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1.
J Am Chem Soc ; 145(24): 13261-13272, 2023 06 21.
Article in English | MEDLINE | ID: covidwho-20240992

ABSTRACT

Activating antigen-presenting cells is essential to generate adaptive immunity, while the efficacy of conventional activation strategies remains unsatisfactory due to suboptimal antigen-specific priming. Here, in situ polymerization-mediated antigen presentation (IPAP) is described, in which antigen-loaded nanovaccines are spontaneously formed and efficiently anchored onto the surface of dendritic cells in vivo through co-deposition with dopamine. The resulting chemically bound nanovaccines can promote antigen presentation by elevating macropinocytosis-based cell uptake and reducing lysosome-related antigen degradation. IPAP is able to prolong the duration of antigen reservation in the injection site and enhance subsequent accumulation in the draining lymph nodes, thereby eliciting robust antigen-specific cellular and humoral immune responses. IPAP is also applicable for different antigens and capable of circumventing the disadvantages of complicated preparation and purification. By implementation with ovalbumin, IPAP induces a significant protective immunity against ovalbumin-overexpressing tumor cell challenge in a prophylactic murine model. The use of the SARS-CoV-2 Spike protein S1 subunit also remarkably increases the production of S1-specific immunoglobulin G in mice. IPAP offers a unique strategy for stimulating antigen-presenting cells to boost antigen-specific adaptive responses and proposes a facile yet versatile method for immunization against various diseases.


Subject(s)
Antigen Presentation , COVID-19 , Mice , Humans , Animals , Ovalbumin , Polymerization , Dendritic Cells , COVID-19/metabolism , SARS-CoV-2 , Antigens , Mice, Inbred C57BL
2.
Front Cell Infect Microbiol ; 13: 1105872, 2023.
Article in English | MEDLINE | ID: covidwho-20238927

ABSTRACT

Tuberculosis (TB) caused by the complex Mycobacterium tuberculosis (Mtb) is the main cause of death by a single bacterial agent. Last year, TB was the second leading infectious killer after SARS-CoV-2. Nevertheless, many biological and immunological aspects of TB are not completely elucidated, such as the complex process of immunoregulation mediated by regulatory T cells (Treg cells) and the enzymes indoleamine 2,3-dioxygenase (IDO) and heme oxygenase 1 (HO-1). In this study, the contribution of these immunoregulatory factors was compared in mice infected with Mtb strains with different levels of virulence. First Balb/c mice were infected by intratracheal route, with a high dose of mild virulence reference strain H37Rv or with a highly virulent clinical isolate (strain 5186). In the lungs of infected mice, the kinetics of Treg cells during the infection were determined by cytofluorometry and the expression of IDO and HO-1 by RT-PCR and immunohistochemistry. Then, the contribution of immune-regulation mediated by Treg cells, IDO and HO-1, was evaluated by treating infected animals with specific cytotoxic monoclonal antibodies for Treg cells depletion anti-CD25 (PC61 clone) or by blocking IDO and HO-1 activity using specific inhibitors (1-methyl-D,L-tryptophan or zinc protoporphyrin-IX, respectively). Mice infected with the mild virulent strain showed a progressive increment of Treg cells, showing this highest number at the beginning of the late phase of the infection (28 days), the same trend was observed in the expression of both enzymes being macrophages the cells that showed the highest immunostaining. Animals infected with the highly virulent strain showed lower survival (34 days) and higher amounts of Treg cells, as well as higher expression of IDO and HO-1 one week before. In comparison with non-treated animals, mice infected with strain H37Rv with depletion of Treg cells or treated with the enzymes blockers during late infection showed a significant decrease of bacilli loads, higher expression of IFN-g and lower IL-4 but with a similar extension of inflammatory lung consolidation determined by automated morphometry. In contrast, the depletion of Treg cells in infected mice with the highly virulent strain 5186 produced diffuse alveolar damage that was similar to severe acute viral pneumonia, lesser survival and increase of bacillary loads, while blocking of both IDO and HO-1 produced high bacillary loads and extensive pneumonia with necrosis. Thus, it seems that Treg cells, IDO and HO-1 activities are detrimental during late pulmonary TB induced by mild virulence Mtb, probably because these factors decrease immune protection mediated by the Th1 response. In contrast, Treg cells, IDO and HO-1 are beneficial when the infection is produced by a highly virulent strain, by regulation of excessive inflammation that produced alveolar damage, pulmonary necrosis, acute respiratory insufficiency, and rapid death.


