Extracellular Vesicles In Patients Research Articles

Expression and Diagnostic Value of tRNA-Derived Fragments Secreted by Extracellular Vesicles in Hypopharyngeal Carcinoma.

ObjectiveTo detect the expression level of tRNA-derived fragments secreted by extracellular vesicles in hypopharyngeal cancer and explore the influence of tRNA-derived fragments on the occurrence of hypopharyngeal cancer and lung metastasis.MethodsAfter high-speed centrifugation, tRNA, which was extracted from the extracellular vesicles of patients with hypopharyngeal cancer and healthy subjects, was sequenced using microarrays. The expression of three differentially expressed tRNAs in hypopharyngeal cancer, healthy subjects, human normal laryngeal epithelial cells and hypopharyngeal cancer line was detected by qRT-PCR. The correlation between the upregulated tRNA, as identified by qRT-PCR, and the clinicopathological features of the non-lung metastatic patients was further analyzed. Finally, the expressions of upregulated tRNA were compared between the non-lung metastatic and lung metastatic patients. The risk factors of hypopharyngeal cancer with lung metastatic were identified by the Cox regression analysis.ResultsBy high-speed centrifugation, extracellular vesicles were extracted successfully. It was found that a variety of tRNAs in the extracellular vesicles from patients with hypopharyngeal cancer by sequencing. qRT-PCR validation indicated that tRF-1:30-Lys-CTT-1-M2 was significantly overexpressed in hypopharyngeal cancer patients and tumor cells, especially in lung metastatic patients. It was indicated that tRF-1:30-Lys-CTT-1-M2 overexpression was closely related to such pathological features as tumor staging, differentiation grade, smoking history and drinking history. According to the Cox regression analysis, stage III–IV, smoking history, drinking history and tRF-1:30-Lys-CTT-1-M2 overexpression were independent risk factors for metastasis of hypopharyngeal cancer.ConclusiontRF-1:30-Lys-CTT-1-M2 was overexpressed in patients with hypopharyngeal cancer and was identified as an independent risk factor for lung metastasis. It can be used as a novel biomarker for the diagnosis and lung metastasis monitoring of hypopharyngeal cancer.

Effects of Lactobacillus plantarum CJLP55 on Clinical Improvement, Skin Condition and Urine Bacterial Extracellular Vesicles in Patients with Acne Vulgaris: A Randomized, Double-Blind, Placebo-Controlled Study.

Lactobacillus plantarum CJLP55 has anti-pathogenic bacterial and anti-inflammatory activities in vitro. We investigated the dietary effect of CJLP55 supplement in patients with acne vulgaris, a prevalent inflammatory skin condition. Subjects ingested CJLP55 or placebo (n = 14 per group) supplements for 12 weeks in this double-blind, placebo-controlled randomized study. Acne lesion count and grade, skin sebum, hydration, pH and surface lipids were assessed. Metagenomic DNA analysis was performed on urine extracellular vesicles (EV), which indirectly reflect systemic bacterial flora. Compared to the placebo supplement, CJLP55 supplement improved acne lesion count and grade, decreased sebum triglycerides (TG), and increased hydration and ceramide 2, the major ceramide species that maintains the epidermal lipid barrier for hydration. In addition, CJLP55 supplement decreased the prevalence of Proteobacteria and increased Firmicutes, which were correlated with decreased TG, the major skin surface lipid of sebum origin. CJLP55 supplement further decreased the Bacteroidetes:Firmicutes ratio, a relevant marker of bacterial dysbiosis. No differences in skin pH, other skin surface lipids or urine bacterial EV phylum were noted between CJLP55 and placebo supplements. Dietary Lactobacillus plantarum CJLP55 was beneficial to clinical state, skin sebum, and hydration and urine bacterial EV phylum flora in patients with acne vulgaris.

Proteomic profile of melanoma cell-derived small extracellular vesicles in patients' plasma: a potential correlate of melanoma progression.

