International Society for Extracellular Vesicles Workshop. QuantitatEVs: multiscale analyses, from bulk to single extracellular vesicle.

The 'QuantitatEVs: multiscale analyses, from bulk to single vesicle' workshop aimed to discuss quantitative strategies and harmonized wet and computational approaches toward the comprehensive analysis of extracellular vesicles (EVs) from bulk to single vesicle analyses with a special focus on emerging technologies. The workshop covered the key issues in the quantitative analysis of different EV-associated molecular components and EV biophysical features, which are considered the core of EV-associated biomarker discovery and validation for their clinical translation. The in-person-only workshop was held in Trento, Italy, from January 31st to February 2nd, 2023, and continued in Milan on February 3rd with "Next Generation EVs", a satellite event dedicated to early career researchers (ECR). This report summarizes the main topics and outcomes of the workshop.

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

MIBlood-EV: Minimal information to enhance the quality and reproducibility of blood extracellular vesicle research.

Blood is the most commonly used body fluid for extracellular vesicle (EV) research. The composition of a blood sample and its derivatives (i.e., plasma and serum) are not only donor-dependent but also influenced by collection and preparation protocols. Since there are hundreds of pre-analytical protocols and over forty variables, the development of standard operating procedures for EV research is very challenging. To improve the reproducibility of blood EV research, the International Society for Extracellular Vesicles (ISEV) Blood EV Task Force proposes standardized reporting of (i) the applied blood collection and preparation protocol and (ii) the quality of the prepared plasma and serum samples. Gathering detailed information will provide insight into the performance of the protocols and more effectively identify potential confounders in the prepared plasma and serum samples. To collect this information, the ISEV Blood EV Task Force created the Minimal Information for Blood EV research (MIBlood-EV), a tool to record and report information about pre-analytical protocols used for plasma and serum preparation as well as assays used to assess the quality of these preparations. This tool does not require modifications of established local pre-analytical protocols and can be easily implemented to enhance existing databases thereby enabling evidence-based optimization of pre-analytical protocols through meta-analysis. Taken together, insight into the quality of prepared plasma and serum samples will (i) improve the quality of biobanks for EV research, (ii) guide the exchange of plasma and serum samples between biobanks and laboratories, (iii) facilitate inter-laboratory comparative EV studies, and (iv) improve the peer review process.

Plasma Extracellular Vesicle Characteristics as Biomarkers of Resectability and Radicality of Surgical Resection in Pancreatic Cancer-A Prospective Cohort Study.

Due to possible diagnostic misjudgment of tumor resectability, patients with pancreatic ductal adenocarcinoma (PDAC) might be exposed to non-radical resection or unnecessary laparotomy. With small extracellular vesicles (sEV) obtained by liquid biopsy, we aimed to evaluate their potential as biomarkers of tumor resectability, radicality of resection and overall survival (OS). Our prospective study included 83 PDAC patients undergoing surgery with curative intent followed-up longitudinally. sEV were isolated from plasma, and their concentration and size were determined. Fifty patients underwent PDAC resection, and thirty-three had no resection. Preoperatively, patients undergoing resection had higher sEV concentrations than those without resection (p = 0.023). Resection was predicted at the cutoff value of 1.88 × 109/mL for preoperative sEV concentration (p = 0.023) and the cutoff value of 194.8 nm for preoperative mean diameter (p = 0.057). Furthermore, patients with R0 resection demonstrated higher preoperative plasma sEV concentrations than patients with R1/R2 resection (p = 0.014). If sEV concentration was above 1.88 × 109/mL or if the mean diameter was below 194.8 nm, patients had significantly longer OS (p = 0.018 and p = 0.030, respectively). Our proof-of-principle study identified preoperative sEV characteristics as putative biomarkers of feasibility and radicality of PDAC resection that also enable discrimination of patients with worse OS. Liquid biopsy with sEV could aid in PDAC patient stratification and treatment optimization in the future.

Isolation and characterization of extracellular vesicles from biotechnologically important fungus Aureobasidium pullulans.

