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1.
Molecules ; 26(21)2021 Nov 08.
Article in English | MEDLINE | ID: covidwho-1512511

ABSTRACT

This work describes an untargeted analytical approach for the screening, identification, and characterization of the trans-epithelial transport of green tea (Camellia sinensis) catechin extracts with in vitro inhibitory effect against the SARS-CoV-2 papain-like protease (PLpro) activity. After specific catechin extraction, a chromatographic separation obtained six fractions were carried out. The fractions were assessed in vitro against the PLpro target. Fraction 5 showed the highest inhibitory activity against the SARS-CoV-2 PLpro (IC50 of 0.125 µg mL-1). The untargeted characterization revealed that (-)-epicatechin-3-gallate (ECG) was the most abundant compound in the fraction and the primary molecule absorbed by differentiated Caco-2 cells. Results indicated that fraction 5 was approximately 10 times more active than ECG (IC50 value equal to 11.62 ± 0.47 µg mL-1) to inhibit the PLpro target. Overall, our findings highlight the synergistic effects of the various components of the crude extract compared to isolated ECG.


Subject(s)
Catechin/pharmacology , Coronavirus Papain-Like Proteases/metabolism , Tea/metabolism , Antiviral Agents/chemistry , COVID-19/drug therapy , COVID-19/metabolism , Caco-2 Cells , Camellia sinensis/metabolism , Catechin/analogs & derivatives , Catechin/chemistry , Catechin/metabolism , Coronavirus Papain-Like Proteases/drug effects , Epithelium/drug effects , Epithelium/metabolism , Humans , Mass Spectrometry/methods , Plant Extracts/chemistry , Plant Extracts/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Tea/chemistry , Tea/physiology
2.
Molecules ; 26(21)2021 Nov 06.
Article in English | MEDLINE | ID: covidwho-1502470

ABSTRACT

The normal function of the airway epithelium is vital for the host's well-being. Conditions that might compromise the structure and functionality of the airway epithelium include congenital tracheal anomalies, infection, trauma and post-intubation injuries. Recently, the onset of COVID-19 and its complications in managing respiratory failure further intensified the need for tracheal tissue replacement. Thus far, plenty of naturally derived, synthetic or allogeneic materials have been studied for their applicability in tracheal tissue replacement. However, a reliable tracheal replacement material is missing. Therefore, this study used a tissue engineering approach for constructing tracheal tissue. Human respiratory epithelial cells (RECs) were isolated from nasal turbinate, and the cells were incorporated into a calcium chloride-polymerized human blood plasma to form a human tissue respiratory epithelial construct (HTREC). The quality of HTREC in vitro, focusing on the cellular proliferation, differentiation and distribution of the RECs, was examined using histological, gene expression and immunocytochemical analysis. Histological analysis showed a homogenous distribution of RECs within the HTREC, with increased proliferation of the residing RECs within 4 days of investigation. Gene expression analysis revealed a significant increase (p < 0.05) in gene expression level of proliferative and respiratory epithelial-specific markers Ki67 and MUC5B, respectively, within 4 days of investigation. Immunohistochemical analysis also confirmed the expression of Ki67 and MUC5AC markers in residing RECs within the HTREC. The findings show that calcium chloride-polymerized human blood plasma is a suitable material, which supports viability, proliferation and mucin secreting phenotype of RECs, and this suggests that HTREC can be a potential candidate for respiratory epithelial tissue reconstruction.


Subject(s)
Respiratory Mucosa/metabolism , Tissue Engineering/methods , Trachea/transplantation , Cell Differentiation , Cell Proliferation , Epithelial Cells/metabolism , Epithelium/metabolism , Feasibility Studies , Humans , Ki-67 Antigen/analysis , Ki-67 Antigen/genetics , Mucin 5AC/analysis , Mucin 5AC/genetics , Mucous Membrane/metabolism , Primary Cell Culture/methods , Respiratory Mucosa/physiology , Trachea/metabolism , Trachea/physiology
3.
Viruses ; 12(10)2020 10 16.
Article in English | MEDLINE | ID: covidwho-1389518

