Momordica charantia L.-derived exosome-like nanovesicles stabilize p62 expression to ameliorate doxorubicin cardiotoxicity.

BACKGROUND: Doxorubicin (DOX) is a first-line chemotherapeutic drug for various malignancies that causes cardiotoxicity. Plant-derived exosome-like nanovesicles (P-ELNs) are growing as novel therapeutic agents. Here, we investigated the protective effects in DOX cardiotoxicity of ELNs from Momordica charantia L. (MC-ELNs), a medicinal plant with antioxidant activity. RESULTS: We isolated MC-ELNs using ultracentrifugation and characterized them with canonical mammalian extracellular vesicles features. In vivo studies proved that MC-ELNs ameliorated DOX cardiotoxicity with enhanced cardiac function and myocardial structure. In vitro assays revealed that MC-ELNs promoted cell survival, diminished reactive oxygen species, and protected mitochondrial integrity in DOX-treated H9c2 cells. We found that DOX treatment decreased the protein level of p62 through ubiquitin-dependent degradation pathway in H9c2 and NRVM cells. However, MC-ELNs suppressed DOX-induced p62 ubiquitination degradation, and the recovered p62 bound with Keap1 promoting Nrf2 nuclear translocation and the expressions of downstream gene HO-1. Furthermore, both the knockdown of Nrf2 and the inhibition of p62-Keap1 interaction abrogated the cardioprotective effect of MC-ELNs. CONCLUSIONS: Our findings demonstrated the therapeutic beneficials of MC-ELNs via increasing p62 protein stability, shedding light on preventive approaches for DOX cardiotoxicity.

Exosomes as therapeutic and drug delivery vehicle for neurodegenerative diseases.

Neurodegenerative disorders are complex, progressive, and life-threatening. They cause mortality and disability for millions of people worldwide. Appropriate treatment for neurodegenerative diseases (NDs) is still clinically lacking due to the presence of the blood-brain barrier (BBB). Developing an effective transport system that can cross the BBB and enhance the therapeutic effect of neuroprotective agents has been a major challenge for NDs. Exosomes are endogenous nano-sized vesicles that naturally carry biomolecular cargoes. Many studies have indicated that exosome content, particularly microRNAs (miRNAs), possess biological activities by targeting several signaling pathways involved in apoptosis, inflammation, autophagy, and oxidative stress. Exosome content can influence cellular function in healthy or pathological ways. Furthermore, since exosomes reflect the features of the parental cells, their cargoes offer opportunities for early diagnosis and therapeutic intervention of diseases. Exosomes have unique characteristics that make them ideal for delivering drugs directly to the brain. These characteristics include the ability to pass through the BBB, biocompatibility, stability, and innate targeting properties. This review emphasizes the role of exosomes in alleviating NDs and discusses the associated signaling pathways and molecular mechanisms. Furthermore, the unique biological features of exosomes, making them a promising natural transporter for delivering various medications to the brain to combat several NDs, are also discussed.

Oxaliplatin-induced upregulation of exosomal miR-424-3p derived from human bone marrow mesenchymal stem cells attenuates progression of gastric cancer cells.

Chemotherapy, particularly with oxaliplatin, is a key treatment for advanced gastric cancer (GC), and exosomes derived from human bone marrow mesenchymal stem cells (hBM-MSCs) play a vital role in the tumor microenvironment. The study aims to elucidate the previously unexplored role of exosomes derived from hBM-MSCs in GC tumorigenesis, especially under the influence of chemotherapy. We conducted an experimental study, utilizing miRNA sequencing and biological experiments, to analyze the tumorigenicity of exosomal miR-424-3p secreted by hBM-MSCs and its target gene RHOXF2 in GC cell lines. The results were confirmed through experimentation using a xenograft mouse model. This study demonstrated the role of hBM-MSCs in the GC microenvironment, focusing on their epithelial-mesenchymal transition (EMT) facilitation through exosomes, which led to enhanced tumorigenicity in GC cells. Intriguingly, this pro-tumor effect was abrogated when hBM-MSCs were treated with oxaliplatin. Exosomal miRNA sequencing revealed that oxaliplatin can upregulate the levels of miR-424-3p in exosomes secreted by hBM-MSCs, thereby inhibiting the EMT process in GC cells. Furthermore, miR-424-3p was identified to target and downregulate RHOXF2 expression, impeding the malignant behavior of GC cells both in vitro and in the mouse model. These findings uncover a potential hidden mechanism of oxaliplatin's anti-tumor action and propose the delivery of miR-424-3p via exosomes as a promising avenue for anti-tumor therapy.