Extracellular vesicles (EVs) are increasingly recognized as an important mechanism for cell-cell interactions. Their role in fungi is still poorly understood and they have been isolated from only a handful of species. Here, we isolated and characterized EVs from Aureobasidium pullulans, a biotechnologically important black yeast-like fungus that is increasingly used for biocontrol of phytopathogenic fungi and bacteria. After optimization of the isolation protocol, characterization of EVs from A. pullulans by transmission electron microscopy (TEM) revealed a typical cup-shaped morphology and different subpopulations of EVs. These results were confirmed by nanoparticle tracking analysis (NTA), which revealed that A. pullulans produced 6.1 × 108 nanoparticles per milliliter of culture medium. Proteomic analysis of EVs detected 642 proteins. A small fraction of them had signal peptides for secretion and transmembrane domains. Proteins characteristic of different synthesis pathways were found, suggesting that EVs are synthesized by multiple pathways in A. pullulans. Enrichment analysis using Gene Ontology showed that most of the proteins found in the EVs were associated with primary metabolism. When sequencing the small RNA fraction of A. pullulans EVs, we found two hypothetical novel mil-RNAs. Finally, we tested the biocontrol potential of EVs from A. pullulans. The EVs did not inhibit the germination of spores of three important phytopathogenic fungi-Botrytis cinerea, Colletotrichum acutatum, and Penicillium expansum. However, exposure of grown cultures of C. acutatum and P. expansum to A. pullulans EVs resulted in visible changes in morphology of colonies. These preliminary results suggest that EVs may be part of the antagonistic activity of A. pullulans, which is so far only partially understood. Thus, the first isolation and characterization of EVs from A. pullulans provides a starting point for further studies of EVs in the biotechnologically important traits of the biocontrol black fungus A. pullulans in particular and in the biological role of fungal EVs in general.

Extracellular vesicle-bound DNA in urine is indicative of kidney allograft injury.

Extracellular vesicle-bound DNA (evDNA) is an understudied extracellular vesicle (EV) cargo, particularly in cancer-unrelated research. Although evDNA has been detected in urine, little is known about its characteristics, localization, and biomarker potential for kidney pathologies. To address this, we enriched EVs from urine of well-characterized kidney transplant recipients undergoing allograft biopsy, characterized their evDNA and its association to allograft injury. The SEC-based method enriched pure EVs from urine of kidney transplant recipients, regardless of the allograft injury. Urinary evDNA represented up to 29.2 ± 8% (mean ± SD) of cell-free DNA (cfDNA) and correlated with cfDNA in several characteristics but was less fragmented (P < 0.001). Importantly, using DNase treatment and immunogold labelling TEM, we demonstrated that evDNA was bound to the surface of urinary EVs. Normalised evDNA yield (P = 0.042) and evDNA copy number (P = 0.027) significantly differed between patients with normal histology, rejection injury and non-rejection injury, the later groups having significantly larger uEVs (mean diameter, P = 0.045) and more DNA bound per uEV. ddDNA is detectable in uEV samples of kidney allograft recipients, but its quantity is highly variable. In a proof-of-principle study, several evDNA characteristics correlated with clinical and histological parameters (P = 0.040), supporting that the potential of evDNA as a biomarker for kidney allograft injury should be further investigated.

Unveiling the Native Morphology of Extracellular Vesicles from Human Cerebrospinal Fluid by Atomic Force and Cryogenic Electron Microscopy.

Extracellular vesicles (EVs) are membranous structures in biofluids with enormous diagnostic/prognostic potential for application in liquid biopsies. Any such downstream application requires a detailed characterization of EV concentration, size and morphology. This study aimed to observe the native morphology of EVs in human cerebrospinal fluid after traumatic brain injury. Therefore, they were separated by gravity-driven size-exclusion chromatography (SEC) and investigated by atomic force microscopy (AFM) in liquid and cryogenic transmission electron microscopy (cryo-TEM). The enrichment of EVs in early SEC fractions was confirmed by immunoblot for transmembrane proteins CD9 and CD81. These fractions were then pooled, and the concentration and particle size distribution were determined by Tunable Resistive Pulse Sensing (around 1010 particles/mL, mode 100 nm) and Nanoparticle Tracking Analysis (around 109 particles/mL, mode 150 nm). Liquid AFM and cryo-TEM investigations showed mode sizes of about 60 and 90 nm, respectively, and various morphology features. AFM revealed round, concave, multilobed EV structures; and cryo-TEM identified single, double and multi-membrane EVs. By combining AFM for the surface morphology investigation and cryo-TEM for internal structure differentiation, EV morphological subpopulations in cerebrospinal fluid could be identified. These subpopulations should be further investigated because they could have different biological functions.

