Phospholipase A2 (PLA2) as an Early Indicator of Envenomation in Australian Elapid Snakebites (ASP-27)

Early diagnosis of snake envenomation is essential, especially neurotoxicity and myotoxicity. We investigated the diagnostic value of serum phospholipase (PLA2) in Australian snakebites. In total, 115 envenomated and 80 non-envenomated patients were recruited over 2 years, in which an early blood sample was available pre-antivenom. Serum samples were analyzed for secretory PLA2 activity using a Cayman sPLA2 assay kit (#765001 Cayman Chemical Company, Ann Arbor MI, USA). Venom concentrations were measured for snake identification using venom-specific enzyme immunoassay. The most common snakes were Pseudonaja spp. (33), Notechis scutatus (24), Pseudechis porphyriacus (19) and Tropidechis carinatus (17). There was a significant difference in median PLA2 activity between non-envenomated (9 nmol/min/mL; IQR: 7–11) and envenomated patients (19 nmol/min/mL; IQR: 10–66, p < 0.0001) but Pseudonaja spp. were not different to non-envenomated. There was a significant correlation between venom concentrations and PLA2 activity (r = 0.71; p < 0.0001). PLA2 activity was predictive for envenomation; area under the receiver-operating-characteristic curve (AUC-ROC), 0.79 (95% confidence intervals [95%CI]: 0.72–0.85), which improved with brown snakes excluded, AUC-ROC, 0.88 (95%CI: 0.82–0.94). A cut-point of 16 nmol/min/mL gives a sensitivity of 72% and specificity of 100% for Australian snakes, excluding Pseudonaja. PLA2 activity was a good early predictor of envenomation in most Australian elapid bites. A bedside PLA2 activity test has potential utility for early case identification but may not be useful for excluding envenomation.

Cannabidiol: Assessing preclinical safety in ovarian and endometrial carcinoma cell lines.

e24130 Background: Cancer patients use cannabidiol (CBD) for chemotherapy and cancer symptoms, though research of CBD safety and efficacy for these conditions are ongoing and mixed. We sought to determine endometrial (ECC1) and epithelial ovarian cancer (Kuramochi) cell proliferation when exposed to different concentrations of CBD, for the broader goal to establish if CBD can safely be utilized to treat the symptoms of cancer, including those caused by chemotherapy. Methods: ECC1 and Kuramochi cells were kept in media (RPMI with 10% bovine serum and 1% penicillin/streptomycin). We passaged cells when > 90% confluent by adding tryspin-EDTA, incubating at 37C for 3 minutes, then spinning down with media to harvest the cell pellet. Cells were re-suspended in media, counted and apportioned to 96 well plates. Plates were incubated at 37C x 24 hours. CBD (from Cayman Chemical) was suspended in DMSO per manufacturer instruction then used to treat cells x72 hours at different concentrations (2.5-50uM). MTT was added to cells, cells incubated at 37C x 3 hours, media and MTT were removed and DMSO was added. Optical depth (OD) was calculated for plates using SoftMaxPro version 6.2.2. ODs were used to calculate inhibitory concentration for 50% cell death (IC50). Results: Kuramochi and ECC1 demonstrated decreased cell proliferation when exposed to CBD for 72hours. ECC1 IC50 fell between 2.5-5uM. Kuramochi IC50 fell between 15-20uM. Nearly all ECC1 growth was inhibited at concentrations 10uM or greater. Kuramochi proliferation was 15% that of controls at concentrations of 40 and 50uM CBD. Conclusions: ECC1 and Kuramochi cells demonstrated decreased proliferation in the presence of CBD. This bodes well for future studies of concurrent exposure to CBD and cytotoxic chemotherapy. Further preclinical research needed on CBD effects in endometrial and ovarian cancer, as patients turn to CBD for symptomatic relief from cancer and chemotherapy side effects. [Table: see text]

The Small Molecule Img-98, a Potent and Selective Inhibitor of the Lysine Demethylase Lsd-1, Effectively Augments the Pro-Differentiation Effects of ATRA in a Pre-Clinical Model of AML

