Journal of extracellular biology最新文献

Fungal infections cause approximately 1.6 million deaths annually. Diagnosing and treating fungal infections is difficult due to limited access to diagnostic tests and rising antifungal resistance. Extracellular vesicles (EVs) facilitate interactions between fungal cells and hosts, significantly influencing the pathogen-host relationship. Owing to the complexity of fungal EVs and the lack of clinical studies on their roles in human infections, we analysed EVs from serum and urine samples of patients with infections caused by Candida albicans, Cryptococcus neoformans, and Paracoccidioides brasiliensis to determine their roles. Using mass spectrometry, we identified sterols, sphingolipids, and fatty acids as key metabolites in the EVs. We quantified cholesterol and ergosterol, confirming the presence of both host and fungal EVs in clinical samples. Our research investigated whether these EVs could modulate the host immune response. We observed a proinflammatory response in murine and human macrophages, characterized by increased cytokines, such as tumour necrosis factor-α, interferon-γ, and interleukin-6, and elevated expression of the inducible nitric oxide synthase gene, a marker of M1 macrophage response. Thus, circulating EVs in patients with fungal infections likely play a role in disease pathophysiology. These findings enhance our understanding of EVs in fungal infections, suggesting potential therapeutic targets for systemic mycoses.

The development of novel treatments that restore brain function and improve patient outcomes for Alzheimer's disease (AD) is necessary, given the complications and lack of improvement in recently approved amyloid beta (Aβ)-targeting drugs. Cell-derived extracellular vesicles (EVs) have been found to improve cognitive function through reduced inflammation, oxidative stress, and apoptosis, restoring neuronal and blood-brain barrier function, and inhibiting Aβ and phosphorylated tau build-up in the brain. Given the recent emergence of EVs into clinical trials, it is essential to provide the field with an update on proposed mechanisms of action, gaps in knowledge for further study, and recommendations for producing EVs with high therapeutic efficacy to ensure success in subsequent clinical trials. This systematic review summarizes original research to date that reports effects of mammalian cell-derived EVs for the treatment of AD. Evidence of therapeutic benefits and reported mechanisms of action are discussed. Further, methods for engineering EVs to increase their therapeutic efficacy and produce high-quality EVs relevant to the AD field are outlined. The quality of evidence is discussed in terms of reporting guidelines from the Minimal Information for Studies of Extracellular Vesicles (MISEV). The review further discusses current preclinical AD models and provides direction to improve the quality of AD models for testing novel therapeutics.

Small extracellular vesicles (sEVs) derived from natural killer (NK) cells possess inherent anti-tumour activity and offer the advantages of cell-free therapy. In this study, we genetically engineered NK-sEVs to express interleukin 15 (IL15), an anti-tumour cytokine, and the monoclonal antibody cetuximab on their surface, creating a potent anti-tumour immunotherapy with enhanced tumour-targeting capabilities. These IL15- and cetuximab-tethered NK-sEVs (eEVs) were generated using lentivirus-based modification. eEVs selectively bound to EGFR⁺ cancer cells in vitro, confirming cetuximab-mediated targeting. Compared to control NK-sEVs, eEVs exhibited significantly enhanced cytotoxicity by directly inducing cancer cell death and promoting NK cell-mediated killing. In a lung cancer mouse model, eEVs selectively accumulated in tumours and exhibited significant anti-tumour efficacy. Notably, their administration, alone or in combination with anti-PD-1 antibody therapy, effectively suppressed tumour growth. Overall, our results indicate that genetically engineered NK-sEVs, equipped with IL15 and cetuximab, exhibit potent anti-tumour activity and tumour-targeting capabilities. These findings suggest that eEVs hold significant potential as a novel immunotherapeutic strategy for cancer treatment.

