An extracellular vesicle epitope profile is associated with acute myocardial infarction

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Identification of a serum and urine extracellular vesicle signature predicting renal outcome after kidney transplant

Background A long-standing effort is dedicated towards the identification of biomarkers allowing the prediction of graft outcome after kidney transplant. Extracellular vesicles (EVs) circulating in body fluids represent an attractive candidate, as their cargo mirrors the originating cell and its pathophysiological status. The aim of the study was to investigate EV surface antigens as potential predictors of renal outcome after kidney transplant. Methods We characterized 37 surface antigens by flow cytometry, in serum and urine EVs from 58 patients who were evaluated before, and at 10-14 days, 3 months and 1 year after transplant, for a total of 426 analyzed samples. The outcome was defined according to estimated glomerular filtration rate (eGFR) at 1 year. Results Endothelial cells and platelets markers (CD31, CD41b, CD42a and CD62P) in serum EVs were higher at baseline in patients with persistent kidney dysfunction at 1 year, and progressively decreased after kidney transplant. Conversely, mesenchymal progenitor cell marker (CD1c, CD105, CD133, SSEEA-4) in urine EVs progressively increased after transplant in patients displaying renal recovery at follow-up. These markers correlated with eGFR, creatinine and proteinuria, associated with patient outcome at univariate analysis and were able to predict patient outcome at receiver operating characteristics curves analysis. A specific EV molecular signature obtained by supervised learning correctly classified patients according to 1-year renal outcome. Conclusions An EV-based signature, reflecting the cardiovascular profile of the recipient, and the repairing/regenerative features of the graft, could be introduced as a non-invasive tool for a tailored management of follow-up of patients undergoing kidney transplant

A dynamic clamping approach using in silico IK1 current for discrimination of chamber-specific hiPSC-derived cardiomyocytes

: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) constitute a mixed population of ventricular-, atrial-, nodal-like cells, limiting the reliability for studying chamber-specific disease mechanisms. Previous studies characterised CM phenotype based on action potential (AP) morphology, but the classification criteria were still undefined. Our aim was to use in silico models to develop an automated approach for discriminating the electrophysiological differences between hiPSC-CM. We propose the dynamic clamp (DC) technique with the injection of a specific IK1 current as a tool for deriving nine electrical biomarkers and blindly classifying differentiated CM. An unsupervised learning algorithm was applied to discriminate CM phenotypes and principal component analysis was used to visualise cell clustering. Pharmacological validation was performed by specific ion channel blocker and receptor agonist. The proposed approach improves the translational relevance of the hiPSC-CM model for studying mechanisms underlying inherited or acquired atrial arrhythmias in human CM, and for screening anti-arrhythmic agents

Risk stratification of patients with SARS-CoV-2 by tissue factor expression in circulating extracellular vesicles

Inflammatory response following SARS-CoV-2 infection results in substantial increase of amounts of intravascular pro-coagulant extracellular vesicles (EVs) expressing tissue factor (CD142) on their surface. CD142-EV turned out to be useful as diagnostic biomarker in COVID-19 patients. Here we aimed at studying the prognostic capacity of CD142-EV in SARS-CoV-2 infection. Expression of CD142-EV was evaluated in 261 subjects admitted to hospital for pneumonia and with a positive molecular test for SARS-CoV-2. The study population consisted of a discovery cohort of selected patients (n = 60) and an independent validation cohort including unselected consecutive enrolled patients (n = 201). CD142-EV levels were correlated with post-hospitalization course of the disease and compared to the clinically available 4C Mortality Score as referral. CD142-EV showed a reliable performance to predict patient prognosis in the discovery cohort (AUC = 0.906) with an accuracy of 81.7%, that was confirmed in the validation cohort (AUC = 0.736). Kaplan-Meier curves highlighted a high discrimination power in unselected subjects with CD142-EV being able to stratify the majority of patients according to their prognosis. We obtained a comparable accuracy, being not inferior in terms of prediction of patients' prognosis and risk of mortality, with 4C Mortality Score. The expression of surface vesicular CD142 and its reliability as prognostic marker was technically validated using different immunocapture strategies and assays. The detection of CD142 on EV surface gains considerable interest as risk stratification tool to support clinical decision making in COVID-19