Molecular profiling of small extracellular vesicles (sEV) isolated from plasma of cancer patients emerges as promising strategy for biomarkers discovery. We investigated the proteomic profiles of sEV immunoselected using anti‐CSPG4 antibodies from 15 melanoma patients’ plasma. The proteomes of sEV separated into melanoma cell‐derived (MTEX) and non‐malignant cell‐derived (NMTEX) were compared using high‐resolution mass spectrometry. Paired analysis identified the MTEX‐associated profile of 16 proteins that discriminated MTEX from NMETEX. We also identified the MTEX profile that discriminated between seven patients with no evidence of melanoma (NED) after therapy and eight with progressive disease (PD). Among 75 MTEX proteins overexpressed in PD patients, PDCD6IP (ALIX) had the highest discriminating value, while CNTN1 (contactin‐1) was upregulated only in MTEX of NED patients. This is the first report documenting that proteomes of tumour‐derived sEV in patients’ plasma discriminate cancer from non‐cancer and identify proteins with potential to serve as prognostic biomarkers in melanoma.

Microbiome as a potential diagnostic and predictive biomarker in severe alcoholic hepatitis

Severe alcoholic hepatitis (AH) is the most aggressive form of alcohol-related liver disease with high mortality. The microbiome is an emerging therapeutic target in alcohol-related liver disease. To investigate the microbiome composition in patients with severe AH, and to determine microbiome recovery after rifaximin treatment in gut bacteria and bacteria derived-extracellular vesicles. We enrolled 24 patients with severe AH and 24 healthy controls. Additional faecal samples were collected after 4weeks in 8 patients with severe AH who completed rifaximin treatment. Treatment response was defined based on Lille score model after 7days of treatment. Metagenomic profiling was performed using 16S ribosomal RNA amplicon sequencing. Faecal microbiomes of patients with severe AH had lower alpha diversity and higher beta diversity than those of healthy controls in both gut bacteria and extracellular vesicles. Bacilli, Lactobacillales and Veillonella were significantly increased in the gut bacteria of patients with severe AH, and Veillonella, Veillonella parvula group and Lactobacillales were significantly increased in the extracellular vesicles of patients with severe AH. Eubacterium_g23, Oscillibacter and Clostridiales decreased in the gut bacteria of patients with severe AH, and Eubacterium_g23, Oscillibacter and Christensenellaceae decreased in the extracellular vesicles of patients with severe AH. After rifaximin treatment, 17 taxa in the gut bacteria and 23 taxa in extracellular vesicles were significantly restored in patients with severe AH. In common, Veillonella and Veillonella parvula group increased in patients with severe AH and decreased after rifaximin treatment, and Prevotella and Prevotellaceae decreased in patients with severe AH and increased after rifaximin treatment. Treatment non-responders showed a significantly lower abundance of Prevotella at baseline than did treatment responders. Dysbiosis was confirmed in severe AH but was alleviated by rifaximin treatment. Taxa associated with severe AH can be candidate biomarkers or therapeutic targets.

Detection of EGFR Mutations Using Bronchial Washing-Derived Extracellular Vesicles in Patients with Non-Small-Cell Lung Carcinoma.

