Melbourne Medical School Collected Works - Research Publications
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ItemMitochondrial dysfunction-related lipid changes occur in nonalcoholic fatty liver disease progression(ELSEVIER, 2018-10)Nonalcoholic fatty liver disease (NAFLD) comprises fat-accumulating conditions within hepatocytes that can cause severe liver damage and metabolic comorbidities. Studies suggest that mitochondrial dysfunction contributes to its development and progression and that the hepatic lipidome changes extensively in obesity and in NAFLD. To gain insight into the relationship between lipid metabolism and disease progression through different stages of NAFLD, we performed lipidomic analysis of plasma and liver biopsy samples from obese patients with nonalcoholic fatty liver (NAFL) or nonalcoholic steatohepatitis (NASH) and from those without NAFLD. Congruent with earlier studies, hepatic lipid levels overall increased with NAFLD. Lipid species that differed with NAFLD severity were related to mitochondrial dysfunction; specifically, hepatic cardiolipin and ubiquinone accumulated in NAFL, and levels of acylcarnitine increased with NASH. We propose that increased levels of cardiolipin and ubiquinone may help to preserve mitochondrial function in early NAFLD, but that mitochondrial function eventually fails with progression to NASH, leading to increased acylcarnitine. We also found a negative association between hepatic odd-chain phosphatidylcholine and NAFLD, which may result from mitochondrial dysfunction-related impairment of branched-chain amino acid catabolism. Overall, these data suggest a close link between accumulation of specific hepatic lipid species, mitochondrial dysfunction, and the progression of NAFLD.
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ItemStimulating the Release of Exosomes Increases the Intercellular Transfer of Prions(ELSEVIER, 2016-03-04)Exosomes are small extracellular vesicles released by cells and play important roles in intercellular communication and pathogen transfer. Exosomes have been implicated in several neurodegenerative diseases, including prion disease and Alzheimer disease. Prion disease arises upon misfolding of the normal cellular prion protein, PrP(C), into the disease-associated isoform, PrP(Sc). The disease has a unique transmissible etiology, and exosomes represent a novel and efficient method for prion transmission. The precise mechanism by which prions are transmitted from cell to cell remains to be fully elucidated, although three hypotheses have been proposed: direct cell-cell contact, tunneling nanotubes, and exosomes. Given the reported presence of exosomes in biological fluids and in the lipid and nucleic acid contents of exosomes, these vesicles represent an ideal mechanism for encapsulating prions and potential cofactors to facilitate prion transmission. This study investigates the relationship between exosome release and intercellular prion dissemination. Stimulation of exosome release through treatment with an ionophore, monensin, revealed a corresponding increase in intercellular transfer of prion infectivity. Conversely, inhibition of exosome release using GW4869 to target the neutral sphingomyelinase pathway induced a decrease in intercellular prion transmission. Further examination of the effect of monensin on PrP conversion revealed that monensin also alters the conformational stability of PrP(C), leading to increased generation of proteinase K-resistant prion protein. The findings presented here provide support for a positive relationship between exosome release and intercellular transfer of prion infectivity, highlighting an integral role for exosomes in facilitating the unique transmissible nature of prions.
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ItemSecreted primary human malignant mesothelioma exosome signature reflects oncogenic cargo(NATURE PORTFOLIO, 2016-09-08)Malignant mesothelioma (MM) is a highly-aggressive heterogeneous malignancy, typically diagnosed at advanced stage. An important area of mesothelioma biology and progression is understanding intercellular communication and the contribution of the secretome. Exosomes are secreted extracellular vesicles shown to shuttle cellular cargo and direct intercellular communication in the tumour microenvironment, facilitate immunoregulation and metastasis. In this study, quantitative proteomics was used to investigate MM-derived exosomes from distinct human models and identify select cargo protein networks associated with angiogenesis, metastasis, and immunoregulation. Utilising bioinformatics pathway/network analyses, and correlation with previous studies on tumour exosomes, we defined a select mesothelioma exosomal signature (mEXOS, 570 proteins) enriched in tumour antigens and various cancer-specific signalling (HPGD/ENO1/OSMR) and secreted modulators (FN1/ITLN1/MAMDC2/PDGFD/GBP1). Notably, such circulating cargo offers unique insights into mesothelioma progression and tumour microenvironment reprogramming. Functionally, we demonstrate that oncogenic exosomes facilitate the migratory capacity of fibroblast/endothelial cells, supporting the systematic model of MM progression associated with vascular remodelling and angiogenesis. We provide biophysical and proteomic characterisation of exosomes, define a unique oncogenic signature (mEXOS), and demonstrate the regulatory capacity of exosomes in cell migration/tube formation assays. These findings contribute to understanding tumour-stromal crosstalk in the context of MM, and potential new diagnostic and therapeutic extracellular targets.
