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1.
Transfus Apher Sci ; 61(3): 103459, 2022 Jun.
Article in English | MEDLINE | ID: covidwho-1852173

ABSTRACT

Platelets are at the crossroads between thrombosis and inflammation. When activated, platelets can shed bioactive extracellular vesicles [pEVs] that share the hemostatic potential of their parent cells and act as bioactive shuttles of their granular contents. In a viral infection, platelets are activated, and pEVs are generated with occasional virion integration. Both platelets and pEVs are engaged in a bidirectional interaction with neutrophils and other cells of the immune system and the hemostatic pathways. Severe COVID-19 infection is characterized by a stormy thromboinflammatory response with platelets and their EVs at the center stage of this reaction. This review sheds light on the interactions of platelets, pEVS and SARS-CoV-2 infection and prognostic and potential therapeutic role of pEVs. The review also describes the role of pEVs in the rare adenovirus-based COVID-19 vaccine-induced thrombosis thrombocytopenia.


Subject(s)
COVID-19 , Extracellular Vesicles , Hemostatics , Thrombosis , Blood Platelets/metabolism , COVID-19 Vaccines , Extracellular Vesicles/metabolism , Hemostatics/metabolism , Humans , SARS-CoV-2
2.
Biotechnol Adv ; 59: 107983, 2022 Oct.
Article in English | MEDLINE | ID: covidwho-1850715

ABSTRACT

In recent years, extracellular vesicles (EVs), specifically exosomes, have emerged as a promising strategy for treating a wide spectrum of pathologies, such as cancer and COVID-19, as well as promoting tissue regeneration in various conditions, including cardiomyopathies and spinal cord injuries. Despite the great potential of EV-based therapies, poor yield and unscalable production of EVs remain big challenges to overcome to translate these types of treatment to clinical practices. Here, we review different strategies for enhancing EV yield by physical, biological or chemical means. Some of these novel approaches can lead to about 100-fold increase in EV production yield, thus bringing closer the clinical translation with regard to scalability and efficiency.


Subject(s)
COVID-19 , Exosomes , Extracellular Vesicles , Neoplasms , Humans
4.
J Cell Biol ; 221(6)2022 06 06.
Article in English | MEDLINE | ID: covidwho-1806200

ABSTRACT

The endolysosome system plays central roles in both autophagic degradation and secretory pathways, including the release of extracellular vesicles and particles (EVPs). Although previous work reveals important interconnections between autophagy and EVP-mediated secretion, our understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition, which mediates EVP-associated release of autophagic cargo receptors, including p62/SQSTM1. This secretion is highly regulated and dependent on multiple ATGs required for autophagosome formation, as well as the small GTPase Rab27a. Furthermore, disrupting autophagosome maturation, either via genetic inhibition of autophagosome-to-autolysosome fusion or expression of SARS-CoV-2 ORF3a, is sufficient to induce EVP secretion of autophagy cargo receptors. Finally, ATG-dependent EVP secretion buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Thus, we propose secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis during endolysosomal dysfunction or impaired autophagosome maturation.


Subject(s)
Autophagy , Extracellular Vesicles , Lysosomes , Proteostasis , Autophagosomes/metabolism , Extracellular Vesicles/metabolism , Humans , Lysosomes/metabolism , SARS-CoV-2 , Sequestosome-1 Protein , Viroporin Proteins , rab27 GTP-Binding Proteins
5.
Int J Mol Sci ; 23(7)2022 Mar 30.
Article in English | MEDLINE | ID: covidwho-1785735

