Maximizing cancer therapy via complementary mechanisms of immune activation: PD-1 blockade, neoantigen vaccination, and Tregs depletion.

BACKGROUND: A number of different immune pathways are involved in the effective killing of cancer cells, collectively named as the 'Cancer Immunity Cycle'. Anti-PD-1 checkpoint blockade (CPB) therapy is active on one of these pathways and reinvigorates anticancer T cell immunity, leading to long-term responses in a limited fraction of patients with cancer. We have previously shown that neoantigens-based adenovirus vectored vaccine in combination with anti-PD-1 further expands pre-existing anticancer immunity and elicits novel neoantigen-specific T cells thereby increasing efficacy to 50% of tumor clearance in mice. Here we added a third component to the CPB plus vaccine combination, which is able to modify the suppressive tumor microenvironment by reducing the number of tumor-infiltrating regulatory T cells (Tregs), as strategy for improving the therapeutic efficacy and overcoming resistance. METHODS: The antitumor efficacy of anti-PD-1, neoantigen vaccine and Treg modulating agents, either Bempegaldesleukin (BEMPEG: NKTR-214) or an anti-CTLA-4 mAb with Treg-depleting activity, was investigated in murine tumor models. We evaluated tumor growth in treated animals, neoantigen-specific T cells in tumors, tumor-infiltrating lymphocytes (TILs) and intratumoral Tregs. RESULTS: The addition of BEMPEG or anti-CTLA-4 to the combination of vaccine and anti-PD-1 led to complete eradication of large tumors in nearby 100% of treated animals, in association with expansion and activation of cancer neoantigen-specific T cells and reduction of tumor-infiltrating Tregs. CONCLUSION: These data support the notion that the integrated regulation of three steps of the cancer immunity cycle, including expansion of neoantigen-specific T cells, reversal of the exhausted T cell phenotype together with the reduction of intratumoral Tregs may represent a novel rationally designed drug combination approach to achieve higher cure rates.

A simple microsatellite-based method for hazelnut oil DNA analysis.

Molecular food traceability requires continuous updates to identify more robust, efficient and affordable methodologies to guarantee food quality and safety and especially consumers' health. Available commercial kits are often unsatisfactory and require modifications to successfully detect single components on complex and transformed food matrices. Here we report a simple method for molecular traceability of cold-pressed hazelnut oil based on microsatellite DNA markers. Different genomic extraction methodologies were tested and a total genome pre-amplification step was applied on PCR-negative samples. PCR-capillary electrophoresis using nine microsatellites demonstrates the accuracy of the fingerprint analysis even for filtered oil.

Vitamin C supplementation modulates gene expression in peripheral blood mononuclear cells specifically upon an inflammatory stimulus: a pilot study in healthy subjects.

In order to study the effects of vitamin C supplementation on gene expression and compare its action between physiological and inflammatory conditions, a pilot study was set up utilizing microarray and qPCR technologies. Five healthy volunteers were supplemented with 1 g vitamin C (Redoxon(®)) per day for five consecutive days. Peripheral blood mononuclear cells (PBMNC) were isolated before and just after the last supplementation, and RNA was isolated for the Affymetrix gene 1.0 ST chip analysis. PBMNC were also, ex vivo, treated with LPS, and gene expression was quantified by means of a "Human NFkB Signaling" qPCR array. Only a very moderate effect on the baseline gene expression modulation was associated with vitamin C supplementation. However, in spite of the limited number of subjects analyzed, vitamin C supplementation resulted in a markedly different modulation of gene expression upon the inflammatory stimulus, specifically at the level of the MyD88-dependent pathway and of the anti-inflammatory cytokine IL-10 synthesis. This study suggests that vitamin C supplementation in healthy subjects, not selected according to a specific genetic profile, consuming an adequate amount of vitamin C, and having a satisfactory vitamin C plasma concentration at the baseline, does not result in a significant modification of gene expression profile. Under this satisfactory micronutrient status, supplementation of vitamin C is "buffered" within a homeostatic physiological equilibrium. Differently, following a second "hit" constituted of an inflammatory stimulus such as LPS, able to trigger a critical burst to the normal physiological state, the higher availability of ascorbic acid emerges, and results in a significant modulation of cell response.

Absorption, metabolism, and effects at transcriptome level of a standardized French oak wood extract, Robuvit, in healthy volunteers: pilot study.

The consumption of wine and spirits, traditionally aged in oak barrels, exposes humans to roburin ingestion. These molecules belong to a class of ellagitannins (ETs), and their only known source is oak wood. Very little is currently known about roburin bioavailability and biological activity. We reported for the first time human absorption of roburins from a French oak wood (Quercus robur) water extract (Robuvit) by measuring the increase of total phenols (from 0.63 ± 0.06 to 1.26 ± 0.18 µg GAE equiv/mL plasma) and the appearance of roburin metabolites (three different glucoronidate urolithins and ellagic acid), in plasma, after 5 days of supplementation. Robuvit supplementation induced also the increase of plasma antioxidant capacity from 1.8 ± 0.05 to 1.9 ± 0.01 nmol Trolox equiv/mL plasma. Moreover, utilizing a combined ex vivo cell culture approach, we assessed the effect of Q. robur metabolites (present in human serum after supplementation) on gene expression modulation, utilizing an Affymetrix array matrix, in endothelial, neuronal, and keratinocyte cell lines. The functional analysis reveals that Robuvit metabolites affect ribosome, cell cycle, and spliceosome pathways.

Intracellular zinc is required for intestinal cell survival signals triggered by the inflammatory cytokine TNFα.

The essential micronutrient zinc has long been known to be a functional component of diverse structural proteins and enzymes. More recently, important roles for free or loosely bound intracellular zinc as a signaling factor have been reported. Insufficient zinc intake was shown to exacerbate symptoms in mouse models of inflammation such as experimental colitis, while zinc supplementation was found to improve intestinal barrier function. Herein, we provide evidence that intracellular zinc is essential for maintaining intestinal epithelial integrity when cells are exposed to the inflammatory cytokine Tumor Necrosis Factor (TNF)α. Using the human intestinal Caco-2/TC7 cell line as an in vitro model, we demonstrate that depletion of intracellular zinc affects TNFα-triggered signaling by shifting intestinal cell fate from survival to death. The mechanism underlying this effect was investigated. We show that TNFα promotes a zinc-dependent survival pathway that includes modulation of gene expression of transcription factors and signaling proteins. We have identified multiple regulatory steps regulated by zinc availability which include the induction of cellular Inhibitor of APoptosis (cIAP2) mRNA, possibly through activation of Nuclear Factor-Kappa B (NF-κB), as both nuclear translocation of the p65 subunit of NF-κB and up-regulation of cIAP2 mRNA were impaired following zinc depletion. Moreover, X-linked inhibitor of apoptosis protein level was profoundly reduced by zinc depletion. Our results provide a possible molecular explanation for the clinical observation that zinc supplements ameliorate Crohn's disease symptoms and decrease intestinal permeability in experimental colitis.