ABSTRACT

To address the expression pattern of the SARS-CoV-2 receptor ACE2 and the viral priming protease TMPRSS2 in the respiratory tract, this study investigated RNA sequencing transcriptome profiling of samples of airway and oral mucosa. As shown, ACE2 has medium levels of expression in both small airway epithelium and masticatory mucosa, and high levels of expression in nasal epithelium. The expression of ACE2 is low in mucosal-associated invariant T (MAIT) cells and cannot be detected in alveolar macrophages. TMPRSS2 is highly expressed in small airway epithelium and nasal epithelium and has lower expression in masticatory mucosa. Our results provide the molecular basis that the nasal mucosa is the most susceptible locus in the respiratory tract for SARS-CoV-2 infection and consequently for subsequent droplet transmission and should be the focus for protection against SARS-CoV-2 infection.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/genetics , Peptidyl-Dipeptidase A/biosynthesis , Pneumonia, Viral/genetics , Serine Endopeptidases/biosynthesis , Virus Internalization , Angiotensin-Converting Enzyme 2 , COVID-19 , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Epithelium/metabolism , Epithelium/virology , Gene Expression , Gene Expression Profiling , Humans , Nasal Mucosa/metabolism , Nasal Mucosa/virology , Pandemics , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/metabolism , Pneumonia, Viral/virology , Respiratory System/metabolism , Respiratory System/virology , SARS-CoV-2 , Serine Endopeptidases/genetics
4.
Eur J Med Res ; 26(1): 52, 2021 Jun 09.
Article in English | MEDLINE | ID: covidwho-1262517

ABSTRACT

BACKGROUND: We are laryngologists. We observe natural phonatory and swallowing functions in clinical examinations with a trans-nasal laryngeal fiberscope (TNLF). Before each observation, we use epinephrine to enlarge and smooth the common nasal meatus (bottom of nostril) and then insert a wet swab inside the nose, as in taking a swab culture in the nasopharynx. During the current COVID-19 pandemic situation, this careful technique prevents any complications, including nasal bleeding, painfulness, and induced sneezing. Here, we introduce our routine to observe esophageal movement in swallowing in a natural (sitting) position without anesthesia. CASE PRESENTATION: The case was a 70-year-old female who complained that something was stuck in her esophagus; there was a strange sensation below the larynx and pharynx. After enlarging and smoothing the common nasal meatus, we inserted the TNLF (slim type ⌀2.9 mm fiberscope, VNL8-J10, PENTAX Medical, Tokyo, Japan.) in the normal way. We then observed the phonatory and swallowing movements of the vocal folds. As usual, to not interfere with natural movements, we used no anesthesia. We found no pathological condition in the pyriform sinus. We asked the patient to swallow the fiberscope. During the swallow, we pushed the TNLF and inserted the tip a bit deeper, which made the fiberscope easily enter the esophagus, like in the insertion of a nasogastric tube. We then asked the patient to swallow a sip of water or saliva to clear and enlarge the lumen of the esophagus. This made it possible to observe the esophagus easily without any air supply. With tone enhancement scan, the esophagus was found to be completely normal except for glycogenic acanthosis. CONCLUSIONS: The advantage of this examination is that it is easily able to perform without anesthesia and with the patient in sitting position. It is quick and minimally invasive, enabling observation the physiologically natural swallowing. It is also possible to observe without anesthesia down to the level of the esophagogastric junction using with a thin type flexible bronchoscope. In the future, gastric fiberscopes might be thinner, even with narrow band imaging (NBI) function. Before that time, physicians should remember to just insert along the bottom of the nose.


