rapid-isolation-of-human-breast-milk-derived-extracellular-vesicles

Rapid Isolation of Human Breast Milk-Derived Extracellular Vesicles

Extracellular vesicles (EVs), nanosized particles approximately 20-1000 nm in size, are rapidly garnering attention for their therapeutic potential. ...

Alabama State University

Membrane-bound vesicles that are released from cells are increasingly being studied as a medium of intercellular communication, as these act to shuttle functional proteins, such as lipids, DNA, rRNA, and miRNA, between cells during essential physiological processes. Extracellular vesicles (EVs), most commonly exosomes, are consistently produced by virus-infected cells, and they play crucial roles in mediating communication between infected and uninfected cells. Notably, pathophysiological roles for EVs have been established in various viral infections, including human immune deficiency virus (HIV), coronavirus (CoV), and human adenovirus (HAdv). Retroviruses, such as HIV, modulate the production and composition of EVs, and critically, these viruses can exploit EV formation, secretion, and release pathways to promote infection, transmission, and intercellular spread. Consequently, EV production has been investigated as a potential tool for the development of improved viral infection diagnostics and therapeutics. This review will summarize our present knowledge of EV-virus relationships, focusing on their known roles in pathophysiological pathways, immunomodulatory mechanisms, and utility for biomarker discovery. This review will also discuss the potential for EVs to be exploited as diagnostic and treatment tools for viral infection.

Extracellular Vesicles: Roles in Human Viral Infections, Immune-Diagnostic, and Therapeutic Applications.

Adenovirus (Ad) is a major causal agent of acute respiratory infections. However, they are a powerful delivery system for gene therapy and vaccines. Some Ad serotypes antagonize the immune system leading to meningitis, conjunctivitis, gastroenteritis, and/or acute hemorrhagic cystitis. Studies have shown that the release of small, membrane-derived extracellular vesicles (EVs) may offer a mechanism by which viruses can enter cells via receptor-independent entry and how they influence disease pathogenesis and/or host protection considering their existence in almost all bodily fluids. We proposed that Ad3 could alter EV biogenesis, composition, and trafficking and may stimulate various immune responses . In the present study, we evaluated the impact of  infection with Ad3 vector on EV biogenesis and composition in the human adenocarcinoma lung epithelial cell line A549. Cells were infected in an exosome-free media at different multiplicity of infections (MOIs) and time points. The cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and fluorometric calcein-AM. EVs were isolated via ultracentrifugation. Isolated EV proteins were quantified and evaluated via nanoparticle tracking, transmission electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting assays. The cell viability significantly decreased with an increase in MOI and incubation time. A significant increase in particle mean sizes, concentrations, and total EV protein content was detected at higher MOIs when compared to uninfected cells (control group). A549 cell-derived EVs revealed the presence of TSG101, tetraspanins CD9 and CD63, and heat shock proteins 70 and 100 with significantly elevated levels of Rab5, 7, and 35 at higher MOIs (300, 750, and 1500) when compared to the controls. Our findings suggested Ad3 could modulate EV biogenesis, composition, and trafficking which could impact infection pathogenesis and disease progression. This study might suggest EVs could be diagnostic and therapeutic advancement to Ad infections and other related viral infections. However, further investigation is warranted to explore the underlying mechanism(s).

Human Adenovirus Serotype 3 Infection Modulates the Biogenesis and Composition of Lung Cell-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) released by regenerative cells such as mesenchymal stem cells are effective facilitators of healing, therapy, and repair. Conversely, EVs released from infected and/or diseased cells could be useful as markers in the detection and diagnosis of disease conditions such as cancer at their earliest most detectable, and treatable stage. A very important type of EVs, termed exosomes offer a hypothetical new paradigm in disease detection, diagnosis, and treatment. A broad range of exosome-based biomedical and therapeutic applications are now being evaluated in recent clinical trials. Exosomes are found in virtually all bodily fluids and cells and are capable of crossing tight junctions and toughly regulated boundaries such as the blood-brain barrier. Exosomes' expedition ends when they are taken up by bystander cells which corroborates the fact that they are conduits for cells releasing them. Exosomes released by diseased cells have been associated with cell-to-cell progression of diseases like viral disease, neurodegeneration, and certain cancers. Due to high discrimination in most disease conditions, exosome uptake is usually cell-specific. Lots of research evidence have revealed that infusion of exosomes derived from regenerative cells such as stem cells could impede the development of certain infections and age-related diseases by activating self-repair machinery through RNA, DNA, protein, and lipid transfer between cells in patients. They have also been demonstrated in the restoration of the circulating population of exosomes in tissues and the fluid of recipients. The first human clinical trials of exosome therapies are now underway, establishing the future of regenerative exosome in regenerative medicine. This article is categorized under: Cancer > Stem Cells and Development Immune System Diseases > Stem Cells and Development Immune System Diseases > Molecular and Cellular Physiology.