Acute Promyelocytic Leukemia (APL) is a cytogenetically unique subtype of acute myeloid leukemia (AML), characterized by the presence of the t(15;17)-associated PML-RARA fusion gene. This disease is curable in most patients with all-trans-retinoic acid (ATRA) based therapies, which effectively differentiate malignant promyelocytes. In patients with non-APL AML, most patients with die from their disease and ATRA has little activity. Therefore, research strategies that seek to extend the efficacy of ATRA-based treatment in AML are key avenues of investigation. From our previous studies, an epigenetic analysis of primary AML samples revealed that relative to normal CD33+ cells, loss of RARα2 expression in AML is associated with a reduction in H3K4me2 on the RARA2 promoter (a modification that is associated with transcriptional activation). The mono- and di-methyl lysine demethylase LSD1 (KDM1A) is highly expressed in patients with AML, and its overexpression has been implicated in various other tumors. Based on these data we correctly predicted that the use of small-molecule inhibitors targeting LSD1 (LSD1i) could result in epigenetic reprogramming that enhanced or facilitated the execution of the ATRA-induced differentiation program in AML cells. In the current study, we characterized a range of small molecule inhibitors of LSD-1. All the agents tested (RN-1, GSKi, SP2509, TCP, IMG-98 and OG-L002) led to inhibition of LSD-1 in a biochemical assay with varying degrees of potency. From this study, we further characterized the anti-tumor effects of IMG-98 alone and in combination with ATRA. IMG-98 is a novel LSD1 inhibitor relative to drugs of this class with comparatively different specificity, potency, pharmacokinetics, and metabolism. Its greater heavy atom count and chemical complexity contribute to these properties. By fluorine nuclear magnetic resonance (fNMR) and florescent spectrophotometry, the molecule rapidly reacts irreversibly with the FAD co-factor of LSD1 and this polypeptide is necessary to catalyze the reaction. Thermal stability shifts show the inactivated form of the enzyme becomes much more stable suggesting significant structural changes. Treatment with IMG-98 promoted the expression of the cell surface marker CD11b, associated with a differentiated immunophenotype, in both AML cell lines and primary patient material. IMG-98 produced a potent anti-proliferative effect across a range of AML cell lines and also led to growth inhibition of AML blast colony forming ability. In combination studies with ATRA, IMG-98 re-sensitized AML cells to ATRA by reactivating ATRA driven differentiation programs. Post-differentiation apoptosis was more significant for combined therapy (ATRA + IMG-98) than with either agent alone. Heatmap display of unsupervised hierarchical clustering of genes in AML cell lines differentially expressed in response to treatment with combinations of ATRA, IMG-98 or the combination, confirmed that ATRA combined with IMG-98 enhanced the expression of a subset of genes associated with the myeloid differentiation program. Updated studies on mechanisms underpinning mode of action of IMG-98 in this model will be presented. Taken together, these data demonstrate that ATRA combined with pharmacological inhibition of LSD1, may provide a promising treatment for AML by promoting differentiation and subsequent growth inhibition of AML blasts. A closely related molecule to IMG-98 is currently being optimized in late preclinical development, and clinical trials with this compound are anticipated to start in 2016. Figure 1. Comparative screening assay for LSD1 inhibition with commercially available agents (LSD1 Inhibitor Screening Assay, Cayman Chemical, Cat# 700120) Figure 1. Comparative screening assay for LSD1 inhibition with commercially available agents (LSD1 Inhibitor Screening Assay, Cayman Chemical, Cat# 700120) Disclosures Rienhoff: Imago: Employment.

Abstract T P80: Inhibition of Ezh2 Leads to Decreased M1 and Enhanced M2 Microglia Phenotypes