Exosomal miRNAs in prenatal diagnosis: Recent advances.

Exosomes, small membranous microvesicles released by cells, contain a range of bioactive molecules, including proteins and miRNAs, which play critical roles in intercellular communication and physiological and pathological processes. Current research suggests that exosomal miRNAs could serve as valuable biomarkers for prenatal diseases, offering a noninvasive method for early detection and monitoring. Studies linking exosomal miRNAs to various birth defects, including fetal growth restriction, urinary tract malformations, cardiovascular system malformations, and hereditary diseases like Down syndrome, were discussed. However, there are some conflicting study findings due to different exosome separation methods. Here, we also discussed exosome separation methods, emphasizing the importance of method selection based on specific purposes and sample types. Further studies are needed to standardize isolation techniques, understand the specific mechanisms underlying exosomal miRNA function, and develop reliable noninvasive prenatal diagnostic indicators. Overall, exosomal miRNAs show promise as potential biomarkers for prenatal diagnosis, but further research is necessary to validate their clinical utility.

Exosomes derived from human urine-derived stem cells ameliorate IL-1ß-induced intervertebral disk degeneration.

BACKGROUND: Human intervertebral disk degeneration (IVDD) is a sophisticated degenerative pathological process. A key cause of IVDD progression is nucleus pulposus cell (NPC) degeneration, which contributes to excessive endoplasmic reticulum stress in the intervertebral disk. However, the mechanisms underlying IVDD and NPC degeneration remain unclear. METHODS: We used interleukin (IL)-1ß stimulation to establish an NPC-degenerated IVDD model and investigated whether human urine-derived stem cell (USC) exosomes could prevent IL-1ß-induced NPC degeneration using western blotting, quantitative real-time polymerase chain reaction, flow cytometry, and transcriptome sequencing techniques. RESULTS: We successfully extracted and identified USCs and exosomes from human urine. IL-1ß substantially downregulated NPC viability and induced NPC degeneration while modulating the expression of SOX-9, collagen II, and aggrecan. Exosomes from USCs could rescue IL-1ß-induced NPC degeneration and restore the expression levels of SOX-9, collagen II, and aggrecan. CONCLUSIONS: USC-derived exosomes can prevent NPCs from degeneration following IL-1ß stimulation. This finding can aid the development of a potential treatment strategy for IVDD.

Exosome Therapy: A Novel Approach for Enhancing Estrogen Levels in Perimenopause.

Perimenopause significantly impacts women's health globally, often managed with hormone replacement therapy (HRT) despite the associated risks. This study explores a novel alternative exosome therapy, aimed at stimulating estrogen production in ovarian tissues, thus offering a potential non-hormonal treatment for perimenopausal symptoms. Employing ex vivo methodologies, ovarian cortex specimens from perimenopausal women were treated with exosomes derived from human umbilical cord mesenchymal stem cells and cultured under specific conditions (patent number: PCT/US2022/073467). The exosomes were produced under cyclic guanosine monophosphate (cGMP) conditions, ensuring high safety standards. Estrogen levels were quantified using enzyme-linked immunosorbent assay (ELISA), and gene expression changes in estrogen and follicle-stimulating hormone (FSH) receptors were assessed via quantitative polymerase chain reaction (PCR). Immunohistochemistry (IHC) was utilized to evaluate cellular proliferation and apoptotic markers. The results indicated a significant increase in estrogen levels and estrogen receptor-alpha (Erα) expression in treated tissues compared to controls. Additionally, a decrease in apoptotic markers and an increase in cellular proliferation markers were observed. These findings suggest that exosome therapy can effectively enhance estrogen production and modulate receptor sensitivity in perimenopausal ovarian tissues. This approach could serve as a safer alternative to HRT, aligning with the body's natural regulatory mechanisms and potentially offering a more effective treatment option for managing perimenopausal symptoms.

