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![Methods used to detect and quantify EVs. (A) Nanoparticle tracking analysis (NTA) analyzes laser scattering in purified vesicle suspensions to track the Brownian motion of the vesicles and determine their absolute size (reproduced with permission from [95], copyright 2011 Elsevier). (B) Resistive pulse sensing (RPS) measures the "resistive pulse" rate produced when purified vesicle suspensions migrate across a membrane in response to applied voltage to determine vesicle concentration relative to a reference standard (adapted with permission from [96], copyright 2012 American Chemical Society). (C) Alternating current electrokinetic (ACE) microarrays use the electrical properties of EVs to isolate these particles from plasma sample and then detect the immunofluorescent signal from target EV proteins. The schematic shows a cross-section and a top-down view of a single electrode well on the array (reproduced with permission from [97], copyright 2017 American Chemical Society).](publication/324671242/figure/fig2/AS:627808896307205@1526692713392/Methods-used-to-detect-and-quantify-EVs-A-Nanoparticle-tracking-analysis-NTA.png){kind=tracking}
Methods used to detect and quantify EVs. (A) Nanoparticle tracking analysis (NTA) analyzes laser scattering in purified vesicle suspensions to track the Brownian motion of the vesicles and determine their absolute size (reproduced with permission from [95], copyright 2011 Elsevier). (B) Resistive pulse sensing (RPS) measures the "resistive pulse" rate produced when purified vesicle suspensions migrate across a membrane in response to applied voltage to determine vesicle concentration relative to a reference standard (adapted with permission from [96], copyright 2012 American Chemical Society). (C) Alternating current electrokinetic (ACE) microarrays use the electrical properties of EVs to isolate these particles from plasma sample and then detect the immunofluorescent signal from target EV proteins. The schematic shows a cross-section and a top-down view of a single electrode well on the array (reproduced with permission from [97], copyright 2017 American Chemical Society).
Source publication
Extracellular vesicles (EVs), or exosomes, are nanovesicles of endocytic origin that carry host and pathogen-derived protein, nucleic acid, and lipid cargos. They are secreted by most cell types and play important roles in normal cell-to-cell communications but can also spread pathogen- and host-derived molecules during infections to alter immune r...
Context in source publication
Context 1
... tracking analysis (NTA) employs a laser beam to illuminate all vesicles in a sample suspension, a light microscope to record the scattered light, and software to measure vesicle sizes as determined by the Brownian motion track of each particle [94,95]. These instruments are commercially available and can measure the number and absolute size distribution of vesicles in a solution, and quantitate EVs based on their unique size profile. Resistive pulse sensing (RPS) can determine the absolute size distribution of vesicles in sample suspensions via the Coulter principle [96], and at least one company has developed an instrument to exploit this approach. This system consists of two fluid cells divided by a non-conductive nanoporous membrane. A particle moving through one of these nanopores in response to a voltage applied across the cell membrane alters the ion flow, resulting in a brief "resistive pulse", which is recorded for calculation against a reference standard made with beads of known diameter and concentration [94,96]. Finally, alternating current electrokinetic (ACE) microarrays can isolate EVs from plasma samples to allow on-chip immunofluorescent detection of EV proteins and to provide mRNA for RT-PCR analysis [97], providing a potential means to isolate and analyze total EV populations without a separate EV isolation step (Figure 3). None of these approaches can quantitate disease-specific EV populations from the general EV ...
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Citations
... When tissue environments are perturbed or cells become damaged as occurs during a viral infection, EV content changes based on cellular reprogramming in response to pathological stress (11,12). EVs can either activate or inhibit innate and adaptive immune cell responses based on their content (13)(14)(15). ...
... EVs have been demonstrated to express major histocompatibility complex (MHC) class I or II and directly activate innate antigen presenting cells (APCs) or adaptive T and B cells in an antigen/self-antigenspecific manner (16,17). Tetraspanins like CD9, CD63 and CD81, which are commonly used to characterize EVs, bind factors on innate immune cells like integrins (i.e., CD11b) that are important in activating and modulating immune responses (12,18). ...