Regenerative mesenchymal stem cell-derived extracellular vesicles: A potential alternative to cell-based therapy in viral infection and disease damage control.

Coronavirus (CoV) has persistently become a global health concern causing various diseases in a wide variety of hosts, including humans, birds, and companion animals. However, the virus-mediated responses in animal hosts have not been studied extensively due to pathogenesis complexity and disease developments. Extracellular vesicles (EVs) are widely explored in viral infections for their intercellular communication, nanocarrier, and immunomodulatory properties. We proposed that coronavirus hijacks the host exosomal pathway and modulates the EV biogenesis, composition, and protein trafficking in the host. In the present study, Crandell-Rees feline kidney (CRFK) cells were infected with canine coronavirus (CCoV) in an exosome-free medium at the multiplicity of infection (MOI) of 400 infectious units (IFU) at various time points. The cell viability was significantly decreased over time, as determined by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Post-infection EVs were isolated, and transmission electron microscopy (TEM) showed the presence of small EVs (sEVs) after infection. NanoSight particle tracking analysis (NTA) revealed that EV sizes averaged between 100 and 200 nm at both incubation times; however, the mean size of infection-derived EVs was significantly decreased at 48 h when compared to uninfected control EVs. Quantitative analysis of protein levels performed by dot blot scanning showed that the expression levels of ACE-2, annexin-V, flotillin-1, TLR-7, LAMP, TNF-α, caspase-1, caspase-8, and others were altered in EVs after infection. Our findings suggested that coronavirus infection impacts cell viability, modulates EV biogenesis, and alters cargo composition and protein trafficking in the host, which could impact viral progression and disease development. Future experiments with different animal CoVs will provide a detailed understanding of host EV biology in infection pathogenesis and progression. Hence, EVs could offer a diagnostic and therapeutic tool to study virus-mediated host responses that could be extended to study the interspecies jump of animal CoVs to cause infection in humans.

Canine Coronavirus Infection Modulates the Biogenesis and Composition of Cell-Derived Extracellular Vesicles.

Exosomes also termed small extracellular vesicles (sEVs) are important mediators of intercellular communication in many physiological and pathological processes such as protein clearance, immunity, infections, signaling, and cancer. Elevated circulating levels of exosomes have been linked to some viral infections, aggressive cancer, and neurodegenerative diseases. Some pharmacological compounds have been demonstrated to effectively inhibit exosome production pathways. There are very few studies on exosome inhibition and how they influence pathophysiological conditions.

Selective pharmacological inhibition alters human carcinoma lung cell-derived extracellular vesicle formation.

The scientific interest in cannabis plants' beneficial properties has recently sparked certain interest in the possible functional characterization of plant-derived extracellular vesicles (PDEVs). Establishing the most appropriate and efficient isolation procedure for PDEVs remains a challenge due to vast differences in the physio-structural characteristics of different plants within the same genera and species. In this study, we employed a crude but standard isolation procedure for the extraction of apoplastic wash fluid (AWF) which is known to contain the PDEVs. This method includes a detailed stepwise process of PDEV extraction from five (5) cultivars of cannabis plants, namely: Citrus (C), Henola (HA), Bialobrezenski (BZ), Southern-Sunset (SS), and Cat-Daddy (CAD). Approximately, 150 leaves were collected from each plant strain. In order to collect PDEV pellets, apoplastic wash fluid (AWF) was extracted from plants via negative pressure permeabilization and infiltration followed by high-speed differential ultracentrifugation. Particle tracking analysis of PDEVs revealed particle size distribution in the range of 20 to 200 nm from all plant strains, while PDEV total protein concentration from HA was higher than that of SS. Although HA-PDEVs' total protein was higher than SS-PDEVs, SS-PDEVs' RNA yield was higher than that of HA-PDEVs. Our result suggests that the cannabis plant strains contain EVs, and PDEV concentration from the cannabis plant could be age or strain dependent. Overall, the results provide a guide for the selection and optimization of PDEV isolation methods for future studies.