Updating MISEV: Evolving the minimal requirements for studies of extracellular vesicles.

The minimal information for studies of extracellular vesicles (EVs, MISEV) is a field-consensus rigour initiative of the International Society for Extracellular Vesicles (ISEV). The last update to MISEV, MISEV2018, was informed by input from more than 400 scientists and made recommendations in the six broad topics of EV nomenclature, sample collection and pre-processing, EV separation and concentration, characterization, functional studies, and reporting requirements/exceptions. To gather opinions on MISEV and ideas for new updates, the ISEV Board of Directors canvassed previous MISEV authors and society members. Here, we share conclusions that are relevant to the ongoing evolution of the MISEV initiative and other ISEV rigour and standardization efforts.

Blood Nanoparticles - Influence on Extracellular Vesicle Isolation and Characterization.

Blood is a rich source of disease biomarkers, which include extracellular vesicles (EVs). EVs are nanometer-to micrometer-sized spherical particles that are enclosed by a phospholipid bilayer and are secreted by most cell types. EVs reflect the physiological cell of origin in terms of their molecular composition and biophysical characteristics, and they accumulate in blood even when released from remote organs or tissues, while protecting their cargo from degradation. The molecular components (e.g., proteins, miRNAs) and biophysical characteristics (e.g., size, concentration) of blood EVs have been studied as biomarkers of cancers and neurodegenerative, autoimmune, and cardiovascular diseases. However, most biomarker studies do not address the problem of contaminants in EV isolates from blood plasma, and how these might affect downstream EV analysis. Indeed, nonphysiological EVs, protein aggregates, lipoproteins and viruses share many molecular and/or biophysical characteristics with EVs, and can therefore co-isolate with EVs from blood plasma. Consequently, isolation and downstream analysis of EVs from blood plasma remain a unique challenge, with important impacts on the outcomes of biomarker studies. To help improve rigor, reproducibility, and reliability of EV biomarker studies, we describe here the major contaminants of EV isolates from blood plasma, and we report on how different EV isolation methods affect their levels, and how contaminants that remain can affect the interpretation of downstream EV analysis.

Optimization of isolation protocol and characterization of urinary extracellular vesicles as biomarkers of kidney allograft injury.

AIMS: Long-term kidney allograft survival requires a personalized approach to allograft injury recognition in a timely and reliable manner. Kidney biopsy is invasive and unsuitable for continuous function assessment. Alternatively, in urine, we find extracellular vesicles (uEVs), stable carriers of kidney pathology signals. Analysis of uEVs and their cargo could allow for more frequent and non-invasive assessment of allograft function. We aimed to optimize the uEVs isolation method applicable for kidney allograft injury biomarker studies. MATERIALS AND METHODS: To this end, we optimized several steps of size-exclusion chromatography (SEC)-based method for uEVs isolation from second morning urine of kidney allograft recipients. uEVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), western analysis, and quantitative PCR. RESULTS: According to TEM and NTA, SEC isolated high concentrations (8.64 × 108 EVs/mL of urine) of EVs that showed typical morphology and mean size (171 nm), but addition of EDTA and filtration step were needed to remove impurities. Additionally, typical EV proteins Hsc70, CD63, flotillin, tubulin, GAPDH, and miR hsa-let-7i were detected in isolated uEVs, further confirming their identity. CONCLUSION: Optimized method based on SEC was effective and adequate in isolating pure EVs from urine of kidney allograft recipients and could be used in future biomarker studies.

Extracellular Vesicle Enriched miR-625-3p Is Associated with Survival of Malignant Mesothelioma Patients.

