ORCID Profile
0000-0002-4566-5036
Current Organisation
Flinders University
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Publisher: MDPI AG
Date: 24-02-2021
Abstract: Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human blood and present the intriguing potential for a ‘liquid biopsy’ to track disease and the effectiveness of interventions. Recently, we have further demonstrated the potential for EV derived biomarkers to account for variability in drug exposure. This study sought to evaluate the variability in abundance and cargo of global and liver-specific circulating sEV, within (diurnal) and between in iduals in a cohort of healthy subjects (n = 10). We present normal ranges for EV concentration and size and expression of generic EV protein markers and the liver-specific asialoglycoprotein receptor 1 (ASGR1) in s les collected in the morning and afternoon. EV abundance and cargo was generally not affected by fasting, except CD9 which exhibited a statistically significant increase (p = 0.018). Diurnal variability was observed in the expression of CD81 and ASGR1, which significantly decreased (p = 0.011) and increased (p = 0.009), respectively. These results have potential implications for study s ling protocols and normalisation of biomarker data when considering the expression of sEV derived cargo as a biomarker strategy. Specifically, the novel finding that liver-specific EVs exhibit diurnal variability in healthy subjects should have broad implications in the study of drug metabolism and development of minimally invasive biomarkers for liver disease.
Publisher: Wiley
Date: 16-09-2022
DOI: 10.1002/CPT.2734
Publisher: BMJ
Date: 07-04-2020
DOI: 10.1136/THORAXJNL-2019-214027
Abstract: Mutations in the cystic fibrosis transmembrane regulator ( CFTR ) gene form the basis of cystic fibrosis (CF). There remains an important knowledge gap in CF as to how diminished CFTR activity leads to the dominant inflammatory response within CF airways. To investigate if extracellular vesicles (EVs) contribute to inflammatory signalling in CF. EVs released from CFBE41o-, CuFi-5, 16HBE14o- and NuLi-1 cells were characterised by nanoparticle tracking analysis (NTA). EVs isolated from bronchoalveolar lavage fluid (BALF) from 30 people with CF (PWCF) were analysed by NTA and mass spectrometry and compared with controls. Neutrophils were isolated from the blood of 8 PWCF to examine neutrophil migration in the presence of CFBE41o- EVs. A significantly higher level of EVs were released from CFBE41o- (p .0001) and CuFi-5 (p=0.0209) relative to control cell lines. A significantly higher level of EVs were detected in BALF of PWCF, in three different age groups relative to controls (p=0.01, 0.001, 0.002). A significantly lower level of EVs were released from CFBE41o- (p .001) and CuFi-5 (p=0.0002) cell lines treated with CFTR modulators. Significant changes in the protein expression of 126 unique proteins was determined in EVs obtained from the BALF of PWCF of different age groups (p .001–0.05). A significant increase in chemotaxis of neutrophils derived from PWCF was observed in the presence of CFBE41o EVs (p=0.0024) compared with controls. This study demonstrates that EVs are produced in CF airway cells, have differential protein expression at different ages and drive neutrophil recruitment in CF.
Publisher: Wiley
Date: 18-04-2021
DOI: 10.1002/CPT.2244
Abstract: Liver‐derived small extracellular vesicles (sEVs), prepared from small sets of banked serum s les using a novel two‐step protocol, were deployed as liquid biopsy to study the induction of cytochromes P450 (CYP3A4, CYP3A5, and CYP2D6) and organic anion transporting polypeptides (OATP1B1 and OATP1B3) during pregnancy (nonpregnant (T0), first, second, and third (T3) trimester women N = 3 each) and after administration of rif icin (RIF) to healthy male subjects. Proteomic analysis revealed induction (mean fold‐increase, 90% confidence interval) of sEV CYP3A4 after RIF 300 mg × 7 days (3.5, 95% CI = 2.5–4.5, N = 4, P = 0.029) and 600 mg × 14 days (3.7, 95% CI = 2.1–6.0, N = 5, P = 0.018) consistent with the mean oral midazolam area under the plasma concentration time curve (AUC) ratio in the same subjects (0.28, 95% CI = 0.22–0.34, P 0.0001 and 0.17, 95% CI = 0.13–0.22, P 0.0001). Compared with CYP3A4, liver sEV CYP3A5 protein (subjects genotyped CYP3A5*1/*3 ) was weakly induced (≤ 1.5‐fold). It was also possible to measure liver sEV‐catalyzed dextromethorphan (DEX) O‐demethylation to dextrorphan (DXO), correlated with sEV CYP2D6 expression ( r = 0.917, P = 0.0001 N = 10) and 3‐hour plasma DXO‐to‐DEX concentration ratio ( r = 0.843, P = 0.002, N = 10), and show that CYP2D6 was not induced by RIF. Nonparametric analysis of liver sEV revealed significantly higher CYP3A4 (3.2‐fold, P = 0.003) and CYP2D6 (3.7‐fold, P = 0.03) protein expression in T3 vs. T0 women. In contrast, expression of both OATPs in liver sEV was unaltered by RIF administration and pregnancy.