For many decades viral infections have been suspected as ‘triggers’ of autoimmune disease, but mechanisms for how this could occur have been difficult to establish. Recent studies have shown that viral infections that are commonly associated with viral myocarditis and other autoimmune diseases such as coxsackievirus B3 (CVB3) and SARS-CoV-2 target mitochondria and are released from cells in mitochondrial vesicles that are able to activate the innate immune response. Studies have shown that Toll-like receptor (TLR)4 and the inflammasome pathway are activated by mitochondrial components. Autoreactivity against cardiac myosin and heart-specific immune responses that occur after infection with viruses where the heart is not the primary site of infection (e.g., CVB3, SARS-CoV-2) may occur because the heart has the highest density of mitochondria in the body. Evidence exists for autoantibodies against mitochondrial antigens in patients with myocarditis and dilated cardiomyopathy. Defects in tolerance mechanisms like autoimmune regulator gene (AIRE) may further increase the likelihood of autoreactivity against mitochondrial antigens leading to autoimmune disease. The focus of this review is to summarize current literature regarding the role of viral infection in the production of extracellular vesicles containing mitochondria and virus and the development of myocarditis.
... Most cell types release extracellular vesicles (EVs), and EVs with a diameter of 30-150 nm released through the endocytic pathway are defined as exosomes. Bacterial infection affects the release of exosomes, and the cargo loaded in exosomes may exert regulatory effects on recipient cells [41]. For example, exosomes released from macrophages infected by mycobacteria contain mycobacterial antigens to stimulate immune responses [42]. ...
Background
A large number of Fusobacterium nucleatum (Fn) are present in colorectal cancer (CRC) tissues of patients who relapse after chemotherapy, and Fn has been reported to promote oxaliplatin and 5-FU chemoresistance in CRC. Pathogens such as bacteria and parasites stimulate exosome production in tumor cells, and the regulatory mechanism of exosomal circRNA in the transmission of oxaliplatin and 5-FU chemotherapy resistance in Fn-infected CRC remains unclear.
Methods
Hsa_circ_0004085 was screened by second-generation sequencing of CRC tissues. The correlation between hsa_circ_0004085 and patient clinical response to oxaliplatin/5-FU was analyzed. Exosome tracing experiments and live imaging systems were used to test the effect of Fn infection in CRC on the distribution of hsa_circ_0004085. Colony formation, ER tracking analysis and immunofluorescence were carried out to verify the regulatory effect of exosomes produced by Fn-infected CRC cells on chemotherapeutic resistance and ER stress. RNA pulldown, LC–MS/MS analysis and RIP were used to explore the regulatory mechanism of downstream target genes by hsa_circ_0004085.
Results
First, we screened out hsa_circ_0004085 with abnormally high expression in CRC clinical samples infected with Fn and found that patients with high expression of hsa_circ_0004085 in plasma had a poor clinical response to oxaliplatin/5-FU. Subsequently, the circular structure of hsa_circ_0004085 was identified. Fn infection promoted hsa_circ_0004085 formation by hnRNP L and packaged hsa_circ_0004085 into exosomes by hnRNP A1. Exosomes produced by Fn-infected CRC cells transferred hsa_circ_0004085 between cells and delivered oxaliplatin/5-FU resistance to recipient cells by relieving ER stress. Hsa_circ_0004085 enhanced the stability of GRP78 mRNA by binding to RRBP1 and promoted the nuclear translocation of ATF6p50 to relieve ER stress.
Conclusions
Plasma levels of hsa_circ_0004085 are increased in colon cancer patients with intracellular Fn and are associated with a poor response to oxaliplatin/5-FU. Fn infection promoted hsa_circ_0004085 formation by hnRNP L and packaged hsa_circ_0004085 into exosomes by hnRNP A1. Exosomes secreted by Fn-infected CRC cells deliver hsa_circ_0004085 between cells. Hsa_circ_0004085 relieves ER stress in recipient cells by regulating GRP78 and ATF6p50, thereby delivering resistance to oxaliplatin and 5-FU.
