Extracellular vesicles and circulating nucleic acids最新文献

Urinary tract infections (UTIs) pose a significant public health challenge, affecting approximately 407 million people worldwide and causing substantial morbidity and approximately 237,000 deaths. Bacteria and fungi represent the most frequent causative microbes, leading to symptoms such as low abdominal pain, fever, frequent urination, hematuria, sepsis, inflammation of the bladder and kidney, and even death. In recent years, extracellular vesicles (EVs) have emerged as critical mediators of UTI pathogenesis. EVs are lipid bilayer nanoscale particles that carry DNA, RNA, enzymes, and other biomolecules. They can facilitate microbial colonization, immune modulation and evasion, tissue invasion, and antimicrobial agent resistance. This review summarizes current knowledge on the role of bacterial and fungal-derived EVs in UTIs, their mechanisms of action, and their potential therapeutic implications.

Aim: Autologous blood transfusions (ABT), especially those involving stored erythrocyte concentrates (ECs), are known to be misused as performance enhancers in recreational and competitive athletes. EC storage not only increases the release of extracellular vesicles (EVs), but also significantly alters the microRNA profiles. Since re-transfused EVs also appear in urine, this study was designed to evaluate whether the urinary EV-associated microRNA load could serve as a valuable indicator in the challenging detection of ABT. Methods: Thirty healthy, recreationally active males were included and equally divided into three groups. The control group did not donate or receive any blood components. Group 1 donated about 500 mL of whole blood once, which was subsequently processed into ECs. These were stored for six weeks and then re-infused into the respective donor. Group 2 donated about 500 mL of whole blood twice, at an interval of two weeks. The obtained ECs were stored for six or four weeks, respectively, until parallel re-infusion. In all groups, urine samples were collected over three consecutive weeks before whole-blood donation to establish each individual's baseline, as well as before re-transfusion and several hours and days afterward. Urine samples were processed and analyzed for general urinary health and creatinine levels. Urinary EVs were further isolated by immunoaffinity and characterized using transmission electron microscopy, fluorescence nanoparticle tracking analysis, and western blotting, as well as an established multiplexed bead-based flow cytometry method, followed by RNA isolation and in-depth small RNA profiling using next-generation sequencing and comprehensive data analyses. Results: Urinary EVs presented with typical morphology of small EVs (< 200 nm) and an overall concentration of 8.79 ± 7.00 × 10¹⁰ particles/g U_Crea (urinary creatinine). Significant increases in urinary EV concentrations were detected up to three days after ABT. Apart from Alix, Syntenin, and tumor susceptibility gene 101 (TSG101), surface markers CD63, CD9, CD133/1, CD24, CD326, CD81, and CD31 were also shown to be highly abundant on urinary EVs. Impurities or contaminations were not detected. Cluster analysis based on surface markers showed a clear separation between the control and ABT group. Furthermore, microRNA profiling revealed 13 microRNAs differently regulated upon ABT with miR-155-5p, miR-320b, and miR-6869-5p being the most abundant. Conclusion: This proof-of-concept study suggests an impact of ABT on the urinary EV-microRNA cargo and yields comprehensive findings into surface markers of urinary EVs. While the adoption of urinary EV-associated microRNAs in routine doping tests requires further exploration, these data serve as a valuable basis for a variety of subsequent investigations.

Aim: Liver fibrosis (LF) is a major pathological stage that may progress to end-stage chronic liver injury but currently lacks effective treatment strategies. Previous studies have shown that adipose-derived stem cell extracellular vesicles (ADSC-EVs) play crucial roles in tissue repair, immune regulation, and anti-inflammatory effects. This study aims to elucidate the therapeutic effect of ADSC-EVs in LF and reveal their regulation mechanisms in gut-liver axis dysregulation. Methods: The LF mouse model was established by intraperitoneal injection of diethylnitrosamine/CCl₄. LF mice for ADSC-EV treatment received ADSC-EVs (200 μg per mouse) twice a week for three weeks. Then, hepatic function tests, liver and gut histopathology, and gut microbiota analyses were performed. Results: ADSC-EVs effectively improved hepatic function, reduced collagen deposition and suppressed hepatic stellate cell activation, exhibiting potent anti-fibrotic potential in LF mice. Additionally, they significantly restored intestinal barrier integrity by reducing intestinal permeability and reinforcing the mucus barrier. Furthermore, ADSC-EV treatment regulated gut microbiota dysbiosis, increased the abundance of beneficial intestinal bacteria such as Akkermansia muciniphila. ADSC-EV intervention also elevated the level of butyric acid in cecal contents and significantly reduced systemic inflammation. Conclusion: Our findings suggest that ADSC-EVs represent a promising novel therapeutic strategy for LF, promoting liver tissue repair, enhancing intestinal barrier function, and maintaining gut homeostasis to establish a virtuous circle within the liver-gut axis.

