Lung Myofibroblasts Promote Macrophage Profibrotic Activity through Lactate-induced Histone Lactylation.

Augmented glycolysis due to metabolic reprogramming in lung myofibroblasts is critical to their profibrotic phenotype. The primary glycolysis byproduct, lactate, is also secreted into the extracellular milieu, together with which myofibroblasts and macrophages form a spatially restricted site usually described as fibrotic niche. Therefore, we hypothesized that myofibroblast glycolysis might have a non-cell autonomous effect through lactate regulating the pathogenic phenotype of alveolar macrophages. Here, we demonstrated that there was a markedly increased lactate in the conditioned media of TGF-ß1 (transforming growth factor-ß1)-induced lung myofibroblasts and in the BAL fluids (BALFs) from mice with TGF-ß1- or bleomycin-induced lung fibrosis. Importantly, the media and BALFs promoted profibrotic mediator expression in macrophages. Mechanistically, lactate induced histone lactylation in the promoters of the profibrotic genes in macrophages, consistent with the upregulation of this epigenetic modification in these cells in the fibrotic lungs. The lactate inductions of the histone lactylation and profibrotic gene expression were mediated by p300, as evidenced by their diminished concentrations in p300-knockdown macrophages. Collectively, our study establishes that in addition to protein, lipid, and nucleic acid molecules, a metabolite can also mediate intercellular regulations in the setting of lung fibrosis. Our findings shed new light on the mechanism underlying the key contribution of myofibroblast glycolysis to the pathogenesis of lung fibrosis.

Cocaine-Specific Effects on Exosome Biogenesis in Microglial Cells.

Cocaine is a highly addictive stimulant and a well-known drug, with multiple effects on physiology. Cocaine can have direct effects on all cell types in the brain, including microglia. Microglia can be activated by other conditions, such as infection, inflammation, or injury. However, how cocaine regulates microglia and the influence of cocaine on microglial-derived exosomes remains unknown. Exosomes are nanovesicles that are responsible for intercellular communications, signaling, and trafficking necessary cargo for cell homeostasis. In this study, we hypothesized that cocaine affects exosome biogenesis and composition in BV2 microglial cells. BV2 microglial cells were cultured in exosome-depleted RPMI-1640 media and were treated according to the experimental designs. We observed that cell viability decreased by 11% at 100 µM cocaine treatment but was unaffected at other concentrations. After treatments, the exosomes were isolated from the condition media. Purified exosomes were characterized and quantified using transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA). By NTA, there was a significant decrease in particles/mL after cocaine treatment. There was a 39.5%, 58.1%, 32.3% and 28.1% decrease in particles/mL at 100 nM, 1 µM, 10 µM and 100 µM cocaine, respectively. The characterization of exosomes and exosomal protein was performed by western/dot blot analyses. Tetraspanins CD11b, CD18 and CD63 were relatively unchanged after cocaine treatment. The heat shock proteins (Hsps), Hsp70 and Hsp90, were both significantly increased at 10 µM and 100 µM, but only hsp70 was significantly increased at 10 nM. The Rab proteins were assessed to investigate their role in cocaine-mediated exosomal decrease. Rab11 was significantly decreased at 10 nM, 100 nM, 1 µM, 10 µM and 100 µM by 15%, 28%, 25%, 38% and 22%, respectively. Rab27 was decreased at all concentrations but only significantly decreased at 100 nM, 1 µM and 100 µM cocaine by 21%, 24% and 23%, respectively. Rab35 had no significant changes noted when compared to control. Rab7 increased at all cocaine concentrations but only a significant increase in expression at 100 nM and 10 µM by 1.32-fold and 1.4-fold increase. Cocaine was found to alter exosome biogenesis and composition in BV2 microglial cells. Western and dot blot analyses verified the identities of purified exosomes, and the specific protein compositions of exosomes were found to change in the presence of cocaine. Furthermore, cocaine exposure modulated the expression of exosomal proteins, such as Hsps and Rab GTPases, suggesting the protein composition and formation of microglial-derived exosomes were regulated by cocaine.

Lipopolysaccharide Administration Alters Extracellular Vesicles in Cell Lines and Mice.

