Effect of Prior ChAdOx1 COVID-19 Immunisation on T-Cell Responses to ChAdOx1-HBV.

There are varying data concerning the effect of prior anti-vector immunity on the T-cell response induced by immunisation with an identical vectored vaccine containing a heterologous antigen insert. To determine whether prior exposure to ChAdOx1-SARS-CoV2 immunisation (Vaxzevria®) impacts magnitudes of antigen-specific T-cell responses elicited by subsequent administration of the same viral vector (encoding HBV antigens, ChAdOx1-HBV), healthy volunteers that had received Vaxzevria® (n = 15) or the Pfizer or Moderna mRNA COVID-19 vaccine (n = 11) between 10 and 18 weeks prior were recruited to receive a single intramuscular injection of ChAdOx1-HBV. Anti-ChAdOx1-neutralising antibody titers were determined, and vector or insert-specific T-cell responses were measured by a gamma-interferon ELISpot and intracellular cytokine staining (ICS) assay using multiparameter flow cytometry. Participants were followed for three months after the ChAdOx1-HBV injection, which was well-tolerated, and no dropouts occurred. The baseline ChAdOx1 neutralisation titers were higher in the Vaxzevria® cohort (median of 848) than in the mRNA cohort (median of 25). T-cell responses to HBV antigens, measured by ELISpot, were higher on day 28 in the mRNA group (p = 0.013) but were similar between groups on day 84 (p = 0.441). By ICS, these differences persisted at the last time point. There was no clear correlation between the baseline responses to the adenoviral hexon and the subsequent ELISpot responses. As vaccination within 3 months using the same viral vector backbone affected the insert-specific T-cell responses, a greater interval after prior adenoviral immunisation using heterologous antigens may be warranted in settings in which these cells play critical roles.

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.

Intravenous administration of viral vectors expressing prostate cancer antigens enhances the magnitude and functionality of CD8+ T cell responses.

BACKGROUND: The use of immunotherapeutic vaccination in prostate cancer is a promising approach that likely requires the induction of functional, cytotoxic T cells . The experimental approach described here uses a well-studied adenovirus-poxvirus heterologous prime-boost regimen, in which the vectors encode a combination of prostate cancer antigens, with the booster dose delivered by either the intravenous or intramuscular (IM) route. This prime-boost regimen was investigated for antigen-specific CD8+ T cell induction. METHODS: The coding sequences for four antigens expressed in prostate cancer, 5T4, PSA, PAP, and STEAP1, were inserted into replication-incompetent chimpanzee adenovirus Oxford 1 (ChAdOx1) and into replication-deficient modified vaccinia Ankara (MVA). In four strains of mice, ChAdOx1 prime was delivered intramuscularly, with an MVA boost delivered by either IM or intravenous routes. Immune responses were measured in splenocytes using ELISpot, multiparameter flow cytometry, and a targeted in vivo killing assay. RESULTS: The prime-boost regimen was highly immunogenic, with intravenous administration of the boost resulting in a sixfold increase in the magnitude of antigen-specific T cells induced and increased in vivo killing relative to the intramuscular boosting route. Prostate-specific antigen (PSA)-specific responses were dominant in all mouse strains studied (C57BL/6, BALBc, CD-1 and HLA-A2 transgenic). CONCLUSION: This quadrivalent immunotherapeutic approach using four antigens expressed in prostate cancer induced high magnitude, functional CD8+ T cells in murine models. The data suggest that comparing the intravenous versus intramuscular boosting routes is worthy of investigation in humans.

