Therapeutic application of extracellular vesicular EGFR isoform D as a co-drug to target squamous cell cancers with tyrosine kinase inhibitors.

Targeting wild-type epidermal growth factor receptor (EGFR) using tyrosine kinase inhibitors (TKIs) never achieved its purported success in cancers such as head and neck squamous cell carcinoma, which are largely EGFR-dependent. We had previously shown that exceptional responders to TKIs have a genetic aberration that results in overexpression of an EGFR splice variant, isoform D (IsoD). IsoD lacks an integral transmembrane and kinase domain and is secreted in extracellular vesicles (EVs) in TKI-sensitive patient-derived cultures. Remarkably, the exquisite sensitivity to TKIs could be transferred to TKI-resistant tumor cells, and IsoD protein in the EV is necessary and sufficient to transfer the phenotype in vitro and in vivo across multiple models and drugs. This drug response requires an intact endocytic mechanism, binding to full-length EGFR, and signaling through Src-phosphorylation within the endosomal compartment. We propose a therapeutic strategy using EVs containing EGFR IsoD as a co-drug to expand the use of TKI therapy to EGFR-driven cancers.

Exosomal AFAP1-AS1 Promotes the Growth, Metastasis, and Glycolysis of Pituitary Adenoma by Inhibiting HuR Degradation.

Exosomal long noncoding RNAs (lncRNAs), which are highly expressed in tumor-derived exosomes, regulate various cellular behaviors such as cell proliferation, metastasis, and glycolysis by facilitating intercellular communication. Here, we explored the role and regulatory mechanism of tumor-derived exosomal lncRNAs in pituitary adenomas (PA). We isolated exosomes from PA cells, and performed in vitro and in vivo assays to examine their effect on the proliferation, metastasis, and glycolysis of PA cells. In addition, we conducted RNA pull-down, RNA immunoprecipitation, co-immunoprecipitation, and ubiquitination assays to investigate the downstream mechanism of exosomal AFAP1-AS1. Exosomes from PA cells augmented the proliferation, mobility, and glycolysis of PA cells. Moreover, AFAP1-AS1 was significantly enriched in these exosomes and stimulated the growth, migration, invasion, and glycolysis of PA cells in vitro, as well as tumor metastasis in vivo. It also enhanced the binding affinity between Hu antigen R (HuR) and SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1), resulting in HuR ubiquitination and degradation accompanied by enhanced expression of hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2). Moreover, HuR overexpression alleviated the exosomal AFAP1-AS1-mediated promotion of growth, metastasis, and glycolysis effects. These findings indicate that tumor-derived exosomal AFAP1-AS1 modulated SMURF1-mediated HuR ubiquitination and degradation to upregulate HK2 and PKM2 expression, thereby enhancing PA cell growth, metastasis, and glucose metabolism. This suggests targeting exosomal AFAP1-AS1 may be a potential strategy for the treatment of PA.

Regulation of lipid metabolism in grass carp primary hepatocytes by exosomes derived from fatty hepatocytes though GRP78.

Exosomes regulate lipid metabolism by carrying miRNAs, nucleic acids, and proteins, thereby influencing the function of receptor cells. Glucose-regulated protein 78 (GRP78) is also involved in the regulation of lipid metabolism. However, it remains unclear whether exosomes derived from fatty hepatocytes (OA-Exo) regulate lipid metabolism through the enrichment of GRP78. In this study, we observed the expression of GRP78 was significantly increased in fatty hepatocytes (incubating hepatocytes with oleic acid (OA) for 24 h) and OA-Exo (P < 0.05). In addition, OA-Exo (50 µg/mL) and GRP78 protein (1 µg/mL) significant increased the content of triacylglycerol (TG) and total cholesterol (TC), as well as up-regulated the expression of GRP78 and inositol-requiring enzyme-1alpha (IRE1α) protein (P < 0.05). We further used YUM70 (an inhibitor of GRP78) to inhibit endogenous GRP78, and compared with the YUM70 group, OA-Exo reversed the effect of YUM70 and increased the content of TG, TC, and the expression of GRP78 protein in hepatocytes (P < 0.05). Furthermore, the inhibition of the IRE1α pathway with 4µ8C resulted in a significant decrease in TG content compared to the control group (P < 0.05). However, when compared with the 4µ8C group, OA-Exo and GRP78 reversed the effect of 4µ8C and significantly increased TG content (P < 0.05). Taken together, these results indicated that OA-Exo activated IRE1α to promote lipid accumulation in hepatocytes through the enrichment of GRP78. This study provided a new perspective for further exploration of exosomal lipid metabolism in fish.