Subject(s)
COVID-19 , Mycobacterium tuberculosis , Tuberculosis, Pulmonary , Mice , Animals , Heme Oxygenase-1 , Mycobacterium tuberculosis/metabolism , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , T-Lymphocytes, Regulatory , Virulence , COVID-19/metabolism , SARS-CoV-2/metabolism , Lung/microbiology , Necrosis/metabolism
3.
Molecules ; 28(11)2023 May 30.
Article in English | MEDLINE | ID: covidwho-20238682

ABSTRACT

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) induces a severe cytokine storm that may cause acute lung injury/acute respiratory distress syndrome (ALI/ARDS) with high clinical morbidity and mortality in infected individuals. Cepharanthine (CEP) is a bisbenzylisoquinoline alkaloid isolated and extracted from Stephania cepharantha Hayata. It exhibits various pharmacological effects, including antioxidant, anti-inflammatory, immunomodulatory, anti-tumor, and antiviral activities. The low oral bioavailability of CEP can be attributed to its poor water solubility. In this study, we utilized the freeze-drying method to prepare dry powder inhalers (DPI) for the treatment of acute lung injury (ALI) in rats via pulmonary administration. According to the powder properties study, the aerodynamic median diameter (Da) of the DPIs was 3.2 µm, and the in vitro lung deposition rate was 30.26; thus, meeting the Chinese Pharmacopoeia standard for pulmonary inhalation administration. We established an ALI rat model by intratracheal injection of hydrochloric acid (1.2 mL/kg, pH = 1.25). At 1 h after the model's establishment, CEP dry powder inhalers (CEP DPIs) (30 mg/kg) were sprayed into the lungs of rats with ALI via the trachea. Compared with the model group, the treatment group exhibited a reduced pulmonary edema and hemorrhage, and significantly reduced content of inflammatory factors (TNF-α, IL-6 and total protein) in their lungs (p < 0.01), indicating that the main mechanism of CEP underlying the treatment of ALI is anti-inflammation. Overall, the dry powder inhaler can deliver the drug directly to the site of the disease, increasing the intrapulmonary utilization of CEP and improving its efficacy, making it a promising inhalable formulation for the treatment of ALI.


Subject(s)
Acute Lung Injury , Benzylisoquinolines , COVID-19 , Rats , Animals , Administration, Inhalation , Dry Powder Inhalers , COVID-19/metabolism , SARS-CoV-2 , Respiratory Aerosols and Droplets , Lung/metabolism , Acute Lung Injury/drug therapy , Acute Lung Injury/metabolism , Benzylisoquinolines/pharmacology , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Anti-Inflammatory Agents/analysis , Particle Size , Powders/analysis
4.
Front Immunol ; 14: 1123155, 2023.
Article in English | MEDLINE | ID: covidwho-20238534

ABSTRACT

Introduction: Natural killer (NK) cells plays a pivotal role in the control of viral infections, and their function depend on the balance between their activating and inhibitory receptors. The immune dysregulation observed in COVID-19 patients was previously associated with downregulation of NK cell numbers and function, yet the mechanism of inhibition of NK cell functions and the interplay between infected cells and NK cells remain largely unknown. Methods: In this study we show that SARS-CoV-2 infection of airway epithelial cells can directly influence NK cell phenotype and functions in the infection microenvironment. NK cells were co-cultured with SARS-CoV-2 infected epithelial cells, in a direct contact with A549ACE2/TMPRSS2 cell line or in a microenvironment of the infection in a 3D ex vivo human airway epithelium (HAE) model and NK cell surface expression of a set of most important receptors (CD16, NKG2D, NKp46, DNAM-1, NKG2C, CD161, NKG2A, TIM-3, TIGIT, and PD-1) was analyzed. Results: We observed a selective, in both utilized experimental models, significant downregulation the proportion of CD161 (NKR-P1A or KLRB1) expressing NK cells, and its expression level, which was followed by a significant impairment of NK cells cytotoxicity level against K562 cells. What is more, we confirmed that SARS-CoV-2 infection upregulates the expression of the ligand for CD161 receptor, lectin-like transcript 1 (LLT1, CLEC2D or OCIL), on infected epithelial cells. LLT1 protein can be also detected not only in supernatants of SARS-CoV-2 infected A549ACE2/TMPRSS2 cells and HAE basolateral medium, but also in serum of COVID-19 patients. Finally, we proved that soluble LLT1 protein treatment of NK cells significantly reduces i) the proportion of CD161+ NK cells, ii) the ability of NK cells to control SARS-CoV-2 infection in A549ACE2/TMPRSS2 cells and iii) the production of granzyme B by NK cells and their cytotoxicity capacity, yet not degranulation level. Conclusion: We propose a novel mechanism of SARS-CoV-2 inhibition of NK cell functions via activation of the LLT1-CD161 axis.