ABSTRACT

Acute kidney injury (AKI) is a sudden decline of renal function and represents a global clinical problem due to an elevated morbidity and mortality. Despite many efforts, currently there are no treatments to halt this devastating condition. Extracellular vesicles (EVs) are nanoparticles secreted by various cell types in both physiological and pathological conditions. EVs can arise from distinct parts of the kidney and can mediate intercellular communication between various cell types along the nephron. Besides their potential as diagnostic tools, EVs have been proposed as powerful new tools for regenerative medicine and have been broadly studied as therapeutic mediators in different models of experimental AKI. In this review, we present an overview of the basic features and biological relevance of EVs, with an emphasis on their functional role in cell-to-cell communication in the kidney. We explore versatile roles of EVs in crucial pathophysiological mechanisms contributing to AKI and give a detailed description of the renoprotective effects of EVs from different origins in AKI. Finally, we explain known mechanisms of action of EVs in AKI and provide an outlook on the potential clinical translation of EVs in the setting of AKI.


Subject(s)
Acute Kidney Injury , Extracellular Vesicles , Mesenchymal Stem Cells , Acute Kidney Injury/pathology , Extracellular Vesicles/metabolism , Humans , Kidney/metabolism , Mesenchymal Stem Cells/metabolism
6.
J Extracell Vesicles ; 11(4): e12204, 2022 04.
Article in English | MEDLINE | ID: covidwho-1772724

ABSTRACT

Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil-derived extracellular vesicles (NDEVs) involved in immune and thrombo-inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID-19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta.


Subject(s)
COVID-19 , Extracellular Vesicles , Extracellular Vesicles/chemistry , Female , Flow Cytometry/methods , Humans , Neutrophils , Pregnancy , Protein Transport
7.
J Control Release ; 345: 214-230, 2022 05.
Article in English | MEDLINE | ID: covidwho-1747828

ABSTRACT

Mesenchymal stem cell-derived small extracellular vesicles (MSC-EVs) are promising nanotherapeutic agent for pneumonia (bacterial origin, COVID-19), but the optimal administration route and potential mechanisms of action remain poorly understood. This study compared the administration of MSC-EVs via inhalation and tail vein injection for the treatment of acute lung injury (ALI) and determined the host-derived mechanisms that may contribute to the therapeutic effects of MSC-EVs in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells (macrophage cell line) and animal models. Luminex liquid chip and hematoxylin and eosin (HE) staining revealed that, compared with the vehicle control, inhaled MSC-EVs outperformed those injected via the tail vein, by reducing the expression of pro-inflammatory cytokines, increasing the expression of anti-inflammatory cytokine, and decreasing pathological scores in ALI. MSC-EV administration promoted the polarization of macrophages towards a M2 phenotype in vitro and in vivo (via inhalation). RNA sequencing revealed that immune and redox mediators, including TLR4, Arg1, and HO-1, were associated with the activity MSC-EVs against ALI mice. Western blotting and immunofluorescence revealed that correlative inflammatory and oxidative mediators were involved in the therapeutic effects of MSC-EVs in LPS-stimulated cells and mice. Moreover, variable expression of Nrf2 was observed following treatment with MSC-EVs in cell and animal models, and knockdown of Nrf2 attenuated the anti-inflammatory and antioxidant activities of MSC-EVs in LPS-stimulated macrophages. Together, these data suggest that inhalation of MSC-EVs as a noninvasive strategy for attenuation of ALI, and the adaptive regulation of Nrf2 may contribute to their anti-inflammatory and anti-oxidant activity in mice.


Subject(s)
Acute Lung Injury , COVID-19 , Extracellular Vesicles , Mesenchymal Stem Cells , Acute Lung Injury/therapy , Animals , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/therapeutic use , Antioxidants , Cytokines/metabolism , Disease Models, Animal , Extracellular Vesicles/metabolism , Lipopolysaccharides , Mesenchymal Stem Cells/metabolism , Mice , NF-E2-Related Factor 2/metabolism
9.
J Extracell Vesicles ; 11(3): e12192, 2022 03.
Article in English | MEDLINE | ID: covidwho-1739175