Publisher: Springer Science and Business Media LLC
Date: 09-2016
Publisher: Springer Science and Business Media LLC
Date: 04-11-2020
DOI: 10.1038/S41598-020-75817-9
Abstract: MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary.
Publisher: Oxford University Press (OUP)
Date: 30-06-2016
DOI: 10.1189/JLB.5AB1115-493RR
Abstract: The role of NK cells in visceral adipose tissue (VAT) and liver inflammation in obesity is not fully understood. This study investigated the frequency, cytokine expression, chemokine receptor, and cytotoxicity receptor profile of NK cells in the blood, omentum, and liver of patients with the obesity-associated cancer, oesophageal adenocarcinoma (OAC). The effect of chronically inflamed tissue microenvironments on NK cell viability and function was also examined. We identified significantly lower NK cell frequencies in the liver of OAC patients compared with healthy controls and within the omentum and liver of OAC patients compared with blood, whereas IL-10-producing populations were significantly higher. Interestingly, our data suggest that reduced frequencies of NK cells in omentum and liver of OAC patients are not a result of impaired NK cell chemotaxis to these tissues. In fact, our functional data revealed that secreted factors from omentum and liver of OAC patients induce significant levels of NK cell death and lead to reduced percentages of TNF-α+ and NKP46+ NK cells and higher frequencies of IL-10-producing NK cells. Together, these data suggest that the omental and hepatic microenvironments of OAC patients alter the NK cell phenotype to a more anti-inflammatory homeostatic role.
Publisher: Wiley
Date: 09-2022
DOI: 10.1002/JEX2.56
Abstract: Extracellular vesicles (EVs) are membrane‐bound nanosized particles released by cells into bodily fluids containing an array of molecular cargo. Several characteristics, including stability and accessibility in biofluids such as blood and urine, make EVs and associated cargo attractive biomarkers and therapeutic tools. To promote robust characterisation of EV isolates, the minimal requirements for the study of extracellular vesicles (MISEV) guidelines recommend the analysis of proteins in EV s les, including positive EV‐associated markers and negative contaminant markers based on commonly co‐isolated components of the starting material. Western blot is conventionally used to address the guidelines however, this approach is limited in terms of quantitation and throughput and requires larger volumes than typically available for patient s les. The increasing application of EVs as liquid biopsy in clinical contexts requires a high‐throughput multiplexed approach for analysis of protein markers from small volumes of starting material. Here, we document the development and validation of a targeted liquid chromatography tandem mass spectrometry (LC‐MS/MS) assay for the quantification of markers associated with EVs and non‐vesicle contaminants from human blood s les. The assay was highly sensitive, requiring only a fraction of the s le consumed for immunoblots, fully quantitative and high throughput. Application of the assay to EVs isolated by size exclusion chromatography (SEC) and precipitation revealed differences in yield, purity and recovery of subpopulations.
Publisher: MDPI AG
Date: 17-01-2022
DOI: 10.3390/BIOMEDICINES10010195
Abstract: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably .78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 05-08-2021
Abstract: Extracellular vesicles (EVs) are small, nonreplicating, lipid-encapsulated particles that contain a myriad of protein and nucleic acid cargo derived from their tissue of origin. The potential role of EV-derived biomarkers to the study of drug metabolism and disposition (DMD) has gained attention in recent years. The key trait that makes EVs an attractive biomarker source is their capacity to provide comparable insights to solid organ biopsy through an appreciably less invasive collection procedure. Blood-derived EVs exist as a heterogenous milieu of biologically distinct particles originating from different sources through different biogenesis pathways. Furthermore, blood (plasma and serum) contains an array of vesicular and nonvesicular contaminants, such as apoptotic bodies, plasma proteins, and lipoproteins that are routinely coisolated with EVs, albeit to a different extent depending on the isolation technique. The following minireview summarizes current studies reporting DMD biomarkers and addresses elements of EV isolation and quantification relevant to the application of EV-derived DMD biomarkers. Evidence based-best practice guidance aligned to Minimum Information for the Study of Extracellular Vesicles and EV-TRACK reporting standards are summarized in the context of DMD studies. SIGNIFICANCE STATEMENT: Extracellular vesicle (EV)-derived protein and nucleic acid cargo represent a potentially game-changing source of novel DMD biomarkers with the capacity to define within- and between-in idual variability in drug exposure irrespective of etiology. However, robust translation of EV-derived biomarkers requires the generation of transparent reproducible evidence. This review outlines the critical elements of data generation and reporting relevant to achieving this evidence in a drug metabolism and disposition context.
Publisher: Springer US
Date: 2023
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