Simple SummaryConsidering the spatiotemporal heterogeneity, more frequent monitoring of the disease progress using less-invasive liquid biopsy technologies is highly desired. Here, we demonstrate that epidermal growth factor receptor (EGFR) mutations could be readily detected from minimally invasive bronchial washing (BW)-derived EVs with good accuracy. The acquisition of T790M resistance mutation was detected earlier in BW-derived EVs than in plasma or tissue samples. The longitudinal analysis of BW-derived EVs showed excellent correlation with the disease progression measured by CT images. We demonstrate the clinical potential of BW-derived EVs as a liquid-biopsy sample for prognosis and precision medicine in patients with lung cancer.The detection of epidermal growth factor receptor (EGFR) mutation, based on tissue biopsy samples, provides a valuable guideline for the prognosis and precision medicine in patients with lung cancer. In this study, we aimed to examine minimally invasive bronchial washing (BW)-derived extracellular vesicles (EVs) for EGFR mutation analysis in patients with lung cancer. A lab-on-a-disc equipped with a filter with 20-nm pore diameter, Exo-Disc, was used to enrich EVs in BW samples. The overall detection sensitivity of EGFR mutations in 55 BW-derived samples was 89.7% and 31.0% for EV-derived DNA (EV-DNA) and EV-excluded cell free-DNA (EV-X-cfDNA), respectively, with 100% specificity. The detection rate of T790M in 13 matched samples was 61.5%, 10.0%, and 30.8% from BW-derived EV-DNA, plasma-derived cfDNA, and tissue samples, respectively. The acquisition of T790M resistance mutation was detected earlier in BW-derived EVs than plasma or tissue samples. The longitudinal analysis of BW-derived EVs showed excellent correlation with the disease progression measured by CT images. The EGFR mutations can be readily detected in BW-derived EVs, which demonstrates their clinical potential as a liquid-biopsy sample that may aid precise management, including assessment of the treatment response and drug resistance in patients with lung cancer.

Extracellular vesicles in patients in the acute phase of psychosis and after clinical improvement: an explorative study.

Extracellular vesicles (EVs) are cell-derived structures that transport proteins, lipids and nucleic acids between cells, thereby affecting the phenotype of the recipient cell. As the content of EVs reflects the status of the originating cell, EVs can have potential as biomarkers. Identifying EVs, including their cells of origin and their cargo, may provide insights in the pathophysiology of psychosis. Here, we present an in-depth analysis and proteomics of EVs from peripheral blood in patients (n = 25) during and after the acute phase of psychosis. Concentration and protein content of EVs in psychotic patients were twofold higher than in 25 age- and sex-matched healthy controls (p < 0.001 for both concentration and protein content), and the diameter of EVs was larger in patients (p = 0.02). Properties of EVs did not differ significantly in blood sampled during and after the acute psychotic episode. Proteomic analyses on isolated EVs from individual patients revealed 1,853 proteins, whereof 45 were brain-elevated proteins. Of these, five proteins involved in regulation of plasticity of glutamatergic synapses were significantly different in psychotic patients compared to controls; neurogranin (NRGN), neuron-specific calcium-binding protein hippocalcin (HPCA), kalirin (KALRN), beta-adducin (ADD2) and ankyrin-2 (ANK2). To summarize, our results show that peripheral EVs in psychotic patients are different from those in healthy controls and point at alterations on the glutamatergic system. We suggest that EVs allow investigation of blood-borne brain-originating biological material and that their role as biomarkers in patients with psychotic disorders is worthy of further exploration.

Changes in Circulating Extracellular Vesicles in Patients with ST-Elevation Myocardial Infarction and Potential Effects of Remote Ischemic Conditioning-A Randomized Controlled Trial.

(1) Background: Extracellular vesicles (EVs) have been recognized as a cellular communication tool with cardioprotective properties; however, it is unknown whether cardioprotection by remote ischemic conditioning (RIC) involves EVs. (2) Methods: We randomized patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI) to additionally receive a protocol of RIC or a sham-intervention. Blood was taken before and immediately, 24 h, four days and one month after PCI. Additionally, we investigated EVs from healthy volunteers undergoing RIC. EVs were characterized by a high-sensitive flow cytometer (Beckman Coulter Cytoflex S, Krefeld, Germany). (3) Results: We analyzed 32 patients (16 RIC, 16 control) and five healthy volunteers. We investigated platelet-, endothelial-, leukocyte-, monocyte- and granulocyte-derived EVs and their pro-thrombotic sub-populations expressing superficial phosphatidylserine (PS+). We did not observe a significant effect of RIC on the numbers of circulating EVs, although granulocyte-derived EVs were significantly higher in the RIC group. In line, RIC had not impact on EVs in healthy volunteers. Additionally, we observed changes of PS+/PEV, EEVs and PS+/CD15+ EVs irrespective of RIC with time following STEMI. 4) Conclusion: We provide further insights into the course of different circulating EVs during the acute and sub-acute phases of STEMI. With respect to the investigated EV populations, RIC seems to have no effect, with only minor differences found for granulocyte EVs.