Graphical Abstract
... After being released into the extracellular space, these exosomes interact with the corresponding recipient cells either through direct membrane fusion, micropinocytosis, or via receptor-mediated endocytosis [38]. Following exocytosis, the exosomes still retain several endosome-related proteins, like annexins, lipid raft-associated proteins, and ESCRT accessory proteins, which are further used for intracellular membrane fusion and their transport to the recipient cell [39]. Exosomal membrane proteins include tetraspanins such as CD63 and CD81, fusion proteins like CD9, lysosomal protein LAMP2b, as well as heat shock proteins like HSP70. ...
... Exosomal membrane proteins include tetraspanins such as CD63 and CD81, fusion proteins like CD9, lysosomal protein LAMP2b, as well as heat shock proteins like HSP70. Among these, tetraspanins are known to play a vital role in mediating exosome formation as well as fusion with the target cells [39,40]. Two additional exosomal proteins include RAB27A, which help in the determination of exosome size, and RAB27B, which are small GTPases, involved in modulating the docking of the MVBs at the plasma membrane during the process of exosome formation [1,38]. ...
Extracellular vesicles (EVs) are small membrane-enclosed structures that have gained much attention from researchers across varying scientific fields in the past few decades. Cells secrete diverse types of EVs into the extracellular milieu which include exosomes, microvesicles, and apoptotic bodies. These EVs play a crucial role in facilitating intracellular communication via the transport of proteins, lipids, DNA, rRNA, and miRNAs. It is well known that a number of viruses hijack several cellular pathways involved in EV biogenesis to aid in their replication, assembly, and egress. On the other hand, EVs can also trigger host antiviral immune responses by carrying immunomodulatory molecules and viral antigens on their surface. Owing to this intricate relationship between EVs and viruses, intriguing studies have identified various EV-mediated viral infections and interrogated how EVs can alter overall viral spread and longevity. This review provides a comprehensive overview on the EV-virus relationship, and details various modes of EV-mediated viral spread in the context of clinically relevant enveloped and non-enveloped viruses.
... EVs can be used either in their naturally secreted state or in an engineered form for tailored therapeutic purposes [153][154][155]. While EVs are excellent as carriers for viral antigens, presenting them in their unaltered form to evoke an effective immune response, they also have the capacity to transport host-derived antiviral compounds and immune enhancers [156,157]. Notwithstanding, the challenges associated with employing EVs as therapeutics, their favorable biological attributes and natural carrier capabilities for small molecules render them a compelling choice for vaccine development. Current EVs isolation and purification techniques can be harnessed with the utmost potential to adhere to cGMP requirements. ...
Extracellular vesicles (EVs) have a significant impact on the pathophysiological processes associated with various diseases such as tumors, inflammation, and infection. They exhibit molecular, biochemical, and entry control characteristics similar to viral infections. Viruses, on the other hand, depend on host metabolic machineries to fulfill their biosynthetic requirements. Due to potential advantages such as biocompatibility, biodegradation, and efficient immune activation, EVs have emerged as potential therapeutic targets against the SARS-CoV-2 infection. Studies on COVID-19 patients have shown that they frequently have dysregulated lipid profiles, which are associated with an increased risk of severe repercussions. Lipid droplets (LDs) serve as organelles with significant roles in lipid metabolism and energy homeostasis as well as having a wide range of functions in infections. The down-modulation of lipids, such as sphingolipid ceramide and eicosanoids, or of the transcriptional factors involved in lipogenesis seem to inhibit the viral multiplication, suggesting their involvement in the virus replication and pathogenesis as well as highlighting their potential as targets for drug development. Hence, this review focuses on the role of modulation of lipid metabolism and EVs in the mechanism of immune system evasion during SARS-CoV-2 infection and explores the therapeutic potential of EVs as well as application for delivering therapeutic substances to mitigate viral infections.
