Levels Of Extracellular Vesicles Research Articles

Procoagulant effect of extracellular vesicles in patients after transcatheter aortic valve replacement or transcatheter aortic valve replacement with percutaneous coronary intervention.

Patients with severe aortic stenosis (AS) after replacement of the transcatheter aortic valve (TAVR) are more likely to develop thrombotic complications such as cerebral embolism and artificial valve thrombosis. However, the mechanism is not yet well defined. We aimed to explore the plasma extracellular vesicles (EVs) levels and their role in the induction of procoagulant activity (PCA) in patients receiving TAVR alone or TAVR with percutaneous coronary intervention (PCI). EVs were analyzed with flow cytometer. Markers of platelet and endothelial cell activation were quantified using selective enzyme-linked immunosorbent assay (ELISA) kits. Procoagulant activity (PCA) was assessed by clotting time, purified clotting complex assays, and fibrin production assays. Our results confirmed that EVs with positive phosphatedylserin (PS+EV), platelet EVs (PEVs) and positive tissue factor EVs (TF+EVs) were higher in patients following TAVR than before TAVR, particularly in TAVR with PCI. Furthermore, endothelial-derived EVs (EEVs) were also higher in patients after TAVR with PCI than pre-TAVR, however, the EEVs levels in TAVR alone patients were gradually reduce than pre-TAVR. In addition, we further proved that total EVs contributed to dramatically shortened coagulation time, increased intrinsic/extrinsic factor Xa and thrombin generation in patients after TAVR, especially in TAVR with PCI. The PCA was markedly attenuated by approximately 80% with lactucin. Our study reveals a previously unrecognized link between plasma EV levels and hypercoagulability in patients after TAVR, especially TAVR with PCI. Blockade of PS+EVs may improve the hypercoagulable state and prognosis of patients.

Src family kinases engage differential pathways for encapsulation into extracellular vesicles.

Extracellular vesicles (EVs) are heterogeneous biological nanoparticles secreted by all cell types. Identifying the proteins preferentially encapsulated in secreted EVs will help understand their heterogeneity. Src family kinases including Src and Fyn are a group of tyrosine kinases with fatty acylation modifications and/or multiple lysine residues (contributing charge interaction) at their N-terminus. Here, we demonstrate that Src and Fyn kinases were preferentially encapsulated in EVs and fatty acylation including myristoylation and palmitoylation facilitated their encapsulation. Genetic loss or pharmacological inhibition of myristoylation suppressed Src and/or Fyn kinase levels in EVs. Similarly, loss of palmitoylation reduced Fyn levels in EVs. Additionally, mutation of lysine at sites 5, 7, and 9 of Src kinase also inhibited the encapsulation of myristoylated Src into EVs. Knockdown of TSG101, which is a protein involved in the endosomal sorting complexes required for transport (ESCRT) protein complex mediated EVs biogenesis and led to a reduction of Src levels in EVs. In contrast, filipin III treatment, which disturbed the lipid raft structure, reduced Fyn kinase levels, but not Src kinase levels in EVs. Finally, elevated levels of Src protein were detected in the serum EVs of host mice carrying constitutively active Src-mediated prostate tumors in vivo. Collectively, the data suggest that different EVs biogenesis pathways exist and can regulate the encapsulation of specific proteins into EVs. This study provides an understanding of the EVs heterogeneity created by different EVs biogenesis pathways.

P170 Trikafta® modulates release of extracellular vesicles in cystic fibrosis

Dysregulation of CFTR leads to chronic inflammation in Cystic Fibrosis (CF) airways. Extracellular vesicles (EVs) have emerged as an important biomarkers of disease. We previously reported elevated levels of EVs in CF with distinct age-related protein profiles. The levels of EVs and its associated proteins in CF in the presence of CF modulators Trikafta (elexacaftor, tezacaftor, ivacaftor), however remains unexplored. The study aims to characterise and study the effect of Trikafta on EVs and its associated protein targets in CF airway models and serum samples from patients with cystic fibrosis (PWCF). Bronchial brushings from CF patients were passaged and differentiated by air liquid interface (ALI) cultures. After 21 days, TEER was recorded and differentiation of mucociliary layer was confirmed with MUC5AC staining. Further the cultures were treated with Trikafta. EVs released into the apical and basal compartments before and after treatment were extracted by differential centrifugation and characterised by NTA and mass spectrometry (MS). The therapeutic restoration of CFTR was examined using immunoblotting. Serum samples from PWCF on Trikafta were also examined for EV release and content before and after 12 months treatment. After 21 days, of ALI culture. EVs were isolated before and after treatment with Trikafta showed a significant 1.5-fold decrease in EVs in both apical and basal compartments respectively (PWCF (Apical) Control: 4.2E+07 ± 3E+06; Treated 3.2E+07 ± 2E+06; (Basal) Control: 1.3E +08 ± 1.6E+07; Treated 8.1E+07 ± 5E+06) demonstrating that Trikafta treatment diminishes epithelial EV releases. MS based proteomics revealed unique EV protein fingerprints post Trikafta treatment for both epithelial EVs and serum EVs with changes in expression of immune proteins were observed. The study demonstrates that Trikafta can modulate EV release and content in PWCF demonstrating novel mechanisms by which CFTR modulators can regulate the inflammatory landscape in CF.