Malignant mesothelioma (MM) is characterized by poor prognosis and short survival. Extracellular vesicles (EVs) are membrane-bound particles released from cells into various body fluids, and their molecular composition reflects the characteristics of the origin cell. Blood EVs or their miRNA cargo might serve as new minimally invasive biomarkers that would enable earlier detection of MM or treatment outcome prediction. Our aim was to evaluate miRNAs enriched in serum EVs as potential prognostic biomarkers in MM patients in a pilot longitudinal study. EVs were isolated from serum samples obtained before and after treatment using ultracentrifugation on 20% sucrose cushion. Serum EV-enriched miR-103-3p, miR-126-3p and miR-625-3p were quantified using qPCR. After treatment, expression of miR-625-3p and miR-126-3p significantly increased in MM patients with poor treatment outcome (p = 0.012 and p = 0.036, respectively). A relative increase in miR-625-3p expression after treatment for more than 3.2% was associated with shorter progression-free survival (7.5 vs. 19.4 months, HR = 3.92, 95% CI = 1.20-12.80, p = 0.024) and overall survival (12.5 vs. 49.1 months, HR = 5.45, 95% CI = 1.06-28.11, p = 0.043) of MM patients. Bioinformatic analysis showed enrichment of 33 miR-625-3p targets in eight biological pathways. Serum EV-enriched miR-625-3p could therefore serve as a prognostic biomarker in MM and could contribute to a more personalized treatment.

Seven Years at High Salinity-Experimental Evolution of the Extremely Halotolerant Black Yeast Hortaea werneckii.

The experimental evolution of microorganisms exposed to extreme conditions can provide insight into cellular adaptation to stress. Typically, stress-sensitive species are exposed to stress over many generations and then examined for improvements in their stress tolerance. In contrast, when starting with an already stress-tolerant progenitor there may be less room for further improvement, it may still be able to tweak its cellular machinery to increase extremotolerance, perhaps at the cost of poorer performance under non-extreme conditions. To investigate these possibilities, a strain of extremely halotolerant black yeast Hortaea werneckii was grown for over seven years through at least 800 generations in a medium containing 4.3 M NaCl. Although this salinity is well above the optimum (0.8-1.7 M) for the species, the growth rate of the evolved H. werneckii did not change in the absence of salt or at high concentrations of NaCl, KCl, sorbitol, or glycerol. Other phenotypic traits did change during the course of the experimental evolution, including fewer multicellular chains in the evolved strains, significantly narrower cells, increased resistance to caspofungin, and altered melanisation. Whole-genome sequencing revealed the occurrence of multiple aneuploidies during the experimental evolution of the otherwise diploid H. werneckii. A significant overrepresentation of several gene groups was observed in aneuploid regions. Taken together, these changes suggest that long-term growth at extreme salinity led to alterations in cell wall and morphology, signalling pathways, and the pentose phosphate cycle. Although there is currently limited evidence for the adaptive value of these changes, they offer promising starting points for future studies of fungal halotolerance.

Urinary Extracellular Vesicles and Their miRNA Cargo in Patients with Fabry Nephropathy.

Current biomarkers of Fabry nephropathy lack sensitivity in detecting early kidney damage and do not predict progression of nephropathy. Urinary extracellular vesicles (uEVs) and their molecular cargo could reflect early changes in renal impairment as they are secreted by the cells lining the urinary tract. We aimed to conduct a proof-of-concept study to investigate whether analysis of uEV characteristics and expression of uEV-derived microRNAs (miRNAs) could be applicable in studies to predict the development and progression of nephropathy in Fabry disease. A total of 20 Fabry patients were divided into two groups, depending on the presence of nephropathy. Chronological urine samples collected during 10-year follow-up were used for uEVs isolation with size exclusion chromatography. Nanoparticle tracking analysis was used to determine concentration and size of uEVs. We evaluated the expression of five uEV-derived miRNAs by qPCR (miR-23a-3p, miR-29a-3p, miR-30b-5p, miR-34a-5p, miR-200a-3p). There was no difference in the concentration and size of uEVs between patients with and without nephropathy at last follow-up or longitudinally. However, we found increased expression of miR-29a-3p and miR-200a-3p in uEVs isolated from chronological samples of patients with Fabry nephropathy. This may indicate an attempt by the organism to prevent the progression of renal damage leading to end-stage renal disease as previously reported in type 1 diabetes. In addition, we found an increased expression of miR-30b-5p in the 10-year period in uEVs of patients without renal dysfunction. miR-30b-5 was reported to have a protective role in podocyte injury and may possibly be important in Fabry nephropathy. These findings indicate that uEVs and their molecular cargo could be a promising target of studies focusing on elucidation of Fabry nephropathy. Nevertheless, total concentration and size of uEVs were neither indicative of the presence nor progression of Fabry nephropathy, while the role of the analyzed miRNAs in Fabry nephropathy progression was merely indicated and needs further in-depth studies.