Subject(s)
COVID-19 , Receptors, Cell Surface , Humans , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Killer Cells, Natural , Receptors, Cell Surface/metabolism , SARS-CoV-2/metabolism
5.
Front Immunol ; 14: 1188079, 2023.
Article in English | MEDLINE | ID: covidwho-20237314

ABSTRACT

Background: Immune cell recruitment, endothelial cell barrier disruption, and platelet activation are hallmarks of lung injuries caused by COVID-19 or other insults which can result in acute respiratory distress syndrome (ARDS). Basement membrane (BM) disruption is commonly observed in ARDS, however, the role of newly generated bioactive BM fragments is mostly unknown. Here, we investigate the role of endostatin, a fragment of the BM protein collagen XVIIIα1, on ARDS associated cellular functions such as neutrophil recruitment, endothelial cell barrier integrity, and platelet aggregation in vitro. Methods: In our study we analyzed endostatin in plasma and post-mortem lung specimens of patients with COVID-19 and non-COVID-19 ARDS. Functionally, we investigated the effect of endostatin on neutrophil activation and migration, platelet aggregation, and endothelial barrier function in vitro. Additionally, we performed correlation analysis for endostatin and other critical plasma parameters. Results: We observed increased plasma levels of endostatin in our COVID-19 and non-COVID-19 ARDS cohort. Immunohistochemical staining of ARDS lung sections depicted BM disruption, alongside immunoreactivity for endostatin in proximity to immune cells, endothelial cells, and fibrinous clots. Functionally, endostatin enhanced the activity of neutrophils, and platelets, and the thrombin-induced microvascular barrier disruption. Finally, we showed a positive correlation of endostatin with soluble disease markers VE-Cadherin, c-reactive protein (CRP), fibrinogen, and interleukin (IL)-6 in our COVID-19 cohort. Conclusion: The cumulative effects of endostatin on propagating neutrophil chemotaxis, platelet aggregation, and endothelial cell barrier disruption may suggest endostatin as a link between those cellular events in ARDS pathology.


Subject(s)
COVID-19 , Respiratory Distress Syndrome , Humans , Endostatins/adverse effects , Endostatins/metabolism , Capillary Permeability , Endothelial Cells/metabolism , COVID-19/metabolism , Respiratory Distress Syndrome/pathology , Inflammation/metabolism
6.
Viruses ; 15(5)2023 04 23.
Article in English | MEDLINE | ID: covidwho-20236769

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) canonically utilizes clathrin-mediated endocytosis (CME) and several other endocytic mechanisms to invade airway epithelial cells. Endocytic inhibitors, particularly those targeting CME-related proteins, have been identified as promising antiviral drugs. Currently, these inhibitors are ambiguously classified as chemical, pharmaceutical, or natural inhibitors. However, their varying mechanisms may suggest a more realistic classification system. Herein, we present a new mechanistic-based classification of endocytosis inhibitors, in which they are segregated among four distinct classes including: (i) inhibitors that disrupt endocytosis-related protein-protein interactions, and assembly or dissociation of complexes; (ii) inhibitors of large dynamin GTPase and/or kinase/phosphatase activities associated with endocytosis; (iii) inhibitors that modulate the structure of subcellular components, especially the plasma membrane, and actin; and (iv) inhibitors that cause physiological or metabolic alterations in the endocytosis niche. Excluding antiviral drugs designed to halt SARS-CoV-2 replication, other drugs, either FDA-approved or suggested through basic research, could be systematically assigned to one of these classes. We observed that many anti-SARS-CoV-2 drugs could be included either in class III or IV as they interfere with the structural or physiological integrity of subcellular components, respectively. This perspective may contribute to our understanding of the relative efficacy of endocytosis-related inhibitors and support the optimization of their individual or combined antiviral potential against SARS-CoV-2. However, their selectivity, combined effects, and possible interactions with non-endocytic cellular targets need more clarification.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , COVID-19/metabolism , Endocytosis , Antiviral Agents/pharmacology , Antiviral Agents/metabolism , Cell Membrane/metabolism
7.
Cell Mol Immunol ; 20(7): 835-849, 2023 Jul.
Article in English | MEDLINE | ID: covidwho-20235826

ABSTRACT

Early and strong interferon type I (IFN-I) responses are usually associated with mild COVID-19 disease, whereas persistent or unregulated proinflammatory cytokine responses are associated with severe disease outcomes. Previous work suggested that monocyte-derived macrophages (MDMs) are resistant and unresponsive to SARS-CoV-2 infection. Here, we demonstrate that upon phagocytosis of SARS-CoV-2-infected cells, MDMs are activated and secrete IL-6 and TNF. Importantly, activated MDMs in turn mediate strong activation of plasmacytoid dendritic cells (pDCs), leading to the secretion of high levels of IFN-α and TNF. Furthermore, pDC activation promoted IL-6 production by MDMs. This kind of pDC activation was dependent on direct integrin-mediated cell‒cell contacts and involved stimulation of the TLR7 and STING signaling pathways. Overall, the present study describes a novel and potent pathway of pDC activation that is linked to the macrophage-mediated clearance of infected cells. These findings suggest that a high infection rate by SARS-CoV-2 may lead to exaggerated cytokine responses, which may contribute to tissue damage and severe disease.