Predicting ICANS by means of plasma CAR‐T cell derived extracellular vesicles in patients undergoing infusion of anti‐CD19 CAR‐T cells

Introduction: Immune effector cell-associated neurotoxicity syndrome (ICANS) is a life-threatening adverse effect of anti-CD19 chimeric antigen receptor (CAR) T-cell therapy that usually occurs within 5–7 days after cell infusion. Although several clinical and biochemical parameters have been associated with ICANS, it is still a matter of debate how to predict its onset at the patient level. We here tested the hypothesis that CAR-T cell derived extracellular vesicles (EV) carrying the engineered CAR protein and produced early after CAR-T cell activation can be used as predictive biomarker of ICANS. Purposely, we measured plasma CAR+ EV in lymphoma patients underwent anti-CD19 CAR-T cell therapy. Methods: Seventy-one patients with aggressive r/r B-cell lymphomas were admitted to the advanced cell therapy unit of IRCCS AOU of Bologna (NCT04892433) for anti-CD19 CAR-T cell infusion. Included patients received tisa-cel (n = 27), axi-cel (n = 34), or brexu-cel (n = 10) after a median number of 3 prior lines of treatment (2–11); median age was 62 years (19–76) and no patients had CNS disease at the time of CAR-T cell infusion. Twenty out of 71 patients (28%) had ICANS of any grade: 5 patients (7%) ICANS grade 1, 7 patients (10%) ICANS grade 2 and 8 patients (11%) ICANS grade ≥3 (3 patients ICANS grade 3, 3 patients ICANS grade 4 and 2 patients ICANS grade 5 with diffuse cerebral edema). ICANS was classified according to Lee et al. The median time from CAR-T cell infusion to ICANS onset was 5 days (3–12). Available plasma samples at day +1 after CAR-T cell infusion were analyzed for CAR+ EV by FACS analysis. Data analysis was performed with Prism software v9.1.3 (GraphPad). Results: CAR+ EV were already detectable +1 day after CAR-T cell infusion in 58 patients. The median onset of ICANS was at day +5 (3–12). Patients with ICANS of any grade showed higher CAR+ EV level compared to no-ICANS ones (p < 0.0001). CAR+ EV anticipated the median ICANS onset of 2 to 11 days. CAR+ EV ROC analysis showed that a concentration >187.5 CAR+ EV/μl at day +1 after infusion predicts ICANS onset with sensitivity of 100% and specificity of 83.33% (p < 0.0001). Conclusions: These findings lead us to hypothesize that the plasma level of CAR+ EV mirrors target engagement by CAR-T cells, and their massive release is related to ICANS. Thus, CAR+ EV level could be considered a putative early predictor of ICANS onset; further analyses in larger cohorts are warranted to confirm this finding. Keywords: Cellular therapies, Diagnostic and Prognostic Biomarkers Conflicts of interests pertinent to the abstract. P. L. Zinzani Consultant or advisory role: Secura Bio, Celltrion, Gilead, Janssen-Cilag, BMS, Servier, Sandoz, MSD, AstraZeneca, Takeda, Roche, EUSA Pharma, Kyowa Kirin, Novartis, ADC Therapeutics, Incyte, BeiGene Other remuneration: Speakers bureau: Celltrion, Gilead, Janssen-Cilag, BMS, Servier, MSD, AstraZeneca, Takeda, Roche, EUSA Pharma, Kyowa Kirin, Novartis, Incyte, BeiGene F. Bonifazi Consultant or advisory role: Novartis, Gilead

17β-estradiol promotes extracellular vesicle release and selective miRNA loading in ERα-positive breast cancer

The causes and consequences of abnormal biogenesis of extracellular vesicles (EVs) are not yet well understood in malignancies, including in breast cancers (BCs). Given the hormonal signaling dependence of estrogen receptor-positive (ER+) BC, we hypothesized that 17β-estradiol (estrogen) might influence EV production and microRNA (miRNA) loading. We report that physiological doses of 17β-estradiol promote EV secretion specifically from ER+ BC cells via inhibition of miR-149-5p, hindering its regulatory activity on SP1, a transcription factor that regulates the EV biogenesis factor nSMase2. Additionally, miR-149-5p downregulation promotes hnRNPA1 expression, responsible for the loading of let-7's miRNAs into EVs. In multiple patient cohorts, we observed increased levels of let-7a-5p and let-7d-5p in EVs derived from the blood of premenopausal ER+ BC patients, and elevated EV levels in patients with high BMI, both conditions associated with higher levels of 17β-estradiol. In brief, we identified a unique estrogen-driven mechanism by which ER+ BC cells eliminate tumor suppressor miRNAs in EVs, with effects on modulating tumor-associated macrophages in the microenvironment.