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

Immune profiling of plasma-derived extracellular vesicles identifies Parkinson disease

Objective To develop a diagnostic model based on plasma-derived extracellular vesicle (EV) subpopulations in Parkinson disease (PD) and atypical parkinsonism (AP), we applied an innovative flow cytometric multiplex bead-based platform. Methods Plasma-derived EVs were isolated from PD, matched healthy controls, multiple system atrophy (MSA), and AP with tauopathies (AP-Tau). The expression levels of 37 EV surface markers were measured by flow cytometry and correlated with clinical scales. A diagnostic model based on EV surface markers expression was built via supervised machine learning algorithms and validated in an external cohort. Results Distinctive pools of EV surface markers related to inflammatory and immune cells stratified patients according to the clinical diagnosis. PD and MSA displayed a greater pool of overexpressed immune markers, suggesting a different immune dysregulation in PD and MSA vs AP-Tau. The receiver operating characteristic curve analysis of a compound EV marker showed optimal diagnostic performance for PD (area under the curve [AUC] 0.908; sensitivity 96.3%, specificity 78.9%) and MSA (AUC 0.974; sensitivity 100%, specificity 94.7%) and good accuracy for AP-Tau (AUC 0.718; sensitivity 77.8%, specificity 89.5%). A diagnostic model based on EV marker expression correctly classified 88.9% of patients with reliable diagnostic performance after internal and external validations. Conclusions Immune profiling of plasmatic EVs represents a crucial step toward the identification of biomarkers of disease for PD and AP

Profiling Inflammatory Extracellular Vesicles in Plasma and Cerebrospinal Fluid: An Optimized Diagnostic Model for Parkinson's Disease.

Extracellular vesicles (EVs) play a central role in intercellular communication, which is relevant for inflammatory and immune processes implicated in neurodegenerative disorders, such as Parkinson's Disease (PD). We characterized and compared distinctive cerebrospinal fluid (CSF)-derived EVs in PD and atypical parkinsonisms (AP), aiming to integrate a diagnostic model based on immune profiling of plasma-derived EVs via artificial intelligence. Plasma- and CSF-derived EVs were isolated from patients with PD, multiple system atrophy (MSA), AP with tauopathies (AP-Tau), and healthy controls. Expression levels of 37 EV surface markers were measured by a flow cytometric bead-based platform and a diagnostic model based on expression of EV surface markers was built by supervised learning algorithms. The PD group showed higher amount of CSF-derived EVs than other groups. Among the 17 EV surface markers differentially expressed in plasma, eight were expressed also in CSF of a subgroup of PD, 10 in MSA, and 6 in AP-Tau. A two-level random forest model was built using EV markers co-expressed in plasma and CSF. The model discriminated PD from non-PD patients with high sensitivity (96.6%) and accuracy (92.6%). EV surface marker characterization bolsters the relevance of inflammation in PD and it underscores the role of EVs as pathways/biomarkers for protein aggregation-related neurodegenerative diseases

Osteonecrosis of the Jaws (ONJ) after Bisphosphonate Treatment in Patients with Multiple Myeloma: Decreasing ONJ Incidence after Adoption of Preventive Measures