Subject(s)
COVID-19 , Interferon Type I , Humans , SARS-CoV-2/metabolism , Interleukin-6/metabolism , COVID-19/metabolism , Interferon-alpha/metabolism , Macrophages/metabolism , Cytokines/metabolism , Phagocytosis , Interferon Type I/metabolism , Dendritic Cells/metabolism
8.
Int J Mol Sci ; 24(11)2023 May 31.
Article in English | MEDLINE | ID: covidwho-20233259

ABSTRACT

The human gut microbiome contains the largest number of bacteria in the body and has the potential to greatly influence metabolism, not only locally but also systemically. There is an established link between a healthy, balanced, and diverse microbiome and overall health. When the gut microbiome becomes unbalanced (dysbiosis) through dietary changes, medication use, lifestyle choices, environmental factors, and ageing, this has a profound effect on our health and is linked to many diseases, including lifestyle diseases, metabolic diseases, inflammatory diseases, and neurological diseases. While this link in humans is largely an association of dysbiosis with disease, in animal models, a causative link can be demonstrated. The link between the gut and the brain is particularly important in maintaining brain health, with a strong association between dysbiosis in the gut and neurodegenerative and neurodevelopmental diseases. This link suggests not only that the gut microbiota composition can be used to make an early diagnosis of neurodegenerative and neurodevelopmental diseases but also that modifying the gut microbiome to influence the microbiome-gut-brain axis might present a therapeutic target for diseases that have proved intractable, with the aim of altering the trajectory of neurodegenerative and neurodevelopmental diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, autism spectrum disorder, and attention-deficit hyperactivity disorder, among others. There is also a microbiome-gut-brain link to other potentially reversible neurological diseases, such as migraine, post-operative cognitive dysfunction, and long COVID, which might be considered models of therapy for neurodegenerative disease. The role of traditional methods in altering the microbiome, as well as newer, more novel treatments such as faecal microbiome transplants and photobiomodulation, are discussed.


Subject(s)
Autism Spectrum Disorder , COVID-19 , Microbiota , Neurodegenerative Diseases , Animals , Humans , Brain-Gut Axis , Neurodegenerative Diseases/metabolism , Autism Spectrum Disorder/metabolism , Dysbiosis/metabolism , Post-Acute COVID-19 Syndrome , COVID-19/metabolism , Brain/metabolism
9.
Biomolecules ; 13(5)2023 05 15.
Article in English | MEDLINE | ID: covidwho-20232245

ABSTRACT

Plant cells release tiny membranous vesicles called extracellular vesicles (EVs), which are rich in lipids, proteins, nucleic acids, and pharmacologically active compounds. These plant-derived EVs (PDEVs) are safe and easily extractable and have been shown to have therapeutic effects against inflammation, cancer, bacteria, and aging. They have shown promise in preventing or treating colitis, cancer, alcoholic liver disease, and even COVID-19. PDEVs can also be used as natural carriers for small-molecule drugs and nucleic acids through various administration routes such as oral, transdermal, or injection. The unique advantages of PDEVs make them highly competitive in clinical applications and preventive healthcare products in the future. This review covers the latest methods for isolating and characterizing PDEVs, their applications in disease prevention and treatment, and their potential as a new drug carrier, with special attention to their commercial viability and toxicological profile, as the future of nanomedicine therapeutics. This review champions the formation of a new task force specializing in PDEVs to address a global need for rigor and standardization in PDEV research.


Subject(s)
COVID-19 , Extracellular Vesicles , Neoplasms , Humans , COVID-19/metabolism , Extracellular Vesicles/metabolism , Drug Delivery Systems/methods , Drug Carriers/metabolism , Neoplasms/metabolism
10.
Int J Mol Sci ; 24(10)2023 May 20.
Article in English | MEDLINE | ID: covidwho-20244196

