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1.
J Cell Biol ; 221(7)2022 07 04.
Article in English | MEDLINE | ID: covidwho-2082890

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal pathogen of the ongoing global pandemic of coronavirus disease 2019 (COVID-19). Loss of smell and taste are symptoms of COVID-19, and may be related to cilia dysfunction. Here, we found that the SARS-CoV-2 ORF10 increases the overall E3 ligase activity of the CUL2ZYG11B complex by interacting with ZYG11B. Enhanced CUL2ZYG11B activity by ORF10 causes increased ubiquitination and subsequent proteasome-mediated degradation of an intraflagellar transport (IFT) complex B protein, IFT46, thereby impairing both cilia biogenesis and maintenance. Further, we show that exposure of the respiratory tract of hACE2 mice to SARS-CoV-2 or SARS-CoV-2 ORF10 alone results in cilia-dysfunction-related phenotypes, and the ORF10 expression in primary human nasal epithelial cells (HNECs) also caused a rapid loss of the ciliary layer. Our study demonstrates how SARS-CoV-2 ORF10 hijacks CUL2ZYG11B to eliminate IFT46 and leads to cilia dysfunction, thereby offering a powerful etiopathological explanation for how SARS-CoV-2 causes multiple cilia-dysfunction-related symptoms specific to COVID-19.


Subject(s)
Cilia , SARS-CoV-2 , Ubiquitin-Protein Ligases , Animals , Cells, Cultured , Cilia/metabolism , Cilia/pathology , Cytoskeletal Proteins , Epithelial Cells/metabolism , Epithelial Cells/virology , Humans , Mice , SARS-CoV-2/pathogenicity , Smell , Ubiquitin-Protein Ligases/metabolism
2.
Sci Rep ; 12(1): 1724, 2022 02 02.
Article in English | MEDLINE | ID: covidwho-1663979

ABSTRACT

This study introduces localized surface plasmon resonance (L-SPR) mediated heating filter membrane (HFM) for inactivating universal viral particles by using the photothermal effect of plasmonic metal nanoparticles (NPs). Plasmonic metal NPs were coated onto filter membrane via a conventional spray-coating method. The surface temperature of the HFM could be controlled to approximately 40-60 °C at room temperature, owing to the photothermal effect of the gold (Au) NPs coated on them, under irradiation by visible light-emitting diodes. Due to the photothermal effect of the HFMs, the virus titer of H1Npdm09 was reduced by > 99.9%, the full inactivation time being < 10 min, confirming the 50% tissue culture infective dose (TCID50) assay. Crystal violet staining showed that the infectious samples with photothermal inactivation lost their infectivity against Mardin-Darby Canine Kidney cells. Moreover, photothermal inactivation could also be applied to reduce the infectivity of SARS-CoV-2, showing reduction rate of 99%. We used quantitative reverse transcription polymerase chain reaction (qRT-PCR) techniques to confirm the existence of viral genes on the surface of the HFM. The results of the TCID50 assay, crystal violet staining method, and qRT-PCR showed that the effective and immediate reduction in viral infectivity possibly originated from the denaturation or deformation of membrane proteins and components. This study provides a new, simple, and effective method to inactivate viral infectivity, leading to its potential application in various fields of indoor air quality control and medical science.


Subject(s)
COVID-19/virology , Hot Temperature , Light , Metal Nanoparticles , Micropore Filters , SARS-CoV-2 , Surface Plasmon Resonance/methods , Virion , Virus Inactivation , Air Pollution, Indoor , Animals , Cells, Cultured , Dogs , Gold/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity
3.
J Vis Exp ; (186)2022 08 17.
Article in English | MEDLINE | ID: covidwho-2024393

ABSTRACT

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. Efficient and safe delivery systems must be included in the mRNA vaccines due to the fragile properties of mRNA. A self-assembled peptide-poloxamine nanoparticle (PP-sNp) gene delivery system is specifically designed for the pulmonary delivery of nucleic acids and displays promising capabilities in mediating successful mRNA transfection. Here, an improved method for preparing PP-sNp is described to elaborate on how the PP-sNp encapsulates Metridia luciferase (MetLuc) mRNA and successfully transfects cultured cells. MetLuc-mRNA is obtained by an in vitro transcription process from a linear DNA template. A PP-sNp is produced by mixing synthetic peptide/poloxamine with mRNA solution using a microfluidic mixer, allowing for the self-assembly of PP-sNp. The charge of PP-sNp is subsequently evaluated by measuring the zeta potential. Meanwhile, the polydispersity and hydrodynamic size of PP-sNp nanoparticles are measured using dynamic light scattering. The mRNA/PP-sNp nanoparticles are transfected into cultured cells, and supernatants from the cell culture are assayed for luciferase activity. The representative results demonstrate their capacity for in vitro transfection. This protocol may shed light on developing next-generation mRNA vaccine delivery systems.