Extracellular vesicles (EVs) play a fundamental role in cell and infection biology and have the potential to act as biomarkers for novel diagnostic tools. In this study, we explored the in vitro impact of bacterial lipopolysaccharide administration on cell lines that represents a target for bacterial infection in the host. Administration of lipopolysaccharide at varying concentrations to A549 and BV-2 cell lines caused only modest changes in cell death, but EV numbers were significantly changed. After treatment with the highest concentration of lipopolysaccharide, EVs derived from A549 cells packaged significantly less interleukin-6 and lysosomal-associated membrane protein 1. EVs derived from BV-2 cells packaged significantly less tumor necrosis factor after administration of lipopolysaccharide concentrations of 0.1 µg/mL and 1 µg/mL. We also examined the impact of lipopolysaccharide administration on exosome biogenesis and cargo composition in BALB/c mice. Serum-isolated EVs from lipopolysaccharide-treated mice showed significantly increased lysosomal-associated membrane protein 1 and toll-like receptor 4 levels compared with EVs from control mice. In summary, this study demonstrated that EV numbers and cargo were altered using these in vitro and in vivo models of bacterial infection.

Tetraspanin blockage reduces exosome-mediated HIV-1 entry.

HIV-1 is one of the most studied retroviruses. The role of exosomes in HIV-1 entry and pathogenesis are beginning to be appreciated. Exosomes can incorporate host proteins that are also contained in viruses (e.g., tetraspanins).

Differential binding of the HIV-1 envelope to phosphatidylserine receptors.

Prior work has shown that the HIV-1 envelope of the human immunodeficiency virus (HIV) interacts directly with T-cell immunoglobulin mucin (TIM) family proteins. Herein, we demonstrate that HIV-1 envelope glycoproteins from varying HIV-1 clades bind differentially to TIM proteins and functionally similar proteins acting as phosphatidylserine (PtdSer) receptors. Using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) technology, we show that lysate containing HIV-1 envelope and recombinant HIV-1 envelope glycoproteins bind TIM-4 and advanced glycosylation end product-specific receptor (AGER). The complex binding of HIV-1 UG21 gp140 to TIM-4 or AGER suggests a biphasic interaction with these proteins.

Monitoring of biodistribution and persistence of conditionally replicative adenovirus in a murine model of ovarian cancer using capsid-incorporated mCherry and expression of human somatostatin receptor subtype 2 gene.

A significant limiting factor to the human clinical application of conditionally replicative adenovirus (CRAd)-based virotherapy is the inability to noninvasively monitor these agents and their potential persistence. To address this issue, we proposed a novel imaging approach that combines transient expression of the human somatostatin receptor (SSTR) subtype 2 reporter gene with genetic labeling of the viral capsid with mCherry fluorescent protein. To test this dual modality system, we constructed the Ad5/3Δ24pIXcherry/SSTR CRAd and validated its capacity to generate fluorescent and nuclear signals in vitro and following intratumoral injection. Analysis of 64Cu-CB-TE2A-Y3-TATE biodistribution in mice revealed reduced uptake in tumors injected with the imaging CRAd relative to the replication-incompetent, Ad-expressing SSTR2 but significantly greater uptake compared to the negative CRAd control. Optical imaging demonstrated relative correlation of fluorescent signal with virus replication as determined by viral genome quantification in tumors. Positron emission tomography/computed tomography studies demonstrated that we can visualize radioactive uptake in tumors injected with imaging CRAd and the trend for greater uptake by standardized uptake value analysis compared to control CRAd. In the aggregate, the plasticity of our dual imaging approach should provide the technical basis for monitoring CRAd biodistribution and persistence in preclinical studies while offering potential utility for a range of clinical applications.

A recombinant adenovirus-based vector elicits a specific humoral immune response against the V3 loop of HIV-1 gp120 in mice through the "Antigen Capsid-Incorporation" strategy.

BACKGROUND: Due to potential advantages, human adenoviral vectors have been evaluated pre-clinically as recombinant vaccine vectors against several cancers and infectious diseases, including human immunodeficiency virus (HIV) infection. The V3 loop of HIV-1 glycoprotein 120 (gp120) contains important neutralizing epitopes and plays key roles in HIV entry and infectivity. METHODS: In order to investigate the humoral immune response development against portions of the V3 loop, we sought to generate four versions of adenovirus (Ad)-based V3 vectors by incorporating four different antigen inserts into the hypervariable region 1 (HVR1) of human adenovirus type 5 (hAd5) hexon. The strategy whereby antigens are incorporated within the adenovirus capsid is known as the "Antigen Capsid-Incorporation" strategy. RESULTS: Of the four recombinant vectors, Ad-HVR1-lgs-His6-V3 and Ad-HVR1-long-V3 had the capability to present heterologous antigens on capsid surface, while maintaining low viral particle to infectious particle (VP/IP) ratios. The VP/IP ratios indicated both high viability and stability of these two vectors, as well as the possibility that V3 epitopes on these two vectors could be presented to immune system. Furthermore, both Ad-HVR1-lgs-His6-V3 and Ad-HVR1-long-V3 could, to some extent escape the neutralization by anti-adenovirus polyclonal antibody (PAb), but rather not the immunity by anti-gp120 (902) monoclonal antibody (MAb). The neutralization assay together with the whole virus enzyme-linked immunosorbent assay (ELISA) suggested that these two vectors could present V3 epitopes similar to the natural V3 presence in native HIV virions. However, subsequent mice immunizations clearly showed that only Ad-HVR1-lgs-His6-V3 elicited strong humoral immune response against V3. Isotype ELISAs identified IgG2a and IgG2b as the dominant IgG isotypes, while IgG1 comprised the minority. CONCLUSIONS: Our findings demonstrated that human adenovirus (hAd) vectors which present HIV antigen via the "Antigen Capsid-Incorporation" strategy could successfully elicit antigen-specific humoral immune responses, which could potentially open an avenue for the development of Ad-based HIV V3 vaccines.