Additional comments on extracellular vesicles derived from mesenchymal stem cells mediate extracellular matrix remodeling in osteoarthritis.

Recently, we read an article published by the Yang et al. The results of this study indicated that engineered exosomes loaded with microRNA-29a (miR-29a) alleviate knee inflammation and maintain extracellular matrix stability in Sprague Dawley rats. The study's results provide useful information for treating knee osteoarthritis (KOA). This letter, shares our perspectives on treating KOA using engineered exosomes for miR-29a.

Comparative genomics between Trichomonas tenax and Trichomonas vaginalis: CAZymes and candidate virulence factors.

Introduction: The oral trichomonad Trichomonas tenax is increasingly appreciated as a likely contributor to periodontitis, a chronic inflammatory disease induced by dysbiotic microbiota, in humans and domestic animals and is strongly associated with its worst prognosis. Our current understanding of the molecular basis of T. tenax interactions with host cells and the microbiota of the oral cavity are still rather limited. One laboratory strain of T. tenax (Hs-4:NIH/ATCC 30207) can be grown axenically and two draft genome assemblies have been published for that strain, although the structural and functional annotation of these genomes is not available. Methods: GenSAS and Galaxy were used to annotate two publicly available draft genomes for T. tenax, with a focus on protein-coding genes. A custom pipeline was used to annotate the CAZymes for T. tenax and the human sexually transmitted parasite Trichomonas vaginalis, the most well-characterized trichomonad. A combination of bioinformatics analyses was used to screen for homologs of T. vaginalis virulence and colonization factors within the T. tenax annotated proteins. Results: Our annotation of the two T. tenax draft genome sequences and their comparison with T. vaginalis proteins provide evidence for several candidate virulence factors. These include candidate surface proteins, secreted proteins and enzymes mediating potential interactions with host cells and/or members of the oral microbiota. The CAZymes annotation identified a broad range of glycoside hydrolase (GH) families, with the majority of these being shared between the two Trichomonas species. Discussion: The presence of candidate T. tenax virulence genes supports the hypothesis that this species is associated with periodontitis through direct and indirect mechanisms. Notably, several GH proteins could represent potential new virulence factors for both Trichomonas species. These data support a model where T. tenax interactions with host cells and members of the oral microbiota could synergistically contribute to the damaging inflammation characteristic of periodontitis, supporting a causal link between T. tenax and periodontitis.

α-Synuclein in speculative neuronal extracellular vesicles: A marker for Parkinson's disease risk?

This opinion piece describes major limitations of using α-synuclein in speculative neuronally enriched for diagnosing or predicting Parkinson's disease risk from prodromal conditions such as REM behaviour disorder. It concludes that such an approach is unreliable and recommends that future researchers divert away to more widely accepted approaches such as seed amplification assays.

Intracellularly Synthesized Artificial Exosome Treats Acute Lung Injury.

Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), induce high morbidity and mortality rates, which challenge the present approaches for the treatment of ALI/ARDS. The clinically used photosensitizer verteporfin (VER) exhibits great potential in the treatment of acute lung injury and acute respiratory distress syndrome (ALI/ARDS) by regulating macrophage polarization and reducing inflammation. Nevertheless, its hydrophobic characteristics, nonspecificity, and constrained bioavailability hinder its therapeutic efficacy. In this work, we developed a type of VER-cored artificial exosome (EVM), which was produced by using mesoporous silica nanoparticles (MSNs) to load VER, followed by the exocytosis of internalized VER-MSNs from mouse bone marrow-derived mesenchymal stem cells (mBMSCs) without further modification. Both in vitro and in vivo assessments confirmed the powerful anti-inflammation induced by EVM. EVM also showed significant higher accumulation to inflammatory lungs compared with healthy ones, which was beneficial to the treatment of ALI/ARDS. EVM improved pulmonary function, attenuated lung injury, and reduced mortality in ALI mice with high levels of biocompatibility, exhibiting a 5-fold higher survival rate than the control. This type of artificial exosome emitted near-infrared light in the presence of laser activation, which endowed EVM with trackable ability both in vitro and in vivo. Our work developed a type of clinically used photosensitizer-loaded artificial exosome with membrane integrity and traceability. To the best of our knowledge, this kind of intracellularly synthesized artificial exosome was developed and showed great potential in ALI/ARDS therapy.