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 (TCR) binding domain and an anti-CD3 T-cell engaging domain, which redirects T cells to kill gp100-expressing tumor cells. Here, we report a multicenter phase I/II trial of tebentafusp in metastatic melanoma (NCT01211262) focusing on the mechanism of action of tebentafusp. PATIENTS AND METHODS: Eighty-four patients with advanced melanoma received tebentafusp. Treatment efficacy, treatment-related adverse events, 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 patients with metastatic uveal melanoma and patients with metastatic cutaneous melanoma. A 1-year overall survival rate of 65% was achieved for both patient cohorts. On-treatment cytokine measurements were consistent with the induction of IFNγ 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 gp100-expressing skin melanocytes) showed a positive association with patient survival. CONCLUSIONS: These data suggest that redirecting T cells using a gp100-targeting TCR/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.

MicroRNAs as potential biomarkers in congenital heart surgery.

OBJECTIVE: Pediatric congenital heart surgery (CHS) involves intracardiac, valvular, and vascular repairs. Accurate tools to aid short-term outcome prediction in pediatric CHS are lacking. Clinical scores, such as the vasoactive-inotrope score and ventilation index, are used to define outcome in clinical studies. MicroRNA-1-3p (miR-1) is expressed by both cardiomyocytes and vascular cells and is regulated by hypoxia. In adult patients, miR-1 increases in the circulation after open-heart cardiac surgery, suggesting its potential as a clinical biomarker. Thus, we investigated whether perioperative circulating miR-1 measurements can help predict post-CHS short-term outcomes in pediatric patients. METHODS: Plasma miR-1 was retrospectively measured in a cohort of 199 consecutive pediatric CHS patients (median age 1.2 years). Samples were taken before surgery and at the end of the operation. Plasma miR-1 concentration was measured by reverse transcription-quantitative polymerase chain reaction and expressed as miR-1 copies/µL and as relative expression to spiked-in exogenous cel-miR-39. RESULTS: Baseline plasma miR-1 did not vary across different diagnoses, increased during surgery (204-fold median relative increase, P < .001), and was associated with aortic crossclamp duration postoperatively (P < .001). Importantly, miR-1 levels at the end of the operation positively correlated with intensive care stay (P < .001), early severe cardiovascular events (P = .01), and with high vasoactive-inotrope score (P = .001) and ventilation index (P < .001), suggesting that miR-1 could accelerate the identification of patients with cardiopulmonary bypass-related ischemic complications, requiring more intensive support. CONCLUSIONS: Our study suggests miR-1 as a novel potential circulating biomarker to predict early postoperative outcome and inform clinical management in pediatric heart surgery.

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.

OBJECTIVE: Gestational diabetes mellitus (GDM) produces fetal hyperglycemia with increased lifelong risks for the exposed offspring of cardiovascular and other diseases. Epigenetic mechanisms induce long-term gene expression changes in response to in utero environmental perturbations. Moreover, microRNAs (miRs) control the function of endothelial cells (ECs) under physiological and pathological conditions and can target the epigenetic machinery. We investigated the functional and expressional effect of GDM on human fetal ECs of the umbilical cord vein (HUVECs). We focused on miR-101 and 1 of its targets, enhancer of zester homolog-2 (EZH2), which trimethylates the lysine 27 of histone 3, thus repressing gene transcription. EZH2 exists as isoforms α and ß. APPROACH AND RESULTS: HUVECs were prepared from GDM or healthy pregnancies and tested in apoptosis, migration, and Matrigel assays. GDM-HUVECs demonstrated decreased functional capacities, increased miR-101 expression, and reduced EZH2- ß and trimethylation of histone H3 on lysine 27 levels. MiR-101 inhibition increased EZH2 expression and improved GDM-HUVEC function. Healthy HUVECs were exposed to high or normal d-glucose concentration for 48 hours and then tested for miR-101 and EZH2 expression. Similar to GDM, high glucose increased miR-101 expression. Chromatin immunoprecipitation using an antibody for EZH2 followed by polymerase chain reaction analyses for miR-101 gene promoter regions showed that both GDM and high glucose concentration reduced EZH2 binding to the miR-101 locus in HUVECs. Moreover, EZH2-ß overexpression inhibited miR-101 promoter activity in HUVECs. CONCLUSIONS: 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.

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.