ABSTRACT

Several vaccines have been introduced to combat the coronavirus infectious disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current SARS-CoV-2 vaccines include mRNA-containing lipid nanoparticles or adenoviral vectors that encode the SARS-CoV-2 Spike (S) protein of SARS-CoV-2, inactivated virus, or protein subunits. Despite growing success in worldwide vaccination efforts, additional capabilities may be needed in the future to address issues such as stability and storage requirements, need for vaccine boosters, desirability of different routes of administration, and emergence of SARS-CoV-2 variants such as the Delta variant. Here, we present a novel, well-characterized SARS-CoV-2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium that are decorated with the mammalian cell culture-derived Spike receptor-binding domain (RBD). RBD-conjugated outer membrane vesicles (RBD-OMVs) were used to immunize the golden Syrian hamster (Mesocricetus auratus) model of COVID-19. Intranasal immunization resulted in high titres of blood anti-RBD IgG as well as detectable mucosal responses. Neutralizing antibody activity against wild-type and Delta variants was evident in all vaccinated subjects. Upon challenge with live virus, hamsters immunized with RBD-OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titres in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV-based vaccine approaches.


Subject(s)
COVID-19 , Extracellular Vesicles , Viral Vaccines , Animals , Antibodies, Neutralizing , COVID-19/prevention & control , COVID-19 Vaccines , Humans , Liposomes , Mammals , Nanoparticles , SARS-CoV-2
10.
Sci Rep ; 12(1): 3794, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1735281

ABSTRACT

SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism. However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2. Our data reveal, with unprecedented resolution, the presence of: (1) long tunneling nanotubes that connect two or more host cells over submillimeter distances; (2) large scale multiple cell fusion events (syncytia); and (3) abundant extracellular vesicles of various sizes. Taken together, these ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Such route may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host.


Subject(s)
Microscopy/methods , SARS-CoV-2/physiology , Virus Internalization , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/transmission , COVID-19/virology , Chlorocebus aethiops , Cytoplasm/chemistry , Cytoplasm/ultrastructure , Cytoplasm/virology , Extracellular Vesicles/chemistry , Extracellular Vesicles/ultrastructure , Giant Cells/chemistry , Giant Cells/physiology , Helium/chemistry , Humans , Ions/chemistry , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
11.
Viruses ; 12(5)2020 04 26.
Article in English | MEDLINE | ID: covidwho-1726007

ABSTRACT

In January 2020, Chinese health agencies reported an outbreak of a novel coronavirus-2 (CoV-2) which can lead to severe acute respiratory syndrome (SARS). The virus, which belongs to the coronavirus family (SARS-CoV-2), was named coronavirus disease 2019 (COVID-19) and declared a pandemic by the World Health Organization (WHO). Full-length genome sequences of SARS-CoV-2 showed 79.6% sequence identity to SARS-CoV, with 96% identity to a bat coronavirus at the whole-genome level. COVID-19 has caused over 133,000 deaths and there are over 2 million total confirmed cases as of April 15th, 2020. Current treatment plans are still under investigation due to a lack of understanding of COVID-19. One potential mechanism to slow disease progression is the use of antiviral drugs to either block the entry of the virus or interfere with viral replication and maturation. Currently, antiviral drugs, including chloroquine/hydroxychloroquine, remdesivir, and lopinavir/ritonavir, have shown effective inhibition of SARS-CoV-2 in vitro. Due to the high dose needed and narrow therapeutic window, many patients are experiencing severe side effects with the above drugs. Hence, repurposing these drugs with a proper formulation is needed to improve the safety and efficacy for COVID-19 treatment. Extracellular vesicles (EVs) are a family of natural carriers in the human body. They play a critical role in cell-to-cell communications. EVs can be used as unique drug carriers to deliver protease inhibitors to treat COVID-19. EVs may provide targeted delivery of protease inhibitors, with fewer systemic side effects. More importantly, EVs are eligible for major aseptic processing and can be upscaled for mass production. Currently, the FDA is facilitating applications to treat COVID-19, which provides a very good chance to use EVs to contribute in this combat.