ABSTRACT

The effectiveness of the antiviral immune response largely depends on the activation of cytotoxic T cells. The heterogeneous group of functionally active T cells expressing the CD56 molecule (NKT-like cells), that combines the properties of T lymphocytes and NK cells, is poorly studied in COVID-19. This work aimed to analyze the activation and differentiation of both circulating NKT-like cells and CD56- T cells during COVID-19 among intensive care unit (ICU) patients, moderate severity (MS) patients, and convalescents. A decreased proportion of CD56+ T cells was found in ICU patients with fatal outcome. Severe COVID-19 was accompanied by a decrease in the proportion of CD8+ T cells, mainly due to the CD56- cell death, and a redistribution of the NKT-like cell subset composition with a predominance of more differentiated cytotoxic CD8+ T cells. The differentiation process was accompanied by an increase in the proportions of KIR2DL2/3+ and NKp30+ cells in the CD56+ T cell subset of COVID-19 patients and convalescents. Decreased percentages of NKG2D+ and NKG2A+ cells and increased PD-1 and HLA-DR expression levels were found in both CD56- and CD56+ T cells, and can be considered as indicators of COVID-19 progression. In the CD56- T cell fraction, increased CD16 levels were observed in MS patients and in ICU patients with lethal outcome, suggesting a negative role for CD56-CD16+ T cells in COVID-19. Overall, our findings suggest an antiviral role of CD56+ T cells in COVID-19.


Subject(s)
CD8-Positive T-Lymphocytes , COVID-19 , Humans , COVID-19/metabolism , T-Lymphocyte Subsets , Killer Cells, Natural , Cell Differentiation
11.
PLoS Biol ; 21(6): e3002097, 2023 06.
Article in English | MEDLINE | ID: covidwho-20243340

ABSTRACT

Identifying host genes essential for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has the potential to reveal novel drug targets and further our understanding of Coronavirus Disease 2019 (COVID-19). We previously performed a genome-wide CRISPR/Cas9 screen to identify proviral host factors for highly pathogenic human coronaviruses. Few host factors were required by diverse coronaviruses across multiple cell types, but DYRK1A was one such exception. Although its role in coronavirus infection was previously undescribed, DYRK1A encodes Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A and is known to regulate cell proliferation and neuronal development. Here, we demonstrate that DYRK1A regulates ACE2 and DPP4 transcription independent of its catalytic kinase function to support SARS-CoV, SARS-CoV-2, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) entry. We show that DYRK1A promotes DNA accessibility at the ACE2 promoter and a putative distal enhancer, facilitating transcription and gene expression. Finally, we validate that the proviral activity of DYRK1A is conserved across species using cells of nonhuman primate and human origin. In summary, we report that DYRK1A is a novel regulator of ACE2 and DPP4 expression that may dictate susceptibility to multiple highly pathogenic human coronaviruses.


Subject(s)
COVID-19 , Virus Internalization , Animals , Humans , Angiotensin-Converting Enzyme 2 , COVID-19/genetics , COVID-19/metabolism , Dipeptidyl Peptidase 4 , Middle East Respiratory Syndrome Coronavirus/genetics , SARS-CoV-2/genetics , Severe acute respiratory syndrome-related coronavirus/genetics
12.
Nutrients ; 15(11)2023 May 23.
Article in English | MEDLINE | ID: covidwho-20243208

ABSTRACT

Childhood obesity is a global public health problem. Worldwide, 41 million children under 5 years and 340 million children and adolescents between 5 and 19 years are overweight. In addition, the recent COVID-19 epidemic has further amplified this social phenomenon. Obesity is a condition associated with various comorbidities, such as nonalcoholic fatty liver disease (NAFLD). The pathophysiology of NAFLD in obesity is intricate and involves the interaction and dysregulation of several mechanisms, such as insulin resistance, cytokine signaling, and alteration of the gut microbiota. NAFLD is defined as the presence of hepatic steatosis in more than 5% of hepatocytes, evaluated by histological analysis. It can evolve from hepatic steatosis to steatohepatitis, fibrosis, cirrhosis, hepatocellular carcinoma, and end-stage liver failure. Body weight reduction through lifestyle modification remains the first-line intervention for the management of pediatric NAFLD. Indeed, studies suggest that diets low in fat and sugar and conversely rich in dietary fibers promote the improvement of metabolic parameters. This review aims to evaluate the existing relationship between obesity and NAFLD in the pediatric population and to assess the dietary patterns and nutritional supplementations that can be recommended to prevent and manage obesity and its comorbidities.