Feline coronavirus influences the biogenesis and composition of extracellular vesicles derived from CRFK cells.

Introduction: 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. Methods: In the current research, Crandell-Rees feline kidney (CRFK) cells were infected with feline coronavirus (FCoV) in an exosome-free media at a multiplicity of infection (MOI) of 2,500 infectious units (IFU) at 48 h and 72 h time points. Cell viability was analyzed and found to be significantly decreased by 9% (48 h) and 15% (72 h) due to FCoV infection. EVs were isolated by ultracentrifugation, and the surface morphology of isolated EVs was analyzed via Scanning Electron Microscope (SEM). Results: NanoSight particle tracking analysis (NTA) confirmed that the mean particle sizes of control EVs were 131.9 nm and 126.6 nm, while FCoV infected-derived EVs were 143.4 nm and 120.9 nm at 48 and 72 h, respectively. Total DNA, RNA, and protein levels were determined in isolated EVs at both incubation time points; however, total protein was significantly increased at 48 h. Expression of specific protein markers such as TMPRSS2, ACE2, Alix, TSG101, CDs (29, 47, 63), TLRs (3, 6, 7), TNF-α, and others were altered in infection-derived EVs when compared to control-derived EVs after FCoV infection. Discussion: Our findings suggested that FCoV infection could alter the EV production and composition in host cells, which affects the infection progression and disease evolution. One purpose of studying EVs in various animal coronaviruses that are in close contact with humans is to provide significant information about disease development, transmission, and adaptation. Hence, this study suggests that EVs could provide diagnostic and therapeutic applications in animal CoVs, and such understanding could provide information to prevent future coronavirus outbreaks.

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

Introduction: 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. Methods: 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. Results: 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), (p < 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 (p < 0.001) and exo-RNA (p < 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 (p < 0.001). Discussion: 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.

A SARS-CoV-2: Companion Animal Transmission and Variants Classification.

The continuous emergence of novel viruses and their diseases are a threat to global public health as there have been three outbreaks of coronaviruses that are highly pathogenic to humans in the span of the last two decades, severe acute respiratory syndrome (SARS)-CoV in 2002, Middle East respiratory syndrome (MERS)-CoV in 2012, and novel SARS-CoV-2 which emerged in 2019. The unprecedented spread of SARS-CoV-2 worldwide has given rise to multiple SARS-CoV-2 variants that have either altered transmissibility, infectivity, or immune escaping ability, causing diseases in a broad range of animals including human and non-human hosts such as companion, farm, zoo, or wild animals. In this review, we have discussed the recent SARS-CoV-2 outbreak, potential animal reservoirs, and natural infections in companion and farm animals, with a particular focus on SARS-CoV-2 variants. The expeditious development of COVID-19 vaccines and the advancements in antiviral therapeutics have contained the COVID-19 pandemic to some extent; however, extensive research and surveillance concerning viral epidemiology, animal transmission, variants, or seroprevalence in diverse hosts are essential for the future eradication of COVID-19.

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

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.