Subject(s)
COVID-19/prevention & control , Esophagus/metabolism , Glycogen/metabolism , Intubation, Intratracheal/methods , Aged , Anesthesia , COVID-19/epidemiology , COVID-19/virology , Epithelium/metabolism , Female , Humans , Intubation, Intratracheal/instrumentation , Mucous Membrane/metabolism , Nasal Cavity , Pandemics , Reproducibility of Results , SARS-CoV-2/physiology , Sneezing
5.
PLoS Pathog ; 17(4): e1009041, 2021 04.
Article in English | MEDLINE | ID: covidwho-1231262

ABSTRACT

Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus with latent and lytic cycles. EBV replicates in the stratified epithelium but the nasopharynx is also composed of pseudostratified epithelium with distinct cell types. Latent infection is associated with nasopharyngeal carcinoma (NPC). Here, we show with nasopharyngeal conditionally reprogrammed cells cultured at the air-liquid interface that pseudostratified epithelial cells are susceptible to EBV infection. Donors varied in susceptibility to de novo EBV infection, but susceptible cultures also displayed differences with respect to pathogenesis. The cultures from one donor yielded lytic infection but cells from two other donors were positive for EBV-encoded EBERs and negative for other lytic infection markers. All cultures stained positive for the pseudostratified markers CK7, MUC5AC, α-tubulin in cilia, and the EBV epithelial cell receptor Ephrin receptor A2. To define EBV transcriptional programs by cell type and to elucidate latent/lytic infection-differential changes, we performed single cell RNA-sequencing on one EBV-infected culture that resulted in alignment with many EBV transcripts. EBV transcripts represented a small portion of the total transcriptome (~0.17%). All cell types in the pseudostratified epithelium had detectable EBV transcripts with suprabasal cells showing the highest number of reads aligning to many EBV genes. Several restriction factors (IRF1, MX1, STAT1, C18orf25) known to limit lytic infection were expressed at lower levels in the lytic subcluster. A third of the differentially-expressed genes in NPC tumors compared to an uninfected pseudostratified ALI culture overlapped with the differentially-expressed genes in the latent subcluster. A third of these commonly perturbed genes were specific to EBV infection and changed in the same direction. Collectively, these findings suggest that the pseudostratified epithelium could harbor EBV infection and that the pseudostratified infection model mirrors many of the transcriptional changes imposed by EBV infection in NPC.


Subject(s)
Epithelial Cells/virology , Epstein-Barr Virus Infections/virology , Host-Pathogen Interactions/immunology , Nasopharyngeal Neoplasms/virology , Carcinoma/metabolism , Carcinoma/virology , Epithelial Cells/metabolism , Epithelium/metabolism , Epithelium/virology , Epstein-Barr Virus Infections/metabolism , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/pathogenicity , Humans , Nasopharyngeal Carcinoma/virology , RNA, Viral/genetics
6.
Cell Rep ; 35(5): 109055, 2021 05 04.
Article in English | MEDLINE | ID: covidwho-1179291

ABSTRACT

Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Although infection initiates in the proximal airways, severe and sometimes fatal symptoms of the disease are caused by infection of the alveolar type 2 (AT2) cells of the distal lung and associated inflammation. In this study, we develop primary human lung epithelial infection models to understand initial responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface (ALI) cultures of proximal airway epithelium and alveosphere cultures of distal lung AT2 cells are readily infected by SARS-CoV-2, leading to an epithelial cell-autonomous proinflammatory response with increased expression of interferon signaling genes. Studies to validate the efficacy of selected candidate COVID-19 drugs confirm that remdesivir strongly suppresses viral infection/replication. We provide a relevant platform for study of COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and emergent respiratory pathogens.


Subject(s)
Alveolar Epithelial Cells/virology , COVID-19/drug therapy , COVID-19/pathology , Lung/virology , SARS-CoV-2/drug effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Adult , Aged , Alanine/analogs & derivatives , Alanine/pharmacology , Alveolar Epithelial Cells/metabolism , COVID-19/metabolism , COVID-19/virology , Child, Preschool , Drug Discovery/methods , Epithelial Cells/virology , Epithelium/metabolism , Epithelium/virology , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Lung/pathology , Male , Middle Aged , Models, Biological , Primary Cell Culture , Respiratory Mucosa/virology , SARS-CoV-2/physiology , Virus Replication/drug effects
7.
PLoS One ; 15(12): e0243959, 2020.
Article in English | MEDLINE | ID: covidwho-1067398

