The effect of cardioplegic supplementation with sildenafil on cardiac energetics in a piglet model of cardiopulmonary bypass and cardioplegic arrest with warm or cold cardioplegia

Cardioplegic cardioprotection strategies used during paediatric open-heart surgery remain suboptimal. Sildenafil, a phosphodiesterase 5 (PDE-5) inhibitor, has been shown to be cardioprotective against ischemia/reperfusion injury in a variety of experimental models and this study therefore tested the efficacy of supplementation of cardioplegia with sildenafil in a piglet model of cardiopulmonary bypass and arrest, using both cold and warm cardioplegia protocols. Piglets were anaesthetized and placed on coronary pulmonary bypass (CPB), the aorta cross-clamped and the hearts arrested for 60 min with cardioplegia with or without sildenafil (10 nM). Twenty minutes after removal of cross clamp (reperfusion), attempts were made to wean the pigs from CPB. Termination was carried out after 60 min reperfusion. Throughout the protocol blood and left ventricular tissue samples were taken for analysis of selected metabolites (using HPLC) and troponin I. In both the cold and warm cardioplegia protocols there was evidence that sildenafil supplementation resulted in faster recovery of ATP levels, improved energy charge (a measure of metabolic flux) and altered release of hypoxanthine and inosine, two purine catabolites. There was no effect on troponin release within the studied short timeframe. In conclusion, sildenafil supplementation of cardioplegia resulted in improved cardiac energetics in a translational animal model of paediatric CPB surgery

A Robust, state-of-the-art amperometric microbiosensor for glutamate detection

Scientific knowledge of glutamate (GLU) neurobiology is severely hampered by the inadequacy of the available in vivo brain sampling techniques. Due to the crucial role of GLU in central nervous system function and pathology, the development of a reliable sampling device is mandatory. GLU biosensor holds potential to address many of the known issues of in vivo GLU measurement. We report here on the development and test of a labor- and cost-effective microbiosensor, suitable to be applied for measuring brain GLU. A glycerol-based cryopreservation method was also tested. Needle type Pt biosensors were coated with a permselective Nafion–Poly(o-phenylenediamine) layer and cross-linked to l-glutamate oxidase with poly(ethylene glycol) diglycidyl ether. Tested in vitro, the device shows high sensitivity and specificity for GLU, while being poorly influenced by common interfering substances such as ascorbate, dopamine and dihydroxyphenylacetic acid. Further, the cryopreservation procedure kept sensitivity unaltered for 30 days and possibly longer. We conclude that a highly efficient GLU biosensor of minimal dimensions can be consistently and affordably constructed with relative ease. Together with the possibility of cryopreservation this shall foster diffusion and exploitation of GLU biosensors technology

Coumaric acid induces mitochondrial damage and oxidative-mediated cell death of human endothelial cells

Evidence that higher natural antioxidants (NA) intake provides cardiovascular protection is contradictory. The endothelium plays a pivotal role in cardiovascular homeostasis, and for this reason, the molecular events resulting from the interaction of NA with endothelial cells (ECs) are actively investigated. Here, we show that moderately high doses of coumaric acid (CA) induced intracellular reactive oxygen species (ROS) production, mitochondrial membrane depolarization and ECs death. Treatment of ECs with cyclosporine A, a mitochondrial permeability transition pore inhibitor, prevented the oxidative-mediated cell damage indicating mitochondrial involvement in CA-induced ECs impairment. CA-induced intracellular ROS generation was counteracted by the specific cytochrome P450 (CYP) 2C9 inhibitor sulfaphenazole (SPZ). SPZ also prevented CA-induced mitochondrial membrane depolarization and ECs death, implicating CYP2C9 in mediating the cellular response upon CA treatment. Our results indicate that moderately high doses of CA can promote CYP2C9-mediated oxidative stress eliciting mitochondrial-dependent ECs death and may pave the way toward mechanistic insight into NA effects on cardiovascular cells

Resveratrol alters human endothelial cells redox state and causes mitochondrial-dependent cell death

Studies analyzing the impact of natural antioxidants (NA) on Endothelial Cells (ECs) have dramatically increased during the last years, since a deregulated ECs redox state is at the base of the onset and progression of several cardiovascular diseases. However, whether NA can provide cardiovascular benefits is still a controversial area of debate. Resveratrol (RES), a natural polyphenol found in grapes, is believed to provide cardiovascular benefits by virtue of its antioxidant effect on the endothelium. Here, we report that tissue-attainable doses of resveratrol increased the intracellular oxidative state, thus affecting mitochondrial membrane depolarization and inducing EC death. Cyclosporine A, a mitochondrial permeability transition pore inhibitor, prevented oxidative-mediated cell death, thus implicating mitochondria in resveratrol-induced EC impairment. The specific cytochrome P450 (CYP) 2C9 inhibitor, sulfaphenazole, counteracted both oxidative stress and mitochondrial membrane depolarization, providing EC protection against resveratrol-elicited pro-oxidant effects. Our findings strongly suggest that CYP2C9 mediates resveratrol-induced oxidative stress leading to mitochondria impairment and EC death

Gestational diabetes mellitus impairs fetal endothelial cell functions through a mechanism involving microRNA-101 and histone methyltransferase enhancer of zester homolog-2

GDM impairs HUVEC function via miR-101 upregulation. EZH2 is both a transcriptional inhibitor and a target gene of miR-101 in HUVECs, and it contributes to some of the miR-101-induced defects of GDM-HUVECs

Tebentafusp, a TCR/anti-CD3 bispecific fusion protein targeting gp100, potently activated antitumor immune responses in patients with metastatic melanoma

Purpose: Tebentafusp is a first-in-class bispecific fusion protein designed to target gp100 (a melanoma-associated antigen) through a high affinity T cell receptor binding domain, and an anti-CD3 T cell engaging domain which redirects T cells to kill gp100-expressing tumor cells. Here we report from a multicentre Phase 1/2 trial of tebentafusp in metastatic melanoma (NCT01211262) focusing on the mechanism of action of tebentafusp Experimental Design: 84 patients with advanced melanoma received tebentafusp. Treatment efficacy, treatment-related AEs and biomarker assessments were performed for blood-derived and tumor biopsy samples obtained at baseline and on-treatment Results: Tebentafusp was generally well tolerated and active in both metastatic uveal melanoma (mUM) and metastatic cutaneous melanoma (mCM) patients. A 65% 1-year overall survival rate was achieved for both patient cohorts. On-treatment cytokine measurements were consistent with the induction of IFNg pathway-related markers in the periphery and tumor. Notably, tebentafusp induced an increase in serum CXCL10 (a T cell attractant), and a reduction in circulating CXCR3+ CD8+ T cells together with an increase in cytotoxic T cells in the tumor microenvironment. Furthermore, increased serum CXCL10 or the appearance of rash, (likely due to cytotoxic T cells targeting of gp100-expressing skin melanocytes) showed a positive association with patient survival. Conclusions: These data suggest that re-directing T cells using a gp100-targeting T cell receptor/anti-CD3 bispecific fusion protein may provide benefit to patients with metastatic melanoma. Furthermore, the activity observed in these two molecularly disparate melanoma classes hints at the broad therapeutic potential of tebentafusp