Subject(s)
COVID-19 , Nanoparticles , Cells, Cultured , Humans , Luciferases/genetics , Peptides/genetics , RNA, Messenger/genetics , Transfection , Vaccines, Synthetic , mRNA Vaccines
4.
Int J Mol Sci ; 23(16)2022 Aug 18.
Article in English | MEDLINE | ID: covidwho-2023739

ABSTRACT

The European mink (Mustela lutreola) is one of Europe's most endangered species, and it is on the brink of extinction in the Iberian Peninsula. The species' precarious situation requires the application of new ex situ conservation methodologies that complement the existing ex situ and in situ conservation measures. Here, we report for the first time the establishment of a biobank for European mink mesenchymal stem cells (emMSC) and oocytes from specimens found dead in the Iberian Peninsula, either free or in captivity. New emMSC lines were isolated from different tissues: bone marrow (emBM-MSC), oral mucosa (emOM-MSc), dermal skin (emDS-MSC), oviduct (emO-MSc), endometrium (emE-MSC), testicular (emT-MSC), and adipose tissue from two different adipose depots: subcutaneous (emSCA-MSC) and ovarian (emOA-MSC). All eight emMSC lines showed plastic adhesion, a detectable expression of characteristic markers of MSCs, and, when cultured under osteogenic and adipogenic conditions, differentiation capacity to these lineages. Additionally, we were able to keep 227 Cumulus-oocyte complexes (COCs) in the biobank, 97 of which are grade I or II. The European mink MSC and oocyte biobank will allow for the conservation of the species' genetic variability, the application of assisted reproduction techniques, and the development of in vitro models for studying the molecular mechanisms of infectious diseases that threaten the species' precarious situation.


Subject(s)
Mesenchymal Stem Cells , Mink , Animals , Cell Differentiation , Cells, Cultured , Endangered Species , Female , Mink/genetics , Oocytes , Osteogenesis
5.
Front Immunol ; 13: 943333, 2022.
Article in English | MEDLINE | ID: covidwho-2022722

ABSTRACT

Mesenchymal stromal cell (MSC) therapy has seen increased attention as a possible option to treat a number of inflammatory conditions including COVID-19 acute respiratory distress syndrome (ARDS). As rates of obesity and metabolic disease continue to rise worldwide, increasing proportions of patients treated with MSC therapy will be living with obesity. The obese environment poses critical challenges for immunomodulatory therapies that should be accounted for during development and testing of MSCs. In this review, we look to cancer immunotherapy as a model for the challenges MSCs may face in obese environments. We then outline current evidence that obesity alters MSC immunomodulatory function, drastically modifies the host immune system, and therefore reshapes interactions between MSCs and immune cells. Finally, we argue that obese environments may alter essential features of allogeneic MSCs and offer potential strategies for licensing of MSCs to enhance their efficacy in the obese microenvironment. Our aim is to combine insights from basic research in MSC biology and clinical trials to inform new strategies to ensure MSC therapy is effective for a broad range of patients.