ABSTRACT

There has been significant concern regarding fertility and reproductive outcomes during the SARS-CoV2 pandemic. Recent data suggests a high concentration of SARS-Cov2 receptors, ACE2 or TMPRSS2, in nasal epithelium and cornea, which explains person-to-person transmission. We investigated the prevalence of SARS-CoV2 receptors among reproductive tissues by exploring the single-cell sequencing datasets from uterus, myometrium, ovary, fallopian tube, and breast epithelium. We did not detect significant expression of either ACE2 or TMPRSS2 in the normal human myometrium, uterus, ovaries, fallopian tube, or breast. Furthermore, none of the cell types in the female reproductive organs we investigated, showed the co-expression of ACE2 with proteases, TMPRSS2, Cathepsin B (CTSB), and Cathepsin L (CTSL) known to facilitate the entry of SARS2-CoV2 into the host cell. These results suggest that myometrium, uterus, ovaries, fallopian tube, and breast are unlikely to be susceptible to infection by SARS-CoV2.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , Cathepsin B/genetics , Cathepsin L/genetics , SARS-CoV-2/genetics , Serine Endopeptidases/genetics , Angiotensin-Converting Enzyme 2/metabolism , Breast/metabolism , Breast/virology , COVID-19/epidemiology , COVID-19/transmission , COVID-19/virology , Epithelium/metabolism , Epithelium/virology , Fallopian Tubes/metabolism , Fallopian Tubes/virology , Female , Fertility/genetics , High-Throughput Nucleotide Sequencing , Humans , Myometrium/metabolism , Myometrium/virology , Ovary/metabolism , Ovary/virology , RNA, Viral/genetics , RNA, Viral/isolation & purification , Reproductive Tract Infections/genetics , Reproductive Tract Infections/virology , SARS-CoV-2/pathogenicity , Serine Endopeptidases/metabolism , Single-Cell Analysis , Uterus/metabolism , Uterus/virology
8.
Mol Cell Biochem ; 476(1): 93-107, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-737128

ABSTRACT

Mesenchymal stem cells (MSCs) can alleviate acute respiratory distress syndrome (ARDS), but the mechanisms involved are unclear, especially about their specific effects on cellular mitochondrial respiratory function. Thirty mice were allocated into the Control, LPS, and LPS + Bone marrow mesenchymal stem cell (BMSC) group (n = 10/group). Mouse alveolar epithelial cells (MLE-12) and macrophage cells (RAW264.7) were divided into the same groups. Pathological variation, inflammation-related factors, reactive oxygen species (ROS), ATP levels, and oxygen consumption rate (OCR) were analyzed. Pathologic features of ARDS were observed in the LPS group and were significantly alleviated by BMSCs. The trend in inflammation-related factors among the three groups was the LPS group > LPS + BMSC group > Control group. In the MLE-12 co-culture system, IL-6 was increased in the LPS group but not significantly reduced in the LPS + BMSC group. In the RAW264.7 co-culture system, IL-1ß, TNF-α, and IL-10 levels were all increased in the LPS group, IL-1ß and TNF-α levels were reduced by BMSCs, while IL-10 level kept increasing. ROS and ATP levels were increased and decreased respectively in both MLE-12 and RAW264.7 cells in the LPS groups but reversed by BMSCs. Basal OCR, ATP-linked OCR, and maximal OCR were lower in the LPS groups. Impaired basal OCR and ATP-linked OCR in MLE-12 cells were partially restored by BMSCs, while impaired basal OCR and maximal OCR in RAW264.7 cells were restored by BMSCs. BMSCs improved the mitochondrial respiration dysfunction of macrophages and alveolar epithelial cells induced by LPS, alleviated lung tissue injury, and inflammatory response in a mouse model of ARDS.