Subject(s)
Coronavirus Infections/drug therapy , Drug Repositioning , Extracellular Vesicles/chemistry , HIV Protease Inhibitors/administration & dosage , Pneumonia, Viral/drug therapy , Betacoronavirus/genetics , Betacoronavirus/metabolism , COVID-19 , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Drug Approval , Drug Delivery Systems , Humans , Pandemics , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , SARS-CoV-2
12.
Int J Mol Sci ; 23(4)2022 Feb 19.
Article in English | MEDLINE | ID: covidwho-1715402

ABSTRACT

Platelets, which are small anuclear cell fragments, play important roles in thrombosis and hemostasis, but also actively release factors that can both suppress and induce viral infections. Platelet-released factors include sCD40L, microvesicles (MVs), and alpha granules that have the capacity to exert either pro-inflammatory or anti-inflammatory effects depending on the virus. These factors are prime targets for use in extracellular vesicle (EV)-based therapy due to their ability to reduce viral infections and exert anti-inflammatory effects. While there are some studies regarding platelet microvesicle-based (PMV-based) therapy, there is still much to learn about PMVs before such therapy can be used. This review provides the background necessary to understand the roles of platelet-released factors, how these factors might be useful in PMV-based therapy, and a critical discussion of current knowledge of platelets and their role in viral diseases.


Subject(s)
Blood Coagulation Factors/metabolism , Blood Platelets/metabolism , Extracellular Vesicles/metabolism , Virus Diseases/metabolism , Animals , Cell-Derived Microparticles/metabolism , Humans , Platelet Activation/physiology
13.
FASEB J ; 36(3): e22234, 2022 03.
Article in English | MEDLINE | ID: covidwho-1702985

ABSTRACT

The transmembrane protease angiotensin converting enzyme 2 (ACE2) is a protective regulator within the renin angiotensin system and additionally represents the cellular receptor for SARS-CoV. The release of soluble ACE2 (sACE2) from the cell surface is hence believed to be a crucial part of its (patho)physiological functions, as both, ACE2 protease activity and SARS-CoV binding ability, are transferred from the cell membrane to body fluids. Yet, the molecular sources of sACE2 are still not completely investigated. In this study, we show different sources and prerequisites for the release of sACE2 from the cell membrane. By using inhibitors as well as CRISPR/Cas9-derived cells, we demonstrated that, in addition to the metalloprotease ADAM17, also ADAM10 is an important novel shedding protease of ACE2. Moreover, we observed that ACE2 can also be released in extracellular vesicles. The degree of either ADAM10- or ADAM17-mediated ACE2 shedding is dependent on stimulatory conditions and on the expression level of the pro-inflammatory ADAM17 regulator iRhom2. Finally, by using structural analysis and in vitro verification, we determined for the first time that the susceptibility to ADAM10- and ADAM17-mediated shedding is mediated by the collectrin-like part of ACE2. Overall, our findings give novel insights into sACE2 release by several independent molecular mechanisms.


Subject(s)
ADAM10 Protein/metabolism , ADAM17 Protein/metabolism , Amyloid Precursor Protein Secretases/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Extracellular Vesicles/metabolism , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , SARS Virus/metabolism , ADAM10 Protein/genetics , ADAM17 Protein/genetics , Amyloid Precursor Protein Secretases/genetics , Angiotensin-Converting Enzyme 2/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Extracellular Vesicles/genetics , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Glycoproteins/genetics , Membrane Proteins/genetics , SARS Virus/genetics , SARS-CoV-2
14.
Nat Cell Biol ; 23(12): 1240-1254, 2021 12.
Article in English | MEDLINE | ID: covidwho-1699219