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ItemPodoplanin is a component of extracellular vesicles that reprograms cell-derived exosomal proteins and modulates lymphatic vessel formation(IMPACT JOURNALS LLC, 2016-03-29)Podoplanin (PDPN) is a transmembrane glycoprotein that plays crucial roles in embryonic development, the immune response, and malignant progression. Here, we report that cells ectopically or endogenously expressing PDPN release extracellular vesicles (EVs) that contain PDPN mRNA and protein. PDPN incorporates into membrane shed microvesicles (MVs) and endosomal-derived exosomes (EXOs), where it was found to colocalize with the canonical EV marker CD63 by immunoelectron microscopy. We have previously found that expression of PDPN in MDCK cells induces an epithelial-mesenchymal transition (EMT). Proteomic profiling of MDCK-PDPN cells compared to control cells shows that PDPN-induced EMT is associated with upregulation of oncogenic proteins and diminished expression of tumor suppressors. Proteomic analysis of exosomes reveals that MDCK-PDPN EXOs were enriched in protein cargos involved in cell adhesion, cytoskeletal remodeling, signal transduction and, importantly, intracellular trafficking and EV biogenesis. Indeed, expression of PDPN in MDCK cells stimulated both EXO and MV production, while knockdown of endogenous PDPN in human HN5 squamous carcinoma cells reduced EXO production and inhibited tumorigenesis. EXOs released from MDCK-PDPN and control cells both stimulated in vitro angiogenesis, but only EXOs containing PDPN were shown to promote lymphatic vessel formation. This effect was mediated by PDPN on the surface of EXOs, as demonstrated by a neutralizing specific monoclonal antibody. These results contribute to our understanding of PDPN-induced EMT in association to tumor progression, and suggest an important role for PDPN in EV biogenesis and/or release and for PDPN-EXOs in modulating lymphangiogenesis.
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ItemDeep Sequencing of RNA from Three Different Extracellular Vesicle (EV) Subtypes Released from the Human LIM1863 Colon Cancer Cell Line Uncovers Distinct Mirna-Enrichment Signatures(PUBLIC LIBRARY SCIENCE, 2014-10-17)Secreted microRNAs (miRNAs) enclosed within extracellular vesicles (EVs) play a pivotal role in intercellular communication by regulating recipient cell gene expression and affecting target cell function. Here, we report the isolation of three distinct EV subtypes from the human colon carcinoma cell line LIM1863--shed microvesicles (sMVs) and two exosome populations (immunoaffinity isolated A33-exosomes and EpCAM-exosomes). Deep sequencing of miRNA libraries prepared from parental LIM1863 cells/derived EV subtype RNA yielded 254 miRNA identifications, of which 63 are selectively enriched in the EVs--miR-19a/b-3p, miR-378a/c/d, and miR-577 and members of the let-7 and miR-8 families being the most prominent. Let-7a-3p*, let-7f-1-3p*, miR-451a, miR-574-5p*, miR-4454 and miR-7641 are common to all EV subtypes, and 6 miRNAs (miR-320a/b/c/d, miR-221-3p, and miR-200c-3p) discern LIM1863 exosomes from sMVs; miR-98-5p was selectively represented only in sMVs. Notably, A33-Exos contained the largest number (32) of exclusively-enriched miRNAs; 14 of these miRNAs have not been reported in the context of CRC tissue/biofluid analyses and warrant further examination as potential diagnostic markers of CRC. Surprisingly, miRNA passenger strands (star miRNAs) for miR-3613-3p*, -362-3p*, -625-3p*, -6842-3p* were the dominant strand in A33-Exos, the converse to that observed in parental cells. This finding suggests miRNA biogenesis may be interlinked with endosomal/exosomal processing.
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ItemErratum: Factors determining the stability, size distribution, and cellular accumulation of small, monodisperse chitosan nanoparticles as candidate vectors for anticancer drug delivery: application to the passive encapsulation of [(14)C]-doxorubicin [Corrigendum].(Informa UK Limited, 2016)[This corrects the article on p. 67 in vol. 8, PMID: 26715842.].