Subject(s)
Epithelium/metabolism , Mesenchymal Stem Cells/cytology , Mitochondria/metabolism , Pulmonary Alveoli/metabolism , Respiratory Distress Syndrome/metabolism , Adenosine Triphosphate/metabolism , Animals , Bone Marrow Cells/cytology , Coculture Techniques , Inflammation , Interleukin-10/metabolism , Interleukin-6/metabolism , Lipopolysaccharides/metabolism , Lung Injury/metabolism , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Oxygen Consumption , RAW 264.7 Cells
9.
Biochem Biophys Res Commun ; 533(4): 1276-1282, 2020 12 17.
Article in English | MEDLINE | ID: covidwho-885206

ABSTRACT

BACKGROUND: The whole world was hit hard by the coronavirus disease-19 (COVID-19). Given that angiotensin I converting enzyme 2 (ACE2) is the viral entry molecule, understanding ACE2 has become a major focus of current COVID-19 research. ACE2 is highly expressed in the gut, but its role has not been fully understood and thus COVID-19 treatments intending to downregulate ACE2 level may cause untoward side effects. Gaining insight into the functions of ACE2 in gut homeostasis therefore merits closer examination, and is beneficial to find potential therapeutic alternatives for COVID-19. METHODS: We took advantage of Ace2 knockout out mice and isolated intestinal organoids to examine the role of ACE2 in intestinal stemness. Inflammatory bowel disease (IBD) mouse model was established by 4% dextran sodium sulfate. LGR5 and KI67 levels were quantitated to reflect the virtue of intestinal stem cells (ISCs). FITC-dextran 4 (FD-4) assay was used to assess intestinal barrier function. RESULTS: Western blotting identified the expression of ACE2 in colon, which was consistent with the results of immunofluorescence and RT-PCR. Moreover, Ace2-/- organoids showed decreased LRG5 and KI67 levels, and elevated calcium concentration. Furthermore, the permeability of ace2-/- organoids was markedly increased compared with ace2+/+ organoids. Collectively, ace2-/- mice were more susceptible than ace2+/+ mice to IBD, including earlier bloody stool, undermined intestinal architecture and more pronounced weight loss. CONCLUSIONS: Our data reveal that ACE2 contributes to the proliferation of intestinal stem cells and hence orchestrates the mucosal homeostasis.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Epithelium/metabolism , Angiotensin-Converting Enzyme 2/deficiency , Animals , Calcium/metabolism , Cell Membrane Permeability , Inflammatory Bowel Diseases/enzymology , Inflammatory Bowel Diseases/pathology , Intestines/pathology , Mice, Inbred C57BL , Mice, Knockout , Organoids/metabolism , Stem Cells/cytology , Stem Cells/metabolism
12.
Acta Physiol (Oxf) ; 231(3): e13551, 2021 03.
Article in English | MEDLINE | ID: covidwho-732095

ABSTRACT

Nicotinamide adenine dinucleotide (NAD) is an abundant cofactor that plays crucial roles in several cellular processes. NAD can be synthesized de novo starting with tryptophan, or from salvage pathways starting with NAD precursors like nicotinic acid (NA), nicotinamide (NAM) or nicotinamide riboside (NR), referred to as niacin/B3 vitamins, arising from dietary supply or from cellular NAD catabolism. Given the interconversion between its oxidized (NAD+ ) and reduced form (NADH), NAD participates in a wide range of reactions: regulation of cellular redox status, energy metabolism and mitochondrial biogenesis. Plus, NAD acts as a signalling molecule, being a cosubstrate for several enzymes such as sirtuins, poly-ADP-ribose-polymerases (PARPs) and some ectoenzymes like CD38, regulating critical biological processes like gene expression, DNA repair, calcium signalling and circadian rhythms. Given the large number of mitochondria present in cardiac tissue, the heart has the highest NAD levels and is one of the most metabolically demanding organs. In several models of heart failure, myocardial NAD levels are depressed and this depression is caused by mitochondrial dysfunction, metabolic remodelling and inflammation. Emerging evidence suggests that regulating NAD homeostasis by NAD precursor supplementation has therapeutic efficiency in improving myocardial bioenergetics and function. This review provides an overview of the latest understanding of the different NAD biosynthesis pathways, as well as its role as a signalling molecule particularly in cardiac tissue. We highlight the significance of preserving NAD equilibrium in various models of heart diseases and shed light on the potential pharmacological interventions aiming to use NAD boosters as therapeutic agents.