Circulating Extracellular Vesicle Levels in Patients with Coronavirus Disease 2019 Coagulopathy: A Prospective Cohort Study.

Coronavirus disease 2019 (COVID-19) is associated with coagulopathy. However, the underlying mechanisms are not completely understood. We evaluated the association between COVID-19 coagulopathy and extracellular vesicle (EV) levels. We hypothesized that several EV levels would be higher in COVID-19 coagulopathy patients than in non-coagulopathy patients. This prospective observational study was conducted in four tertiary care faculties in Japan. We enrolled 99 COVID-19 patients (48 with coagulopathy and 51 without coagulopathy) aged ≥20 years who required hospitalization, and 10 healthy volunteers; we divided the patients into coagulopathy and non-coagulopathy groups according to the D-dimer levels (≥1 μg/mL and <1 μg/mL, respectively). We used flow cytometry to measure the tissue-factor-bearing, endothelium-derived, platelet-derived, monocyte-derived, and neutrophil-derived EV levels in platelet-free plasma. The EV levels were compared between the two COVID-19 groups as well as among the coagulopathy patients, non-coagulopathy patients, and healthy volunteers. No significant difference was found in EV levels between the two groups. Meanwhile, the cluster of differentiation (CD) 41 + EV levels were significantly higher in COVID-19 coagulopathy patients than in healthy volunteers (549.90 [255.05-984.65] vs. 184.3 [150.1-254.1] counts/µL, p = 0.011). Therefore, CD41+ EVs might play an essential role in COVID-19 coagulopathy development.

Circulating extracellular vesicles are associated with the clinical outcomes of sepsis.

Sepsis is associated with endothelial cell (EC) dysfunction, increased vascular permeability and organ injury, which may lead to mortality, acute respiratory distress syndrome (ARDS) and acute renal failure (ARF). There are no reliable biomarkers to predict these sepsis complications at present. Recent evidence suggests that circulating extracellular vesicles (EVs) and their content caspase-1 and miR-126 may play a critical role in modulating vascular injury in sepsis; however, the association between circulating EVs and sepsis outcomes remains largely unknown. We obtained plasma samples from septic patients (n=96) within 24 hours of hospital admission and from healthy controls (n=45). Total, monocyte- or EC-derived EVs were isolated from the plasma samples. Transendothelial electrical resistance (TEER) was used as an indicator of EC dysfunction. Caspase-1 activity in EVs was detected and their association with sepsis outcomes including mortality, ARDS and ARF was analyzed. In another set of experiments, total EVs were isolated from plasma samples of 12 septic patients and 12 non-septic critical illness controls on days 1, and 3 after hospital admission. RNAs were isolated from these EVs and Next-generation sequencing was performed. The association between miR-126 levels and sepsis outcomes such as mortality, ARDS and ARF was analyzed. Septic patients with circulating EVs that induced EC injury (lower transendothelial electrical resistance) were more likely to experience ARDS (p<0.05). Higher caspase-1 activity in total EVs, monocyte- or EC-derived EVs was significantly associated with the development of ARDS (p<0.05). MiR-126-3p levels in EC EVs were significantly decreased in ARDS patients compared with healthy controls (p<0.05). Moreover, a decline in miR-126-5p levels from day 1 to day 3 was associated with increased mortality, ARDS and ARF; while decline in miR-126-3p levels from day 1 to day 3 was associated with ARDS development. Enhanced caspase-1 activity and declining miR-126 levels in circulating EVs are associated with sepsis-related organ failure and mortality. Extracellular vesicular contents may serve as novel prognostic biomarkers and/or targets for future therapeutic approaches in sepsis.

Clinical-Grade Patches as a Medium for Enrichment of Sweat-Extracellular Vesicles and Facilitating Their Metabolic Analysis.

Cell-secreted extracellular vesicles (EVs), carrying components such as RNA, DNA, proteins, and metabolites, serve as candidates for developing non-invasive solutions for monitoring health and disease, owing to their capacity to cross various biological barriers and to become integrated into human sweat. However, the evidence for sweat-associated EVs providing clinically relevant information to use in disease diagnostics has not been reported. Developing cost-effective, easy, and reliable methodologies to investigate EVs' molecular load and composition in the sweat may help to validate their relevance in clinical diagnosis. We used clinical-grade dressing patches, with the aim being to accumulate, purify and characterize sweat EVs from healthy participants exposed to transient heat. The skin patch-based protocol described in this paper enables the enrichment of sweat EVs that express EV markers, such as CD63. A targeted metabolomics study of the sweat EVs identified 24 components. These are associated with amino acids, glutamate, glutathione, fatty acids, TCA, and glycolysis pathways. Furthermore, as a proof-of-concept, when comparing the metabolites' levels in sweat EVs isolated from healthy individuals with those of participants with Type 2 diabetes following heat exposure, our findings revealed that the metabolic patterns of sweat EVs may be linked with metabolic changes. Moreover, the concentration of these metabolites may reflect correlations with blood glucose and BMI. Together our data revealed that sweat EVs can be purified using routinely used clinical patches, setting the foundations for larger-scale clinical cohort work. Furthermore, the metabolites identified in sweat EVs also offer a realistic means to identify relevant disease biomarkers. This study thus provides a proof-of-concept towards a novel methodology that will focus on the use of the sweat EVs and their metabolites as a non-invasive approach, in order to monitor wellbeing and changes in diseases.