P040 Proteomic characterisation of serum extracellular vesicles in patients with inflammatory bowel diseases: a novel approach for biomarker discovery

Abstract Background Our aim was to identify novel biomarkers and molecular pathways that may be involved in the development or progression of inflammatory bowel diseases (IBD) by the comparison of serum extracellular vesicles (EVs) proteomic profile between healthy controls (HC), Crohn’s disease (CD) [active (aCD) and quiescent (qCD)] and ulcerative colitis (UC) [active (aUC) and quiescent (qUC)] patients. Methods A total of 150 individuals with an ileocolonoscopy performed the same day of blood collection were included: 30 HC, 30 aCD, 30 qCD, 30 aUC and 30 qUC. Severity of inflammation was rated based on the Simple Endoscopic Score (SES-CD) in CD and the endoscopic subscore of the Mayo index in UC. Serum EVs were isolated from blood serum and their size, distribution and concentration were determined by nanoparticle tracking analysis with NanoSight LM10 (Malvern, UK). The presence of EVs positive and negative markers were evaluated by immunoblotting. Proteomic differential analysis was performed by Label Free nLC tandem mass spectrometry analysis by nanoACQUITY UPLC (Waters) coupled on-line to a LTQ Orbitrap XL (Thermo Electron, Bremen, Germany). Peptide identification was performed by Mascot (MatrixScience, London, UK) against Uniprot/Swissprot DataBase, and protein quantification by Progenesis LC-MS (Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK). Proteins matching at least two unique peptides were considered for the analysis. Proteins with p ≤ 0.05 and ratio &amp;gt;1.5 in any sense were considered deregulated. Proteomic results and clinical data were further integrated for biomarker discovery. Proteins with p-value cross-validation &amp;lt;0.05 and accuracy ≥0.65 individually or in combination with another molecule were considered to have potential as biomarkers. Results EVs size and concentration are shown in Table 1. There were not differences in EVs concentration and size between groups (Figure 1). A total of 324 proteins were identified, being some of them differentially expressed in different conditions (Table 2). Several proteins met the criteria to be proposed as accurate biomarkers: 17 in UC vs. HC, 9 in aUC vs. HC, 3 in qUC vs. HC, 13 in CD vs. HC, 16 in aCD vs. HC and 14 in qCD vs. HC. These proteins are involved in relevant processes in IBD pathogenesis such as angiogenesis, cell migration, immune response, acute phase response, inflammation and coagulation. Finally, a panel of seven proteins was chosen for validation in an independent cohort (Table 3). Conclusion Serum EVs of patients with IBD (CD and UC) carry differentially expressed proteins compared with HC and also depending on the presence of intestinal inflammation. These proteins are promising biomarkers of IBD that will be validated in an independent cohort. M. Chaparro and M. Azkargorta have equally contributed as first authors. F. Elortza and J.P. Gisbert have equally contributed as senior authors.

Rivaroxaban Reduces the Levels of Extracellular Vesicles in Patients with Venous Thromboembolism and Non-Valvular Atrial Fibrillation

