Objective Cocaine use is associated with arterial thrombosis, including myocardial infarction and stroke. Cocaine use results in increased plasma von Willebrand Factor (VWF), accelerated atherosclerosis, and platelet-rich arterial thrombi, suggesting that cocaine activates the endothelium, promoting platelet-VWF interactions. Approach and Results Human umbilical vein (HUVEC), brain microvasculature (BMVEC), or coronary artery (CAEC) endothelial cells were treated with cocaine or metabolites benzoylecgonine, cocaethylene, norcocaine, or ecgonine methylester. Supernatant VWF concentration and multimer structure were measured, and platelet–VWF strings formed on the endothelial surface under flow were quantified. Cocaine, benzoylecgonine, and cocaethylene induced endothelial VWF release, with the two metabolites being more potent than the parent molecule. BMVEC were more sensitive to cocaine and metabolites than were HUVEC or CAEC. CAEC released VWF into the supernatant but did not form VWF–platelet strings. Intracellular cAMP concentration was not increased after treatment with cocaine or its metabolites. Conclusions Both cocaine and metabolites benzoylecgonine and cocaethylene induced endothelial VWF secretion, possibly explaining thrombotic risk after cocaine ingestion. VWF secretion is likely to vary between vascular beds, with brain endothelial cells being particularly sensitive. These results suggest that clinical management of cocaine-induced ischemia may benefit from therapies aimed at disrupting the VWF–platelet interaction.

Purpose Selenium is an essential trace element that supports animal health through the antioxidant defense system by protecting cells from oxidative-related damage. Using inorganic selenium species, such as sodium selenite (Na Sel), as a food supplement is cost-effective; however, its limitation as a nutritional supplement is its cytotoxicity. One strategy to mitigate this problem is by delivering inorganic selenium using a nanoparticle delivery system (SeNP). Methods Rainbow trout intestinal epithelial cells, bovine turbinate cells and bovine intestinal myofibroblasts were treated with soluble Na Sel or SeNPs. Two SeNP formulations were tested; SeNP-Ionic where inorganic selenium was ionically bound to cationic phytoglycogen (PhG) NPs, and SeNP-Covalent, where inorganic selenium was covalently bound to PhG NPs. Selenium-induced cytotoxicity along with selenium bioavailability were measured. Results SeNPs (SeNP-Ionic or SeNP-Covalent) substantially reduced cytotoxicity in all cell types examined compared to similar doses of soluble inorganic selenium. The SeNP formulations did not affect selenium bioavailability, as selenium-induced glutathione peroxidase (GPx) activity and GPx1 transcript levels were similarly elevated whether cells were treated with soluble Na Sel or SeNPs. This was the case for all three cell types tested. Conclusion Nanoparticle-assisted inorganic selenium delivery, which demonstrated equal bioavailability without causing deleterious cytotoxic side effects, has potential applications for safely supplementing animal diets with inorganic selenium at what are usually toxic doses.

BackgroundOvarian cancer is a leading cause of cancer mortality in women, and only a small percentage of cases are caught at an early stage. Novel treatments with improved e cacy are needed to ght ovarian cancer and to overcome resistance to traditional therapies. Double-stranded (ds) RNA, including the synthetic polyinosinic cytidylic acid (poly (I:C), has shown promise as a cancer therapeutic. Two ovarian cancer cell lines were tested for their ability to produce an immune response to poly (I:C) delivered using a nanoparticle carrier, a biodegradable phytoglycogen derived from sweet corn, called nanodendrix (NDX). SKOV-3 and OVCAR-3 have been previously identi ed as dsRNA-resistant and dsRNA-sensitive, respectively. ResultsFirstly, NDX was found to effectively bind poly (I:C), at a w/w ratio of 2:1 NDX:poly (I:C), the resulting particles, poly (I:C)-NDX, were tested for biological activity through uptake and two therapeutic modes of action, cytotoxicity and stimulation of the innate immune response. Both cell lines bound poly (I:C)-NDX, as observed using immunocytochemistry. In OVCAR-3 poly (I:C)-NDX caused signi cant cell death, even at concentrations as low as 62.5ng/mL, measured using the cell viability indicator dye alamarBlue; no cell death was observed with poly (I:C) alone across all concentrations, up to 5µg/mL in SKOV-3 and 0.5µg/mL in OVCAR-3. In both OVCAR-3 and SKOV-3, poly (I:C)-NDX stimulated the production of an innate immune chemokine, CXCL10, at the transcript and protein levels, at signi cantly higher levels than poly (I:C) alone. Interestingly, in response to poly (I:C)-NDX SKOV-3 produced a more robust immune response compared and higher levels of capase-3/-7 activation compared to OVCAR-3, despite showing no signi cant cell death. ConclusionsPoly (I:C)-NDX represents a robust and multifunctional therapy with demonstrated e cacy against a range of ovarian tumour cells, potentiating poly (I:C) and sensitizing resistant cells. Additionally, the SKOV-3 and OVCAR-3 combination represents a powerful comparative model to help unravel dsRNA-mediated immune responses in ovarian cancer cells.

