Alorro M.G.,La Trobe University |
Pierce T.P.,La Trobe University |
Eissmann M.F.,La Trobe University |
Dijkstra C.,La Trobe University |
And 4 more authors.
Genesis | Year: 2017
Signal transducer and activator of transcription 3 (Stat3) is a transcription factor that has many essential roles during inflammation, development and cancer. Stat3 is therefore an attractive therapeutic target in many diseases. While current Stat3 knockout mouse models led to a better understanding of the role of Stat3, the irreversible nature of Stat3 ablation does not model the effects of transient Stat3 therapeutic inhibition, and does not inform on potential dosage effects of Stat3. Using RNAi technology, we have generated a new mouse model allowing the inducible and reversible silencing of Stat3 in vivo, which mirrors the effects of specific Stat3 therapeutic interference. We showed that upon Doxycycline-mediated activation of the Stat3 short-hairpin RNA, Stat3 expression was efficiently reduced by about 80% in multiple organs and cell types. Moreover, Stat3 reduction was sufficient to reduce tumor burden in a clinically-validated mouse model of gastric cancer. Finally, we demonstrated that Stat3 silencing during embryonic development led to reduced birth rate without leading to complete embryonic lethality, in contrast to full Stat3 ablation. In conclusion, this new mouse model will be invaluable to understand the effects of Stat3 therapeutic interference and Stat3 dosage effects. © 2017 Wiley Periodicals, Inc.
PubMed | Florey Institute of Neurosciences and Mental Health, Northern Health, Austin Health and Australian Center for Blood Diseases
Type: Journal Article | Journal: Journal of thrombosis and thrombolysis | Year: 2016
Cerebral venous thrombosis (CVT) is a rare venous thrombotic event. We review our local experience in the management of CVT in comparison to other venous thromboembolism (VTE) with specific focus on risk factors for thrombotic recurrence. Retrospective evaluation of consecutive CVT presentations from January 2005 to June 2015, at two major tertiary hospitals in Northeast Melbourne, Australia. This population was compared to a separate audit of 1003 consecutive patients with DVT and PE. Fifty-two patients (30 female, 22 male) with a median age of 40 (18-83) years, presented with 53 episodes of CVT. Twenty-nine episodes (55%) were associated with an underlying risk factor, with hormonal risk factors in females being most common. The median duration of anticoagulation was 6months with 11 receiving life-long anticoagulation. Eighty-one percent had residual thrombosis on repeat imaging, which was not associated with recurrence at the same or distant site. Nine (17%) had CVT-related haemorrhagic transformation with two resultant CVT-related deaths (RR 22.5; p=0.04). All three VTE recurrences occured in males with unprovoked events (RR 18.2; p=0.05) who were subsequently diagnosed with myeloproliferative neoplasm (MPN). Compared to the non-cancer VTE population, non-cancer CVT patients were younger, had similar rate of provoked events and VTE recurrence, although with significantly higher rate of MPN diagnosis (RR 9.30 (2.29-37.76); p=0.002) CVT is a rare thrombotic disorder. All recurrences in this audit occurred in male patients with unprovoked events and subsequent diagnosis of MPN, suggesting further evaluation for MPN may be warranted in patients with unprovoked CVT.
Freeman R.,Australian Center for Blood Diseases |
Niego B.,Australian Center for Blood Diseases |
Croucher D.R.,University of Wollongong |
Croucher D.R.,Garvan Institute of Medical Research |
And 2 more authors.