... This complex interaction between the diagnostic/drug (the OV) and the target (the tumor) again benefits from the integration of omic approaches and computational methods during both development and testing. As an example, the analysis of the secretome of tumors treated with OVs provides information on the mechanisms of immune activation induced by the OV itself and has a diagnostic value [147,148]. The optimization of integrated approaches for functional real-time imaging in clinical settings of OV replication, checkpoint expression, and immune cell trafficking is a priority for advancements in the field of OV immunovirotherapies [149]. ...
Oncolytic viruses (OVs) are the frontier therapy for refractory cancers, especially in integration with immunomodulation strategies. In cancer immunovirotherapy, the many available “omics” and systems biology technologies generate at a fast pace a challenging huge amount of data, where apparently clashing information mirrors the complexity of individual clinical situations and OV used. In this review, we present and discuss how currently big data analysis, on one hand and, on the other, simulation, modeling, and computational technologies, provide invaluable support to interpret and integrate “omic” information and drive novel synthetic biology and personalized OV engineering approaches for effective immunovirotherapy. Altogether, these tools, possibly aided in the future by artificial intelligence as well, will allow for the blending of the information into OV recombinants able to achieve tumor clearance in a patient-tailored way. Various endeavors to the envisioned “synthesis” of turning OVs into personalized theranostic agents are presented.
... During viral or bacterial infection, EVs can transport pathogen-or host-derived factors to alter the pathophysiological mechanisms of infection transmission and host immune responses. 11 Therefore, the employment of SEV analysis techniques to disclose the compositional and functional heterogeneity of the host-cellderived EV populations at distinct time points during infectious disease progression is crucial for the application of EVs in guiding infection diagnosis and therapy. ...
Extracellular vesicles (EVs) are extensively dispersed lipid bilayer membrane vesicles involved in the delivery and transportation of molecular payloads to certain cell types to facilitate intercellular interactions. Their significant roles in physiological and pathological processes make EVs outstanding biomarkers for disease diagnosis and treatment monitoring as well as ideal candidates for drug delivery. Nevertheless, differences in the biogenesis processes among EV subpopulations have led to a diversity of biophysical characteristics and molecular cargos. Additionally, the prevalent heterogeneity of EVs has been found to substantially hamper the sensitivity and accuracy of disease diagnosis and therapeutic monitoring, thus impeding the advancement of clinical applications. In recent years, the evolution of single EV (SEV) analysis has enabled an in-depth comprehension of the physical properties, molecular composition, and biological roles of EVs at the individual vesicle level. This review examines the sample acquisition tactics prior to SEV analysis, i.e., EV isolation techniques, and outlines the current state-of-the-art label-free and label-based technologies for SEV identification. Furthermore, the challenges and prospects of biomedical applications based on SEV analysis are systematically discussed.
... and more importantly, the usage of MB-AuNP-I-PCR, which is relatively a more precise method than I-PCR to detect biomarkers in clinical specimens 15 . Of note, there are two distinct populations of EVs released from Mtb infected macrophages, e.g., those enclosing the host EVs cell markers (CD63/CD9) and the Mtb markers, such as lipoglycans (LAM) and lipoproteins (LpqH) that regulate both innate and acquired immune responses 38,39 . Similarly, Mycobacterium bovis BCGinfected human THP-1 and murine J774 macrophages were shown to release exosomes containing LAM, LpqH and Ag85 complex 40 . ...
... Similarly, Mycobacterium bovis BCGinfected human THP-1 and murine J774 macrophages were shown to release exosomes containing LAM, LpqH and Ag85 complex 40 . Moreover, the release of mycobacterial EVs (MEVs) containing LAM/LpqH depends on Mtb's viability, entailing that active Mtb replication is essential for MEVs generation 38,39 , thus suggesting that LAM/LpqH detection within MEVs may indicate active TB disease. Humoral responses to MEVs (containing LAM) with sera of HIV-negative pulmonary TB patients were also documented by ELISA and immunoblot techniques 41 . ...