All approved RNA therapeutics require parenteral delivery. Here, we demonstrate an orally bioavailable formulation wherein synthetic noncoding (nc) RNA, packaged into lipid nanoparticles, is loaded into casein-chitosan (C2) micelles. We used the C2 formulation to deliver TY1, a 24-nucleotide synthetic ncRNA, which targets DNA damage and attenuates inflammation in macrophages. C2-formulated TY1 (TY1^C2) efficiently packages and protects TY1 against degradative enzymes. In healthy mice, oral TY1^C2 was well-tolerated and nontoxic. Oral TY1^C2 exhibited disease-modifying bioactivity in two models of tissue injury: (1) rat myocardial infarction, where a single oral dose of TY1^C2 was cardioprotective, on par with intravenously-delivered TY1; and (2) mouse acute lung injury, where a single dose of TY1^C2 attenuated pulmonary inflammation. Mechanistic dissection revealed that TY1^C2 is taken up by intestinal macrophages, namely those of the lamina propria and Peyer's patches. Afterwards, TY1 could be detected in circulating monocytes for up to 72 h post-ingestion. Unlike TY1, which acts on macrophages, an antisense oligonucleotide against Factor VII, which acts on hepatocytes, is not effective when administered in the C2 formulation. Thus, not all ncRNA drugs are bioactive when delivered by mouth. Oral delivery of macrophage-active RNA opens up a wide range of potential new therapeutic opportunities.

The gut microbiota, a collection of microorganisms residing within the human gastrointestinal tract, exerts profound effects on the health of the host. In recent years, studies have revealed that the gut microbiota influences not only the function of the digestive system but also has close associations with various systemic diseases, including cardiovascular diseases. Myocardial remodelling refers to the structural, functional and molecular changes in the myocardium that occur in response to alterations in load. This process encompasses changes such as myocardial hypertrophy, apoptosis, necrosis and myocardial fibrosis. Bacterial extracellular vesicles (BEVs) are small vesicles secreted by the gut microbiota that can carry bioactive substances such as proteins, lipids and nucleic acids, participating in intercellular communication. BEVs are capable of traversing the gut barrier and entering the bloodstream, thereby influencing the functional status of distant organs, including the heart. Under the condition of Myocardial remodelling, these BEVs may exert protective or detrimental effects on cardiomyocytes by modulating pathways such as inflammation, oxidative stress and apoptosis.

Proteomic and transcriptomic analyses of cerebrospinal fluid (CSF)-derived extracellular vesicles (EVs) offer unique insights into molecular changes associated with central nervous system (CNS) diseases and may result in biomarker identification. No gold standard method to enrich EVs from CSF has been established, and head-to-head comparisons of outputs of different protocols are scarce. Using a large pool of CSF, we characterised the EV preparations resulting from four enrichment protocols and compared them in terms of yield and purity. We found that particles enriched by ultracentrifugation (UC) or a combination of ultrafiltration and size exclusion chromatography (UF-SEC) exhibited the typical morphological and biochemical characteristics of small EVs and were highly enriched in proteins and polyadenylated (polyA) transcripts associated with EV-related biological processes. UF-SEC preparations had higher particle yields, whilst more proteins were identified in UC preparations. Approximately 40% of the EV preparations’ proteome was not identified in unenriched CSF, among which a core proteome of 45 proteins was identified in 30 EV preparations from independent experiments, which may serve as CSF-derived EV markers. Enrichment scores to protein contaminants, albumin and apolipoprotein E were higher in UF-SEC preparations. In conclusion, all protocols analysed here resulted in enrichment of particles with small EV characteristics, with EV enrichments from UF-SEC resulting in the highest yield and purity.