Brain Research | Year: 2014
Tissue-type plasminogen activator (t-PA) is the only thrombolytic treatment available for patients with acute ischaemic stroke. However, t-PA can increase permeability of the blood-brain barrier (BBB). Desmoteplase is a plasminogen activator derived from the common vampire bat, currently under clinical development for ischaemic stroke. We compared how t-PA and desmoteplase influenced BBB permeability using a human in vitro model where primary brain endothelial cells (BEC) and astrocytes are co-cultured on the opposite sides of a porous membrane. Permeability changes were evaluated 6 or 24 h post-stimulation by passage of fluorescent albumin across the membrane. Under normoxic conditions, t-PA, but not desmoteplase, increased BBB permeability. Surprisingly, the ability of t-PA to affect the barrier was lost under conditions of oxygen-glucose deprivation (OGD). Addition of plasminogen re-sensitised the BBB to the action of t-PA under both normoxia and OGD, but did not affect the inert behaviour of desmoteplase, even when digested fibrinogen was added to ensure optimal plasmin generation. These observations coincided with plasmin-dependent changes in astrocyte and BEC morphology and disruption of tight junction proteins in BECs, specifically initiated by t-PA but not by desmoteplase. Finally, inhibition of plasmin post-stimulation with t-PA and plasminogen, especially within 2 h, protected the BBB against t-PA-mediated barrier opening. Hence t-PA, but not desmoteplase, increases BBB permeability under both normoxic and OGD conditions in a reversible, plasmin-dependent process. The inability of desmoteplase to increase permeability despite its capacity to generate plasmin provides further support for its use as thrombolytic in patients with ischaemic stroke. © 2014 Elsevier B.V.
Straub A.,Baker IDI Heart and Diabetes Institute |
Straub A.,University of Tübingen |
Krajewski S.,Baker IDI Heart and Diabetes Institute |
Krajewski S.,University of Tübingen |
And 13 more authors.
Arteriosclerosis, Thrombosis, and Vascular Biology | Year: 2011
Objective-: Hypothermia is used in various clinical settings to inhibit ischemia-related organ damage. However, prothrombotic effects have been described as potential side effects. This study aimed to elucidate the mechanism of hypothermia-induced platelet activation and subsequent prothrombotic events and to develop preventative pharmacological strategies applicable during clinically used hypothermia. Methods and Results-: Platelet function was investigated ex vivo and in vivo at clinically used hypothermia (28°C/18°C). Hypothermic mice demonstrated increased expression of platelet activation marker P-selectin, platelet-leukocyte aggregate formation, and thrombocytopenia. Intravital microscopy of FeCl3-injured murine mesenteric arteries revealed increased platelet thrombus formation with hypothermia. Ex vivo flow chamber experiments indicated increased platelet-fibrinogen adhesion under hypothermia. We show that hypothermia results in reduced ADP hydrolysis via reduction of CD39 (E-NTPDase1) activity, resulting in increased levels of ADP and subsequent augmented primary and secondary platelet activation. In vivo administration of ADP receptor P2Y12 antagonists and recombinant soluble CD39 prevented hypothermia-induced thrombus formation and thrombocytopenia, respectively. Conclusion-: The platelet agonist ADP plays a key role in hypothermia-induced platelet activation. Inhibition of receptor binding or hydrolysis of ADP has the potential to protect platelets against hypothermia-induced activation. Our findings provide a rational basis for further evaluation of novel antithrombotic strategies in clinically applied hypothermia. © 2011 American Heart Association, Inc.
Wang X.,Baker IDI Heart and Diabetes Institute |
Palasubramaniam J.,Baker IDI Heart and Diabetes Institute |
Gkanatsas Y.,Baker IDI Heart and Diabetes Institute |
Hohmann J.D.,Baker IDI Heart and Diabetes Institute |
And 13 more authors.