... Our quantitative SYBR Green MB-AuNP-RT-I-PCR can also monitor the disease dynamics, since a dynamic range of LAM+MPT-64 (400 fg/mL to 11 ng/mL) was detected within urinary EVs of GUTB patients (Fig. 6b). Both LAM and MPT-64 represent biomarkers for active Mtb replication, their enhanced levels within urine/urinary EVs may reflect the progression of active GUTB disease 5,14,39 . We previously demonstrated diminished MPT-64+PstS1 (Rv0934) concentrations by RT-I-PCR in clinical specimens of pulmonary TB patients on therapy and probably the decreased bacterial load 45 . ...
We detected a cocktail of Mycobacterium tuberculosis lipoarabinomannan (LAM) and MPT-64 biomarkers within urine extracellular vesicles (EVs) of genitourinary TB (GUTB) patients by nano-based immuno-PCR (I-PCR) assay, i.e., magnetic bead-coupled gold nanoparticle-based I-PCR (MB-AuNP-I-PCR) and compared the results with I-PCR and Magneto-ELISA. The size (s) of urine EVs ranged between 52.6 and 220.4 nm as analyzed by transmission electron microscopy (TEM) and nanoparticle tracking analysis. Functionalized AuNPs (coupled with detection antibodies/oligonucleotides) were characterized by UV–vis spectroscopy, TEM, ELISA, PCR, Atomic Force Microscopy and Fourier Transform Infrared spectroscopy, while conjugation of capture antibodies with MBs was validated by UV–vis spectroscopy and Magneto-ELISA. Our MB-AuNP-I-PCR exhibited sensitivities of 85% and 87.2% in clinically suspected (n = 40) and total (n = 47) GUTB cases, respectively, with 97.1% specificity in non-TB controls (n = 35). These results were further authenticated by the quantitative SYBR Green MB-AuNP-real-time I-PCR (MB-AuNP-RT-I-PCR). Concurrently, I-PCR and Magneto-ELISA showed sensitivities of 68.1% and 61.7%, respectively in total GUTB cases, which were significantly lower (p < 0.05–0.01) than MB-AuNP-I-PCR. Markedly, a wide range (400 fg/mL–11 ng/mL) of LAM+MPT-64 was quantified within urine EVs of GUTB cases by SYBR Green MB-AuNP-RT-I-PCR, which can assess the disease dynamics. This study will certainly improve the current algorithms used in GUTB diagnostics.
... Extracellular vesicles (EVs) carrying various bioactive molecules including proteins, DNAs, and RNAs, from their source cells, have been used for disease diagnosis and therapeutics [1][2][3]. EVs are present in biological fluids and play a critical role in multiple physiological and pathological processes [4][5][6]. Serving as cell-cell communicators, EVs can be decoded to obtain developmental information about diseased cells or tissues [7-9]. ...
... Furthermore, these devices can be miniaturized and integrated into portable lab-on-a-chip platforms, enabling the development of new disease detection methods as well as new approaches for monitoring disease progression in clinics. (2) Another advancement is the EV-based biomarker discovery for exploring disease etiology. The innovation of sound-powered EV handling may address the current sample preparation and processing challenges for current multi-omics technologies for precise investigations of the biomarkers carried by EVs from patients. ...
Extracellular vesicles are nano- to micro-scale, membrane-bound particles released by cells into extracellular space, and act as carriers of biomarkers and therapeutics, holding promising potential in translational medicine. However, the challenges remain in handling and detecting extracellular vesicles for disease diagnosis as well as exploring their therapeutic capability for disease treatment. Here, we review the recent engineering and technology advances by leveraging the power of sound waves to address the challenges in diagnostic and therapeutic applications of extracellular vesicles and biomimetic nanovesicles. We first introduce the fundamental principles of sound waves for understanding different acoustic-assisted extracellular vesicle technologies. We discuss the acoustic-assisted diagnostic methods including the purification, manipulation, biosensing, and bioimaging of extracellular vesicles. Then, we summarize the recent advances in acoustically enhanced therapeutics using extracellular vesicles and biomimetic nanovesicles. Finally, we provide perspectives into current challenges and future clinical applications of the promising extracellular vesicles and biomimetic nanovesicles powered by sound.