Background: Rivaroxaban is a direct oral anticoagulant (DOAC) with similar efficacy to vitamin K antagonists in the management of non-valvular atrial fibrillation (NVAF) and venous thromboembolism (VTE) but with a more favourable bleeding profile. Recent studies including the landmark COMPASS trial have demonstrated that rivaroxaban possesses cardioprotective and anti-inflammatory properties beyond its well-established anticoagulant effects however, these remain poorly characterized. The effects of FXa inhibition on the generation of circulating extracellular vesicles (EV) are currently unknown. We hypothesize that circulating proinflammatory EV profiles differ in patients treated with rivaroxaban compared to those treated with warfarin and that these changes may represent a novel biomarker for cardioprotective effects mediated by FXa inhibition. Methods: Patients stably anticoagulated with 20 mg Rivaroxaban (n=15) once daily or warfarin (n=15) (at a target INR of 2.0 - 3.0) who had commenced therapy no sooner than 3 months previously for VTE treatment or NVAF were recruited following informed consent at the Mater Misericordiae University Hospital, Dublin. Demographic and clinical data were collected including patient age, body mass index, smoking and alcohol history, medical comorbidities, white cell and platelet count, creatinine clearance, liver profile, medications, detailed indication for anticoagulation and time since last dose of medication. Patients receiving warfarin had a time in therapeutic range of &gt; 55% and had an INR within target range at the time of sampling. Exclusion criteria included severe renal impairment (creatinine clearance &lt; 30 ml/min), significant liver impairment, known proinflammatory conditions (including systemic lupus erythematosus, inflammatory bowel disease, rheumatoid arthritis), recurrent VTE, active malignancy, previous stroke or systemic inflammation, antiphospholipid syndrome, strong thrombophilia (eg antithrombin deficiency), individuals aged &lt; 18, patients receiving inhibitory CYP3A4 and P-glycoprotein medications or platelet inhibitors, bleeding or platelet function disorders, and thrombocytopenia (platelet count &lt; 150 x 109/ml) Total particle counts in platelet poor plasma were measured by Nanoparticle Tracking Analysis (NTA) and flow cytometry. NTA was carried out using a NanoSight NS300 (Malvern) with a camera level of 13 and a detection threshold of 10. 15 x 60 second videos were captured per sample. Flow Cytometry analysis was performed using a CytoFlex LX (Beckman Coulter). Gigamix beads (Biocytex) were used to establish size gates for 100, 300, 500 and 900 nm particles. Samples were analysed in triplicate. Statistical analysis was performed in RStudio (version 1.2.1335) using a two-tailed t-test with a significance level of 5%. Results and Conclusion: Overall, we observed lower circulating EV levels in patients anticoagulated with FXa inhibitors versus vitamin K antagonists (Figure 1). Within our groups, patients with NVAF exhibited overall lower EV particle counts than VTE patients. Interestingly, EV levels were observed to be further diminished in patients treated with rivaroxaban versus warfarin. In this group, we detected a 2.5-fold decrease in the level of circulating EVs in patients with NVAF (1.18x1011±5.03x1010 particles/mL vs 2.92x1011±2.08x1011 particles/mL, p=0.039) and a 1.7-fold decrease in patients with VTE (3.37x1011±2.46x1011 particles/mL vs 5.71x1011±1.471011 particles/mL, p=0.164), in contrast to warfarin treatment. Collectively, these data suggest that FXa inhibition reduces the generation of circulating EVs, independent of the disease. These findings are of translational relevance towards characterizing cardioprotective mechanisms associated with rivaroxaban therapy. Disclosures Ni Ainle: Leo Pharma: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Actelion: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Maguire:Bayer: Research Funding; Leo Pharma: Research Funding; Actelion: Research Funding.

Inflammation-related gene expression profiles of salivary extracellular vesicles in patients with head trauma.

At present, there is no reliable biomarker for the diagnosis of traumatic brain injury (TBI). Studies have shown that extracellular vesicles released by damaged cells into biological fluids can be used as potential biomarkers for diagnosis of TBI and evaluation of TBI severity. We hypothesize that the genetic profile of salivary extracellular vesicles in patients with head trauma differs from that in uninjured subjects. Findings from this hypothesis would help investigate the severity of TBI. This study included 19 subjects, consisting of seven healthy controls who denied history of head trauma, six patients diagnosed with concussion injury from an outpatient concussion clinic, and six patients with TBI who received treatment in the emergency department within 24 hours after injury. Real-time PCR analysis of salivary extracellular vesicles in participants was performed using TaqMan Human Inflammation array. Gene expression analysis revealed nine upregulated genes in emergency department patients (LOX5, ANXA3, CASP1, IL2RG, ITGAM, ITGB2, LTA4H, MAPK14, and TNFRSF1A) and 13 upregulated genes in concussion clinic patients compared with healthy participants (ADRB1, ADRB2, BDKRB1, HRH1, HRH2, LTB4R2, LTB4R, PTAFR, CYSLTR1, CES1, KLK1, MC2R, and PTGER3). Each patient group had a unique profile. Comparison between groups showed that 15 inflammation-related genes had significant expression change. Our results indicate that inflammation biomarkers can be used for diagnosis of TBI and evaluation of disease severity. This study was approved by the Institutional Review Board on December 18, 2015 (approval No. 0078-12) and on June 9, 2016 (approval No. 4093-16).