Subject(s)
COVID-19 , Mesenchymal Stem Cells , COVID-19/therapy , Cells, Cultured , Humans , Immunomodulation , Mesenchymal Stem Cells/metabolism , Obesity/metabolism , Obesity/therapy
6.
Am J Respir Cell Mol Biol ; 67(3): 389-401, 2022 09.
Article in English | MEDLINE | ID: covidwho-2020635

ABSTRACT

The lung epithelium forms the first barrier against respiratory pathogens and noxious chemicals; however, little is known about how more than 90% of this barrier, made of AT1 (alveolar type 1) cells, responds to injury. Using the Sendai virus to model natural infection in mice, we find evidence that AT1 cells have an intermediary role by persisting in areas depleted of AT2 cells, upregulating IFN responsive genes, and receding from invading airway cells. Sendai virus infection mobilizes airway cells to form alveolar SOX2+ (Sry-box 2+) clusters without differentiating into AT1 or AT2 cells. Large AT2 cell-depleted areas remain covered by AT1 cells, which we name "AT2-less regions", and are replaced by SOX2+ clusters spreading both basally and luminally. AT2 cell proliferation and differentiation are largely confined to topologically distal regions and form de novo alveolar surface, with limited contribution to in situ repairs of AT2-less regions. Time-course single-cell RNA sequencing profiling and RNAscope validation suggest enhanced immune responses and altered growth signals in AT1 cells. Our comprehensive spatiotemporal and genomewide study highlights the hitherto unappreciated role of AT1 cells in lung injury-repair.


Subject(s)
Alveolar Epithelial Cells , Respirovirus Infections , Alveolar Epithelial Cells/metabolism , Animals , Cell Differentiation/physiology , Cells, Cultured , Lung , Mice
7.
Turk Neurosurg ; 32(4): 680-687, 2022.
Article in English | MEDLINE | ID: covidwho-2006417

ABSTRACT

AIM: To evaluate the effects of favipiravir (FVP) on cell viability and cytotoxicity in human degenerated primary intervertebral disc (IVD) tissue cell cultures. Furthermore, the protein expressions of hypoxia-inducible factor 1 alpha (HIF-1α), nuclear factor-kappa-b (NF-kB), and interleukin-1 beta (IL-1ß) were also examined. MATERIAL AND METHODS: Untreated cell cultures served as the control group, named group 1. Cell cultures treated with FVP served as the study group, named group 2. Pharmacomolecular analyses were performed in all groups at 0, 24, 48, and 72 hours (h). Obtained data were evaluated statistically. RESULTS: Cell proliferation was suppressed in the FVP-treated samples compared to the control group samples at 24 and 72 h, and this was statistically significant (p < 0.05). Decreased or increased protein expression levels of HIF-1α, NF-κB, and IL-1ß in FVPtreated samples may be an indication of suppression in anabolic events as well as proliferation in IVD cultures. FVP administration showed that AF/NP cells in a culture medium may induce a strong inflammatory response to FVP. This strong inflammatory response is likely to cause slowed proliferation. It may also be a trigger for many catabolic events. NF-κB expression increased within the first 24 h and then decreased rapidly. Based on the data obtained, it may be suggested that the rapidly increasing NF-kB may have stimulated the expression of many antiproliferative genes. CONCLUSION: The suppression of IL-1ß and NF-kB protein expressions in IVD cells treated with FVP is important in the treatment of IVD degeneration (IDD). If the protein expression of HIF-1α could be increased along with the suppression of IL-1ß and NF-kB, FVP would perhaps be a promising pharmacological agent in the treatment of IDD.


Subject(s)
Intervertebral Disc Degeneration , Intervertebral Disc , Amides , Apoptosis , Autophagy , Cells, Cultured , Humans , Interleukin-1beta/metabolism , Intervertebral Disc Degeneration/genetics , NF-kappa B/metabolism , Pyrazines
8.
Microbiol Spectr ; 10(5): e0105622, 2022 Oct 26.
Article in English | MEDLINE | ID: covidwho-2001788

ABSTRACT

Infection by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has posed a severe threat to global public health. The current study revealed that several inhibitors of protein kinases C (PKCs) possess protective activity against SARS-CoV-2 infection. Four pan-PKC inhibitors, Go 6983, bisindolylmaleimide I, enzastaurin, and sotrastaurin, reduced the replication of a SARS-CoV-2 replicon in both BHK-21 and Huh7 cells. A PKCδ-specific inhibitor, rottlerin, was also effective in reducing viral infection. The PKC inhibitors acted at an early step of SARS-CoV-2 infection. Finally, PKC inhibitors blocked the replication of wild-type SARS-CoV-2 in ACE2-expressing A549 cells. Our work highlights the importance of the PKC signaling pathway in infection by SARS-CoV-2 and provides evidence that PKC-specific inhibitors are potential therapeutic agents against SARS-CoV-2. IMPORTANCE There is an urgent need for effective therapeutic drugs to control the pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). We found that several inhibitors of protein kinases C (PKCs) dramatically decrease the replication of SARS-CoV-2 in cultured cells. These PKC inhibitors interfere with an early step of viral infection. Therefore, the rapid and prominent antiviral effect of PKC inhibitors underscores that they are promising antiviral agents and suggests that PKCs are important host factors involved in infection by SARS-CoV-2.