Background Ovarian cancer is a leading cause of cancer mortality in women, and only a small percentage of cases are caught at an early stage. Novel treatments with improved efficacy are needed to fight ovarian cancer and to overcome resistance to traditional therapies. Double-stranded (ds) RNA, including the synthetic polyinosinic cytidylic acid (poly (I:C), has shown promise as a cancer therapeutic. Two ovarian cancer cell lines were tested for their ability to produce an immune response to poly (I:C) delivered using a nanoparticle carrier, a biodegradable phytoglycogen derived from sweet corn, called nanodendrix (NDX). SKOV-3 and OVCAR-3 have been previously identified as dsRNA-resistant and dsRNA-sensitive, respectively. Results Firstly, NDX was found to effectively bind poly (I:C), at a w/w ratio of 2:1 NDX:poly (I:C), the resulting particles, poly (I:C)-NDX, were tested for biological activity through uptake and two therapeutic modes of action, cytotoxicity and stimulation of the innate immune response. Both cell lines bound poly (I:C)-NDX, as observed using immunocytochemistry. In OVCAR-3 poly (I:C)-NDX caused significant cell death, even at concentrations as low as 62.5ng/mL, measured using the cell viability indicator dye alamarBlue; no cell death was observed with poly (I:C) alone across all concentrations, up to 5µg/mL in SKOV-3 and 0.5µg/mL in OVCAR-3. In both OVCAR-3 and SKOV-3, poly (I:C)-NDX stimulated the production of an innate immune chemokine, CXCL10, at the transcript and protein levels, at significantly higher levels than poly (I:C) alone. Interestingly, in response to poly (I:C)-NDX SKOV-3 produced a more robust immune response compared and higher levels of capase-3/-7 activation compared to OVCAR-3, despite showing no significant cell death. Conclusions Poly (I:C)-NDX represents a robust and multifunctional therapy with demonstrated efficacy against a range of ovarian tumour cells, potentiating poly (I:C) and sensitizing resistant cells. Additionally, the SKOV-3 and OVCAR-3 combination represents a powerful comparative model to help unravel dsRNA-mediated immune responses in ovarian cancer cells.

1148 Cocaine ingestion is associated with increased risk of myocardial infarction, stroke, and deep vein thrombosis. Elevated risk for these thrombotic events persists for 7–10 days after cocaine ingestion, despite the fact that cocaine is rapidly cleared from the blood (with a half-life in circulation of less than 1 hour) being rapidly converted into numerous metabolites that circulate for 1–2 weeks. Thus, it is likely that these longer-lived cocaine metabolites account for the long-term thrombotic risk with cocaine use. Cocaine ingestion has been associated with increased plasma VWF concentration, early atherosclerosis, and platelet-rich arterial thrombi. VWF is the major adhesive ligand that attaches platelets to the vessel wall, either to the subendothelium at sites of injury where the endothelium has been denuded or, during inflammation, to intact endothelium, from which it is secreted. VWF is secreted by endothelial cells from intracellular storage granules (Weibel-Palade bodies) in a large, hyperadhesive multimeric form (ultra-large VWF, or ULVWF) that either remains tethered to the endothelial surface or is released into bulk flow. Platelet-VWF adhesion, and subsequent thrombus formation, may be augmented further by cocaine-induced vaso-constriction, increasing shear stress. Thus, we hypothesized that cocaine and/or its metabolites would stimulate endothelial VWF secretion as a mechanism of thrombotic risk. To test this possibility, we exposed cultured human endothelial cells from umbilical vein (HUVEC), brain microvasculature (BMVEC), or coronary artery (CAEC) to cocaine or one of its four major metabolites at concentration ranges reported to occur in plasma following cocaine use. The cocaine metabolites we tested were benzoylecgonine (BE), cocaethylene (CE), norcocaine (NC), and ecgonine methyl ester (EME). We assayed VWF release by platelet-VWF string formation in a parallel-plate flow chamber (2.5 dyne/cm2) and by measuring the concentration of VWF released into the supernatant. Cocaine concentrations as low as 0.1 μg/ml induced VWF release from HUVEC; 1–2 μg/ml cocaine was as effective in releasing VWF as 25 μM histamine or 4 mg/ml dDAVP. Of the cocaine metabolites, only BE and CE induced VWF release from endothelial cells. BMVEC were 10-fold more sensitive to cocaine and metabolites BE and CE than HUVEC in both platelet-string formation and VWF antigen assays. In CAEC, VWF release was slightly reduced compared to HUVEC in response to cocaine, BE, or CE. Consistent with this pattern, staining for intracellular VWF revealed the following hierarchy of intracellular VWF: BMVEC > HUVEC >> CAEC. In BMVEC and HUVEC, VWF staining was restricted to Weibel-Palade bodies, with CAEC also demonstrating a diffuse cytoplasmic pattern. We tested whether intracellular cAMP levels increased after cocaine or cocaine metabolite exposure to explore whether VWF release was dependent on Protein Kinase A, as is the case with dDAVP. Intracellular cAMP did not increase following exposure to cocaine or its metabolites in any of the endothelial cell lines. These results suggest that cocaine contributes to thrombosis by activating endothelial cells to secrete ULVWF via a mechanism that does not involve increased intracellular cAMP. The prolonged thrombotic risk after cocaine ingestion likely relates to the continued action of cocaine metabolites BE and/or CE on endothelial ULVWF secretion. VWF secretion is likely to vary between vascular beds, with brain endothelial cells being particularly sensitive. Furthermore, these results suggest that clinical management of cocaine-induced ischemia or vaso-occlusion may benefit from therapies aimed at disrupting the ULVWF–platelet interaction. Disclosures: No relevant conflicts of interest to declare.