Effect of hemodialysis on extracellular vesicles and circulating submicron particles

BackgroundAlthough hemodialysis is a highly effective treatment for diffusive clearance of low molecular weight uremic toxins, its effect on circulating extracellular vesicles and submicron particles is less clear. The purpose of this study was to examine the impact of hemodialysis on circulating levels of submicron particles.MethodsPlasma samples from patients were collected immediately before and after the mid-week hemodialysis session. Total submicron particles were assessed by nanoparticle tracking analysis and levels of endothelial (CD144+), platelet (CD41+), leukocyte (CD45+), and total (Annexin V+) membrane microparticles (MPs) were assessed by flow cytometry.ResultsTotal submicron particle number was significantly lower post-dialysis with reductions in particles < 40 nm, 40–100 nm, and 100–1000 nm in size. Circulating annexin V+ MPs, platelet MPs, leukocyte MPs, and endothelial MPs were all reduced following dialysis. Assessment of protein markers suggested that extracellular vesicles were not present in the dialysate, but rather adsorbed to the dialysis membrane.ConclusionsIn summary, hemodialysis is associated with reductions in circulating submicron particles including membrane MPs. Accordingly, there may be significant interdialytic variation in circulating submicron particles. Investigators interested in measuring extracellular vesicles in patients undergoing hemodialysis should therefore carefully consider the timing of biosampling.

764 – Protein and Mirna Profiling of Circulating Extracellular Vesicles in Patients with Primary Sclerosing Cholangitis

764 – Protein and Mirna Profiling of Circulating Extracellular Vesicles in Patients with Primary Sclerosing Cholangitis

SAT-077-Intraperitoneal activation of blood coagulation via tissue factor-exposing extracellular vesicles in patients with advanced chronic liver disease

SAT-077-Intraperitoneal activation of blood coagulation via tissue factor-exposing extracellular vesicles in patients with advanced chronic liver disease

Abstract P2-04-01: Human papillomavirus (HPV) DNA detection in breast cancer by liquid biopsy: Something new on the horizon?