Subject(s)
Heart Diseases/metabolism , Mitochondria/metabolism , NAD/biosynthesis , Animals , Energy Metabolism/physiology , Epithelium/metabolism , Humans , Myocardium/metabolism
14.
Am J Physiol Lung Cell Mol Physiol ; 319(1): L115-L120, 2020 07 01.
Article in English | MEDLINE | ID: covidwho-558506

ABSTRACT

COVID-19 can be divided into three clinical stages, and one can speculate that these stages correlate with where the infection resides. For the asymptomatic phase, the infection mostly resides in the nose, where it elicits a minimal innate immune response. For the mildly symptomatic phase, the infection is mostly in the pseudostratified epithelium of the larger airways and is accompanied by a more vigorous innate immune response. In the conducting airways, the epithelium can recover from the infection, because the keratin 5 basal cells are spared and they are the progenitor cells for the bronchial epithelium. There may be more severe disease in the bronchioles, where the club cells are likely infected. The devastating third phase is in the gas exchange units of the lung, where ACE2-expressing alveolar type II cells and perhaps type I cells are infected. The loss of type II cells results in respiratory insufficiency due to the loss of pulmonary surfactant, alveolar flooding, and possible loss of normal repair, since type II cells are the progenitors of type I cells. The loss of type I and type II cells will also block normal active resorption of alveolar fluid. Subsequent endothelial damage leads to transudation of plasma proteins, formation of hyaline membranes, and an inflammatory exudate, characteristic of ARDS. Repair might be normal, but if the type II cells are severely damaged alternative pathways for epithelial repair may be activated, which would result in some residual lung disease.


Subject(s)
Alveolar Epithelial Cells/virology , Betacoronavirus/pathogenicity , Coronavirus Infections/virology , Epithelial Cells/virology , Pneumonia, Viral/virology , Alveolar Epithelial Cells/metabolism , COVID-19 , Coronavirus Infections/diagnosis , Coronavirus Infections/therapy , Epithelial Cells/metabolism , Epithelium/metabolism , Epithelium/virology , Humans , Lung/metabolism , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/therapy , Respiratory Mucosa/metabolism , Respiratory Mucosa/virology , SARS-CoV-2
15.
Obesity (Silver Spring) ; 28(9): 1586-1589, 2020 09.
Article in English | MEDLINE | ID: covidwho-306140

ABSTRACT

OBJECTIVE: Mortality from coronavirus disease 2019 (COVID-19) is increased in patients with chronic obstructive pulmonary disease (COPD). Furthermore, higher BMI is related to severe disease. Severe acute respiratory syndrome coronavirus 2 utilizes angiotensin converting enzyme 2 (ACE2) to gain cellular entry. METHODS: Whether ACE2 bronchial epithelial expression is increased in COPD patients who have overweight compared with those who do not was investigated by RNA sequencing. RESULTS: Increased ACE2 expression was observed in patients with COPD with overweight (mean BMI, 29 kg/m2 ) compared with those without overweight (mean BMI, 21 kg/m2 ) (P = 0.004). CONCLUSIONS: Increased ACE2 expression may cause increased severe acute respiratory syndrome coronavirus 2 infection of the respiratory tract. Overweight COPD patients may be at greater risk for developing severe COVID-19.


Subject(s)
Bronchi , Epithelium/metabolism , Overweight/complications , Peptidyl-Dipeptidase A/genetics , Pulmonary Disease, Chronic Obstructive/genetics , Aged , Angiotensin-Converting Enzyme 2 , Betacoronavirus , COVID-19 , Coronavirus Infections , Female , Gene Expression Regulation , Humans , Male , Middle Aged , Pandemics , Pneumonia, Viral , Pulmonary Disease, Chronic Obstructive/complications , SARS-CoV-2
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