Aim: This study aims to investigate how the gut microbiota communicates with the host via bacterial extracellular vesicles (BEVs), given that direct contact between microbes and the healthy intestinal epithelium is prevented by a sterile mucin gel layer. Understanding these indirect interactions is critical because the specific pathways and mediators of microbiota-host interactions are incompletely understood. Tracking BEVs in vivo however is particularly challenging due to their nanoscale size and complex molecular composition. Methods: To address these challenges, we developed a highly sensitive Nanoluciferase (NanoLuc) system for luminescence-based detection of BEVs produced by the model human commensal bacterium Bacteroides thetaiotaomicron. This approach was evaluated in germ-free and specific-pathogen-free mice, with comparisons between administration routes demonstrating the advantages of this system for in vivo BEV labelling over conventional lipophilic dyes. Results: We report, for the first time, that BEVs endogenously produced in the gastrointestinal tract (GIT) of mice can deliver bioactive NanoLuc protein to multiple organ tissues, including the central nervous system. Our findings establish that naturally occurring BEVs in the GIT are capable of traversing multiple host barriers, including the intestinal epithelium, vascular endothelium, and the blood-brain-barrier, to access tissues such as the brain and eyes. Conclusion: These findings advance our understanding of BEV-mediated microbe-host interactions and demonstrate the potential of BEVs as vehicles for long-distance delivery of bioactive therapeutics.

Aim: Precancerous lesions of gastric cancer (PLGC) represent a critical window for prevention. Developing non-invasive tools that can reliably detect these lesions is therefore a prerequisite for lowering gastric-cancer incidence. Recent work has highlighted the diagnostic promise of plasma extracellular vesicle DNAs (evDNAs) and the 5-hydroxymethylcytosine (5hmC)-Seal epigenomic platform. Here we profiled genome-wide 5hmC patterns in circulating evDNA to discover biomarkers and build a classification model. Methods: We performed whole-genome 5hmC-Seal on plasma evDNAs from 67 PLGC patients and 67 healthy individuals. By identifying trend-expressed differentially hydroxymethylated regions (DhMRs), we used machine learning algorithms to screen for diagnostic biomarkers of PLGC and established a corresponding diagnostic model. Results: We ultimately constructed a diagnostic model comprising nine DhMRs. In the test set, the area under the curve (AUC) value was 0.963, with an accuracy of 0.886, sensitivity of 95.45%, and specificity of 81.82%. These results indicate that DhMRs in evDNA can serve as diagnostic biomarkers for PLGC, with good diagnostic capability and reliability. Correlation analysis showed a strong association between the DhMRs in the diagnostic model and clinical pathological indicators of PLGC. Conclusion: We developed a non-invasive diagnostic model for PLGC by profiling 5hmC in plasma evDNA. In both accuracy and inter-batch robustness, it surpasses all previously reported assays. Our findings establish plasma-evDNA 5hmC profiling as a reliable, minimally invasive strategy for the early detection and precise diagnosis of gastric precancerous lesions, and provide a new translational and clinical framework for future work.

Mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEVs) have emerged as a promising cell-free alternative to MSC-based therapies, offering superior safety, scalability, and stability profiles. These nanosized vesicles are now widely regarded as the principal therapeutic effectors of MSCs, capable of recapitulating many of the benefits attributed to their parental cells. However, their successful clinical translation depends on overcoming key challenges, particularly those related to product variability, viral safety, and the definition of mechanistically relevant potency-associated critical quality attributes (CQAs). This review explores the sources of MSC-sEV variability, including MSC tissue origin, manufacturing parameters, and limitations associated with primary and pluripotent stem cell-derived MSCs. The use of immortalized monoclonal MSC lines is discussed as a potential solution to improve batch consistency. Regulatory frameworks such as the International Council for Harmonisation (ICH) guideline Q5A(R2) are also highlighted for ensuring viral safety in sEV manufacturing processes. A major focus is the critical evaluation of microRNAs (miRNAs) - long regarded as leading candidates for potency CQAs in MSC-sEV products. Despite their prevalence in the extracellular vesicle literature, mounting evidence challenges their functional relevance in therapeutic contexts. Studies consistently show that miRNAs are underrepresented in sEVs, occur at very low copy numbers, and lack essential components (e.g., Argonaute proteins) required for canonical RNA interference. Moreover, the efficiency of EV internalization and endosomal escape remains exceedingly low, rendering miRNA-based gene regulation mechanistically implausible at physiologically relevant doses. These findings call into question the widespread assumption that miRNAs are primary effectors of MSC-sEV activity.

Aim: This study aimed to evaluate the reproducibility of the isolation and characterization of feces-derived bacterial membrane vesicles. Methods: Human fecal samples (n = 12) stored at -80 °C were thawed, sampled, and then refrozen. From these samples, bacterial membrane vesicles were isolated through ultrafiltration, ultracentrifugation and size exclusion chromatography. Vesicle-associated DNA was characterized by marker [16 ribosomal DNA (rDNA)] sequencing to determine composition. The same fecal samples were thawed again after > 6 months of storage at -80 °C to repeat this procedure. Compositions and other vesicle characteristics were compared to investigate effects of storage and freeze/thawing on sample stability. In addition, for four of the fecal aliquots, the bacteria were subjected to marker gene sequencing alongside their derived membrane vesicles. Results: No significant differences were observed in the pre- and post freeze/thawing composition of feces-derived bacterial membrane vesicles [permutational multivariate analysis of variance (PERMANOVA) P = 0.356] or bacteria (PERMANOVA P = 0.721) as determined by 16S rDNA sequencing. Additionally, no significant differences were observed in vesicle size, concentration, and associated protein or DNA content. These results indicate that, long-term storage of feces at -80 °C and an additional freeze/thawing cycle does not induce compositional or qualitative changes to vesicle repertoires. Conclusion: These reproducibility findings hold great relevance for research on (gut)bacteria derived membrane vesicles. Our results indicate that fecal samples can be stably preserved at -80 °C for bacterial and vesicle isolations as their characteristics remain stable over time.

As global aging intensifies, the issue of bone aging has become increasingly prominent. Osteoporosis and osteoarthritis, which are common complications of bone aging, significantly impair patients' quality of life due to their high prevalence and disability rates, thereby presenting a major public health challenge. Extracellular vesicles (EVs), nanoscale particles released by cells, are regarded as an ideal platform for bone aging due to their high biocompatibility, ease of modification, and significant therapeutic efficacy. This review provides a comprehensive summary of the latest advancements in mammalian-, bacterial-, and plant-derived EVs, particularly in the context of bone aging. Furthermore, organoids, as lab-grown models replicating organ physiology, produce organoid-derived EVs that represent an especially promising avenue for therapeutic application. This review focuses on exploring potential therapeutic strategies that capitalize on the unique advantages of each EV type for treating bone aging. It is anticipated that a thorough comprehension of these EV types will unveil new avenues for bone aging treatment.

The crosstalk between the skeletal muscles and the liver is receiving growing attention, as patients with chronic liver disease often develop a loss of skeletal muscle mass. In these patients, particularly those with metabolic dysfunction-associated steatotic liver disease, physical exercise improves insulin sensitivity and hepatic steatosis. However, excessive exercise may impair mitochondrial function, inflammation, and liver health. The study by Liu et al. demonstrates that overtraining promotes liver fibrosis through myocyte-derived small extracellular vesicles. Here, we comment on the novelty of these findings and areas to be developed in the future.