Mesenchymal Stem Cell-Secreted Exosomes and Soluble Signals Regulate Breast Cancer Metastatic Dormancy: Current Progress and Future Outlook.

Breast cancer is most common in women, and in most cases there is no evidence of spread and the primary tumor is removed, resulting in a 'cure'. However, in 10% to 30% of these women, distant metastases recur after years to decades. This is due to breast cancer cells disseminating to distant organs and lying quiescent. This is called metastatic dormancy. Dormant cells are generally resistant to chemotherapy, hormone therapy and immunotherapy as they are non-cycling and receive survival signals from their microenvironment. In this state, they are clinically irrelevant. However, risk factors, including aging and inflammation can awaken dormant cells and cause breast cancer recurrences, which may happen even more than ten years after the primary tumor removal. How these breast cancer cells remain in dormancy is being unraveled. A key element appears to be the mesenchymal stem cells in the bone marrow that have been shown to promote breast cancer metastatic dormancy in recent studies. Indirect co-culture, direct co-culture and exosome extraction were conducted to investigate the modes of signal operation. Multiple signaling molecules act in this process including both protein factors and microRNAs. We integrate these studies to summarize current findings and gaps in the field and suggest future research directions for this field.

miR-493-5p Silenced by DNA Methylation Promotes Angiogenesis via Exosomes and VEGF-A-Mediated Intracellular Cross-Talk Between ESCC Cells and HUVECs.

Background: Exosomal microRNAs (miRNAs) in the tumor microenvironment play crucial roles in tumorigenesis and tumor progression by participating in intercellular cross-talk. However, the functions of exosomal miRNAs and the mechanisms by which they regulate esophageal squamous cell carcinoma (ESCC) progression are unclear. Methods: RNA sequencing and GEO analysis were conducted to identify candidate exosomal miRNAs involved in ESCC development. Receiver operating characteristic curve analysis was performed to assess the diagnostic value of plasma exosomal miR-493-5p. EdU, tube formation and Transwell assays were used to investigate the effects of exosomal miR-493-5p on human umbilical vein endothelial cells (HUVECs). A subcutaneous xenograft model was used to evaluate the antitumor effects of miR-493-5p and decitabine (a DNA methyltransferase inhibitor). The relationship between miR-493-5p and SP1/SP3 was revealed via a dual-luciferase reporter assay. A series of rescue assays were subsequently performed to investigate whether SP1/SP3 participate in exosomal miR-493-5p-mediated ESCC angiogenesis. Results: We found that miR-493-5p expression was notably reduced in the plasma exosomes of ESCC patients, which showed the high potential value in early ESCC diagnosis. Additionally, miR-493-5p, as a candidate tumor suppressor, inhibited the proliferation, migration and tube formation of HUVECs by suppressing the expression of VEGFA and exerted its angiostatic effect via exosomes. Moreover, we found that SP1/SP3 are direct targets of miR-493-5p and that re-expression of SP1/SP3 could reverse the inhibitory effects of miR-493-5p. Further investigation revealed that miR-493-5p expression could be regulated by DNA methyltransferase 3A (DNMT3A) and DNMT3B, and either miR-493-5p overexpression or restoration of miR-493-5p expression with decitabine increased the antitumor effects of bevacizumab. Conclusion: Exosomal miR-493-5p is a highly valuable ESCC diagnosis marker and inhibits ESCC-associated angiogenesis. miR-493-5p can be silenced via DNA methylation, and restoration of miR-493-5p expression with decitabine increases the antitumor effects of bevacizumab, suggesting its potential as a therapeutic target for ESCC treatment.