Aquaporin 5—in Extracellular Vesicles of Human Vitreous as a Potential Marker for Fungal Endophthalmitis

Purpose Extracellular vesicles (EVs) are lipid-bilayered nanoparticles that play an important role in cellular cross-talk, and as received attention for their role as diseases biomarker. Aquaporin-5 (AQP5) is a small integral membrane protein that help in the migration of cells, proliferation, and invasion. However, the association of AQP5 with fungal diseases is still unknown. The aim of this study was to evaluate the expression of AQP5 in EVs (EV-AQP5) extracted from the vitreous of patients with Fungal Endophthalmitis (FE). Methods Vitreous fluid was collected from 20 patients clinically suspected as FE, 10 patients from non-infectious conditions, and 10 patients with bacterial endophthalmitis as controls. EVs were isolated from human vitreous and characterized by dynamic light scattering, and scanning electron microscopy. Human Aquaporin-5 levels were evaluated using a commercial ELISA Kit. The Receiver Operating Characteristic (ROC) curves and its significance were correlated with microbiology data. Results Isolated EVs size were approx.250–380 nm in diameter. The measured levels of EV-AQP5 resulted significantly higher in FE patients (mean=216±15pg/ml; 95% confidence interval (CI): 182–250) in comparison to controls (mean=130±12pg/ml; 95%CI: 111–166)(p = .001). However, AQP5 levels in EVs derived from culture-proven bacteria patients were insignificant compared to controls (mean=169±4 pg/ml; 95%CI: 161–177). ROC curve was used to define the optimal cut-off level of the test at 180 pg/ml with an AUC of 98% (95%CI: 95–100) (p = .03), with a sensitivity of 100% and specificity of 90%. Additionally, the AQP5 level in EVs derived from culture-negative vitreous was above the threshold value (200 ± 10 pg/ml (95%CI: 180–230) in comparison to the control group (p < .001) However, no significant association was found between age or visual acuity and the level of AQP5 in FE. Conclusion Our results reveal that the vitreous EV-AQP5 levels can aid in differentiating FE from non-infectious retinal conditions, mainly when the cultures are negative.

Effect of Genetic Factors, Age and Sex on Levels of Circulating Extracellular Vesicles and Platelets.

Extracellular vesicles (EVs) mediate cell interactions in biological processes, such as receptor activation or molecule transfer. Estimates of variation by age and sex have been limited by small sample size, and no report has assessed the contribution of genetic factors to levels of EVs. Here, we evaluated blood levels of 25 EV and 3 platelet traits in 974 individuals (933 genotyped) and reported the first genome-wide association study (GWAS) on levels of these traits. EV levels all decreased with age, whereas the trend for their surface markers was more heterogeneous. Platelets and CD31dim platelet EVs significantly increased in females compared to males, although CD31 expression on both platelets and platelet EVs decreased in females. Levels of the other EV subsets were similar between sexes. GWAS revealed three statistically significant genetic signals associated with EV levels in the F10 and GBP1 genes and in the intergenic region between LRIG1 and KBTBD8. These add to a signal in the 3'UTR of RHOF associated with CD31 expression on platelets that was previously found to be associated with other platelet traits. These findings suggest that EV formation is not a simple, constant adjunct of metabolism but is under both age-related and genetic control that can be independent of the regulation of the levels of the cells from which the EVs derive.

Metabolomic signatures distinguish extracellular vesicles from osteoclasts and odontoclasts.