Characterization of cannabis strain-plant-derived extracellular vesicles as potential biomarkers.

 Drug repurposing is fast growing and becoming an attractive approach for identifying novel targets, such as exosomes for cancer and antiviral therapy. Exosomes are a specialized class of extracellular vesicles that serve as functional mediators in intercellular communication and signaling that are important in normal physiological functions. A continuously growing body of evidence has established a correlation between the abnormal release of exosomes with various viral disease pathologies including cancer. Cells that are virus-infected release exosomes known to influence the process via the loading and transfer of viral components, such as miRNA, small (s) RNA, DNA, and proteins. Inhibition of exosome release may abate the spread and severity of viral infection, thus making exosomes an attractive target for antiviral therapies. We previously demonstrated the pharmacological inhibition of exosomes.  Herein, we used a cell-based assay to determine the effect of Human adenovirus type 3 (HAdV3) on the exosome inhibition process by azole and Heparin derivatives. HAdV3-infected cells were treated with two concentrations of each inhibitor at different time points.  HAdV3 activities led to increased total sRNA, DNA, and exosome particle concentrations via particle tracking in the presence of Climbazole and Heparin relative to uninfected exosomes. In addition, there was an increased expression of classical markers such as ALG-2 interacting protein X (ALIX), and tetraspanin (CD63), ( < 0.05) and upregulated transcription factor interferon regulatory factor (IRF) 8 in the presence of HAdV3 after 24 hours (h) of treatment. Whereas higher concentrations of Climbazole and Heparin sodium salt were found to inhibit total exosome protein ( < 0.001) and exo-RNA ( < 0.01) content even in the presence of HAdV3 relative to infected exosomes only. Activities of HAdV3 in the presence of selected inhibitors resulted in the positive regulation of exosome related DNA damage/repair signaling proteins. Blocking exosome secretion partially obstructed viral entry. Immunological studies revealed that HAdV3 fiber protein levels in A549 cells were reduced at all concentrations of Climbazole and Heparin and both multiplicities of infections ( < 0.001).  Our findings suggest that while HAdV may bolster inhibited exosome content and release when modulating certain activities of the endosomal pathway mediators, HAdV entry might be constrained by the activities of these pharmacological agents.

Human adenovirus type 3 restores pharmacologically inhibited exosomal cargo in lung carcinoma cells.

Coronavirus (CoV) has become a public health crisis that causes numerous illnesses in humans and certain animals. Studies have identified the small, lipid-bound structures called extracellular vesicles (EVs) as the mechanism through which viruses can enter host cells, spread, and evade the host's immune defenses. EVs are able to package and carry numerous viral compounds, including proteins, genetic substances, lipids, and receptor proteins. We proposed that the coronavirus could alter EV production and content, as well as influence EV biogenesis and composition in host cells.

Article	Year
Extracellular Vesicles: Roles in Human Viral Infections, Immune-Diagnostic, and Therapeutic Applications. Pathogens (Basel, Switzerland)\| PubMed ID: 33348699	2020
Human Adenovirus Serotype 3 Infection Modulates the Biogenesis and Composition of Lung Cell-Derived Extracellular Vesicles. Journal of immunology research\| PubMed ID: 34926703	2021
Regenerative mesenchymal stem cell-derived extracellular vesicles: A potential alternative to cell-based therapy in viral infection and disease damage control. WIREs mechanisms of disease\| PubMed ID: 36065123	2022
Canine Coronavirus Infection Modulates the Biogenesis and Composition of Cell-Derived Extracellular Vesicles. Biomedicines\| PubMed ID: 36979955	2023
Selective pharmacological inhibition alters human carcinoma lung cell-derived extracellular vesicle formation. Heliyon\| PubMed ID: 37303541	2023
Characterization of cannabis strain-plant-derived extracellular vesicles as potential biomarkers. Protoplasma\| PubMed ID: 37330445	2023
Human adenovirus type 3 restores pharmacologically inhibited exosomal cargo in lung carcinoma cells. Frontiers in pharmacology\| PubMed ID: 38449802	2024
Feline coronavirus influences the biogenesis and composition of extracellular vesicles derived from CRFK cells. Frontiers in veterinary science\| PubMed ID: 39091390	2024

Ayodeji Ipinmoroti

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