Dr Lisa Lincz

The interaction of acute myeloid leukaemic (AML) blasts with the bone marrow (BM) microenvironment is a major determinant governing disease progression and resistance to treatment. The constitutive expression of E-selectin in the vascular compartment of BM, a key endothelial cell factor, directly mediates chemoresistance via E-selectin ligand/receptors. Despite the success of hypomethylating agent (HMA)-containing regimens to induce remissions in older AML patients, the development of primary or secondary resistance is common. We report that following treatment with 5-azacitidine, promoter regions regulating the biosynthesis of the E-selectin ligands, sialyl Lewis X, become further hypomethylated. The resultant upregulation of these gene products, in particular a(1,3)-fucosyltransferase VII (FUT7) and a(2,3)-sialyltransferase IV (ST3GAL4), likely causes functional E-selectin binding. When combined with the E-selectin antagonist uproleselan, the adhesion to E-selectin is reversed and the survival of mice transplanted with AML cells is prolonged. Finally, we present clinical evidence showing that BM myeloid cells from higher risk MDS and AML patients have the potential to bind E-selectin, and these cells are more abundant in 5-azacitidine-non-responsive patients. The collective data provide a strong rationale to evaluate 5-azacitidine in combination with the E-selectin antagonist, uproleselan, in this patient population.

Since their introduction several years ago, poly (ADP-ribose) polymerase (PARP) inhibi-tors (PARPi) have become the standard of care for breast and gynaecological cancers with BRCA gene mutations. Given that PARPi act by exploiting defective DNA repair mechanisms within tu-mour cells, they should be ideally suited to combatting haematological malignancies where these pathways are notoriously defective, even though BRCA mutations are rare. To date, despite prom-ising results in vitro, few clinical trials in humans for haematological malignancies have been per-formed, and additional investigation is required. Paradoxically, secondary haematological malignancies have arisen in patients after treatment with PARPi, raising concerns about their potential use as therapies for any blood or bone marrow-related disorders. Here, we provide a comprehensive review of the biological, pre-clinical, and clinical evidence for and against treating individual haematological malignancies with approved and experimental PARPi. We conclude that the prom-ise of effective treatment still exists, but remains limited by the lack of investigation into useful biomarkers unique to these malignancies.

Immunomodulatory therapies after stroke have the potential to provide clinical benefit to a subset of patients, but risk subverting the protective, healing aspects of the innate immune response. Neutrophils clear necrotic cerebral tissue and are important in immunomodulation, but can also contribute to tissue injury. Human trials for immunomodulatory stroke treatments in the sub-acute time frame have attempted to prevent peripheral neutrophil infiltration, but none have been successful and one trial demonstrated harm. These unselected trials had broad inclusion criteria and appear to not have had a specific treatment target. Unfortunately, due to the heterogeneous nature of brain ischemia in humans resulting in variation in clinical severity, the negative effect of thrombolytic drugs on the blood¿brain barrier, and the heterogeneity of immune response, it may only be a subset of stroke patients who can realistically benefit from immunomodulation therapies. Translational research strategies require both an understanding of lab practices which create highly controlled environments in contrast to clinical practice where the diagnosis of stroke does not require the identification of a vessel occlusion. These differences between lab and clinical practices can be resolved through the integration of appropriate patient selection criteria and use of advanced imaging and ridged patient selection practices in clinical trials which will be an important part to the success of any future trials of translational research such as immunomodulation.

Large artery atherosclerotic stroke (LAS) shows substantial heritability not explained by previous genome-wide association studies. Here, we explore the role of coding variation in LAS by analyzing variants on the HumanExome BeadChip in a total of 3,127 cases and 9,778 controls from Europe, Australia, and South Asia. We report on a nonsynonymous single-nucleotide variant in serpin family A member 1 (SERPINA1) encoding alpha-1 antitrypsin [AAT; p.V213A; P = 5.99E-9, odds ratio (OR) = 1.22] and confirm histone deacetylase 9 (HDAC9) as a major risk gene for LAS with an association in the 3?-UTR (rs2023938; P = 7.76E-7, OR = 1.28). Using quantitative microscale thermophoresis, we show that M1 (A213) exhibits an almost twofold lower dissociation constant with its primary target human neutrophil elastase (NE) in lipoprotein-containing plasma, but not in lipid-free plasma. Hydrogen/deuterium exchange combined with mass spectrometry further revealed a significant difference in the global flexibility of the two variants. The observed stronger interaction with lipoproteins in plasma and reduced global flexibility of the Val-213 variant most likely improve its local availability and reduce the extent of proteolytic inactivation by other proteases in atherosclerotic plaques. Our results indicate that the interplay between AAT, NE, and lipoprotein particles is modulated by the gate region around position 213 in AAT, far away from the unaltered reactive center loop (357-360). Collectively, our findings point to a functionally relevant balance between lipoproteins, proteases, and AAT in atherosclerosis.

