEDLINE | ID: covidwho-1252513

ABSTRACT

INTRODUCTION: Olfactory epithelium biopsy has been useful for studying diverse otorhinolaryngological and neurological diseases, including the potential to better understand the pathophysiology behind COVID-19 olfactory manifestations. However, the safety and efficacy of the technique for obtaining human olfactory epithelium are still not fully established. OBJECTIVE: This study aimed to determine the safety and efficacy of harvesting olfactory epithelium cells, nerve bundles, and olfactory epithelium proper for morphological analysis from the superior nasal septum. METHODS: During nasal surgery, 22 individuals without olfactory complaints underwent olfactory epithelium biopsies from the superior nasal septum. The efficacy of obtaining olfactory epithelium, verification of intact olfactory epithelium and the presence of nerve bundles in biopsies were assessed using immunofluorescence. Safety for the olfactory function was tested psychophysically using both unilateral and bilateral tests before and 1 month after the operative procedure. RESULTS: Olfactory epithelium was found in 59.1% of the subjects. Of the samples, 50% were of the quality necessary for morphological characterization and 90.9% had nerve bundles. There was no difference in the psychophysical scores obtained in the bilateral olfactory test (University of Pennsylvania Smell Identification Test [UPSIT®]) between means before biopsy: 32.3 vs. postoperative: 32.5, p = 0.81. Also, no significant decrease occurred in unilateral testing (mean unilateral test scores 6 vs. 6.2, p = 0.46). None out of the 56 different odorant identification significantly diminished (p > 0.05). CONCLUSION: The technique depicted for olfactory epithelium biopsy is highly effective in obtaining neuronal olfactory tissue, but it has moderate efficacy in achieving samples useful for morphological analysis. Olfactory sensitivity remained intact.

20.
Iran J Med Sci ; 46(3): 151-168, 2021 05.
Article in English | MEDLINE | ID: covidwho-1248554

ABSTRACT

Coronavirus disease 2019 (COVID-19) emerged as a new contagion during December 2019, since which time it has triggered a rampant spike in fatality rates worldwide due to insufficient medical treatments and a lack of counteragents and prompted the World Health Organization to declare COVID-19 a public health emergency. It is, therefore, vital to accelerate the screening of new molecules or vaccines to win the battle against this pandemic. Experiences from previous epidemiological data on coronaviruses guide investigators in designing and exploring new compounds for a safe and cost-effective treatment. Several reports on the severe acute respiratory syndrome (SARS) epidemic indicate that severe acute respiratory syndrome coronavirus (SARS-CoV) and the novel COVID-19 use angiotensin-converting enzyme 2 (ACE2) as a receptor for binding to the host cell in the lung epithelia through the spike protein on their virion surface. ACE2 is a mono-carboxypeptidase best known for cleaving major peptides and substrates. Its degree in human airway epithelia positively correlates with coronavirus infection. The treatment approach can be the neutralization of the virus entering lung epithelial cells by using sera containing antibodies collected from COVID-19-recovered patients. Hence, we herein propose a pulmonary aerosolized formulation or a nasal drop using sera, which contain antibodies to prevent, treat, or immunize against COVID-19 infection.


Subject(s)
Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/therapeutic use , COVID-19/therapy , Administration, Inhalation , Administration, Intranasal , Aerosols , COVID-19/prevention & control , Humans , Immunization, Passive , Lung
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