Engineering Exosomes for Therapeutic Applications: Decoding Biogenesis, Content Modification, and Cargo Loading Strategies.

Exosomes emerge from endosomal invagination and range in size from 30 to 200 nm. Exosomes contain diverse proteins, lipids, and nucleic acids, which can indicate the state of various physiological and pathological processes. Studies have revealed the remarkable clinical potential of exosomes in diagnosing and prognosing multiple diseases, including cancer, cardiovascular disorders, and neurodegenerative conditions. Exosomes also have the potential to be engineered and deliver their cargo to a specific target. However, further advancements are imperative to optimize exosomes' diagnostic and therapeutic capabilities for practical implementation in clinical settings. This review highlights exosomes' diagnostic and therapeutic applications, emphasizing their engineering through simple incubation, biological, and click chemistry techniques. Additionally, the loading of therapeutic agents onto exosomes, utilizing passive and active strategies, and exploring hybrid and artificial exosomes are discussed.

Immunomodulatory effect of PLGA-encapsulated mesenchymal stem cells-derived exosomes for the treatment of allergic rhinitis.

Introduction: Allergic rhinitis (AR) is an upper airway inflammatory disease of the nasal mucosa. Conventional treatments such as symptomatic pharmacotherapy and allergen-specific immunotherapy have considerable limitations and drawbacks. As an emerging therapy with regenerative potential and immunomodulatory effect, mesenchymal stem cell-derived exosomes (MSC-Exos) have recently been trialed for the treatment of various inflammatory and autoimmune diseases. Methods: In order to achieve sustained and protected release of MSC-Exos for intranasal administration, we fabricated Poly(lactic-co-glycolic acid) (PLGA) micro and nanoparticles-encapsulated MSC-Exos (PLGA-Exos) using mechanical double emulsion for local treatment of AR. Preclinical in vivo imaging, ELISA, qPCR, flow cytometry, immunohistochemical staining, and multiomics sequencing were used for phenotypic and mechanistic evaluation of the therapeutic effect of PLGA-Exos in vitro and in vivo. Results: The results showed that our PLGA platform could efficiently encapsulate and release the exosomes in a sustained manner. At protein level, PLGA-Exos treatment upregulated IL-2, IL-10 and IFN-γ, and downregulated IL-4, IL-17 and antigen-specific IgE in ovalbumin (OVA)-induced AR mice. At cellular level, exosomes treatment reduced Th2 cells, increased Tregs, and reestablished Th1/Th2 balance. At tissue level, PLGA-Exos significantly attenuated the infiltration of immune cells (e.g., eosinophils and goblet cells) in nasal mucosa. Finally, multiomics analysis discovered several signaling cascades, e.g., peroxisome proliferator-activated receptor (PPAR) pathway and glycolysis pathway, that might mechanistically support the immunomodulatory effect of PLGA-Exos. Discussion: For the first time, we present a biomaterial-facilitated local delivery system for stem cell-derived exosomes as a novel and promising strategy for AR treatment.

Exosome-derived microRNAs: emerging players in vitiligo.

Exosome-derived microRNAs (miRNAs) are biomacromolecules and nanoscale extracellular vesicles originating from intracellular compartments that are secreted by most cells into the extracellular space. This review examines the formation and function of exosomal miRNAs in biological information transfer, explores the pathogenesis of vitiligo, and highlights the relationship between exosomal miRNAs and vitiligo. The aim is to deepen the understanding of how exosomal miRNAs influence immune imbalance, oxidative stress damage, melanocyte-keratinocyte interactions, and melanogenesis disorders in the development of vitiligo. This enhanced understanding may contribute to the development of potential diagnostic and therapeutic options for vitiligo.