Alzheimer's disease (AD) is a major neurodegenerative disorder that affects more than 55 million people, with an incidence that is projected to triple by 2050. Despite continuous advancements in the field, reliable treatment and early detection strategies remain elusive. Extracellular vesicles (EVs) play a major role in cellular communication throughout the body. In this study, we assessed the cargo of neuronal-specific EVs for their potential as AD biomarkers. We isolated EVs released from iPSC-derived excitatory glutamatergic neurons generated from eight AD patients (ADiNEVs) and six healthy controls (iNEVs). We performed RNA-sequencing and identified significant differences in RNA cargo between ADiNEVs and iNEVs. Notably, fewer small nuclear RNAs (snRNAs) were found in ADiNEVs. RNA transcripts significantly more abundant in ADiNEVs included MT-CO1, PRR32 and IGSF8 messenger RNAs. We also observed fewer XIST long noncoding RNAs and miR-7-5p microRNA content in ADiNEVs. These findings suggest that precision medicine approaches, such as characterising the content of EVs from a patient's own cells, could advance early detection and management of AD.

Extracellular vesicles (EVs) offer a minimally invasive approach for cancer detection and monitoring. However, the lack of standardized methods for clinical biospecimen preparation and EV isolation limits the clinical utility of EV-based biomarker assessments. A targeted need exists for detailed analysis of plasma EV content. Our study investigates the impact of clinical sample preparation and our ExoTIC device on the quality of plasma-derived EVs and their RNA/protein cargo in metastatic castration-resistant prostate cancer (mCRPC) patients. We assessed sample preparation variables: blood anti-coagulant choice (EDTA or sodium citrate), type of plasma platelet fraction (platelet-rich or platelet-poor), and use of protease inhibitors. EVs were isolated via ExoTIC device, followed by EV characterization and biomarker analysis using nanoparticle tracking analysis (NTA), cryogenic electron microscopy, Western blot, and digital PCR (dPCR). We detected mCRPC-relevant proteins (ARv7 and PSMA) in EVs from all plasma sample types with different sample preparation variables. Additionally, our findings indicate that platelet-poor plasma (PPP) is optimal for detecting EV- and biologically associated mCRPC biomarker miR-375. In this pilot study (n = 3), elevated EV miR-375 levels in PPP samples from mCRPC patients experiencing disease progression during docetaxel treatment were associated with poor therapeutic response to docetaxel chemotherapy, which aligns with our preceding in vitro and in vivo study. Optimal biospecimen preparation for EV analysis could enhance detection accuracy and patient management, highlighting detection of plasma EV-associated mCRPC-specific marker proteins (ARv7 and PSMA) and microRNA miR-375.

Dysfunction of vascular endothelium is characteristic of many aging-related diseases, including Alzheimer's disease (AD) and AD-related dementias (ADRD). Although it is widely posited that endothelial cell dysfunction contributes to the pathogenesis and/or progression of AD/ADRD, it is not clear how. A plausible hypothesis is that intercellular trafficking of extracellular vesicles (EVs) from senescent vascular endothelial cells promotes vascular endothelial cell dysfunction. To test this hypothesis, we compared the expression of proteins and miRNAs in EVs isolated from four sets of genetically identical early passage non-senescent (EP) versus late passage senescent (SEN) primary human coronary artery endothelial cells (HCAECs) derived from four donors. Proteomics and miRNA libraries constructed from these EV isolates were evaluated using FunRich gene ontology analysis to compare functional enrichment between EP and SEN endothelial cell EVs (ECEVs). Replicative senescence was associated with altered EV abundance and contents independent of changes in EV size. Unique sets of miRNAs and proteins were differentially expressed in SEN-ECEVs, including molecules related to cell adhesion, barrier integrity, receptor signalling, endothelial-mesenchymal transition and cell senescence. miR-181a-5p was the most upregulated miRNA in SEN-ECEVs, increasing >5-fold. SEN-ECEV proteomes supported involvement in several pro-inflammatory pathways consistent with senescence and the senescence-associated secretory phenotype (SASP). These data indicate that SEN-ECEVs are enriched in bioactive molecules implicated in senescence-associated vascular dysfunction, blood–brain barrier impairment, and AD/ADRD pathology. These observations suggest involvement of SEN-ECEVs in the pathogenesis of vascular dysfunction associated with AD/ADRD.