Abstract Background The presence of human papillomavirus (HPV) DNA in breast cancer (BC) tissues has been widely investigated in recent years. HPV DNA has been detected in ductal lavage fluids and in serum-derived extracellular vesicles of patients with benign or in situ breast lesions. However, there are no data attesting to its presence in liquid biopsies of different BC subtypes or to its impact on prognosis. Methods We analyzed a total of 72 serum samples for the presence of circulating HPV DNA, of which 20 were from luminal A BC (5 relapsed, 15 non relapsed), 17 from luminal B BC (5 relapsed, 12 non relapsed), 15 from triple-negative BC (6 relapsed and 9 non relapsed), 12 from HER2-positive BC (3 relapsed, 9 non relapsed) and 8 from healthy subjects. Circulating DNA was extracted and purified from 500 μl of serum by Qiamp DNA minikit (Qiagen, Milan, Italy) according to the manufacturer's instructions. HPV DNA was assessed by a high-throughput MALDI-TOF mass spectrometry-based method (Mass Array Platform, Agena Bioscience, Hamburg, Germany). The DNA target sequence was amplified by a multiplex PCR with HPV E6 or E7 gene-specific primers. A primer for primer extension annealing to the amplified product was extended at its 3′ terminal base for each HPV type. Results HPV DNA was detected in 5 BC patients but in none of the healthy controls. None of the serum samples analyzed showed HPV DNA types 16 or 18. Four of the 5 BC cases had high-risk HPV DNA (type 39,45,52,59) and one had low-risk HPV DNA (type 73). The 4 patients with high risk HPV DNA had low-grade cervical intraepithelial neoplasia (CIN 1) detected by Pap smear prior to the diagnosis of BC. No relation was found between HPV infection and tumor subtype or prognosis. Our in vitro studies also revealed the active release of HPV DNA into the extracellular vesicle compartment of cervical cancer cells. Table 1.Features of BC patients positive for circulating HPV DNA.Sample IDAge (years)GradingSubtypeDeathRelapseHPV type, riskCervical lesion17471Luminal A-No59, highCIN 139783Luminal B20/03/2015No45, highCIN 145462Luminal B-No39, highCIN 160583TN03/01/2011Yes73, lowHyperplasia63523HER2-positive-Yes52, highCIN 1TN, triple negative; CIN 1:low-grade cervical intraepithelial neoplasia Conclusions Our findings support the feasibility of HPV DNA evaluation by liquid biopsy in BC. They also suggest that circulating HPV DNA in BC patients might be of cervical tissue origin and that the presence of HPV DNA in BC may be a consequence of its spreading from virus-infected tissue such as that of the uterine cervix. Citation Format: Bravaccini S, Ravaioli S, Rocca A, Maltoni R, Cristalli C, Marasco E, De Carolis S, Cricca M, Bonafè M. Human papillomavirus (HPV) DNA detection in breast cancer by liquid biopsy: Something new on the horizon? [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-04-01.

Elevated blood plasma levels of tissue factor-bearing extracellular vesicles in patients with atrial fibrillation

BackgroundThe risk of thrombus formation in the left atrial appendage (LAA) in patients with atrial fibrillation (AF) may result from blood stasis, local endocardial changes, and/or changed blood composition. Extracellular vesicles (EVs), especially subtypes exposing tissue factor (TF), have procoagulant capacity. We hypothesized that blood concentrations of TF-bearing EVs and other procoagulant biomarkers are elevated in AF patients, particularly in the LAA lumen. MethodsFrom 13 AF patients and 12 controls a venous blood sample was drawn prior to cardiac surgery. Intraoperatively, venous blood and blood directly from the LAA was drawn. Plasma levels of EVs, including TF- and cell type specific antigen-bearing EVs, were measured using a protein microarray platform. Plasma levels of TF, von Willebrand factor (vWF), cell free deoxyribonucleic acid (cf-DNA), procoagulant phospholipids (PPLs), and total submicron particles were also evaluated. ResultsSignificantly higher EV levels, including a several-fold higher median level of TF-bearing EVs were measured in AF patients compared with controls. Median concentrations of TF and vWF were approximately 40% and 30% higher, respectively, in the AF group than in the control group, while no significant differences in levels of cf-DNA, PPLs, or total submicron particles were observed. No significant differences in levels of any of the measured analytes were observed between intraoperative venous and LAA samples. ConclusionsIncreased plasma concentrations of TF in AF patients are accompanied and probably at least partly explained by increased levels of TF-bearing EVs, which may be mechanistically involved in increased thrombogenicity in AF patients.

Portrait of blood-derived extracellular vesicles in patients with Parkinson’s disease

The production of extracellular vesicles (EV) is a ubiquitous feature of eukaryotic cells but pathological events can affect their formation and constituents. We sought to characterize the nature, profile and protein signature of EV in the plasma of Parkinson's disease (PD) patients and how they correlate to clinical measures of the disease. EV were initially collected from cohorts of PD (n = 60; Controls, n = 37) and Huntington's disease (HD) patients (Pre-manifest, n = 11; manifest, n = 52; Controls, n = 55) – for comparative purposes in individuals with another chronic neurodegenerative condition – and exhaustively analyzed using flow cytometry, electron microscopy and proteomics. We then collected 42 samples from an additional independent cohort of PD patients to confirm our initial results. Through a series of iterative steps, we optimized an approach for defining the EV signature in PD. We found that the number of EV derived specifically from erythrocytes segregated with UPDRS scores corresponding to different disease stages. Proteomic analysis further revealed that there is a specific signature of proteins that could reliably differentiate control subjects from mild and moderate PD patients. Taken together, we have developed/identified an EV blood-based assay that has the potential to be used as a biomarker for PD.