The therapeutic potential of exosomes in immunotherapy.

Exosomes are found in various tissues of the body and carry abundant contents including nucleic acids, proteins, and metabolites, which continuously flow between cells of various tissues and mediate important intercellular communication. In addition, exosomes from different cellular sources possess different physiopathological immunomodulatory effects, which are closely related to the immune regeneration of normal or abnormal organs and tissues. Here, we focus on the mechanistic interactions between exosomes and the human immune system, introduce the immuno-regenerative therapeutic potential of exosomes in common clinical immune-related diseases, such as infectious diseases, autoimmune diseases, and tumors, and reveal the safety and efficacy of exosomes as a novel cell-free immune regenerative therapy.

Mesenchymal stem cell-derived exosomes ameliorate diabetic kidney disease through NOD2 signaling pathway.

BACKGROUND AND AIMS: Diabetic kidney disease (DKD) is one of the most common complications of diabetes. It is reported that mesenchymal stem cells (MSCs) derived exosomes (MSCs-Exo) may have great clinical application potential for the treatment of DKD, but the underlying mechanism has not been illustrated. To clarify the effect of MSC-Exo on NOD2 signaling pathway in podocytes under high glucose (HG) and DKD, we conduct this study. METHODS: We co-cultured podocytes and MSCs-Exo under 30 mM HG and injected MSCs-Exo into DKD mice, then we detected the NOD2 signaling pathway by western blot, qRT-PCT, immunofluorescence, transmission electron microscopy and immunohistochemistry both in vitro and in vivo. RESULTS: In vitro, HG lead to the apoptosis, increased the ROS level and activated the NOD2 signaling pathway in podocytes, while MSCs-Exo protected podocytes from injury reduced the expression of inflammatory factors including TNF-α, IL-6, IL-1ß, and IL-18 and alleviated the inflammatory response, inhibited the activation of NOD2 signaling pathway and the expression of it's downstream protein p-P65, p-RIP2, prevented apoptosis, increased cell viability in podocytes caused by HG. In vivo, MSCs-Exo alleviated renal injury in DKD mice, protected renal function, decreased urinary albumin excretion and inhibited the activation of NOD2 signaling pathway as well as the inflammation in renal tissue. CONCLUSION: MSCs-Exo protected the podocytes and DKD mice from inflammation by mediating NOD2 pathway, MSCs-Exo may provide a new target for the treatment of DKD.

Ultrasound-assisted encapsulating folic acid-based carbon quantum dots within breast cancer cell-derived exosomes as a co-receptors-mediated anticancer nanocarrier for enhanced breast cancer therapy.

The nonspecific nature of cancer drug delivery often results in substantial toxic side effects during treatments for breast cancer. To mitigate these negative outcomes, our approach involves loading methotrexate (MTX) within carbon quantum dots (CQDs) synthesized from folic acid, which are then enveloped in exosomal membranes obtained from breast cancer cells (Ex@MTX-CQDs). Analysis utilizing nanoparticle tracking techniques has demonstrated that these Ex@MTX-CQDs maintain the physical and biochemical properties of their exosomal precursors. The release profile of MTX indicated a restricted release percentage (less than 10%) under normal physiological conditions, which is contrasted by a more consistent release rate (approximately 65%) when emulating the conditions found within tumor tissues. The toxicological assessments have confirmed that the presence of exosomes combined with leftover folic acid significantly improves the delivery efficacy of MTX directly to the cancerous cells through the binding to folate and heparan sulfate proteoglycan receptors. This process results in increased disruption of the mitochondrial membrane potential and subsequently triggers apoptosis, ultimately leading to the destruction of cancerous cells. Our research could potentially contribute to the further innovation and application of nanocarriers derived from biological sources for the targeted treatment of breast cancer.