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.

Osteoclast-derived exosomes influence osteoblast differentiation in osteoporosis progression via the lncRNA AW011738/ miR-24-2-5p/ TREM1 axis.

AIMS: To investigate the molecular mechanism of osteoclast-derived exosomes in osteoporosis. MAIN METHODS: RANKL induced osteoclast model was screened for significantly differentially expressed lncRNAs and mRNAs by whole RNA sequencing. Exosomes were characterized using electron microscopy, western blotting and nanosight. Overexpression or knockdown of AW011738 was performed to explore its function. The degree of osteoporosis in an osteoporosis model was assessed by mirco-CT. The osteoclast model, osteoblast differentiation ability and the molecular mechanism of lncRNA AW011738/miR-24-2-5p/TREM1 axis in osteoporosis were assessed by dual luciferase reporter gene assay, Western blotting (WB), immunofluorescence and ALP staining. Bioinformatics was used to predict interactions of key osteoporosis-related genes with miRNAs, transcription factors, and potential drugs after upregulation of AW011738. To predict the protein-protein interaction (PPI) network associated with key genes, GO and KEGG analyses were performed on the key genes. The ssGSVA was used to predict changes in the immune microenvironment. KEY FINDINGS: Osteoclast-derived exosomes containing lncRNA AW011738 decreased the osteogenesis-related markers and accelerated bone loss in OVX mice. Osteoclast (si-AW011738)-derived exosomes showed a significant increase in biomarkers of osteoblast differentiation in vitro compared to the si-NC group. As analyzed by mirco-CT, tail vein injected si-AW011738 OVX mice were less osteoporotic than the control group. AW011738 inhibited osteoblast differentiation by regulating TREM1 expression through microRNA. Meanwhile, overexpression of miR-24-2-5p inhibited TREM1 expression to promote osteoblast differentiation. SIGNIFICANCE: Osteoclast-derived exosomes containing lncRNA AW011738 inhibit osteogenesis in MC3T3-E1 cells through the lncRNA AW011738/miR-24-2-5p/TREM1 axis and exacerbate osteoporosis in OVX mice.

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.

Modulation of naïve mesenchymal stromal cells by extracellular vesicles derived from insulin-producing cells: an in vitro study.

This study was to determine whether extracellular vesicles (EVs) derived from insulin-producing cells (IPCs) can modulate naïve mesenchymal stromal cells (MSCs) to become insulin-secreting. MSCs were isolated from human adipose tissue. The cells were then differentiated to generate IPCs by achemical-based induction protocol. EVs were retrieved from the conditioned media of undifferentiated (naïve) MSCs (uneducated EVs) and from that of MSC-derived IPCs (educated EVs) by sequential ultracentrifugation. The obtained EVs were co-cultured with naïve MSCs.The cocultured cells were evaluated by immunofluorescence, flow cytometry, C-peptide nanogold silver-enhanced immunostaining, relative gene expression and their response to a glucose challenge.Immunostaining for naïve MSCs cocultured with educated EVs was positive for insulin, C-peptide, and GAD65. By flow cytometry, the median percentages of insulin-andC-peptide-positive cells were 16.1% and 14.2% respectively. C-peptide nanogoldimmunostaining providedevidence for the intrinsic synthesis of C-peptide. These cells released increasing amounts of insulin and C-peptide in response to increasing glucose concentrations. Gene expression of relevant pancreatic endocrine genes, except for insulin, was modest. In contrast, the results of naïve MSCs co-cultured with uneducated exosomes were negative for insulin, C-peptide, and GAD65. These findings suggest that this approach may overcome the limitations of cell therapy.