Circulation Research | Year: 2014
RATIONALE:: Fibrinolysis is a valuable alternative for the treatment of myocardial infarction when percutaneous coronary intervention is not available in a timely fashion. For acute ischemic stroke, fibrinolysis is the only treatment option with a very narrow therapeutic window. Clinically approved thrombolytics have significant drawbacks, including bleeding complications. Thus their use is highly restricted, leaving many patients untreated. OBJECTIVE:: We developed a novel targeted fibrinolytic drug that is directed against activated platelets. METHODS AND RESULTS:: We fused single-chain urokinase plasminogen activator (scuPA) to a small recombinant antibody (scFvSCE5), which targets the activated form of the platelet-integrin glycoprotein IIb/IIIa. Antibody binding and scuPA activity of this recombinant fusion protein were on par with the parent molecules. Prophylactic in vivo administration of scFvSCE5-scuPA (75 U/g body weight) prevented carotid artery occlusion after ferric chloride injury in a plasminogen-dependent process compared with saline (P<0.001), and blood flow recovery was similar to high-dose nontargeted urokinase (500 U/g body weight). Tail bleeding time was significantly prolonged with this high dose of nontargeted urokinase, but not with equally effective targeted scFvSCE5-scuPA at 75 U/g body weight. Real-time in vivo molecular ultrasound imaging demonstrates significant therapeutic reduction of thrombus size after administration of 75 U/g body weight scFvSCE5-scuPA as compared with the same dose of a mutated, nontargeting scFv-scuPA or vehicle. The ability of scFvSCE5-scuPA to lyse thrombi was lost in plasminogen-deficient mice, but could be restored by intravenous injection of plasminogen. CONCLUSIONS:: Targeting of scuPA to activated glycoprotein IIb/IIIa allows effective thrombolysis and the potential novel use as a fibrinolytic agent for thromboprophylaxis without bleeding complications. © 2014 American Heart Association, Inc.
Niederleithner H.,Medical University of Vienna |
Heinz M.,Medical University of Vienna |
Tauber S.,Medical University of Vienna |
Tauber S.,University of Vienna |
And 8 more authors.
Journal of Investigative Dermatology | Year: 2012
Wnt signals contribute to melanoma progression by boosting their proliferation and survival. Initially, we expected that activated Wnt signaling also improves their proficiency to recruit blood and lymph vessels. To assess this, we added cell culture supernatants (SNs) of Wnt1 and Wnt1 melanoma to endothelial spheroids. Whereas SNs of Wnt1 - melanoma cells induced lymphatic sprouts, those of Wnt1 + cells were unable to do so and this was restored by vascular endothelial growth factor C (VEGF-C). Subsequent testing of several human melanoma lines revealed that Wnt1 suppressed their VEGF-C expression. This Wnt1 effect did not depend on glycogen synthase kinase-3Β (GSK3Β), Β-catenin, or activator protein-1, but was blocked by cyclosporine A (CsA). To analyze Wnt1 effects in melanoma in vivo, we selected Wnt1 melanoma cell lines, overexpressed Wnt1, and injected them subepidermally into severe combined immunodeficient (SCID) mice. We found reduced VEGF-C expression, reduced lymphangiogenesis, and delayed metastasis to sentinel nodes in Wnt1 as compared with Wnt1 - melanoma (P<0.05). Concomitant overexpression of VEGF-C or feeding of animals with CsA restored lymphangiogenesis and metastasis in Wnt1 + melanoma. In conclusion, Wnt1 is anti-lymphangiogenic by suppressing melanoma-derived VEGF-C expression. © 2012 The Society for Investigative Dermatology.
Nandurkar H.,University of Melbourne |
Chong B.,University of New South Wales |
Salem H.,Australian Center for Blood Diseases |
Gallus A.,Flinders Medical Center |
And 2 more authors.
Internal Medicine Journal | Year: 2014
A working group of clinicians and scientists was formed to review the clinical considerations for use of low-molecular-weight heparin (LMWH) biosimilars. LMWH are biological molecules of significant complexity; the full complexity of chemical structure is still to be elucidated. LMWH biosimilars are products that are biologically similar to their reference product and rely on clinical data from a reference product to establish safety and efficacy. The complex nature of LMWH molecules means that it is uncertain whether a LMWH biosimilar is chemically identical to its reference product; this introduces the possibility of differences in activity and immunogenicity. The challenge for regulators and clinicians is to evaluate the level of evidence required to demonstrate that a LMWH is sufficiently similar to the reference product. The consensus opinion of the working group is that prior to clinical use a LMWH biosimilar should have proven efficacy and safety, similar to the reference product with prospective studies, which should be confirmed with a proactive post-marketing pharmacovigilance programme. © 2014 Royal Australasian College of Physicians.
Kwa M.Q.,University of Melbourne |
Kwa M.Q.,Royal Melbourne Hospital |
Nguyen T.,Royal Melbourne Hospital |
Nguyen T.,Australian Center for Blood Diseases |
And 11 more authors.