Pathological dental root resorption and alveolar bone loss are often detected only after irreversible damage. Biomarkers in the gingival crevicular fluid or saliva could provide a means for early detection; however, such biomarkers have proven elusive. We hypothesize that a multiomic approach might yield reliable diagnostic signatures for root resorption and alveolar bone loss. Previously, we showed that extracellular vesicles (EVs) from osteoclasts and odontoclasts differ in their protein composition. In this study, we investigated the metabolome of EVs from osteoclasts, odontoclasts and clasts (non-resorbing clastic cells). Mouse haematopoietic precursors were cultured on dentine, bone or plastic, in the presence of recombinant RANKL and CSF-1 to trigger differentiation along the clastic line. On Day 7, the cells were fixed and the differentiation state and resorptive status of the clastic cells were confirmed. EVs were isolated from the conditioned media on Day 7 and characterized by nanoparticle tracking and electron microscopy to ensure quality. Global metabolomic profiling was performed using a Thermo Q-Exactive Orbitrap mass spectrometer with a Dionex UHPLC and autosampler. We identified 978 metabolites in clastic EVs. Of those, 79 are potential biomarkers with Variable Interdependent Parameters scores of 2 or greater. Known metabolites cytidine, isocytosine, thymine, succinate and citrulline were found at statistically higher levels in EVs from odontoclasts compared with osteoclasts. We conclude that numerous metabolites found in odontoclast EVs differ from those in osteoclast EVs, and thus represent potential biomarkers for root resorption and periodontal tissue destruction.

PD17-09 CD70-POSITIVE EXTRACELLULAR VESICLES FOR DETECTION OF CLEAR-CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyCME1 Apr 2023PD17-09 CD70-POSITIVE EXTRACELLULAR VESICLES FOR DETECTION OF CLEAR-CELL RENAL CELL CARCINOMA Rodrigo R. Pessoa, Reza Nabavizadeh, Ali Arafa, Roxane Lavoie, Yohan Kim, Liguo Wang, Jeanette Eckel-Passow, Bradley C. Leibovich, and Fabrice Lucien Rodrigo R. PessoaRodrigo R. Pessoa More articles by this author , Reza NabavizadehReza Nabavizadeh More articles by this author , Ali ArafaAli Arafa More articles by this author , Roxane LavoieRoxane Lavoie More articles by this author , Yohan KimYohan Kim More articles by this author , Liguo WangLiguo Wang More articles by this author , Jeanette Eckel-PassowJeanette Eckel-Passow More articles by this author , Bradley C. LeibovichBradley C. Leibovich More articles by this author , and Fabrice LucienFabrice Lucien More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003272.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: One third of patients treated for localized clear-cell renal cell carcinoma (ccRCC) will eventually develop recurrent disease after surgery. Identification of patients who may benefit from intensification of care with adjuvant therapy is an important unmet need. Tumor-derived extracellular vesicles (EVs), microscopic particles released by tumor cells, have emerged as a potential biomarker in other malignancies but have not been studied in kidney cancer. Herein, we sought to characterize tumor specific EVs levels among patients undergoing radical nephrectomy for ccRCC. METHODS: We employed in silico multi-omic analysis of ccRCC tumors using TCGA KIRC (N=537 patients), CPTAC (N= 103 patients) and GTEx (N= 948 donors) to identify CD70, an integral membrane protein, as the top marker candidate for ccRCC-derived EVs. Expression of CD70+ in ccRCC-derived EVs was confirmed using freshly isolated tumor specimens. Blood levels of circulating CD70+-EVs were measured using high resolution flow cytometry in a cohort of age-matched healthy donors (HD) (N=15) and ccRCC patients (N=29) treated with radical nephrectomy. EVs levels between HD and RCC patients were compared using ANOVA test. Association between patient characteristics and EVs levels was analyzed using Pearson’s correlation and Fisher’s exact test. RESULTS: Blood levels of CD70+-EVs were significantly elevated in patients with pT1-T2 (median: 4.3×106 per mL, p<0.01) and pT3-T4 (median: 6.0×106, p<0.001) ccRCC compared with HD (median: 0.4×106, Figure 1A). High CD70+-EVs levels were associated with pT3-T4 stage (Figure 1B, p<0.0001).There was a strong positive correlation between CD70+-EVs at diagnosis and tumor size (Pearson r=0.76, p<0.0001, Figure 1C). Radical nephrectomy was associated with reduction in CD70+-EVs levels in 83.8% of patients (Figure 1D, p=0.0076). CONCLUSIONS: Patients with ccRCC, in particular those with larger and more advanced stage tumors, have high levels of circulating CD70+-EVs, which decreases significantly after surgery. CD70+-EVs levels are a marker of tumor burden in ccRCC and could be further explored as a biomarker for risk-stratification and selection of patients for additional treatment after radical nephrectomy for ccRCC. Source of Funding: Eagles 5th District Cancer Telethon - Cancer Research Fund (FL) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e499 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rodrigo R. Pessoa More articles by this author Reza Nabavizadeh More articles by this author Ali Arafa More articles by this author Roxane Lavoie More articles by this author Yohan Kim More articles by this author Liguo Wang More articles by this author Jeanette Eckel-Passow More articles by this author Bradley C. Leibovich More articles by this author Fabrice Lucien More articles by this author Expand All Advertisement PDF downloadLoading ...