Essentials Russell's viper envenoming is a major health issue in South Asia and causes coagulopathy. We studied the effect of fresh frozen plasma and two antivenom doses on correcting coagulopathy. Fresh frozen plasma did not hasten recovery of coagulopathy. Low-dose antivenom did not worsen coagulopathy. Summary: Background Russell's viper (Daboia russelii) envenoming is a major health issue in South Asia and causes venom-induced consumption coagulopathy (VICC). Objectives To investigate the effects of fresh frozen plasma (FFP) and two antivenom doses in correcting VICC. Methods We undertook an open-label randomized controlled trial in patients with VICC at two Sri Lankan hospitals. Patients with suspected Russell's viper bites and coagulopathy were randomly allocated (1 : 1) to high-dose antivenom (20 vials) or low-dose antivenom (10 vials) plus 4 U of FFP. The primary outcome was the proportion of patients with an International Normalized Ratio (INR) of < 2 at 6 h after antivenom administration. Secondary outcomes included anaphylaxis, major hemorrhage, death, and clotting factor recovery. Results From 214 eligible patients, 141 were randomized: 71 to high-dose antivenom, and 70 to low-dose antivenom/FFP; five had no post-antivenom blood tests. The groups were similar except for a delay of 1 h in antivenom administration for FFP patients. Six hours after antivenom administration, 23 of 69 (33%) patients allocated to high-dose antivenom had an INR of < 2, as compared with 28 of 67 (42%) allocated to low-dose antivenom/FFP (absolute difference 8%; 95% confidence interval - 8% to 25%). Fifteen patients allocated to FFP did not receive it. Severe anaphylaxis occurred equally frequently in each group. One patient given FFP developed transfusion-related acute lung injury. Three deaths occurred in low-dose antivenom/FFP patients, including one intracranial hemorrhage. There was no difference in recovery rates of INR or fibrinogen, but there was more rapid initial recovery of factor V and FX in FFP patients. Conclusion FFP after antivenom administration in patients with Russell's viper bites did not hasten recovery of coagulopathy. Low-dose antivenom/FFP did not worsen VICC, suggesting that low-dose antivenom is sufficient.

Background Characterization of circulating microvesicles (MV) in healthy subjects in relation to various biological factors is not well studied. Objectives We evaluated the influence of age, gender, smoking status, lipid and hormone profiles on circulating MV in healthy subjects. Methods Platelet free plasma from 143 volunteer blood donors (males¿=¿80, females¿=¿63) was evaluated by standardized flow cytometry for MV expressing CD41 (platelet-derived), CD105 (endothelial-derived), CD235 (red cell-derived), TF (tissue factor) and phosphatidylserine (PS) MV. Procoagulant function was measured by the Xa based assay (XaCT) and endogenous thrombin potential (ETP) using thrombin generation assay. Results Those =¿29¿years and =¿60¿years had higher levels of MV subsets (CD41, CD235, TF and PS) compared to those aged 30¿59¿years. The median CD41, CD105, CD235, TF and PS expressing MV by flow cytometry were similar or lower in females, whilst procoagulant activity by the XaCT assay was higher (p¿=¿0.002). In smokers (n¿=¿21), certain MV subsets (CD41, TF and PS) and functional activity (ETP) was lower (p¿<¿0.05). Regression analysis showed that MV parameters of CD41, CD105, TF and ETP could be predicted independently by age, whilst smoking predicted for CD105, CD235, TF, PS and ETP. Certain MV parameters also correlated with BMI, lipid and hormone levels. The small RNA and miRNA levels did not differ by age group, smoking status or gender. Conclusions It is important to recognize that differences may arise depending on age, gender, BMI, lipid, hormone levels and smoking status in apparently healthy subjects when evaluating MV for pathogenic potential.