... We have concentrated on the physiological functions of extracellular vesicles in the male reproductive tract. But in addition to controlling normal physiology, extracellular vesicles also play a function in pathology [39][40][41]. There is considerable agreement that this reaction is caused by changed protein and nucleic acid cargo carried by extracellular vesicles in the context of directing pathophysiology. ...
The epididymis is responsible for post-testicular sperm maturation as it provides a favorable environment for spermatozoa to gain the ability for movement and fertilization. The recent evidence has shown that, the spermatozoa are vulnerable to dynamic variations driven by various cellular exposure mechanisms mediated by epididymosomes. Exosomes provide new insight into a mechanism of intercellular communication because they provide direct evidence for the transfer of several important bio-active cargo elements (proteins, lipid, DNA, mRNA, microRNA, circular RNA, long noncoding RNA) between epididymis and spermatozoa. In broad sense, proteomic analysis of exosomes from epididymis indicates number of proteins that are involved in sperm motility, acrosomal reaction, prevent pre-mature sperm capacitation and male infertility. Pinpointing, how reproductive disorders are associated with bio-active cargo elements of nano-scale exosome in the male reproductive tract. Therefore, the current review presents evidence regarding the distinctive characteristics and functions of nano-scale exosome in the male reproductive tract in both pathological and physiological developments, and argue that these vesicles serve as an important regulator of male reproduction, fertility, and disease susceptibility.
... Many viruses such as HIV, coxsackievirus, hepatitis B and C, influenza, Epstein-Barr virus, and SARS-CoV-2 highjack cellular and mitochondrial programs to enhance viral replication, package virus into EVs, and use EVs containing virus or viral components to subvert the immune response to obtain a replicative advantage in the host (Figure 1). [45][46][47] This is also true for SARS-CoV-2 RNA, which has been detected in EVs. [48][49][50] Virus-containing EVs could enter the circulation from the lungs or other organs and enter the heart to be taken up by resident APCs like mast cells and macrophages to promote myocarditis ( Figure 1). ...
Viral infections are a leading cause of myocarditis and pericarditis worldwide, conditions that frequently coexist. Myocarditis and pericarditis were some of the early comorbidities associated with SARS-CoV-2 infection and COVID-19. Many epidemiologic studies have been conducted since that time concluding that SARS-CoV-2 increased the incidence of myocarditis/pericarditis at least 15× over pre-COVID levels although the condition remains rare. The incidence of myocarditis pre-COVID was reported at 1 to 10 cases/100 000 individuals and with COVID ranging from 150 to 4000 cases/100 000 individuals. Before COVID-19, some vaccines were reported to cause myocarditis and pericarditis in rare cases, but the use of novel mRNA platforms led to a higher number of reported cases than with previous platforms providing new insight into potential pathogenic mechanisms. The incidence of COVID-19 vaccine-associated myocarditis/pericarditis covers a large range depending on the vaccine platform, age, and sex examined. Importantly, the findings highlight that myocarditis occurs predominantly in male patients aged 12 to 40 years regardless of whether the cause was due to a virus-like SARS-CoV-2 or associated with a vaccine-a demographic that has been reported before COVID-19. This review discusses findings from COVID-19 and COVID-19 vaccine-associated myocarditis and pericarditis considering the known symptoms, diagnosis, management, treatment, and pathogenesis of disease that has been gleaned from clinical research and animal models. Sex differences in the immune response to COVID-19 are discussed, and theories for how mRNA vaccines could lead to myocarditis/pericarditis are proposed. Additionally, gaps in our understanding that need further research are raised.