Subject(s)
Antiviral Agents , Protein Kinase C , SARS-CoV-2 , Humans , Angiotensin-Converting Enzyme 2 , Antiviral Agents/pharmacology , Cells, Cultured , COVID-19/drug therapy , Protein Kinase C/pharmacology , SARS-CoV-2/drug effects
9.
Toxicol Lett ; 350: 143-151, 2021 Oct 10.
Article in English | MEDLINE | ID: covidwho-1321487

ABSTRACT

Remdesivir (RDV) is a novel antiviral drug whose mitochondrial effects are not well known. In vitro effects of RDV on the mitochondrial respiration, individual respiratory complexes, and the activity of monoamine oxidase (MAO-A and MAO-B) were measured in isolated mitochondria. At micromolar RDV concentrations, minimal or no inhibitory effects on the studied mitochondrial enzymes were found. At very high concentrations of RDV, there was partial inhibition of complex I- (IC50 675 µmol/L, residual activity 39.4 %) and complex II-linked (IC50 81.8 µmol/L, residual activity 40.7 %) respiration, without inhibition of complex IV-linked respiration, and partial inhibition both of MAO-A (IC50 26.6 µmol/L, residual activity 35.2 %) and MAO-B (IC50 89.8 µmol/L, residual activity 34.0 %) activity. Individual respiratory complexes (I, II + III, and IV) were partially inhibited at a high drug concentration. The active metabolite of RDV (GS-443902) had very little effect on mitochondrial oxygen consumption rate with residual activity of 87.0 % for complex I-linked respiration, 90.3 % for complex II-linked respiration, and with no inhibition of complex IV-linked respiration. In conclusion, measurement of the effect of RDV and its active metabolite on isolated mitochondria shows that there is very little direct effect on mitochondrial respiration occurs at therapeutic drug concentration.


Subject(s)
Antiviral Agents/pharmacology , Cell Respiration/drug effects , Cells, Cultured/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Monoamine Oxidase/drug effects , Monoamine Oxidase/metabolism , Animals , Humans , Models, Animal , Swine
10.
Inflamm Res ; 71(9): 1003-1009, 2022 Sep.
Article in English | MEDLINE | ID: covidwho-1966115

ABSTRACT

Among the countless endeavours made at elucidating the pathogenesis of COVID-19, those aimed at the histopathological alterations of type 2 alveolar epithelial cells (AT2) are of outstanding relevance to the field of lung physiology, as they are the building blocks of the pulmonary alveoli. A merit of high regenerative and proliferative capacity, exocytotic activity resulting in the release of extracellular vesicles (EVs) is particularly high in AT2 cells, especially in those infected with SARS-CoV-2. These AT2 cell-derived EVs, containing the genetic material of the virus, might enter the bloodstream and make their way into the cardiovascular system, where they may infect cardiomyocytes and bring about a series of events leading to heart failure. As surfactant protein C, a marker of AT2 cell activity and a constituent of the lung surfactant complex, occurs abundantly inside the AT2-derived EVs released during the inflammatory stage of COVID-19, it could potentially be used as a biomarker for predicting impending heart failure in those patients with a history of cardiovascular disease.