ABSTRACT

Extracellular vesicles and exomere nanoparticles are under intense investigation as sources of clinically relevant cargo. Here we report the discovery of a distinct extracellular nanoparticle, termed supermere. Supermeres are morphologically distinct from exomeres and display a markedly greater uptake in vivo compared with small extracellular vesicles and exomeres. The protein and RNA composition of supermeres differs from small extracellular vesicles and exomeres. Supermeres are highly enriched with cargo involved in multiple cancers (glycolytic enzymes, TGFBI, miR-1246, MET, GPC1 and AGO2), Alzheimer's disease (APP) and cardiovascular disease (ACE2, ACE and PCSK9). The majority of extracellular RNA is associated with supermeres rather than small extracellular vesicles and exomeres. Cancer-derived supermeres increase lactate secretion, transfer cetuximab resistance and decrease hepatic lipids and glycogen in vivo. This study identifies a distinct functional nanoparticle replete with potential circulating biomarkers and therapeutic targets for a host of human diseases.


Subject(s)
Extracellular Vesicles/metabolism , MicroRNAs/metabolism , Nanoparticles/metabolism , Alzheimer Disease/pathology , Angiotensin-Converting Enzyme 2/metabolism , Biological Transport/physiology , Biomarkers/metabolism , COVID-19/pathology , Cardiovascular Diseases/pathology , Cell Communication/physiology , Cell Line, Tumor , HeLa Cells , Humans , Lactic Acid/metabolism , MicroRNAs/genetics , Nanoparticles/classification , Neoplasms/pathology , Tumor Microenvironment
15.
Int J Mol Sci ; 22(16)2021 Aug 05.
Article in English | MEDLINE | ID: covidwho-1674661

ABSTRACT

Breast cancers and cancers of the genitourinary tract are the most common malignancies among men and women and are still characterized by high mortality rates. In order to improve the outcomes, early diagnosis is crucial, ideally by applying non-invasive and specific biomarkers. A key role in this field is played by extracellular vesicles (EVs), lipid bilayer-delimited structures shed from the surface of almost all cell types, including cancer cells. Subcellular structures contained in EVs such as nucleic acids, proteins, and lipids can be isolated and exploited as biomarkers, since they directly stem from parental cells. Furthermore, it is becoming even more evident that different body fluids can also serve as sources of EVs for diagnostic purposes. In this review, EV isolation and characterization methods are described. Moreover, the potential contribution of EV cargo for diagnostic discovery purposes is described for each tumor.


Subject(s)
Breast Neoplasms/diagnosis , Extracellular Vesicles/metabolism , Urogenital Neoplasms/diagnosis , Animals , Biomarkers, Tumor/metabolism , Breast Neoplasms/metabolism , Early Detection of Cancer/methods , Female , Humans , Neoplasms/diagnosis , Neoplasms/metabolism , Nucleic Acids/metabolism , Urogenital Neoplasms/metabolism
16.
Viruses ; 14(2)2022 02 06.
Article in English | MEDLINE | ID: covidwho-1674825

ABSTRACT

SARS-CoV-2-specific CD8+ T cell immunity is expected to counteract viral variants in both efficient and durable ways. We recently described a way to induce a potent SARS-CoV-2 CD8+ T immune response through the generation of engineered extracellular vesicles (EVs) emerging from muscle cells. This method relies on intramuscular injection of DNA vectors expressing different SARS-CoV-2 antigens fused at their N-terminus with the Nefmut protein, i.e., a very efficient EV-anchoring protein. However, quality, tissue distribution, and efficacy of these SARS-CoV-2-specific CD8+ T cells remained uninvestigated. To fill the gaps, antigen-specific CD8+ T lymphocytes induced by the immunization through the Nefmut-based method were characterized in terms of their polyfunctionality and localization at lung airways, i.e., the primary targets of SARS-CoV-2 infection. We found that injection of vectors expressing Nefmut/S1 and Nefmut/N generated polyfunctional CD8+ T lymphocytes in both spleens and bronchoalveolar lavage fluids (BALFs). When immunized mice were infected with 4.4 lethal doses of 50% of SARS-CoV-2, all S1-immunized mice succumbed, whereas those developing the highest percentages of N-specific CD8+ T lymphocytes resisted the lethal challenge. We also provide evidence that the N-specific immunization coupled with the development of antigen-specific CD8+ T-resident memory cells in lungs, supporting the idea that the Nefmut-based immunization can confer a long-lasting, lung-specific immune memory. In view of the limitations of current anti-SARS-CoV-2 vaccines in terms of antibody waning and efficiency against variants, our CD8+ T cell-based platform could be considered for a new combination prophylactic strategy.