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ItemBrain-derived neurotrophic factor genetic polymorphism (rs6265) is protective against chemotherapy-associated cognitive impairment in patients with early-stage breast cancer.(Oxford University Press (OUP), 2016-02)BACKGROUND: Brain-derived neurotrophic factor (BDNF), a neurotrophin that regulates neuronal function and development, is implicated in several neurodegenerative conditions. Preliminary data suggest that a reduction of BDNF concentrations may lead to postchemotherapy cognitive impairment. We hypothesized that a single nucleotide polymorphism (rs6265) of the BDNF gene may predispose patients to cognitive impairment. This study aimed to evaluate the effect of BDNF gene polymorphism on chemotherapy-associated cognitive impairment. METHODS: Overall, 145 patients receiving chemotherapy for early-stage breast cancer (mean age: 50.8 ± 8.8 y; 82.1% Chinese) were recruited. Patients' cognitive functions were assessed longitudinally using the validated Functional Assessment of Cancer Therapy-Cognitive Function (v.3) and an objective computerized tool, Headminder. Genotyping was performed using Sanger sequencing. Logistic regression was used to evaluate the association between BDNF Val66Met polymorphism and cognition after adjusting for ethnicity and clinically important covariates. RESULTS: Of the 145 patients, 54 (37%) reported cognitive impairment postchemotherapy. The Met/Met genotype was associated with statistically significant lower odds of developing cognitive impairment (odds ratio [OR] = 0.26; 95% CI: 0.08-0.92; P = .036). The Met carriers were less likely to experience impairment in the domains of verbal fluency (OR = 0.34; 95% CI: 0.12-0.90; P = .031) and multitasking ability (OR = 0.37; 95% CI: 0.15-0.91; P = .030) compared with the Val/Val homozygote. No associations were observed between Headminder and the BDNF Val66Met polymorphism. CONCLUSIONS: This is the first study to provide evidence that carriers of the BDNF Met allele are protected against chemotherapy-associated cognitive impairment. Further studies are required to validate the findings.
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ItemCD83 is a new potential biomarker and therapeutic target for Hodgkin lymphoma(FERRATA STORTI FOUNDATION, 2018-03-23)Chemotherapy and hematopoietic stem cell transplantation are effective treatments for most Hodgkin lymphoma patients, however there remains a need for better tumor-specific target therapy in Hodgkin lymphoma patients with refractory or relapsed disease. Herein, we demonstrate that membrane CD83 is a diagnostic and therapeutic target, highly expressed in Hodgkin lymphoma cell lines and Hodgkin and Reed-Sternberg cells in 29/35 (82.9%) Hodgkin lymphoma patient lymph node biopsies. CD83 from Hodgkin lymphoma tumor cells was able to trogocytose to surrounding T cells and, interestingly, the trogocytosing CD83+T cells expressed significantly more programmed death-1 compared to CD83-T cells. Hodgkin lymphoma tumor cells secreted soluble CD83 that inhibited T-cell proliferation, and anti-CD83 antibody partially reversed the inhibitory effect. High levels of soluble CD83 were detected in Hodgkin lymphoma patient sera, which returned to normal in patients who had good clinical responses to chemotherapy confirmed by positron emission tomography scans. We generated a human anti-human CD83 antibody, 3C12C, and its toxin monomethyl auristatin E conjugate, that killed CD83 positive Hodgkin lymphoma cells but not CD83 negative cells. The 3C12C antibody was tested in dose escalation studies in non-human primates. No toxicity was observed, but there was evidence of CD83 positive target cell depletion. These data establish CD83 as a potential biomarker and therapeutic target in Hodgkin lymphoma.
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ItemDendritic cell immunotherapy: clinical outcomes(WILEY, 2014-07)The use of tumour-associated antigens for cancer immunotherapy studies is exacerbated by tolerance to these self-antigens. Tolerance may be broken by using ex vivo monocyte-derived dendritic cells (DCs) pulsed with self-antigens. Targeting tumour-associated antigens directly to DCs in vivo is an alternative and simpler strategy. The identification of cell surface receptors on DCs, and targeting antigens to DC receptors, has become a popular approach for inducing effective immune responses against cancer antigens. Many years ago, we demonstrated that targeting the mannose receptor on macrophages using the carbohydrate mannan to DCs led to appropriate immune responses and tumour protection in animal models. We conducted Phase I, I/II and II, clinical trials demonstrating the effectiveness of oxidised mannan-MUC1 in patients with adenocarcinomas. Here we summarise DC targeting approaches and their efficacy in human clinical trials.
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ItemSystems Medicine-Complexity Within, Simplicity Without(SPRINGERNATURE, 2017-06)This paper presents a brief history of Systems Theory, progresses to Systems Biology, and its relation to the more traditional investigative method of reductionism. The emergence of Systems Medicine represents the application of Systems Biology to disease and clinical issues. The challenges faced by this transition from Systems Biology to Systems Medicine are explained; the requirements of physicians at the bedside, caring for patients, as well as the place of human-human interaction and the needs of the patients are addressed. An organ-focused transition to Systems Medicine, rather than a genomic-, molecular-, or cell-based effort is emphasized. Organ focus represents a middle-out approach to ease this transition and to maximize the benefits of scientific discovery and clinical application. This method manages the perceptions of time and space, the massive amounts of human- and patient-related data, and the ensuing complexity of information.