Vascular Extracellular Vesicles Indicate Severe Hepatopulmonary Syndrome in Cirrhosis

Hepatopulmonary syndrome (HPS) is a pulmonary vasculature complication in the setting of liver disease that is characterized by pathological vasodilation resulting in arterial oxygenation defects. We investigated the role of extracellular vesicles (EV) in cirrhosis patients with HPS, as well as the functional effect of EV administration in a common bile duct ligation (CBDL) HPS mouse model. A total of 113 cirrhosis patients were studied: 42 (Gr. A) with HPS and 71 (Gr. B) without HPS, as well as 22 healthy controls. Plasma levels of EV associated with endothelial cells, epithelial cells, and hepatocytes were measured. The cytokine cargoes were estimated using ELISA. The effect of EV administered intranasally in the CBDL mouse model was investigated for its functional effect in vascular remodeling and inflammation. We found endothelial cells (EC) associated EV (EC-EV) were elevated in cirrhosis patients with and without HPS (p < 0.001) than controls. EC-EV levels were higher in HPS patients (p = 0.004) than in those without HPS. The epithelial cell EVs were significantly high in cirrhosis patients than controls (p < 0.001) but no changes found in patients with HPS than without. There was a progressive increase in EC-EV levels from mild to severe intrapulmonary shunting in HPS patients (p = 0.02 mild vs. severe), and we were able to predict severe HPS with an AUROC of 0.85; p < 0.001. An inverse correlation of EC-EVs was found with hemoglobin (r = -0.24; p = 0.031) and PaO2 (r = 0.690; p = 0.01) and a direct correlation with MELD (r = 0.32; p = 0.014). Further, both TNF-α (p = 0.001) and IL-1β (p = 0.021) as cargo levels were significantly elevated inside the EVs of HPS patients than without HPS. Interestingly, upon administration of intranasal EVs, there was a significant decrease in Evans blue accumulation and lung wet-dry ratio (p = 0.042; 0.038). A significant reduction was also noticed in inflammation and cholestasis. High levels of plasma EC-EV levels were found in patients with HPS with elevated pro-inflammatory cytokine cargoes. EC-EVs were indicative of severe HPS condition. In the CBDL HPS model, we were able to prove the beneficial effects of improving vascular tone, inflammation, and liver pathogenesis.

NKG2A and circulating extracellular vesicles are key regulators of natural killer cell activity in prostate cancer after prostatectomy.

Extracellular vesicles (EVs) are an important regulatory factor for natural killer cell activity (NKA) in the tumor microenvironment. The relationship between circulating EVs in the peripheral blood and natural killer (NK) cells in prostate cancer (PCa) is unclear. This study aimed at investigating the key regulators in the interaction between circulating EVs and NK cells in PCa patients before and after tumor removal. NK-cell characteristics were prospectively assessed in 79 patients treated with robot-assisted laparoscopic radical prostatectomy preoperatively and postoperatively. Compared with healthy donors, the existence of prostate tumors increased the number of circulating EVs and altered ligand expression of EVs. Circulating EVs extracted from cancer patients significantly decreased NKA of NK cells compared with those extracted from healthy donors. Upon treatment with an inhibiting antibody or small interfering RNA, natural killer cell protein group 2A (NKG2A) was identified as the main NKA regulator in cancer patients for accepting the signal from circulating EVs. After surgery, NKA was increased and NKG2A expression on NK cells was significantly reduced. The expression of ligands for natural killer cell protein group 2D (NKG2D) on EVs and the level of circulation EVs both significantly increased. With the decrease in NKG2A levels on NK cells and the increase in total NKG2D ligands on circulating EVs, which was increased postoperatively, both NKG2A on NK cells and NKG2D ligands on circulating exosomes are main regulators of NKA restoration after prostatectomy.

Heat shock protein A2 is a novel extracellular vesicle-associated protein

70-kDa Heat Shock Proteins (HSPA/HSP70) are chaperones playing a central role in the proteostasis control mechanisms. Their basal expression can be highly elevated as an adaptive response to environmental and pathophysiological stress conditions. HSPA2, one of poorly characterised chaperones of the HSPA/HSP70 family, has recently emerged as epithelial cells differentiation-related factor. It is also commonly expressed in cancer cells, where its functional significance remains unclear. Previously, we have found that proteotoxic stress provokes a decrease in HSPA2 levels in cancer cells. In the present study we found that proteasome inhibition-related loss of HSPA2 from cancer cells neither is related to a block in the gene transcription nor does it relate to increased autophagy-mediated disposals of the protein. Proteotoxic stress stimulated extracellular release of HSPA2 in extracellular vesicles (EVs). Interestingly, EVs containing HSPA2 are also released by non-stressed cancer and normal cells. In human urinary EVs levels of HSPA2 were correlated with the levels of TSG101, one of the main EVs markers. We conclude that HSPA2 may constitute basic components of EVs. Nevertheless, its specific role in EVs and cell-to-cell communication requires further investigation.

SIV Infection Regulates Compartmentalization of Circulating Blood Plasma miRNAs within Extracellular Vesicles (EVs) and Extracellular Condensates (ECs) and Decreases EV-Associated miRNA-128.