Journal of Biological Chemistry | Year: 2014
Epidermal and mucosal epithelial cells are integral to host defense. They not only act as a physical barrier but also utilize pattern recognition receptors, such as the Toll-like receptors (TLRs), to detect and respond to pathogens. Members of the interferon regulatory factor (IRF) family of transcription factors are key components of TLR signaling as they impart specificity to downstream responses. Although IRF6 is a critical regulator of epithelial cell proliferation and differentiation, its role in TLR signaling has not previously been addressed. We show here that IRF6 is activated by IRAK1 as well as by MyD88 but not by TRIF or TBK1. Co-immunoprecipitation experiments further demonstrated that IRF6 can interact with IRAK1. Gene silencing in epithelial cells along with gene promoter reporter assays showed that IRAK1 mediates TLR2-inducible CCL5 gene expression at least in part by promoting IRF6 activation. Conversely, IRAK1 regulated CXCL8 gene expression independently of IRF6, thus identifying a molecular mechanism by which TLR2 signaling differentially regulates the expression of specific chemokines in epithelial cells. Bioinformatics analysis and mutagenesis-based experiments identified Ser-413 and Ser-424 as key regulatory sites in IRF6. Phosphomimetic mutation of these residues resulted in greatly enhanced IRF6 dimerization and trans-activator function. Collectively, our findings suggest that, in addition to its importance for epithelial barrier function, IRF6 also contributes to host defense by providing specificity to the regulation of inflammatory chemokine expression by TLR2 in epithelial cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Tsun Wong A.K.,Australian Center for Blood Diseases
Expert Review of Hematology | Year: 2013
Platelets are anucleated fragments produced by megakaryocytes that circulate in the blood. Platelets are involved in the initial cellular response to damaged endothelium and migrate to this area to prevent excessive bleeding. What is becoming more acknowledged over the last few decades is that blood flow (hemodynamics) plays a critical role in platelet function. The purpose of this review is to summarize the current understanding of platelet biology with particular focus on the role of hemodynamics. The emerging concept of shear microgradients, which are challenging the traditional model of platelet function, will also be introduced in the review. © 2013 Expert Reviews Ltd.
Kwa M.Q.,University of Melbourne |
Kwa M.Q.,Royal Melbourne Hospital |
Huynh J.,University of Melbourne |
Huynh J.,Royal Melbourne Hospital |
And 9 more authors.
Journal of Biological Chemistry | Year: 2014
Receptor-interacting protein kinase 4 (RIPK4) and interferon regulatory factor 6 (IRF6) are critical regulators of keratinocyte differentiation, and their mutation causes the related developmental epidermal disorders Bartsocas-Papas syndrome and popliteal pterygium syndrome, respectively. However, the signaling pathways in which RIPK4 and IRF6 operate to regulate keratinocyte differentiation are poorly defined. Here we identify and mechanistically define a direct functional relationship between RIPK4 and IRF6. Gene promoter reporter and in vitro kinase assays, coimmunoprecipitation experiments, and confocal microscopy demonstrated that RIPK4 directly regulates IRF6 trans-activator activity and nuclear translocation. Gene knockdown and overexpression studies indicated that the RIPK4-IRF6 signaling axis controls the expression of key transcriptional regulators of keratinocyte differentiation, including Grainyhead-like 3 and OVO-like 1. Additionally, we demonstrate that the p.Ile121Asn missense mutation in RIPK4, which has been identified recently in Bartsocas-Papas syndrome, inhibits its kinase activity, thereby preventing RIPK4-mediated IRF6 activation and nuclear translocation. We show, through mutagenesis-based experiments, that Ser-413 and Ser-424 in IRF6 are important for its activation by RIPK4. RIPK4 is also important for the regulation of IRF6 expression by the protein kinase C pathway. Therefore, our findings not only provide important mechanistic insights into the regulation of keratinocyte differentiation by RIPK4 and IRF6, but they also suggest one mechanism by which mutations in RIPK4 may cause epidermal disorders (e.g. Bartsocas-Papas syndrome), namely by the impaired activation of IRF6 by RIPK4. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.