Nebulized milk exosomes loaded with siTGF-ß1 ameliorate pulmonary fibrosis by inhibiting EMT pathway and enhancing collagen permeability.

Pulmonary Fibrosis (PF) is a fatal disease in the interstitial lung associated with high mortality, morbidity, and poor prognosis. Transforming growth factor-ß1 (TGF-ß1) is a fibroblast-activating protein that promotes fibrous diseases. Herein, an inhalable system was first developed using milk exosomes (M-Exos) encapsulating siRNA against TGF-ß1 (MsiTGF-ß1), and their therapeutic potential for bleomycin (BLM)-induced PF was investigated. M-siTGF-ß1 was introduced into the lungs of mice with PF through nebulization. The collagen penetration effect and lysosomal escape ability were verified in vitro. Inhaled MsiTGF-ß1 notably alleviated inflammatory infiltration, attenuated extracellular matrix (ECM) deposition, and increased the survival rate of PF mice by 4.7-fold. M-siTGF-ß1 protected lung tissue from BLM toxicity by efficiently delivering specific siRNA to the lungs, leading to TGF-ß1 mRNA silencing and epithelial mesenchymal transition pathway inhibition. Therefore, M-siTGF-ß1 offers a promising avenue for therapeutic intervention in fibrosis-related disorders.

Developing an advanced diagnostic model for hepatocellular carcinoma through multi-omics integration leveraging diverse cell-death patterns.

Introduction: Hepatocellular carcinoma (HCC), representing more than 80% of primary liver cancer cases, lacks satisfactory etiology and diagnostic methods. This study aimed to elucidate the role of programmed cell death-associated genes (CDRGs) in HCC by constructing a diagnostic model using single-cell RNA sequencing (scRNA-seq) and RNA sequencing (RNA-seq) data. Methods: Six categories of CDRGs, including apoptosis, necroptosis, autophagy, pyroptosis, ferroptosis, and cuproptosis, were collected. RNA-seq data from blood-derived exosomes were sourced from the exoRBase database, RNA-seq data from cancer tissues from the TCGA database, and scRNA-seq data from the GEO database. Subsequently, we intersected the differentially expressed genes (DEGs) of the HCC cohort from exoRBase and TCGA databases with CDRGs, as well as DEGs obtained from single-cell datasets. Candidate biomarker genes were then screened using clinical indicators and a machine learning approach, resulting in the construction of a seven-gene diagnostic model for HCC. Additionally, scRNA-seq and spatial transcriptome sequencing (stRNA-seq) data of HCC from the Mendeley data portal were used to investigate the underlying mechanisms of these seven key genes and their association with immune checkpoint blockade (ICB) therapy. Finally, we validated the expression of key molecules in tissues and blood-derived exosomes through quantitative Polymerase Chain Reaction (qPCR) and immunohistochemistry experiments. Results: Collectively, we obtained a total of 50 samples and 104,288 single cells. Following the meticulous screening, we established a seven-gene diagnostic model for HCC, demonstrating high diagnostic efficacy in both the exoRBase HCC cohort (training set: AUC = 1; testing set: AUC = 0.847) and TCGA HCC cohort (training set: AUC = 1; testing set: AUC = 0.976). Subsequent analysis revealed that HCC cluster 3 exhibited a higher stemness index and could serve as the starting point for the differentiation trajectory of HCC cells, also displaying more abundant interactions with other cell types in the microenvironment. Notably, key genes TRIB3 and NQO1 displayed elevated expression levels in HCC cells. Experimental validation further confirmed their elevated expression in both tumor tissues and blood-derived exosomes of cancer patients. Additionally, stRNA analysis not only substantiated these findings but also suggested that patients with high TRIB3 and NQO1 expression might respond more favorably to ICB therapy. Conclusions: The seven-gene diagnostic model demonstrated remarkable accuracy in HCC screening, with TRIB3 emerging as a promising diagnostic tool and therapeutic target for HCC.