Non-alcoholic fatty liver disease (NAFLD) is an inflammatory condition caused by hepatic lipid accumulation that is associated with insulin resistance, diabetes and metabolic syndrome. Although statins should be used with caution in liver diseases, they are increasingly investigated as a possible treatment for NAFLD. The present study recreated an in vitro model of NAFLD using HepG2 cells exposed to oleic acid (OA), which was used to quantify OA-induced lipid accumulation in HepG2 cells treated with various concentrations of simvastatin. In addition, the effect of simvastatin on HepG2 cell morphology and microparticle generation as a marker of cell apoptosis was assessed. OA-induced lipid accumulation was quantified by Oil Red O staining and extraction for optical density determination. Stained lipid droplets were visualized using phase contrast microscopy. Furthermore, HepG2 cell-derived microparticles were counted by flow cytometry subsequent to staining for Annexin V. HepG2 cells treated with 0-1 mM OA showed dose-dependent lipid accumulation. Treatment of HepG2 cells with increasing concentrations of simvastatin followed by treatment with 1 mM OA showed that low simvastatin concen- trations (4-10 µM) were able to reduce lipid accumulation by ~40%, whereas high simvastatin concentrations (20 and 30 µM) induced apoptotic changes in cell morphology and increased the production of Annexin V+ microparticles. This suggests that low simvastatin doses may have a role in preventing NAFLD. However, further investigations are required to confirm this action in vivo and to determine the underlying mechanism by which simvastatin reduces hepatic steatosis.

Background Animal models are used to test toxic effects of snake venoms/toxins and the antivenom required to neutralise them. However, venoms that cause clinically relevant coagulopathy in humans may have differential effects in animals. We aimed to investigate the effect of different procoagulant snake venoms on various animal plasmas. Methods Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and D-dimer levels were measured in seven animal plasmas (human, rabbit, cat, Guinea pig, pig, cow and rat). In vitro clotting times were then used to calculate the effective concentration (EC50) in each plasma for four snake venoms with different procoagulant toxins: Pseudonaja textilis, Daboia russelli, Echis carinatus and Calloselasma rhodostoma. Results Compared to human, PT and aPTT were similar for rat, rabbit and pig, but double for cat and cow, while Guinea pig had similar aPTT but double PT. Fibrinogen and D-dimer levels were similar for all species. Human and rabbit plasmas had the lowest EC50 for P. textilis (0.1 and 0.4 µg/ml), D. russelli (0.4 and 0.1 µg/ml), E. carinatus (0.6 and 0.1 µg/ml) venoms respectively, while cat plasma had the lowest EC50 for C. rhodostoma (11 µg/ml) venom. Cow, rat, pig and Guinea pig plasmas were highly resistant to all four venoms with EC50 10-fold that of human. Conclusions Different animal plasmas have varying susceptibility to procoagulant venoms, and excepting rabbits, animal models are not appropriate to test procoagulant activity. In vitro assays on human plasma should instead be adopted for this purpose.

Introduction Circulating microvesicles (MV) can be analysed using a number of different techniques. The aim of this study was to evaluate the correlation between functional procoagulant based assays including thrombin generation, factor Xa activation test (XaCT), and phosphatidylserine factor Xa-activity by ELISA with optical MV enumeration by flow cytometry and nanoparticle tracking analysis. Methods Citrated blood samples were collected from 60 healthy volunteer blood donors after informed consent. Platelet free plasma was prepared using a standardized published protocol. MV subsets were enumerated by flow cytometry (BDFACS Canto) after staining with specific antibodies for platelets (CD41), endothelial cells (CD105), red cells (CD235) monocytes (CD14), tissue factor (CD142) and for phosphatidylserine expression by binding to annexin V. A standardized protocol using counting beads was employed. Nanotracking analysis was performed on both scatter and fluorescent settings after MV staining with quantum dot stain, Qdot 655. Procoagulant function was assessed by the XaCT assay on an automated coagulation analyser and by thrombin generation assay measuring endogenous thrombin potential (ETP), lagtime, peak (PEAK) and time to peak (ttPEAK) using a Calibrated Automated Thrombogram (CAT). The statistical analysis was carried out with Statistica 12 software using non-parametric tests (Spearman rank order correlations, with significance set at p¿<¿0.05). Results In normal healthy subjects, thrombin generation parameters correlated with levels of MV measured by flow cytometry. ETP, lagtime, ttPEAK and PEAK correlated with MV expressing phosphatidylserine (rs, Spearman rank order correlation was 0.29, 0.40, 0.31 and 0.34 respectively, p¿<¿0.05), and MV expressing tissue factor (rs was 0.29, 0.40, 0.31 and 0.34 respectively, p¿<¿0.05), whilst red cell derived MV correlated with lagtime, ttPEAK and PEAK (rs, was 0.35,0.30 and 0.3, respectively, p¿<¿0.05). Lagtime and ttPEAK negatively correlated with the clot based XaCT test (rs, was -¿0.34 and -¿0.30 respectively, p¿<¿0.05) and positively correlated with the ELISA MP-activity assay (rs¿=¿0.42 for both, p¿<¿0.05). In addition, endothelial MV levels weakly correlated with white cell counts (rs = 0.27, p¿<¿0.05). Conclusions Thrombin generation and flow cytometry for phosphatidylserine or tissue factor expressing MV correlate well as markers for procoagulant activity. A combination of optical or non-optical enumeration as well as functional methods may be required for a complete profiling of circulating MV.