Subject(s)
Antigens, Viral/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/prevention & control , Extracellular Vesicles/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Animals , Antigens, Viral/administration & dosage , Antigens, Viral/genetics , COVID-19/immunology , Female , Genetic Vectors/administration & dosage , Genetic Vectors/immunology , Humans , Lung/immunology , Lung/virology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Vaccination
17.
J Nanobiotechnology ; 19(1): 287, 2021 Sep 26.
Article in English | MEDLINE | ID: covidwho-1662419

ABSTRACT

Pancreatic cancer, at unresectable advanced stages, presents poor prognoses, which could be prevented by early pancreatic cancer diagnosis methods. Recently, a promising early-stage pancreatic cancer biomarker, extracellular vesicles (EVs) related glypican-1 (GPC1) mRNA, is found to overexpress in pancreatic cancer cells. Current mRNA detection methods usually require expensive machinery, strict preservation environments, and time-consuming processes to guarantee detection sensitivity, specificity, and stability. Herein, we propose a novel two-step amplification method (CHAGE) via the target triggered Catalytic Hairpin Assembly strategy combined with Gold-Enhanced point-of-care-testing (POCT) technology for sensitive visual detection of pancreatic cancer biomarker. First, utilizing the catalyzed hairpin DNA circuit, low expression of the GPC1 mRNA was changed into amplification product 1 (AP1, a DNA duplex) as the next detection targets of the paper strips. Second, the AP1 was loaded onto a lateral flow assay and captured with the gold signal nanoparticles to visualize results. Finally, the detected results can be further enhanced by depositing gold to re-enlarge the sizes of gold nanoparticles in detection zones. As a result, the CHAGE methodology lowers the detection limit of mRNA to 100 fM and provides results within 2 h at 37 °C. Furthermore, we demonstrate the successful application in discriminating pancreatic cancer cells by analyzing EVs' GPC1 mRNA expression levels. Hence, the CHAGE methodology proposed here provides a rapid and convenient POCT platform for sensitive detection of mRNAs through unique probes designs (COVID, HPV, etc.).


Subject(s)
Early Detection of Cancer/methods , Pancreatic Neoplasms/diagnosis , RNA, Messenger/isolation & purification , Biomarkers, Tumor/genetics , COVID-19 , Extracellular Vesicles , Glypicans/genetics , Gold , Humans , Metal Nanoparticles , Pancreatic Neoplasms/genetics
18.
Nat Commun ; 13(1): 405, 2022 01 20.
Article in English | MEDLINE | ID: covidwho-1631967

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of the coronavirus induced disease 2019 (COVID-19) with evolving variants of concern. It remains urgent to identify novel approaches against broad strains of SARS-CoV-2, which infect host cells via the entry receptor angiotensin-converting enzyme 2 (ACE2). Herein, we report an increase in circulating extracellular vesicles (EVs) that express ACE2 (evACE2) in plasma of COVID-19 patients, which levels are associated with severe pathogenesis. Importantly, evACE2 isolated from human plasma or cells neutralizes SARS-CoV-2 infection by competing with cellular ACE2. Compared to vesicle-free recombinant human ACE2 (rhACE2), evACE2 shows a 135-fold higher potency in blocking the binding of the viral spike protein RBD, and a 60- to 80-fold higher efficacy in preventing infections by both pseudotyped and authentic SARS-CoV-2. Consistently, evACE2 protects the hACE2 transgenic mice from SARS-CoV-2-induced lung injury and mortality. Furthermore, evACE2 inhibits the infection of SARS-CoV-2 variants (α, ß, and δ) with equal or higher potency than for the wildtype strain, supporting a broad-spectrum antiviral mechanism of evACE2 for therapeutic development to block the infection of existing and future coronaviruses that use the ACE2 receptor.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , COVID-19/immunology , Extracellular Vesicles/immunology , SARS-CoV-2/immunology , A549 Cells , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/blood , COVID-19/epidemiology , Chlorocebus aethiops , Extracellular Vesicles/genetics , Extracellular Vesicles/metabolism , HEK293 Cells , HeLa Cells , Humans , Mice, Transgenic , Neutralization Tests/methods , Pandemics/prevention & control , Protein Binding , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Survival Analysis , Vero Cells
19.
Int J Mol Sci ; 23(2)2022 Jan 06.
Article in English | MEDLINE | ID: covidwho-1631344