[Regulatory effect of Diwu Yanggan Decoction on lysoglycerophospholipids in circulating exosomes in a mouse model of nonalcoholic fatty liver disease].

OBJECTIVE: To evaluate the regulatory effect of Diwu Yanggan (DWYG) Decoction on lysoglycerophospholipids (Lyso-GPLs) in circulating exosomes in a mouse model of nonalcoholic fatty liver disease (NAFLD). METHODS: Circulating exosomes isolated from mouse serum by size exclusion chromatography were morphologically characterized using transmission electron microscope and examined for surface markers CD9, CD63 and TSG101 using Western blotting. Twenty-four male Kunming mice were randomized into 3 groups for normal feeding (control, n=8) or high-fat diet feeding for 1 week to induce NAFLD, after which the latter mice were given DWYG decoction (treatment group, n=8) or normal saline (model group, n=8) by gavage for 4 weeks. After the last treatment, blood samples were collected from the mice for testing serum TC, HDL-C, LDL-C, ALT and AST levels and isolating circulating exosomes. Using multivariate statistical analysis based on targeted metabolomics strategy, the potential biomarkers for Lyso-GPLs in the exosomes were screened. RESULTS: The isolated exosomes about 100 nm in size had a typical saucer-like structure with distinct double-layer membranes and a mean particle size of 137.5 nm and expressed the specific surface marker proteins CD9, CD63 and TSG101. The mouse models of NAFLD had significantly increased serum levels of TC, HDL-C, LDL-C and AST and lowered serum ALT level. A total of 43 Lyso-GPLs with significant reduction after DWYG Decoction treatment were identified in NAFLD mice. CONCLUSION: DWYG Decoction can regulate Lyso-GPLs in circulating exosomes in NAFLD mice, which provides a new clue for studying the therapeutic mechanism of DWYG Decoction for liver disease.

Extracellular vesicle surface display of αPD-L1 and αCD3 antibodies via engineered late domain-based scaffold to activate T-cell anti-tumor immunity.

Extracellular vesicles (EVs) are emerging as promising carriers for the delivery of therapeutic biologics. Genetic engineering represents a robust strategy for loading proteins of interest into EVs. Identification of EV-enriched proteins facilitates protein cargo loading efficiency. Many EV-enriched proteins are sorted into EVs via an endosomal sorting complex required for transport (ESCRT)-dependent pathway. In parallel, viruses hijack this EV biosynthesis machinery via conserved late domain motifs to promote egress from host cells. Inspired by the similarity of biogenesis between EVs and viruses, we developed a synthetic, Late domain-based EV scaffold protein that enables the display of a set of single chain variable fragments (scFvs) on the EV surface. We named this scaffold the Late domain-based exosomal antibody surface display platform (LEAP). We applied the LEAP scaffold to reprogramme HEK293T cell-derived EVs to elicit T-cell anti-tumor immunity by simultaneously displaying αPD-L1 and αCD3 scFvs on the EV surface (denoted as αPD-L1×αCD3 bispecific T-cell engaging exosomes, BiTExos). We demonstrated that αPD-L1×αCD3 BiTExos actively redirected T cells to bind to PD-L1+ tumor cells, promoting T-cell activation, proliferation and tumoricidal cytokine production. Furthermore, the αPD-L1×αCD3 BiTExos promoted T-cell infiltration into the tumor microenvironment to mitigate the tumor burden in vivo. Our study suggested that the LEAP scaffold may serve as a platform for EV surface display and could be applied for a broad range of EV-based biomedical applications.

HSV-1 and Cellular miRNAs in CSF-Derived Exosomes as Diagnostically Relevant Biomarkers for Neuroinflammation.