Subject(s)
COVID-19 , End Stage Liver Disease , Non-alcoholic Fatty Liver Disease , Pediatric Obesity , Adolescent , Child , Humans , Child, Preschool , Non-alcoholic Fatty Liver Disease/metabolism , Overweight/metabolism , Pediatric Obesity/metabolism , COVID-19/metabolism , Diet , Fibrosis , End Stage Liver Disease/pathology , Liver/metabolism
13.
Front Immunol ; 14: 1052141, 2023.
Article in English | MEDLINE | ID: covidwho-20231212

ABSTRACT

Background: The global outbreak of COVID-19, and the limited availability of clinical treatments, forced researchers around the world to search for the pathogenesis and potential treatments. Understanding the pathogenesis of SARS-CoV-2 is crucial to respond better to the current coronavirus disease 2019 (COVID-19) pandemic. Methods: We collected sputum samples from 20 COVID-19 patients and healthy controls. Transmission electron microscopy was used to observe the morphology of SARS-CoV-2. Extracellular vesicles (EVs) were isolated from sputum and the supernatant of VeroE6 cells, and were characterized by transmission electron microscopy, nanoparticle tracking analysis and Western-Blotting. Furthermore, a proximity barcoding assay was used to investigate immune-related proteins in single EV, and the relationship between EVs and SARS-CoV-2. Result: Transmission electron microscopy images of SARS-COV-2 virus reveal EV-like vesicles around the virion, and western blot analysis of EVs extracted from the supernatant of SARS-COV-2-infected VeroE6 cells showed that they expressed SARS-COV-2 protein. These EVs have the infectivity of SARS-COV-2, and the addition can cause the infection and damage of normal VeroE6 cells. In addition, EVs derived from the sputum of patients infected with SARS-COV-2 expressed high levels of IL6 and TGF-ß, which correlated strongly with expression of the SARS-CoV-2 N protein. Among 40 EV subpopulations identified, 18 differed significantly between patients and controls. The EV subpopulation regulated by CD81 was the most likely to correlate with changes in the pulmonary microenvironment after SARS-CoV-2 infection. Single extracellular vesicles in the sputum of COVID-19 patients harbor infection-mediated alterations in host and virus-derived proteins. Conclusions: These results demonstrate that EVs derived from the sputum of patients participate in virus infection and immune responses. This study provides evidence of an association between EVs and SARS-CoV-2, providing insight into the possible pathogenesis of SARS-CoV-2 infection and the possibility of developing nanoparticle-based antiviral drugs.


Subject(s)
COVID-19 , Extracellular Vesicles , Humans , COVID-19/metabolism , SARS-CoV-2 , Integrins/metabolism , Sputum , Proteomics/methods , Extracellular Vesicles/metabolism , Tetraspanin 28
14.
Redox Biol ; 64: 102769, 2023 08.
Article in English | MEDLINE | ID: covidwho-2328371

ABSTRACT

Cholesterol-24-hydroxylase (CH24H or Cyp46a1) is a reticulum-associated membrane protein that plays an irreplaceable role in cholesterol metabolism in the brain and has been well-studied in several neuro-associated diseases in recent years. In the present study, we found that CH24H expression can be induced by several neuroinvasive viruses, including vesicular stomatitis virus (VSV), rabies virus (RABV), Semliki Forest virus (SFV) and murine hepatitis virus (MHV). The CH24H metabolite, 24-hydroxycholesterol (24HC), also shows competence in inhibiting the replication of multiple viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 24HC can increase the cholesterol concentration in multivesicular body (MVB)/late endosome (LE) by disrupting the interaction between OSBP and VAPA, resulting in viral particles being trapped in MVB/LE, ultimately compromising VSV and RABV entry into host cells. These findings provide the first evidence that brain cholesterol oxidation products may play a critical role in viral infection.


Subject(s)
Virus Internalization , Animals , Mice , Cholesterol/metabolism , COVID-19/metabolism , COVID-19/virology , Homeostasis , SARS-CoV-2/metabolism , Cholesterol 24-Hydroxylase/metabolism
15.
Biochim Biophys Acta Biomembr ; 1865(6): 184174, 2023 Aug.
Article in English | MEDLINE | ID: covidwho-2324713

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID, replicates at intracellular membranes. Bone marrow stromal antigen 2 (BST-2; tetherin) is an antiviral response protein that inhibits transport of viral particles after budding within infected cells. RNA viruses such as SARS-CoV-2 use various strategies to disable BST-2, including use of transmembrane 'accessory' proteins that interfere with BST-2 oligomerization. ORF7a is a small, transmembrane protein present in SARS-CoV-2 shown previously to alter BST-2 glycosylation and function. In this study, we investigated the structural basis for BST-2 ORF7a interactions, with a particular focus on transmembrane and juxtamembrane interactions. Our results indicate that transmembrane domains play an important role in BST-2 ORF7a interactions and mutations to the transmembrane domain of BST-2 can alter these interactions, particularly single-nucleotide polymorphisms in BST-2 that result in mutations such as I28S. Using molecular dynamics simulations, we identified specific interfaces and interactions between BST-2 and ORF7a to develop a structural basis for the transmembrane interactions. Differences in glycosylation are observed for BST-2 transmembrane mutants interacting with ORF7a, consistent with the idea that transmembrane domains play a key role in their heterooligomerization. Overall, our results indicate that ORF7a transmembrane domain interactions play a key role along with extracellular and juxtamembrane domains in modulating BST-2 function.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Cell Membrane/genetics , Cell Membrane/metabolism , COVID-19/metabolism , Membrane Proteins/metabolism , SARS-CoV-2/genetics , Viral Regulatory and Accessory Proteins/metabolism
16.
PLoS Pathog ; 19(5): e1011409, 2023 05.
Article in English | MEDLINE | ID: covidwho-2323254