Background: Ischemic stroke results in the activation of microglia, which may polarize toward a pro-inflammatory (M1) phenotype or an anti-inflammatory, neuroprotective (M2) phenotype. Thus, simultaneously suppressing the M1 response and promoting the M2 response could be beneficial in the treatment of stroke. Recently, the epigenetic modulator Jmjd3 has been shown to be essential for M2 polarization. However, Jmjd3 is antagonized by Ezh2 which is associated with M1 polarization. Thus, we hypothesized that inhibition of Ezh2 tilts the balance between Jmjd3 and Ezh2, thereby enhancing polarization toward an M2 phenotype and improved outcome in ischemic stroke. Methods: Mixed glial cultures were isolated from P0.5-P2 C57BL/6J mice and cultured for 14 days before microglial isolation. Microglia were rested for 24 hours before treatment every other day with 6uM GSK343 (Cayman Chemical) or DMSO vehicle control. After 7 days, microglia were stimulated with LPS or IL-4 and RNA was isolated at 4hr and 24hr post-stimulation for qRT-PCR analysis. Results: LPS-induced IL6 and IL1B expression was significantly abrogated by 71% and 53%, respectively (p<0.05), at 24hr when Ezh2 was inhibited. Additionally, Ezh2 inhibition both increased baseline expression of M2-associated genes ARG1, CD206, and IRF4 by 196%, 257%, and 395%, respectively (p<0.05), and rescued their expression in the presence of LPS at 24hr (p<0.05) in which they were otherwise significantly down-regulated. Conclusion: Pharmacological inhibition of Ezh2 limits microglial M1 polarization and enhances M2 polarization.

147 PREGNANCY RATES OF RECIPIENT ANIMALS FOLLOWING APPLICATION OF A SELECTIVE PROSTAGLANDIN F2α RECEPTOR ANTAGONIST DURING EMBRYO RECOVERY

Companion research presented at this meeting has indicated that addition of a prostaglandin2α (PGF2α) receptor (FPr) antagonist to culture medium prevented the detrimental action of PGF2α on embryo development. The aim of this study was to evaluate addition of an FPr antagonist to the collection medium on pregnancy rates after transfer of bovine embryos to recipient animals. An initial experiment was performed to determine in vitro development of in vivo-derived morula-stage frozen-thawed embryos cultured in KSOM-PVA medium with 1000 nm AL-8810 (Cayman Chemical Inc., Ann Arbor, MI, USA) (AL, n = 94), 1000 nm AL-8810 and 10 ng mL–1 PGF2α (Cayman Chemical Inc.) (AL+PGF, n = 94), 10 ng mL–1 PGF2α (PGF, n = 94), or serving as controls (CON, n = 91). Embryos remained in their treatment for a 30-h period until blastocyst development was recorded. In a subsequent experiment, embryos were recovered (n = 783) from superovulated donors on Day 7 after artificial insemination with medium containing 1000 nm AL-8810 (AL), 100 nM AL-8810 (AL100), or with vehicle (VEH: 1 mL DMSO; Sigma-Aldrich, St. Louis, MO, USA) in a double blind study. Following collection, embryos were classified by stage and quality, and then transferred fresh to recipients or frozen (ethylene glycol, direct transfer). Frozen embryos, following thawing, were transferred during the subsequent breeding period. Pregnancy rates were determined by ultrasonography (28–35 days post-transfer) and confirmed by calving date. Data were analyzed using the GLIMMIX procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Results from the initial experiment indicated that culture of in vivo-derived bovine embryos in medium containing AL-8810 improved blastocyst development compared to PGF (58.5% v. 45.7%; P = 0.05). In addition, a strong tendency to increase embryo development was observed in AL+PGF compared to PGF treatment group (57% v. 45.7%; P = 0.07). Overall pregnancy rates of fresh and frozen embryos were increased in the AL and AL100 groups (55% and 58%, respectively) compared to VEH (43%; P = 0.009). Since AL treatments did not differ in pregnancy rates, subsequent analysis combined AL and AL100 data. Transfer of frozen embryos collected with medium containing AL-8810 (n = 238) increased pregnancy rates (AL, 45%) compared to embryos recovered without (n = 221) AL-8810 (VEH, 34%; P = 0.01). Transfer of fresh embryos collected with medium containing AL-8810 (n = 241) tended to have increased pregnancy rates (AL, 76%) compared to control (n = 83; VEH, 66%; P = 0.09). Although data collection continues, no abnormalities in calf health, birth weight, or weaning weight have been observed between any treatments. In conclusion, recovery of embryos with flushing medium containing an FPr antagonist improved pregnancy rates after transfer. Funding was provided by Ultimate Genetics and the Tennessee Agricultural Experiment Station for completion of these studies.