Virus-associated chronic inflammation may contribute to autoimmunity in a number of diseases. In the brain, autoimmune encephalitis appears related to fluctuating reactivation states of neurotropic viruses. In addition, viral miRNAs and proteins can be transmitted via exosomes, which constitute novel but highly relevant mediators of cellular communication. The current study questioned the role of HSV-1-encoded and host-derived miRNAs in cerebrospinal fluid (CSF)-derived exosomes, enriched from stress-induced neuroinflammatory diseases, mainly subarachnoid hemorrhage (SAH), psychiatric disorders (AF and SZ), and various other neuroinflammatory diseases. The results were compared with CSF exosomes from control donors devoid of any neuroinflammatory pathology. Serology proved positive, but variable immunity against herpesviruses in the majority of patients, except controls. Selective ultrastructural examinations identified distinct, herpesvirus-like particles in CSF-derived lymphocytes and monocytes. The likely release of extracellular vesicles and exosomes was most frequently observed from CSF monocytes. The exosomes released were structurally similar to highly purified stem-cell-derived exosomes. Exosomal RNA was quantified for HSV-1-derived miR-H2-3p, miR-H3-3p, miR-H4-3p, miR-H4-5p, miR-H6-3p, miR-H27 and host-derived miR-21-5p, miR-146a-5p, miR-155-5p, and miR-138-5p and correlated with the oxidative stress chemokine IL-8 and the axonal damage marker neurofilament light chain (NfL). Replication-associated miR-H27 correlated with neuronal damage marker NfL, and cell-derived miR-155-5p correlated with oxidative stress marker IL-8. Elevated miR-138-5p targeting HSV-1 latency-associated ICP0 inversely correlated with lower HSV-1 antibodies in CSF. In summary, miR-H27 and miR-155-5p may constitute neuroinflammatory markers for delineating frequent and fluctuating HSV-1 replication and NfL-related axonal damage in addition to the oxidative stress cytokine IL-8 in the brain. Tentatively, HSV-1 remains a relevant pathogen conditioning autoimmune processes and a psychiatric clinical phenotype.

The Role of Intra-Articular Delivery of BM-MSCs-Derived Exosomes in Improving Osteoarthritis: Implication of circYAP1/miRNA-21/TLR7 Axis.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BM-MSCs) have recently attracted great attention due to their crucial anti-inflammatory and regenerative properties. This work aims to examine the curative impact of intra-articular injection of BM-MSCs-derived exosomes in ameliorating osteoarthritis (OA) progression in rats and to explore the interaction between circular RNA of Yes-associated protein 1 (circYAP1) and microRNA-21 (miRNA-21) in the rat knee joints. METHODOLOGY: Gene expression circYAP1, miRNA-21, toll-like receptor-7 (TLR7), aggrecan, and collagen type II were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in the rat articular tissues. In addition, the Enzyme-linked immunosorbent assay (ELISA) technique was used to estimate the level of the inflammatory markers interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 1ß (IL-1ß), and tumor necrosis factor-alpha (TNF-α); and the oxidative markers glutathione (GSH), malondialdehyde (MDA) and total reactive oxygen species (ROS). Histopathological examination using Hematoxylin and Eosin (H&E) staining of the rat articular tissue was also performed along with an estimation of the articular cartilage thickness. RESULTS: Our results showed that BM-MSCs-derived exosomes significantly elevated circYAP1 gene expression level (p < 0.05) along with subsequent downregulation of miRNA-21 and TLR7 (p < 0.05). These effects impacted the inflammatory milieu of rat articular surfaces, where there was a significant reduction (p < 0.05) of the pro-inflammatory and oxidative markers with significantly increased production of the anti-inflammatory and antioxidative markers (p < 0.05). Marked elevation in aggrecan and collagen type II gene expression was also found in the treated groups (p < 0.05). CONCLUSION: Those data suggest that BM-MSCs-derived exosomes have a crucial role in mitigating OA symptoms and pathology progression and might be regarded as an effective as well as acceptable treatment option for OA.