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

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. Methods: In the current study, we examined how inhibition of extracellular vesicle release and/or uptake would impact the exosome formation pathway. Using a constellation of improved EV experimental approaches, we evaluated the concentration-based cytotoxicity effects of pharmacological agents (ketoconazole, climbazole, and heparin) on Human Lung Carcinoma (A549) cell viability. We investigated the effect of inhibitor dosages on exosome production and release. Analysis of exosome inhibition includes quantitative analysis and total protein expression of exosome release after pharmacological inhibition; we examined exosome protein level after inhibition. Results: Selective inhibition of exosomes altered particle sizes, and heparin significantly reduced the total exosomes released. Climbazole and heparin undermined membrane-bound tetraspanin CD63 expression and significantly disrupted ALIX protein (p ≤ 0.0001) and TSG101 (p ≤ 0.001). Azoles and heparin also disrupt transmembrane trafficking by modulating Ras binding protein (p ≤ 0.001). Conclusion: These findings revealed that pharmacological inhibition of exosomes regulates the endocytic pathway and expression of endosomal sorting complex required for transport mediators, suggesting climbazole and heparin as effective inhibitors of exosome synthesis.

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

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.

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

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.

Capsid-incorporation of antigens into adenovirus capsid proteins for a vaccine approach.

Some viral vectors are potent inducers of cellular and humoral responses; therefore, viral vectors can be used to vaccinate against cancer or infectious diseases. This report will focus on adenovirus (Ad)-based vectors. Traditional viral-vector vaccination embodies the concept that the vector uses the host-cell machinery to express antigens that are encoded as transgenes within the viral vector. Several preclinical successes have used this approach in animal model systems. However, in some instances, these conventional Ad-based vaccines have yielded suboptimal clinical results. These suboptimal results are ascribed, in part, to preexisting Ad serotype 5 (Ad5) immunity. To address this issue, the "antigen capsid-incorporation" strategy has been developed to circumvent the drawbacks associated with conventional transgene expression of antigens by Ad vectors. This strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. Incorporating immunogenic peptides into the Ad capsid offers potential advantages. Importantly, vaccination by means of the antigen capsid-incorporated approach results in a strong humoral response, similar to the response generated by native Ad capsid proteins. This strategy also allows for the boosting of antigenic specific responses. This strategy may be the way forward for improved vaccine schemes, especially for those infections requiring a strong humoral antigenic response.

Biology and Pathogenesis of SARS-CoV-2: Understandings for Therapeutic Developments against COVID-19.

Coronaviruses are positive sense, single-stranded, enveloped, and non-segmented RNA viruses that belong to the Coronaviridae family within the order Nidovirales and suborder Coronavirinae. Two Alphacoronavirus strains: HCoV-229E and HCoV-NL63 and five Betacoronaviruses: HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2 have so far been recognized as Human Coronaviruses (HCoVs). Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 is currently the greatest concern for humanity. Despite the overflow of research on SARS-CoV-2 and other HCoVs published every week, existing knowledge in this area is insufficient for the complete understanding of the viruses and the diseases caused by them. This review is based on the analysis of 210 published works, and it attempts to cover the basic biology of coronaviruses, including the genetic characteristics, life cycle, and host-pathogen interaction, pathogenesis, the antiviral drugs, and vaccines against HCoVs, especially focusing on SARS-CoV-2. Furthermore, we will briefly discuss the potential link between extracellular vesicles (EVs) and SARS-CoV-2/COVID-19 pathophysiology.

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

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 vitro. In the present study, we evaluated the impact of in vitro 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).

Evaluation of adenovirus capsid labeling versus transgene expression.

Adenoviral vectors have been utilized for a variety of gene therapy applications. Our group has incorporated bioluminescent, fluorographic reporters, and/or suicide genes within the adenovirus genome for analytical and/or therapeutic purposes. These molecules have also been incorporated as capsid components. Recognizing that incorporations at either locale yield potential advantages and disadvantages, our report evaluates the benefits of transgene incorporation versus capsid incorporation. To this end, we have genetically incorporated firefly luciferase within the early region 3 or at minor capsid protein IX and compared vector functionality by means of reporter readout.

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

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.

Effects of Cocaine on Human Glial-Derived Extracellular Vesicles.

BACKGROUND: Microglia are important myeloid cells present in the brain parenchyma that serve a surveillance function in the central nervous system. Microglial cell activation results in neuroinflammation that, when prolonged, can disrupt immune homeostasis and neurogenesis. Activated microglia-derived extracellular vesicles (EVs) may be involved in the propagation of inflammatory responses and modulation of cell-to-cell communication. However, a complete understanding of how EVs are regulated by drugs of abuse, such as cocaine, is still lacking. FINDINGS: Cocaine exposure reduced human microglial cell (HMC3) viability, decreased expression of CD63 and dectin-1 in HMC3-derived EVs, and increased expression of the apoptotic marker histone H2A.x in HMC3-derived EVs. CONCLUSION: Cocaine impacts HMC3 cell viability and specific EV protein expression, which could disrupt cellular signaling and cell-to-cell communication.