Extracellular Vesicles: A Novel Tool Facilitating Personalized Medicine and Pharmacogenomics in Oncology.

Biomarkers that can guide cancer therapy based on patients' individual cancer molecular signature can enable a more effective treatment with fewer adverse events. Data on actionable somatic mutations and germline genetic variants, studied by personalized medicine and pharmacogenomics, can be obtained from tumor tissue or blood samples. As tissue biopsy cannot reflect the heterogeneity of the tumor or its temporal changes, liquid biopsy is a promising alternative approach. In recent years, extracellular vesicles (EVs) have emerged as a potential source of biomarkers in liquid biopsy. EVs are a heterogeneous population of membrane bound particles, which are released from all cells and accumulate into body fluids. They contain various proteins, lipids, nucleic acids (miRNA, mRNA, and DNA) and metabolites. In cancer, EV biomolecular composition and concentration are changed. Tumor EVs can promote the remodeling of the tumor microenvironment and pre-metastatic niche formation, and contribute to transfer of oncogenic potential or drug resistance during chemotherapy. This makes them a promising source of minimally invasive biomarkers. A limited number of clinical studies investigated EVs to monitor cancer progression, tumor evolution or drug resistance and several putative EV-bound protein and RNA biomarkers were identified. This review is focused on EVs as novel biomarker source for personalized medicine and pharmacogenomics in oncology. As several pharmacogenes and genes associated with targeted therapy, chemotherapy or hormonal therapy were already detected in EVs, they might be used for fine-tuning personalized cancer treatment.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles.

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.

Plasma Extracellular Vesicle Characteristics Correlate with Tumor Differentiation and Predict Overall Survival in Patients with Pancreatic Ductal Adenocarcinoma Undergoing Surgery with Curative Intent.

Better preoperative characterization of patients with pancreatic ductal adenocarcinoma (PDAC) would aid in treatment optimization. Extracellular vesicles (EV) are promising, largely unexplored biomarkers in PDAC. This study aimed to evaluate if plasma EV characteristics are associated with PDAC clinical characteristics and overall survival (OS). The prospective cohort included 34 PDAC patients undergoing surgery with curative intent. Patient data and plasma samples were collected preoperatively, intraoperatively and one month postoperatively. Small plasma EV (sEV) concentration and size were determined by nanoparticle-tracking analysis. A Mann-Whitney test, Spearman's rho and Cox regression were used in statistical analysis. Preoperatively, patients with poorly differentiated tumors had significantly larger plasma sEVs when compared to patients with well/moderately differentiated tumors (mean diameter 176.9 vs. 149.2 nm, p = 0.021), the sEV size even enabling discrimination of the two groups (AUC = 0.742, 95% CI = 0.560-0.923). Plasma sEV characteristics were also a predictor of OS in multivariable analysis. Patients with a more than 33.8% increase in sEV concentration after one month had 7.2 months shorter median OS (p = 0.002), while patients with a more than 28.0% decrease in sEV size had 9.2 months shorter median OS (p = 0.045). Plasma sEV concentration and size correlate with tumor differentiation and may predict OS in PDAC patients. In the future, plasma sEV characteristics could contribute to improved patient stratification for optimized treatment.

Enrichment of plasma extracellular vesicles for reliable quantification of their size and concentration for biomarker discovery.

Human plasma is a complex fluid, increasingly used for extracellular vesicle (EV) biomarker studies. Our aim was to find a simple EV-enrichment method for reliable quantification of EVs in plasma to be used as biomarker of disease. Plasma of ten healthy subjects was processed using sedimentation rate- (sucrose cushion ultracentrifugation-sUC) and size- (size exclusion chromatography-SEC) based methods. According to nanoparticle tracking analysis (NTA), asymmetrical flow field-flow fractionation coupled to detectors (AF4-UV-MALS), miRNA quantification, transmission electron microscopy and enzyme-linked immunosorbent assay, enrichment of EVs from plasma with sUC method lead to high purity of EVs in the samples. High nanoparticle concentrations after SEC resulted from substantial contamination with lipoproteins and other aggregates of EV-like sizes that importantly affect downstream EV quantification. Additionally, sUC EV-enrichment method linked to quantification with NTA or AF4-UV-MALS is repeatable, as the relative standard deviation of EV size measured in independently processed samples from the same plasma source was 5.4% and 2.1% when analyzed by NTA or AF4-UV-MALS, respectively. In conclusion, the sUC EV-enrichment method is compatible with reliable measurement of concentration and size of EVs from plasma and should in the future be tested on larger cohorts in relation to different diseases. This is one of the first studies using AF4-UV-MALS to quantify EVs in blood plasma, which opens new possible clinical utility for the technique.