Subject(s)
COVID-19 , Extracellular Vesicles , Heart Failure , Alveolar Epithelial Cells , Cells, Cultured , Humans , Inflammation , Protein C , SARS-CoV-2 , Surface-Active Agents
11.
Adv Exp Med Biol ; 1370: 23-29, 2022.
Article in English | MEDLINE | ID: covidwho-1958872

ABSTRACT

The novel coronavirus disease (COVID-19), which is prevalent in the world, develops severe pneumonia, of which 30% have fatal acute respiratory distress and acute lung injury. At present, there is no established treatment method for ARDS, and it is desired to develop a therapeutic drug as soon as possible. While TauCl has been reported to have anti-inflammatory effects on culture cells, little information is available concerning in vivo experiments. In the present study, we evaluated the anti-inflammatory effect of taurine chloramine (TauCl), a taurine derivative, against LPS-induced pneumonia in mouse. The mice were pretreated with TauCl intraperitoneally before intratracheal administration of LPS. Additionally, we evaluated the effect of taurine treatment by maintaining the mice on drinking water containing 0.5% taurine. Two days after LPS injection, body weight was decreased by 9.5 %, while lung weight was increased due to the infiltration of inflammatory cells; TauCl attenuated the gain in lung weight. LPS-induced acute pneumonia caused an increase in cytokine/chemokine mRNA expression, including that of IL-1ß, -6, -17, TNF-α, and MCP-1. However, TauCl treatment attenuated IL-6 expression, but not that of the others although the induction of plasma IL-6 tended to be reduced by TauCl treatment. Importantly, a similar effect against LPS-induced acute lung inflammation was confirmed by taurine pretreatment. These findings suggest that TauCl treatment partially prevents IL-6 production induced by acute pneumonia in vivo.


Subject(s)
COVID-19 , Lipopolysaccharides , Animals , Anti-Inflammatory Agents , Cells, Cultured , Interleukin-6 , Lipopolysaccharides/toxicity , Mice , Taurine/analogs & derivatives , Taurine/pharmacology , Taurine/therapeutic use
12.
Microbiol Spectr ; 10(4): e0163922, 2022 08 31.
Article in English | MEDLINE | ID: covidwho-1950021

ABSTRACT

Human coronavirus NL63 (HCoV-NL63) is commonly associated with mild respiratory tract infections in infants, being that the respiratory epithelial cells are the main target for infection and initial replication of this virus. Standard immortalized cells are highly permissive to HCoV-NL63, and they are routinely used for isolation and propagation of the virus from clinical specimens. However, these cell lines are not the natural cell target of the virus and lack sufficient complexity to mimic the natural infection process in vivo. This study comparatively evaluated the differences on the susceptibility to HCoV-NL63 infection and virus replication efficiency of submerged monolayer cultures of LLC-MK2 and primary human respiratory epithelial cells (HRECs) and organotypic airway cultures of respiratory cells (ALI-HRECs). Productive viral infection and growth kinetics were assessed by morphologic examination of cytopathic effects, immunofluorescence, reverse transcription quantitative real-time PCR, and flow cytometry. Results from this study showed higher susceptibility to HCoV-NL63 infection and replication in LLC-MK2 cells followed by ALI-HRECs, with very low susceptibility and no significant virus replication in HRECs. This susceptibility was associated with the expression levels of angiontensin-converting enzyme 2 (ACE2) receptor protein in LLC-MK2, ALI-HRECs, and HRECs, respectively. Remarkably, organotypic ALI-HREC cultures expressed significantly more ACE2 receptor protein and were more susceptible to HCoV-NL63 infection than monolayer cultures of HREC. The ACE2 receptor is, therefore, a critical factor for susceptibility to HCoV-NL63 infection and replication, as is the type of culture used during infection studies. IMPORTANCE HCoV-NL63 is widespread globally, accounting for a significant number of respiratory infections in children and adults. HCoV-NL63 gains entrance into respiratory epithelial cells via the ACE2 receptor, the same cell receptor used by severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Thus, HCoV-NL63 has been suggested as safe surrogate for studying disease mechanisms and therapeutic interventions against SARS-like CoVs, while working under BSL-2 conditions. The present study not only showed the critical role of ACE2 for effective HCoV-NL63 infection and replication, but also shed light on the need of more refined and complex in vitro organotypic models that recapitulate the proxy of air-liquid respiratory epithelia cell composition, structure, and functionality. These cultures have broaden virological studies toward improving our understanding of how coronaviruses cause disease and transmission not just within humans but also in animal populations.