146 DETRIMENTAL EFFECTS OF PROSTAGLANDIN F2α ON IN VITRO EMBRYO DEVELOPMENT IN BOVINE ARE INHIBITED BY A RECEPTOR ANTAGONIST

Numerous studies have demonstrated negative effects of prostaglandin F2α (PGF2α) on bovine reproduction. Discovery of a PGF2α receptor (FPr) in bovine embryos (Scenna et al. 2006 Reprod. Fertil. Dev. 18, 180) allows for development of new therapeutic strategies to improve success of embryo transfer. Therefore, two experiments were performed to investigate the occurrence of any toxic effect of AL-8810 (Cayman Chemical Inc., Ann Arbor, MI, USA), an FPr antagonist, on in vitro development of bovine embryos. In Exp. 1, pre-compacted embryos were cultured in (1) 100 AL (100 nm AL-8810 in potassium simplex optimized medium with polyvinyl alcohol (KSOM-PVA); n = 94); (2) 50 AL (50 nm AL-8810 in KSOM-PVA; n = 94); (3) 25 AL (25 nm AL-8810 in KSOM-PVA; n = 94); and (4) CON (control: KSOM-PVA; n = 95). In Exp. 2, pre-compacted embryos were cultured in (1) 1000 AL (1000 nm AL-8810 in KSOM-PVA; n = 282); (2) 500 AL (500 nm AL-8810 in KSOM-PVA; n = 274); (3) 250 AL (250 nm AL-8810 in KSOM-PVA; n = 274); and (4) CON (control: KSOM-PVA; n = 278). Embryos remained in treatments until blastocyst assessment. Next, two experiments were performed to determine the efficiency of AL-8810 on preventing detrimental effects of PGF2α on pre-compacted embryos. In Exp. 3, pre-compacted embryos were cultured in (1) 100 AL (100 nm AL-8810 in KSOM-PVA; n = 121); (2) 10 PGF (10 ng mL–1 of PGF2α (Cayman Chemical Inc.) in KSOM-PVA; n = 91); (3) AL100+PGF (100 nm AL-8810 and 10 ng mL–1 of PGF2� in KSOM-PVA; n = 116); (4) CON (control: KSOM-PVA; n = 96). In Exp. 4, embryos were cultured in (1) 1000 AL (1000 nm AL-8810 in KSOM-PVA; n = 87); (2) 10 PGF (10 ng mL–1 of PGF2α in KSOM-PVA; n = 87); (3) AL1000+PGF (1000 nm AL-8810 and 10 ng mL–1 of PGF2α in KSOM-PVA; n = 84); (4) CON (control: KSOM-PVA; n = 84). In Exp. 3 and 4, embryos remained in treatments for 48 h when development to morula was assessed. Data for all experiments were analyzed using the GLIMMIX procedure of SAS (SAS Institute, Inc., Cary, NC, USA). For Exp. 1, results indicated that addition of 100, 50, and 25 nm did not compromise embryonic development to the blastocyst stage compared to controls (60.2%, 55.8%, 55.4%, and 49.9%, respectively). In addition, orthogonal contrasts indicated that 100 nm AL-8810 improved development to the blastocyst stage (100 AL = 61% v. CON = 50.6%, P = 0.01). Similarly for Exp. 2, 1000, 500, and 250 nm AL-8810 did not affect in vitro development to the blastocyst stage compared to controls (40%, 39%, 34.8%, and 37.7%, respectively). In Exp. 3 and 4, addition of 1000 nm AL-8810, but not 100 nm, to culture medium of pre-compacted embryos exposed to PGF2α increased the ability of embryos to undergo compaction 48 h later (1000 AL+PGF = 51% v. PGF = 40%; P = 0.05). In conclusion, AL-8810 at a concentration of 1000 nm inhibits detrimental effects of PGF2α on the development of pre-compacted bovine embryos and may prove beneficial for other assisted reproductive techniques in cattle. Funding was provided by Ultimate Genetics and the Tennessee Agricultural Experiment Station for completion of these studies.