ABSTRACT

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ injury including testicular inflammation, reduced testosterone, and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells, however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury could be initiated by direct virus infection or exposure to systemic inflammatory mediators or viral antigens. We characterized SARS-CoV-2 infection in different human testicular 2D and 3D culture systems including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not productively infect any testicular cell type. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma decreased cell viability and resulted in the death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 Envelope protein caused inflammatory response and cytopathic effects dependent on TLR2, while Spike 1 or Nucleocapsid proteins did not. A similar trend was observed in the K18-hACE2 transgenic mice which demonstrated a disrupted tissue architecture with no evidence of virus replication in the testis that correlated with peak lung inflammation. Virus antigens including Spike 1 and Envelope proteins were also detected in the serum during the acute stage of the disease. Collectively, these data strongly suggest that testicular injury associated with SARS-CoV-2 infection is likely an indirect effect of exposure to systemic inflammation and/or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.


Subject(s)
COVID-19 , Male , Mice , Animals , Humans , COVID-19/metabolism , Testis , SARS-CoV-2 , Bystander Effect , Inflammation/metabolism , Mice, Transgenic
17.
Virol J ; 20(1): 97, 2023 05 19.
Article in English | MEDLINE | ID: covidwho-2322368

ABSTRACT

BACKGROUND: SARS-CoV-2 was reported to induce cell fusions to form multinuclear syncytia that might facilitate viral replication, dissemination, immune evasion, and inflammatory responses. In this study, we have reported the types of cells involved in syncytia formation at different stages of COVID-19 disease through electron microscopy. METHODS: Bronchoalveolar fluids from the mild (n = 8, SpO2 > 95%, no hypoxia, within 2-8 days of infection), moderate (n = 8, SpO2 90% to ≤ 93% on room air, respiratory rate ≥ 24/min, breathlessness, within 9-16 days of infection), and severe (n = 8, SpO2 < 90%, respiratory rate > 30/min, external oxygen support, after 17th days of infection) COVID-19 patients were examined by PAP (cell type identification), immunofluorescence (for the level of viral infection), scanning (SEM), and transmission (TEM) electron microscopy to identify the syncytia. RESULTS: Immunofluorescence studies (S protein-specific antibodies) from each syncytium indicate a very high infection level. We could not find any syncytial cells in mildly infected patients. However, identical (neutrophils or type 2 pneumocytes) and heterotypic (neutrophils-monocytes) plasma membrane initial fusion (indicating initiation of fusion) was observed under TEM in moderately infected patients. Fully matured large-size (20-100 µm) syncytial cells were found in severe acute respiratory distress syndrome (ARDS-like) patients of neutrophils, monocytes, and macrophage origin under SEM. CONCLUSIONS: This ultrastructural study on the syncytial cells from COVID-19 patients sheds light on the disease's stages and types of cells involved in the syncytia formations. Syncytia formation was first induced in type II pneumocytes by homotypic fusion and later with haematopoetic cells (monocyte and neutrophils) by heterotypic fusion in the moderate stage (9-16 days) of the disease. Matured syncytia were reported in the late phase of the disease and formed large giant cells of 20 to 100 µm.


Subject(s)
COVID-19 , Humans , COVID-19/metabolism , SARS-CoV-2 , Microscopy, Electron , Alveolar Epithelial Cells , Macrophages , Giant Cells
18.
Circ Res ; 132(10): 1272-1289, 2023 05 12.
Article in English | MEDLINE | ID: covidwho-2319061

ABSTRACT

COVID-19 is characterized by dysregulated thrombosis and coagulation that can increase mortality in patients. Platelets are fast responders to pathogen presence, alerting the surrounding immune cells and contributing to thrombosis and intravascular coagulation. The SARS-CoV-2 genome has been found in platelets from patients with COVID-19, and its coverage varies according to the method of detection, suggesting direct interaction of the virus with these cells. Antibodies against Spike and Nucleocapsid have confirmed this platelet-viral interaction. This review discusses the immune, prothrombotic, and procoagulant characteristics of platelets observed in patients with COVID-19. We outline the direct and indirect interaction of platelets with SARS-CoV-2, the contribution of the virus to programmed cell death pathway activation in platelets and the consequent extracellular vesicle release. We discuss platelet activation and immunothrombosis in patients with COVID-19, the effect of Spike on platelets, and possible activation of platelets by classical platelet activation triggers as well as contribution of platelets to complement activation. As COVID-19-mediated thrombosis and coagulation are still not well understood in vivo, we discuss available murine models and mouse adaptable strains.