Subject(s)
Angiotensin-Converting Enzyme 2 , Coronavirus NL63, Human , Epithelial Cells , Angiotensin-Converting Enzyme 2/metabolism , Animals , Cells, Cultured , Coronavirus NL63, Human/pathogenicity , Epithelial Cells/metabolism , Epithelial Cells/virology , Humans
13.
Biochim Biophys Acta Mol Basis Dis ; 1868(12): 166496, 2022 Dec 01.
Article in English | MEDLINE | ID: covidwho-1936082

ABSTRACT

Patients with COVID-19 have high prevalence of albuminuria which is used as a marker of progression of renal disease and is associated with severe COVID-19. We hypothesized that SARS-CoV-2 spike protein (S protein) could modulate albumin handling in proximal tubule epithelial cells (PTECs) and, consequently contribute to the albuminuria observed in patients with COVID-19. In this context, the possible effect of S protein on albumin endocytosis in PTECs was investigated. Two PTEC lines were used: HEK-293A and LLC-PK1. Incubation of both cell types with S protein for 16 h inhibited albumin uptake at the same magnitude. This effect was associated with canonical megalin-mediated albumin endocytosis because: (1) DQ-albumin uptake, a marker of the lysosomal degradation pathway, was reduced at a similar level compared with fluorescein isothiocyanate (FITC)-albumin uptake; (2) dextran-FITC uptake, a marker of fluid-phase endocytosis, was not changed; (3) cell viability and proliferation were not changed. The inhibitory effect of S protein on albumin uptake was only observed when it was added at the luminal membrane, and it did not involve the ACE2/Ang II/AT1R axis. Although both cells uptake S protein, it does not seem to be required for modulation of albumin endocytosis. The mechanism underlying the inhibition of albumin uptake by S protein encompasses a decrease in megalin expression without changes in megalin trafficking and stability. These results reveal a possible mechanism to explain the albuminuria observed in patients with COVID-19.


Subject(s)
COVID-19 , Low Density Lipoprotein Receptor-Related Protein-2 , Albumins/metabolism , Albumins/pharmacology , Albuminuria/metabolism , Angiotensin-Converting Enzyme 2 , Cells, Cultured , Dextrans/pharmacology , Endocytosis/physiology , Epithelial Cells/metabolism , Fluorescein-5-isothiocyanate/metabolism , Fluorescein-5-isothiocyanate/pharmacology , Humans , Low Density Lipoprotein Receptor-Related Protein-2/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
14.
Front Immunol ; 13: 835830, 2022.
Article in English | MEDLINE | ID: covidwho-1902993

ABSTRACT

CD8+ T cells have key protective roles in many viral infections. While an overall Th1-biased cellular immune response against SARS-CoV-2 has been demonstrated, most reports of anti-SARS-CoV-2 cellular immunity have evaluated bulk T cells using pools of predicted epitopes, without clear delineation of the CD8+ subset and its magnitude and targeting. In recently infected persons (mean 29.8 days after COVID-19 symptom onset), we confirm a Th1 bias (and a novel IL-4-producing population of unclear significance) by flow cytometry, which does not correlate to antibody responses against the receptor binding domain. Evaluating isolated CD8+ T cells in more detail by IFN-γ ELISpot assays, responses against spike, nucleocapsid, matrix, and envelope proteins average 396, 901, 296, and 0 spot-forming cells (SFC) per million, targeting 1.4, 1.5, 0.59, and 0.0 epitope regions respectively. Nucleocapsid targeting is dominant in terms of magnitude, breadth, and density of targeting. The magnitude of responses drops rapidly post-infection; nucleocapsid targeting is most sustained, and vaccination selectively boosts spike targeting. In SARS-CoV-2-naïve persons, evaluation of the anti-spike CD8+ T cell response soon after vaccination (mean 11.3 days) yields anti-spike CD8+ T cell responses averaging 2,463 SFC/million against 4.2 epitope regions, and targeting mirrors that seen in infected persons. These findings provide greater clarity on CD8+ T cell anti-SARS-CoV-2 targeting, breadth, and persistence, suggesting that nucleocapsid inclusion in vaccines could broaden coverage and durability.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , Nucleocapsid/immunology , SARS-CoV-2/physiology , Antibodies, Viral/metabolism , Broadly Neutralizing Antibodies/metabolism , Cells, Cultured , Enzyme-Linked Immunospot Assay , Humans , Molecular Targeted Therapy , Peptides/genetics , Peptides/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , United States , Vaccination
15.
Int J Mol Sci ; 23(9)2022 May 09.
Article in English | MEDLINE | ID: covidwho-1847345