SIV Infection Regulates Compartmentalization of Circulating Blood Plasma miRNAs within Extracellular Vesicles (EVs) and Extracellular Condensates (ECs) and Decreases EV-Associated miRNA-128.

P117 Low-Intensity Pulsed Ultrasound as a new approach of gut disruptive liquid biopsy to boost the release of mucosal extracellular vesicles in Ulcerative Colitis

Abstract Background Ulcerative Colitis (UC) is a chronic, idiopathic intestinal inflammatory disease whose diagnosis requires colonoscopy with biopsy. There is an unmet need for sensitive, and easy-to-detect biomarkers helping a rapid and non-invasive diagnosis, and suitable for following disease progression. Extracellular vesicles (EVs) have been proposed as promising carriers of biomarkers. Nevertheless, their plasma levels remain very low and thus difficult to detect. We explored Low-Intensity Pulsed Ultrasound (LIPUS) as a novel and safe approach to enhance the mucosal release of EVs in experimental models of UC. Methods LIPUS was applied at a frequency of 38 kHz and at an intensity of 150 mW/cm2 for 3 min, using devices dedicated to in vitro or in vivo studies. Primary intestinal fibroblasts, Human Intestinal microvasculature endothelial cells (HIMEC) and peripheral blood mononuclear cells (PBMC) were isolated from UC patients and healthy volunteers (n=6). Cell viability was tested in Caco-2 and all primary cells using MTT assay. Computer acoustic simulations, allowing a reliable control of the energy dose in the colon, were carried out using k-Wave software to translate in vivo the LIPUS. Acute and chronic colitis models were induced in C57BL/6N mice by administration of dextran sodium sulfate (DSS) respectively 2 and 2.5% ad libitum in their drinking water for seven days for the acute and three cycles of DDS for the chronic. Mice were monitored daily for body weight loss, bleeding and stool consistency. EVs were characterized in supernatants, plasma and mucosa at NanoSight and inflammatory mediators detected 1, 2 and 24 h after LIPUS by ELISA assay. Apoptosis and cell proliferation were evaluated by TUNEL and Ki67 stainings. Results After LIPUS, all cells appeared 100% viable and no pro-inflammatory changes were observed. In Caco-2 cells, fibroblasts and HIMEC, LIPUS significantly decreased the levels of IL-8. The maximum release of EVs, range in size from 100 to 120 nm, was recorded after 60 min in resident cells from both UC and healthy-derived cells. Conversely, PBMC displayed a progressive decrease. In both acute and chronic models, the physiological cellular turnover of the mucosa resulted unchanged after LIPUS. A significative increase of EVs was observed after 2 h of stimulation, and dropped down after 24 h. The levels of EVs were higher (p&amp;lt;0.01) in the chronic rather than acute phase of colitis. Conclusion LIPUS proved to be a safe and non pro-inflammatory approach to boost the release of EVs accumulated into the mucosa increasing their levels in the bloodstream. This feature is transient and more evident in the short term. Overall, the LIPUS could pave the way to a new era of a gut disruptive liquid biopsy.

316Engineered extracellular vesicle-mediated delivery of miR-199a-3p increases the viability of 3D-printed cardiac patches.

In recent years, extrusion-based three-dimensional (3D) bioprinting is employed for engineering cardiac patches (CP) due to its ability to assemble complex structures from hydrogel-based bioinks. However, the cell viability in such CPs is low due to shear forces applied on the cells in the bioink, inducing cellular apoptosis. Herein, we investigated whether the incorporation of extracellular vesicles (EVs) in the bioink, engineered to continually deliver the cell survival factor miR-199a-3p would increase the viability within the CP. EVs from THP-1-derived activated macrophages (MΦ) were isolated and characterized by nanoparticle tracking analysis (NTA), cryogenic electron microscopy (cryo-TEM), and Western blot analysis. MiR-199a-3p mimic was loaded into EVs by electroporation after optimization of applied voltage and pulses. Functionality of the engineered EVs was assessed in neonatal rat cardiomyocyte (NRCM) monolayers using immunostaining for the proliferation markers ki67 and Aurora B kinase. To examine the effect of engineered EVs on 3D-bioprinted CP viability, the EVs were added to the bioink, consisting of alginate-RGD, gelatin, and NRCM. Metabolic activity and expression levels of activated-caspase 3 for apoptosis of the 3D-bioprinted CP were evaluated after 5 days. Electroporation (850 V with 5 pulses) was found to be optimal for miR loading; miR-199a-3p levels in EVs increased fivefold compared to simple incubation, with a loading efficiency of 21.0%. EV size and integrity were maintained under these conditions. Cellular uptake of engineered EVs by NRCM was validated, as 58% of cTnT+ cells internalized EVs after 24 h. The engineered EVs induced CM proliferation, increasing the ratio of cell-cycle re-entry of cTnT+ cells by 30% (Ki67) and midbodies+ cell ratio by twofold (Aurora B) compared with the controls. The inclusion of engineered EVs in bioink yielded CP with threefold greater cell viability compared to bioink with no EVs. The prolonged effect of EVs was evident as the CP exhibited elevated metabolic activities after 5 days, with less apoptotic cells compared to CP with no EVs. The addition of miR-199a-3p-loaded EVs to the bioink improved the viability of 3D-printed CP and is expected to contribute to their integration in vivo.