Bisphosphonates (BPs) are administered to Multiple Myeloma (MM) patients with bone lytic lesion. Osteonecrosis of the Jaw (ONJ) is a complication reported since 2003 in patients treated with intravenous (IV) BPs such as zoledronic acid and pamidronate, with 6%-26.3% frequency in early literature series, before some preventive measures were recommended. We evaluated the occurrence of ONJ with and without dental preventive measures in MM patients treated with BPs in our centre between 1996 and 2015. Since 2005, MM patients (already under treatment or before treatment) underwent a baseline mouth assessment (dental visit, Rx orthopantomography, and eventual tooth avulsion or dental care if necessary) and were followed by a multidisciplinary team. We reviewed the charts of 119 MM patients receiving IV BPs, classified into 3 groups: (a) "historic group" (21 patients who had started BP treatment in years before the awareness of ONJ); (b) "screening group" (20 patients starting BPs without baseline evaluation); and (c) "prevention group" (78 patients starting therapy only after baseline preventive assessment and eventual dental care measures). ONJ was observed in 3/21 patients (14.2%) from group a, in 2/20 patients (10%) from group b, and in no patients from group c (0%). Notably, the median number of IV BP administrations decreased after 2005. Our data confirmed a meaningful reduction of ONJ risk in MM patients treated with BPs if preventive measures are applied. Both implementation of prevention measures and reduction of cumulative doses of IV BPs could have contributed to a decreased incidence of ONJ

Calcification of the Atlanto-Occipital Ligament (Ponticulus Posticus) in Orthodontic Patients: A Retrospective Study

Background: Ponticlus Posticus (PP) is a rare anomaly of the first cervical vertebra easily identifiable in lateral cephalometric radiograph and typically required for orthodontic diagnosis. The aim of this study is to evaluate the PP prevalence in lateral Cephalograms in a cohort of orthodontic patients treated at the Dental School of the University of Bari ‘Aldo Moro’, Italy, and to find possible connection between PP and other dental anomalies, as well as the patient’s cephalometric characteristics. Methods: A total of 150 panoramic radiographs and 150 lateral Cephalograms, obtained for orthodontic use only, were evaluated. No patients were referred to for congenital syndromes or disease, or a history of previously occurred maxillofacial trauma. A detailed cephalometric study was performed for each patient, and the whole cohort was divided according to the common three dental malocclusion classes. The values obtained were analyzed using the Chi-Square Test. Results: PP prevalence was 8% (12 of 150 cases), where the complete and partial forms observed 4.7% and 3.3%, respectively. Although females were more affected (9.6%), no statistically significant sex-related difference was found. Furthermore, no statistically significant relationship regarding age or dental anomalies (dental agenesis/palatal impacted canines) among the groups was observed either. Cephalometric analyses revealed that half of the subjects with PP were sagittal skeletal class I and had vertical hyper-divergence. Conclusions: PP is a frequent anatomical variation of the cervical vertebra, apparently unrelated to the skeletal malocclusion type or dental anomalies. The current study needs to further confirm the congenital hypothesis of PP’s origin already reported in literature

Awake Craniotomy in Conscious Sedation: The Role of A2 Agonists

Background: In Awake Craniotomy (AC), α2-agonists and remifentanil (clonidine and dexmedetomidine) are used in the preoperative phase and throughout the procedure to combine monitored anesthesia care and local anesthesia. The study aims were to specify the key role of α2-agonists administered and to evaluate complication presence/absence in anesthesiologic management. Methods: 42 patients undergoing AC in 3 different centers in the south of Italy (Foggia, San Giovanni Rotondo, and Bari) were recruited. Our protocol involves analgo-sedation by administering Dexmedetomidine and Remifentanil in continuous intravenous infusion, allowing the patient to be sedated and in comfort but contactable and spontaneously breathing. During pre-surgery, the patient is premedicated with intramuscular clonidine (2 µg/kg). In the operating setting, Dexmedetomidine in infusion and Remifentanil in Target Controlled Infusion for effect are started. At the end of the surgical procedure, the infusion of drugs was suspended. Results: There were no intraoperative side effects. The mean duration of interventions was 240 ± 62 min. The average quantity of Remifentanil and Dexmedetomidine infused during interventions were 4.2 ± 1.3 mg and 1.0 ± 0.3 mg, respectively. No significant side effects were described in the post-operative phase. A total of 86% of patients and 93% of surgeons were totally satisfied. Conclusions: Synergy between opioid drugs and α2 agonists plays a fundamental role in ensuring procedure success