ABSTRACT

Fibrosis of extraocular muscles (EOMs) is a marker of end-stage in Graves' orbitopathy (GO). To determine the antifibrotic and anti-inflammatory therapeutic effects and the underlying molecular mechanisms of disulfiram (DSF) on perimysial orbital fibroblasts (pOFs) in a GO model in vitro, primary cultures of pOFs from eight patients with GO and six subjects without GO (NG) were established. CCK-8 and EdU assays, IF, qPCR, WB, three-dimensional collagen gel contraction assays, cell scratch experiments, and ELISAs were performed. After TGF-ß1 stimulation of pOFs, the proliferation rate of the GO group but not the NG group increased significantly. DSF dose-dependently inhibited the proliferation, contraction, and migration of pOFs in the GO group. Additionally, DSF dose-dependently inhibited fibrosis and extracellular matrix production markers (FN1, COL1A1, α-SMA, CTGF) at the mRNA and protein levels. Furthermore, DSF mediates antifibrotic effects on GO pOFs partially through the ERK-Snail signaling pathway. In addition, DSF attenuated HA production and suppressed inflammatory chemokine molecule expression induced by TGF-ß1 in GO pOFs. In this in vitro study, we demonstrate the inhibitory effect of DSF on pOFs fibrosis in GO, HA production, and inflammation. DSF may be a potential drug candidate for preventing and treating tissue fibrosis in GO.


Subject(s)
Graves Ophthalmopathy , Anti-Inflammatory Agents/pharmacology , Cells, Cultured , Disulfiram/metabolism , Disulfiram/pharmacology , Fibroblasts/metabolism , Fibrosis , Graves Ophthalmopathy/drug therapy , Graves Ophthalmopathy/metabolism , Humans , Transforming Growth Factor beta1/metabolism
16.
Front Immunol ; 13: 834988, 2022.
Article in English | MEDLINE | ID: covidwho-1817941

ABSTRACT

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect.


Subject(s)
Blood Platelets/immunology , COVID-19/immunology , Complement C5a/metabolism , Receptor, Anaphylatoxin C5a/metabolism , Receptors, IgG/metabolism , SARS-CoV-2/physiology , Thromboembolism/immunology , Adult , Aminopyridines/pharmacology , Cells, Cultured , Female , Hospitalization , Humans , Male , Morpholines/pharmacology , Platelet Activation , Pyrimidines/pharmacology , Severity of Illness Index , Signal Transduction , Syk Kinase/antagonists & inhibitors
17.
Cell Host Microbe ; 30(4): 409, 2022 04 13.
Article in English | MEDLINE | ID: covidwho-1814245

Subject(s)
Cells, Cultured
18.
Am J Respir Cell Mol Biol ; 66(4): 391-401, 2022 04.
Article in English | MEDLINE | ID: covidwho-1775050

ABSTRACT

Asthma is associated with chronic changes in the airway epithelium, a key target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many epithelial changes, including goblet cell metaplasia, are driven by the type 2 cytokine IL-13, but the effects of IL-13 on SARS-CoV-2 infection are unknown. We found that IL-13 stimulation of differentiated human bronchial epithelial cells (HBECs) cultured at air-liquid interface reduced viral RNA recovered from SARS-CoV-2-infected cells and decreased double-stranded RNA, a marker of viral replication, to below the limit of detection in our assay. An intact mucus gel reduced SARS-CoV-2 infection of unstimulated cells, but neither a mucus gel nor SPDEF, which is required for goblet cell metaplasia, were required for the antiviral effects of IL-13. Bulk RNA sequencing revealed that IL-13 regulated 41 of 332 (12%) mRNAs encoding SARS-CoV-2-associated proteins that were detected in HBECs (>1.5-fold change; false discovery rate < 0.05). Although both IL-13 and IFN-α each inhibit SARS-CoV-2 infection, their transcriptional effects differed markedly. Single-cell RNA sequencing revealed cell type-specific differences in SARS-CoV-2-associated gene expression and IL-13 responses. Many IL-13-induced gene expression changes were seen in airway epithelium from individuals with type 2 asthma and chronic obstructive pulmonary disease. IL-13 effects on airway epithelial cells may protect individuals with type 2 asthma from COVID-19 and could lead to identification of novel strategies for reducing SARS-CoV-2 infection.