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.

Extracellular Vesicles as Mediators of Neuroinflammation in Intercellular and Inter-Organ Crosstalk.

Neuroinflammation, crucial in neurological disorders like Alzheimer's disease, multiple sclerosis, and hepatic encephalopathy, involves complex immune responses. Extracellular vesicles (EVs) play a pivotal role in intercellular and inter-organ communication, influencing disease progression. EVs serve as key mediators in the immune system, containing molecules capable of activating molecular pathways that exacerbate neuroinflammatory processes in neurological disorders. However, EVs from mesenchymal stem cells show promise in reducing neuroinflammation and cognitive deficits. EVs can cross CNS barriers, and peripheral immune signals can influence brain function via EV-mediated communication, impacting barrier function and neuroinflammatory responses. Understanding EV interactions within the brain and other organs could unveil novel therapeutic targets for neurological disorders.

Role of STAT3-FOXO3 Signaling in the Modulation of Neuroplasticity by PD-L1-HGF-Decorated Mesenchymal Stem Cell-Derived Exosomes in a Murine Stroke Model.

The limited therapeutic strategies available for stroke leave many patients disabled for life. This study assessed the potential of programmed death-ligand 1 (PD-L1) and hepatocyte growth factor (HGF)-engineered mesenchymal stem cell-derived exosomes (EXO-PD-L1-HGF) in enhancing neurological recovery post-stroke. EXO-PD-L1-HGF, which efficiently endocytosed into target cells, significantly diminishes the H2O2-induced neurotoxicity and increased the antiapoptotic proteins in vitro. EXO-PD-L1-HGF attenuates inflammation by inhibiting T-cell proliferation and increasing the number of CD8+CD122+IL-10+ regulatory T cells. Intravenous injection of EXO-PD-L1-HGF could target stromal cell-derived factor-1α (SDF-1α+) cells over the peri-infarcted area of the ischemic brain through CXCR4 upregulation and accumulation in neuroglial cells post-stroke. EXO-PD-L1-HGF facilitates endogenous nestin+ neural progenitor cell (NPC)-induced neurogenesis via STAT3-FOXO3 signaling cascade, which plays a pivotal role in cell survival and neuroprotection, thereby mitigating infarct size and enhancing neurological recovery in a murine stroke model. Moreover, increasing populations of the immune-regulatory CD19+IL-10+ and CD8+CD122+IL-10+ cells, together with reducing populations of proinflammatory cells, created an anti-inflammatory microenvironment in the ischemic brain. Thus, innovative approaches employing EXO-PD-L1-HGF intervention, which targets SDF-1α+ expression, modulates the immune system, and enhances the activation of resident nestin+ NPCs, might significantly alter the brain microenvironment and create a niche conducive to inducing neuroplastic regeneration post-stroke.

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.

Updates on the Pathophysiology and Therapeutic Potential of Extracellular Vesicles with Focus on Exosomes in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an incurable autoimmune disease with high morbidity and socioeconomic burden. Advances in therapeutics have improved patients' quality of life, however due to the complex disease pathophysiology and heterogeneity, 30% of patients do not respond to treatment. Understanding how different genetic and environmental factors contribute to disease initiation and development as well as uncovering the interactions of immune components is key to the implementation of effective and safe therapies. Recently, the role of extracellular vesicles (EVs) in RA development and possible treatment has been an area of interest. EVs are small lipid-bound entities, often containing genetic material, proteins, lipids and amino acids, facilitating paracrine intercellular communication. They are secreted by all cells, and it is believed that they possess regulatory functions due to high complexity and functional diversity. Although it has been shown that EVs participate in RA pathophysiology, through immune modulation, their exact role remains elusive. Furthermore, EVs could be a promising therapeutic agent in various diseases including RA, due to their biocompatibility, low toxicity and possible manipulation, but further research is required in this area. This review provides a comprehensive discussion of disease pathophysiology and summarizes the latest knowledge regarding the role and therapeutic potential of EVs in RA.