Towards defining reference materials for measuring extracellular vesicle refractive index, epitope abundance, size and concentration.

Accurate characterization of extracellular vesicles (EVs) is critical to explore their diagnostic and therapeutic applications. As the EV research field has developed, so too have the techniques used to characterize them. The development of reference materials are required for the standardization of these techniques. This work, initiated from the ISEV 2017 Biomarker Workshop in Birmingham, UK, and with further discussion during the ISEV 2019 Standardization Workshop in Ghent, Belgium, sets out to elucidate which reference materials are required and which are currently available to standardize commonly used analysis platforms for characterizing EV refractive index, epitope abundance, size and concentration. Due to their predominant use among EV researchers, a particular focus is placed on the optical methods nanoparticle tracking analysis and flow cytometry.

Characterization of Plasma-Derived Small Extracellular Vesicles Indicates Ongoing Endothelial and Platelet Activation in Patients with Thrombotic Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is a systemic autoimmune disease, characterized by thrombosis, obstetric complications and the presence of antiphospholipid antibodies (aPL), which drive endothelial injury and thrombophilia. Extracellular vesicles (EVs) have been implicated in endothelial and thrombotic pathologies. Here, we characterized the quantity, cellular origin and the surface expression of biologically active molecules in small EVs (sEVs) isolated from the plasma of thrombotic APS patients (n = 14), aPL-negative patients with idiopathic thrombosis (aPL-neg IT, n = 5) and healthy blood donors (HBD, n = 7). Nanoparticle tracking analysis showed similar sEV sizes (110-170 nm) between the groups, with an increased quantity of sEVs in patients with APS and aPL-neg IT compared to HBD. MACSPlex analysis of 37 different sEV surface markers showed endothelial (CD31), platelet (CD41b and CD42a), leukocyte (CD45), CD8 lymphocyte and APC (HLA-ABC) cell-derived sEVs. Except for CD8, these molecules were comparably expressed in all study groups. sEVs from APS patients were specifically enriched in surface expression of CD62P, suggesting endothelial and platelet activation in APS. Additionally, APS patients exhibited increased CD133/1 expression compared to aPL-neg IT, suggesting endothelial damage in APS patients. These findings demonstrate enhanced shedding, and distinct biological properties of sEVs in thrombotic APS.

The Neurotropic Black Yeast Exophiala dermatitidis Induces Neurocytotoxicity in Neuroblastoma Cells and Progressive Cell Death.

The neurotropic and extremophilic black yeast Exophiala dermatitidis (Herpotrichellaceae) inhabits diverse indoor environments, in particular bathrooms, steam baths, and dishwashers. Here, we show that the selected strain, EXF-10123, is polymorphic, can grow at 37 °C, is able to assimilate aromatic hydrocarbons (toluene, mineral oil, n-hexadecane), and shows abundant growth with selected neurotransmitters (acetylcholine, gamma-aminobutyric acid, glycine, glutamate, and dopamine) as sole carbon sources. We have for the first time demonstrated the effect of E. dermatitidis on neuroblastoma cell model SH-SY5Y. Aqueous and organic extracts of E. dermatitidis biomass reduced SH-SY5Y viability by 51% and 37%, respectively. Melanized extracellular vesicles (EVs) prepared from this strain reduced viability of the SH-SY5Y to 21%, while non-melanized EVs were considerably less neurotoxic (79% viability). We also demonstrated direct interactions of E. dermatitidis with SH-SY5Y by scanning electron and confocal fluorescence microscopy. The observed invasion and penetration of neuroblastoma cells by E. dermatitidis hyphae presumably causes the degradation of most neuroblastoma cells in only three days. This may represent a so far unknown indirect or direct cause for the development of some neurodegenerative diseases such as Alzheimer's.