Background Russell's viper envenoming is a major problem in South Asia and causes venom induced consumption coagulopathy. This study aimed to investigate the kinetics and dynamics of venom and clotting function in Russell's viper envenoming. Methodology/Principal Findings In a prospective cohort of 146 patients with Russell's viper envenoming, we measured venom concentrations, international normalised ratio [INR], prothrombin time (PT), activated partial thromboplastin time (aPTT), coagulation factors I, II, V, VII, VIII, IX and X, and von Willebrand factor antigen. The median age was 39y (16¿82y) and 111 were male. The median peak INR was 6.8 (interquartile range[IQR]:3.7 to >13), associated with low fibrinogen [median,<0.01g/L;IQR:<0.01¿0.9g/L), low factor V levels [median,<5%;IQR:<5¿4%], low factor VIII levels [median,40%;IQR:12¿79%] and low factor X levels [median,48%; IQR:29¿67%]. There were smaller reductions in factors II, IX and VII over time. All factors recovered over 48h post-antivenom. The median INR remained >3 at 6h post-antivenom but had reduced to <2, by 24h. The aPTT had also returned to close to normal (<50sec) at 24h. Factor VII, VIII and IX levels were unusually high pre-antivenom, median peak concentrations of 393%, 307% and 468% respectively. Pre-antivenom venom concentrations and the INR (r = 0.20, p = 0.02) and aPTT (r = 0.19, p = 0.03) were correlated (non-parametric Spearman analysis). Conclusions Russell's viper coagulopathy results in prolonged aPTT, INR, low fibrinogen, factors V, VIII and X which recover over 48h. Severity of clotting abnormalities was associated with venom concentrations.

The Hippo pathway is emerging as a critical nexus that balances self-renewal of progenitors against differentiation; however, upstream elements in vertebrate Hippo signalling are poorly understood. High expression of Fat1 cadherin within the developing neuroepithelium and the manifestation of severe neurological phenotypes in Fat1-knockout mice suggest roles in neurogenesis. Using the SH-SY5Y model of neuronal differentiation and employing gene silencing techniques, we show that FAT1 acts to control neurite outgrowth, also driving cells towards terminal differentiation via inhibitory effects on proliferation. FAT1 actions were shown to be mediated through Hippo signalling where it activated core Hippo kinase components and antagonised functions of the Hippo effector TAZ. Suppression of FAT1 promoted the nucleocytoplasmic shuttling of TAZ leading to enhanced transcription of the Hippo target gene CTGF together with accompanying increases in nuclear levels of Smad3. Silencing of TAZ reversed the effects of FAT1 depletion thus connecting inactivation of TAZ-TGFbeta signalling with Hippo signalling mediated through FAT1. These findings establish FAT1 as a new upstream Hippo element regulating early stages of differentiation in neuronal cells.