ABSTRACT

Since the first evidence that stem cells can provide pro-resolving effects via paracrine secretion of soluble factors, growing interest has been addressed to define the most ideal cell source for clinical translation. Leftover or clinical waste samples of human amniotic fluid obtained following prenatal screening, clinical intervention, or during scheduled caesarean section (C-section) delivery at term have been recently considered an appealing source of mesenchymal progenitors with peculiar regenerative capacity. Human amniotic fluid stem cells (hAFSC) have been demonstrated to support tissue recovery in several preclinical models of disease by exerting paracrine proliferative, anti-inflammatory and regenerative influence. Small extracellular vesicles (EVs) concentrated from the hAFSC secretome (the total soluble trophic factors secreted in the cell-conditioned medium, hAFSC-CM) recapitulate most of the beneficial cell effects. Independent studies in preclinical models of either adult disorders or severe diseases in newborns have suggested a regenerative role of hAFSC-EVs. EVs can be eventually concentrated from amniotic fluid (hAF) to offer useful prenatal information, as recently suggested. In this review, we focus on the most significant aspects of EVs obtained from either hAFSC and hAF and consider the current challenges for their clinical translation, including isolation, characterization and quantification methods.


Subject(s)
Amniotic Fluid , Extracellular Vesicles , Precision Medicine , Stem Cells , Humans
20.
J Extracell Vesicles ; 11(1): e12179, 2022 01.
Article in English | MEDLINE | ID: covidwho-1605805

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry is mediated by the interaction of the viral spike (S) protein with angiotensin-converting enzyme 2 (ACE2) on the host cell surface. Although a clinical trial testing soluble ACE2 (sACE2) for COVID-19 is currently ongoing, our understanding of the delivery of sACE2 via small extracellular vesicles (sEVs) is still rudimentary. With excellent biocompatibility allowing for the effective delivery of molecular cargos, sEVs are broadly studied as nanoscale protein carriers. In order to exploit the potential of sEVs, we design truncated CD9 scaffolds to display sACE2 on the sEV surface as a decoy receptor for the S protein of SARS-CoV-2. Moreover, to enhance the sACE2-S binding interaction, we employ sACE2 variants. sACE2-loaded sEVs exhibit typical sEVs characteristics and bind to the S protein. Furthermore, engineered sEVs inhibit the entry of wild-type (WT), the globally dominant D614G variant, Beta (K417N-E484K-N501Y) variant, and Delta (L452R-T478K-D614G) variant SARS-CoV-2 pseudovirus, and protect against authentic SARS-CoV-2 and Delta variant infection. Of note, sACE2 variants harbouring sEVs show superior antiviral efficacy than WT sACE2 loaded sEVs. Therapeutic efficacy of the engineered sEVs against SARS-CoV-2 challenge was confirmed using K18-hACE2 mice. The current findings provide opportunities for the development of new sEVs-based antiviral therapeutics.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , COVID-19/immunology , Extracellular Vesicles/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Female , HEK293 Cells , Humans , Mice , Protein Binding , Protein Interaction Domains and Motifs
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