Subject(s)
Asthma , COVID-19 , Cells, Cultured , Epithelial Cells , Epithelium , Humans , Interleukin-13/pharmacology , SARS-CoV-2
19.
J Immunol ; 207(5): 1275-1287, 2021 09 01.
Article in English | MEDLINE | ID: covidwho-1771322

ABSTRACT

The airway epithelial cells (AECs) lining the conducting passageways of the lung secrete a variety of immunomodulatory factors. Among these, PGE2 limits lung inflammation and promotes bronchodilation. By contrast, IL-6 drives intense airway inflammation, remodeling, and fibrosis. The signaling that differentiates the production of these opposing mediators is not understood. In this study, we find that the production of PGE2 and IL-6 following stimulation of human AECs by the damage-associated molecular pattern extracellular ATP shares a common requirement for Ca2+ release-activated Ca2+ (CRAC) channels. ATP-mediated synthesis of PGE2 required activation of metabotropic P2Y2 receptors and CRAC channel-mediated cytosolic phospholipase A2 signaling. By contrast, ATP-evoked synthesis of IL-6 occurred via activation of ionotropic P2X receptors and CRAC channel-mediated calcineurin/NFAT signaling. In contrast to ATP, which elicited the production of both PGE2 and IL-6, the uridine nucleotide, UTP, stimulated PGE2 but not IL-6 production. These results reveal that human AECs employ unique receptor-specific signaling mechanisms with CRAC channels as a signaling nexus to regulate release of opposing immunomodulatory mediators. Collectively, our results identify P2Y2 receptors, CRAC channels, and P2X receptors as potential intervention targets for airway diseases.


Subject(s)
Dinoprostone/metabolism , Inflammation/immunology , Interleukin-6/metabolism , Respiratory Mucosa/metabolism , Adenosine Triphosphate/pharmacokinetics , Alarmins/metabolism , Calcium Release Activated Calcium Channels/metabolism , Cells, Cultured , Humans , Immunomodulation , Interleukin-6/genetics , NFATC Transcription Factors/metabolism , Phospholipases A2/metabolism , Receptors, Purinergic P2X/metabolism , Respiratory Mucosa/pathology , Signal Transduction , Uracil Nucleotides/metabolism
20.
Viruses ; 14(2)2022 02 21.
Article in English | MEDLINE | ID: covidwho-1744920

ABSTRACT

Involvement of macrophages in the SARS-CoV-2-associated cytokine storm, the excessive secretion of inflammatory/anti-viral factors leading to the acute respiratory distress syndrome (ARDS) in COVID-19 patients, is unclear. In this study, we sought to characterize the interplay between the virus and primary human monocyte-derived macrophages (MDM). MDM were stimulated with recombinant IFN-α and/or infected with either live or UV-inactivated SARS-CoV-2 or with two reassortant influenza viruses containing external genes from the H1N1 PR8 strain and heterologous internal genes from a highly pathogenic avian H5N1 or a low pathogenic human seasonal H1N1 strain. Virus replication was monitored by qRT-PCR for the E viral gene for SARS-CoV-2 or M gene for influenza and TCID50 or plaque assay, and cytokine levels were assessed semiquantitatively with qRT-PCR and a proteome cytokine array. We report that MDM are not susceptible to SARS-CoV-2 whereas both influenza viruses replicated in MDM, albeit abortively. We observed a modest cytokine response in SARS-CoV-2 exposed MDM with notable absence of IFN-ß induction, which was instead strongly induced by the influenza viruses. Pre-treatment of MDM with IFN-α enhanced proinflammatory cytokine expression upon exposure to virus. Together, the findings concur that the hyperinflammation observed in SARS-CoV-2 infection is not driven by macrophages.


Subject(s)
Inflammation/virology , Macrophages/immunology , Macrophages/virology , SARS-CoV-2/immunology , Virus Replication/genetics , Cell Line , Cell Line, Tumor , Cells, Cultured , Cytokines/analysis , Cytokines/immunology , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/immunology , Interferon-alpha/pharmacology , Macrophages/drug effects , Male , SARS-CoV-2/genetics , SARS-CoV-2/physiology
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