Nerve infiltration is essential to prostate cancer progression, but the mechanism by which nerves are attracted to prostate tumors remains unknown. We report that the precursor of nerve growth factor (proNGF) is overexpressed in prostate cancer and involved in the ability of prostate cancer cells to induce axonogenesis. A series of 120 prostate cancer and benign prostate hyperplasia (BPH) samples were analyzed by IHC for proNGF. ProNGF was mainly localized in the cytoplasm of epithelial cells, with marked expression in cancer compared with BPH. Importantly, the proNGF level positively correlated with the Gleason score (n = 104, tB = 0.51). A higher level of proNGF was observed in tumors with a Gleason score of =8 compared with a Gleason score of 7 and 6 (P < 0.001). In vitro, proNGF was detected in LNCaP, DU145, and PC-3 prostate cancer cells and BPH-1 cells but not in RWPE-1 immortalized nontumorigenic prostate epithelial cells or primary normal prostate epithelial cells. Co-culture of PC12 neuronal-like cells or 50B11 neurons with PC-3 cells resulted in neurite outgrowth in neuronal cells that was inhibited by blocking antibodies against proNGF, indicating that prostate cancer cells can induce axonogenesis via secretion of proNGF. These data reveal that ProNGF is a biomarker associated with high-risk prostate cancers and a potential driver of infiltration by nerves.

BACKGROUND AND PURPOSE - : Studies in rodent models suggest that upregulating angiopoietin-1 (Angpt1) improves stroke outcomes. The aims of this study were to assess the association of plasma Angpt1 with stroke occurrence and outcome. METHODS - : Plasma Angpt1 was measured in 336 patients who had experienced a recent stroke and 321 healthy controls with no stroke history. Patients with stroke (n=285) were reassessed at 3 months and plasma Angpt1 concentration on admission compared between those with severe and minor disability as assessed by the modified Rankin scale. In a separate cohort of 4032 community-acquired older men prospectively followed for a minimum of 6 years, the association of plasma Angpt1 with stroke incidence was examined. RESULTS - : Median plasma Angpt1 was 3-fold lower in patients who had experienced a recent stroke (6.42, interquartile range, 4.26-9.53 compared with 17.36; interquartile range, 14.01-22.46 ng/mL; P<0.001) and remained associated with stroke after adjustment for other risk factors. Plasma Angpt1 concentrations on admission were lower in patients who had severe disability or died at 3 months (median, 5.52; interquartile range, 3.81-8.75 ng/mL for modified Rankin scale 3-6; n=91) compared with those with minor disability (median, 7.04; interquartile range, 4.75-9.92 ng/mL for modified Rankin scale 0-2; n=194), P=0.012, and remained negatively associated with severe disability or death after adjusting for other risk factors. Plasma Angpt1 was not predictive of stroke incidence in community-dwelling older men. CONCLUSIONS - : Plasma Angpt1 concentrations are low after ischemic stroke particularly in patients with poor stroke outcomes at 3 months. Interventions effective at upregulating Angpt1 could potentially improve stroke outcomes. © 2014 American Heart Association, Inc.

Background: LCn-3PUFA comprised of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) offer cardioprotection involving a decrease in coagulant activity; however, the evidence is equivocal. We have previously demonstrated that the acute (24. h) effects and chronic (4. weeks) effects of LCn-3PUFA supplementation on platelet aggregation in human subjects are sex specific. This study investigated the mechanisms of the sex-dependent effects of LCn-3PUFA with 4. weeks supplementation of EPA-rich vs. DHA-rich oils on procoagulant and platelet activity in healthy subjects. Design: A double-blinded, placebo-controlled randomised trial was conducted in 94 healthy adults: male (. n=41) and female (. n=53). Platelet coagulation parameters including factors I, II, V, VII, VIII, IX, X, vWF:Ag and endogenous thrombin potential were measured at baseline and 4. weeks postsupplementation with EPA-rich or DHA-rich oil capsules. Results: We have previously reported that platelet aggregation is specifically reduced by supplementation with EPA in males and DHA in females. This sex-specific effect was also observed for decreases in plasma levels of Factor II (-7.9±3.8%, P=.026), Factor V (-6.5±4.5%, P=.022) and vWF:Ag (-7.3±2.1%, P=.034) and was most pronounced in males supplemented with EPA. In contrast, DHA-mediated reduction in platelet aggregation in females was not accompanied by any significant changes in the coagulation parameters tested. Conclusion: Significant interactions between sex and specific LCn-3PUFA exist to reduce procoagulant activity differentially in males vs. females and could have profound effects on managing risk of thrombotic disease.