Changes in the urinary extracellular vesicle proteome are associated with nephronophthisis-related ciliopathies

Nephronophthisis is one of the leading genetic causes of end-stage renal disease in childhood. Early diagnostics and prognostics for nephronophthisis are currently limited. We aimed to identify non-invasive protein biomarkers for nephronophthisis in urinary extracellular vesicles. Extracellular vesicles were isolated from urine of 12 patients with a nephronophthisis-related ciliopathy and 12 age- and gender-matched controls, followed by in-depth label-free LC-MS/MS proteomics analysis of gel fractionated extracellular vesicle proteins. Supervised cluster analysis of proteomic profiles separated patients from controls. We identified 156 differentially expressed proteins with fold change ≥4 in patients compared to controls (P < .05). Importantly, expression levels of discriminating proteins were correlated with chronic kidney disease stage, suggesting possible applications for urinary extracellular vesicle biomarkers in prognostics for nephronophthisis. Enrichment analysis of gene ontology terms revealed GO terms including signaling, actin cytoskeleton and endocytosis among the downregulated proteins in patients, whereas terms related to response to wounding and extracellular matrix organization were enriched among upregulated proteins. Our findings represent the first step towards a non-invasive diagnostic test for nephronophthisis. Further research is needed to determine specificity of the candidate biomarkers. In conclusion, proteomic profiles of urinary extracellular vesicles differentiate nephronophthisis-related ciliopathy patients from healthy controls. SignificanceNephronophthisis is an important cause of end-stage renal disease in children and is associated with an average diagnostic delay of 3.5 years. This is the first study investigating candidate biomarkers for nephronophthisis using global proteomics analysis of urinary extracellular vesicles in patients with nephronophthisis compared to control individuals. We show that measuring protein markers in urinary extracellular vesicles is a promising approach for non-invasive early diagnostics of nephronophthisis.

Decreased diversity of nasal microbiota and their secreted extracellular vesicles in patients with chronic rhinosinusitis based on a metagenomic analysis

Decreased diversity of nasal microbiota and their secreted extracellular vesicles in patients with chronic rhinosinusitis based on a metagenomic analysis

Differential miRNA expression in pleural effusions derived from extracellular vesicles of patients with lung cancer, pulmonary tuberculosis, or pneumonia.

MicroRNAs (miRNAs) have been found to play important regulatory roles in various physiological and pathological processes. MiRNAs also exhibit high stability and are present at high concentrations in human bodily fluids. Consequently, miRNAs may represent attractive and novel diagnostic biomarkers for certain clinical conditions. Recently, the capacity for extracellular vesicles, including microvesicles and exosomes, to carry miRNAs that participate in cell-to-cell communication has been described. In the present study, the miRNA expression patterns for three kinds of pleural effusions that were obtained from patients with pneumonia (group A), pulmonary tuberculosis (group B), and lung cancer (group C) were detected with high-throughput sequencing. When the expression levels of these miRNAs were compared among the three groups, three differentially expressed miRNAs were detected between groups A and B, while 27 differentially expressed miRNAs were detected between groups A and C. Notably, miR-378i was significantly elevated only in group B, while miR-205-5p and miR-200b were markedly increased only in group C (p<0.01). Further studies are needed to confirm whether these differentially expressed miRNAs may serve as prospective diagnostic markers for pulmonary diseases.

Individual extracellular vesicles in patients with acute coronary syndromes

Individual extracellular vesicles in patients with acute coronary syndromes