Subject(s)
COVID-19 , Thrombosis , Mice , Animals , COVID-19/metabolism , SARS-CoV-2 , Blood Platelets/metabolism , Platelet Activation
19.
Nat Methods ; 20(6): 860-870, 2023 Jun.
Article in English | MEDLINE | ID: covidwho-2318342

ABSTRACT

Modeling flexible macromolecules is one of the foremost challenges in single-particle cryogenic-electron microscopy (cryo-EM), with the potential to illuminate fundamental questions in structural biology. We introduce Three-Dimensional Flexible Refinement (3DFlex), a motion-based neural network model for continuous molecular heterogeneity for cryo-EM data. 3DFlex exploits knowledge that conformational variability of a protein is often the result of physical processes that transport density over space and tend to preserve local geometry. From two-dimensional image data, 3DFlex enables the determination of high-resolution 3D density, and provides an explicit model of a flexible protein's motion over its conformational landscape. Experimentally, for large molecular machines (tri-snRNP spliceosome complex, translocating ribosome) and small flexible proteins (TRPV1 ion channel, αVß8 integrin, SARS-CoV-2 spike), 3DFlex learns nonrigid molecular motions while resolving details of moving secondary structure elements. 3DFlex can improve 3D density resolution beyond the limits of existing methods because particle images contribute coherent signal over the conformational landscape.


Subject(s)
COVID-19 , Humans , Cryoelectron Microscopy/methods , COVID-19/metabolism , SARS-CoV-2 , Proteins/chemistry , Ribosomes/metabolism
20.
Front Immunol ; 14: 1148268, 2023.
Article in English | MEDLINE | ID: covidwho-2317599

ABSTRACT

Introduction: COVID-19 and autoinflammatory diseases, such as Adult-onset Still's Disease (AOSD), are characterized by hyperinflammation, in which it is observed massive production and uncontrolled secretion of pro-inflammatory cytokines. The specialized pro-resolving lipid mediators (SPMs) family is one the most important processes counteracting hyperinflammation inducing tissue repair and homeostasis restoration. Among SPMs, Protectin D1 (PD1) is able to exert antiviral features, at least in animal models. The aim of this study was to compare the transcriptome of peripheral blood mononuclear cells (PBMCs) from patients with AOSD and COVID-19 and to evaluate the role of PD1 on those diseases, especially in modulating macrophages polarization. Methods: This study enrolled patients with AOSD, COVID-19, and healthy donors HDs, undergoing clinical assessment and blood sample collection. Next-generation deep sequencing was performed to identify differences in PBMCs transcripts profiles. Plasma levels of PD1 were assessed by commercial ELISA kits. Monocyte-derived macrophages were polarized into M1 and M2 phenotypes. We analyzed the effect of PD1 on macrophages differentiation. At 10 days, macrophages were analyzed for surface expression of subtypes markers by flow cytometry. Cytokines production was measured in supernatants by Bio-Plex Assays. Results: In the transcriptomes from AOSD patients and COVID-19 patients, genes involved in inflammation, lipid catabolism, and monocytes activation were specifically dysregulated in AOSD and COVID-19 patients when compared to HDs. Patients affected by COVID-19, hospitalized in intensive care unit (ICU), showed higher levels of PD1 when compared to not-ICU hospitalized patients and HDs (ICU COVID-19 vs not-ICU COVID-19, p= 0.02; HDs vs ICU COVID-19, p= 0.0006). PD1 levels were increased in AOSD patients with SS ≥1 compared to patients with SS=0 (p=0.028) and HDs (p=0.048). In vitro treatment with PD1 of monocytes-derived macrophages from AOSD and COVID-19 patients induced a significant increase of M2 polarization vs control (p<0.05). Furthermore, a significant release of IL-10 and MIP-1ß from M2 macrophages was observed when compared to controls (p<0.05). Discussion: PD1 is able to induce pro-resolutory programs in both AOSD and COVID-19 increasing M2 polarization and inducing their activity. In particular, PD1-treated M2 macrophages from AOSD and COVID-19 patients increased the production of IL-10 and enhanced homeostatic restoration through MIP-1ß production.


Subject(s)
COVID-19 , Still's Disease, Adult-Onset , Humans , Transcriptome , Interleukin-10/metabolism , Leukocytes, Mononuclear/metabolism , Chemokine CCL4/metabolism , COVID-19/metabolism , Cytokines/metabolism , Docosahexaenoic Acids/metabolism , Macrophages , Cell Differentiation/genetics
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