Peripheral blood cellular profile at pre-lymphodepletion is associated with CD19-targeted CAR-T cell-associated neurotoxicity.

Infusion of second generation autologous CD19-targeted chimeric antigen receptor (CAR) T cells in patients with R/R relapsed/refractory B-cell lymphoma (BCL) is affected by inflammatory complications, such as Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS). Current literature suggests that the immune profile prior to CAR-T infusion modifies the chance to develop ICANS. This is a monocenter prospective study on 53 patients receiving approved CAR T-cell products (29 axi-cel, 24 tisa-cel) for R/R-BCL. Clinical, biochemical, and hematological variables were analyzed at the time of pre-lymphodepletion (pre-LD). In a subset of 21 patients whose fresh peripheral blood sample was available, we performed cytofluorimetric analysis of leukocytes and extracellular vesicles (EVs). Moreover, we assessed a panel of soluble plasma biomarkers (IL-6/IL-10/GDF-15/IL-15/CXCL9/NfL) and microRNAs (miR-146a-5p, miR-21-5p, miR-126-3p, miR-150-5p) which are associated with senescence and inflammation. Multivariate analysis at the pre-LD time-point in the entire cohort (n=53) showed that a lower percentage of CD3+CD8+ lymphocytes (38.6% vs 46.8%, OR=0.937 [95% CI: 0.882-0.996], p=0.035) and higher levels of serum C-reactive protein (CRP, 4.52 mg/dl vs 1.00 mg/dl, OR=7.133 [95% CI: 1.796-28], p=0.005) are associated with ICANS. In the pre-LD samples of 21 patients, a significant increase in the percentage of CD8+CD45RA+CD57+ senescent cells (median % value: 16.50% vs 9.10%, p=0.009) and monocytic-myeloid derived suppressor cells (M-MDSC, median % value: 4.4 vs 1.8, p=0.020) was found in ICANS patients. These latter also showed increased levels of EVs carrying CD14+ and CD45+ myeloid markers, of the myeloid chemokine CXCL-9, as well of the MDSC-secreted cytokine IL-10. Notably, the serum levels of circulating neurofilament light chain, a marker of neuroaxonal injury, were positively correlated with the levels of senescent CD8+ T cells, M-MDSC, IL-10 and CXCL-9. No variation in the levels of the selected miRNAs was observed between ICANS and no-ICANS patients. Our data support the notion that pre-CAR-T systemic inflammation is associated with ICANS. Higher proportion of senescence CD8+ T cells and M-MDSC correlate with early signs of neuroaxonal injury at pre-LD time-point, suggesting that ICANS may be the final event of a process that begins before CAR-T infusion, consequence to patient clinical history.

H-ferritin in sows' colostrum- and milk-derived extracellular vesicles: a novel iron delivery concept.

Iron deficiency anemia is a significant problem in piglets, as they are born with insufficient iron stores for supporting their rapid body growth. Further, sows' milk contains inadequate iron levels for meeting the demands of piglet rapid growth in the pre-wean stage. The forms of iron present in the milk are essential to understanding bioavailability and potential routes for supplementing iron to mitigate iron deficiency anemia in piglets. Recently, our studies showed that H-ferritin (FTH1) is involved in iron transport to different tissues and can be used as an oral iron supplement to correct iron deficiency in rats and monkeys. In this study, we investigate the FTH1 levels in colostrum and milk in Yorkshires-crossbred sows (n = 27) and collected samples at the 1st, 15th, and 28th days of lactation to measure FTH1. Colostrum and milk were found to have FTH1, but there is no significant difference between the different days of lactation. FTH1 has been observed to be enriched in extracellular vesicles (EVs) of other species, and therefore examined the EVs in the samples. Colostrum-derived EVs were enriched with L-ferritin compared to FTH1, while in milk-derived EVs, only FTH1 was detected (P = 0.04). In milk-derived EVs, FTH1 was significantly higher (P = 0.021; P = 006) than FTH1 in colostrum-derived EVs. Furthermore, FTH1 levels of milk-derived EVs were significantly higher (P = 0.0002; P = 0004) than whole milk and colostrum FTH1. These results indicate that FTH1 is enriched in the milk-derived EVs and suggest that EVs play a predominant role in the FTH1 delivery mechanism for the piglet. The extent to which FTH1 in EVs accounts for the overall iron delivery mechanism in piglets is yet to be determined.