Suppression of the host antiviral response by non-infectious varicella zoster virus extracellular vesicles.

Varicella zoster virus (VZV) reactivates from ganglionic sensory neurons to produce herpes zoster (shingles) in a unilateral dermatomal distribution, typically in the thoracic region. Reactivation not only heightens the risk of stroke and other neurological complications but also increases susceptibility to co-infections with various viral and bacterial pathogens at sites distant from the original infection. The mechanism by which VZV results in complications remote from the initial foci remains unclear. Small extracellular vesicles (sEVs) are membranous signaling structures that can deliver proteins and nucleic acids to modify the function of distal cells and tissues during normal physiological conditions. Although viruses have been documented to exploit the sEV machinery to propagate infection, the role of non-infectious sEVs released from VZV-infected neurons in viral spread and disease has not been studied. Using multi-omic approaches, we characterized the content of sEVs released from VZV-infected human sensory neurons (VZV sEVs). One viral protein was detected (immediate-early 62), as well as numerous immunosuppressive and vascular disease-associated host proteins and miRNAs that were absent in sEVs from uninfected neurons. Notably, VZV sEVs are non-infectious yet transcriptionally altered primary human cells, suppressing the antiviral type 1 interferon response and promoting neuroinvasion of a secondary pathogen in vivo. These results challenge our understanding of VZV infection, proposing that the virus may contribute to distant pathologies through non-infectious sEVs beyond the primary infection site. Furthermore, this study provides a previously undescribed immune-evasion mechanism induced by VZV that highlights the significance of non-infectious sEVs in early VZV pathogenesis. IMPORTANCE: Varicella zoster virus (VZV) is a ubiquitous human virus that predominantly spreads by direct cell-cell contact and requires efficient and immediate host immune evasion strategies to spread. The mechanisms of immune evasion prior to virion entry have not been fully elucidated and represent a critical gap in our complete understanding of VZV pathogenesis. This study describes a previously unreported antiviral evasion strategy employed by VZV through the exploitation of the infected host cell's small extracellular vesicle (sEV) machinery. These findings suggest that non-infectious VZV sEVs could travel throughout the body, affecting cells remote from the site of infection and challenging the current understanding of VZV clinical disease, which has focused on local effects and direct infection. The significance of these sEVs in early VZV pathogenesis highlights the importance of further investigating their role in viral spread and secondary disease development to reduce systemic complications following VZV infections.

Combinational therapy of mesenchymal stem cell-derived extracellular vesicles and azithromycin improves clinical and histopathological recovery in CLP sepsis model.

BACKGROUND: Sepsis is a syndrome that occurs following an infection and marked by severe inflammatory responses, and if not treated in time, it can lead to multi-organ failure syndrome and death. This study examines the effects of a novel combination therapy using azithromycin and mesenchymal stem cell-derived extracellular vesicles (EVs) on a cecal ligation and puncture (CLP) model of sepsis. METHODS: Human Wharton's jelly-mesenchymal stem cells were cultured, characterized, and used to extract EVs. The CLP sepsis model was induced in mice, followed by treatments: saline, AZM, EVs, and combination therapy (A+E). Clinical sepsis scores were recorded 24 h post-treatment. Serum, peritoneal fluid, and organ tissues (kidney, liver, lung) were collected and analyzed for biochemical parameters (AST ALT, and creatinine), inflammatory markers, bacterial load, and histopathological changes. RESULTS: The A+E combined treatment improved the clinical scores of septic mice. The administration of A+E reduced bacterial loads in the peritoneum of septic mice, contributing to effective control of infection. Inflammatory markers of neutrophils-to-lymphocytes ratio (NLR) and TNF-α serum levels were significantly lower in the combinational therapy group, indicating significant anti-inflammatory effect of this combination. Additionally, combination of AZM and EVs alleviated organ damage mainly within liver, kidneys and lungs. Based on histopathological assessments and biochemical parameters, there was diminished tissue damage as well as reduced inflammation, which is correlated with improved functions of these vital organs. CONCLUSION: The combined use of azithromycin and EVs offers a promising therapeutic approach for sepsis by effectively controlling infection and modulating the inflammatory response.