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Background Although Bismuth-Corlette (BC) type 4 perihilar cholangiocarcinoma (pCCA) is no longer considered a contraindication for curative surgery, few data are available from Western series to indicate the outcomes for these patients. This study aimed to compare the short- and long-term outcomes for patients with BC type 4 versus BC types 2 and 3 pCCA undergoing surgical resection using a multi-institutional international database. Methods Uni- and multivariable analyses of patients undergoing surgery at 20 Western centers for BC types 2 and 3 pCCA and BC type 4 pCCA. Results Among 1138 pCCA patients included in the study, 826 (73%) had BC type 2 or 3 disease and 312 (27%) had type 4 disease. The two groups demonstrated significant differences in terms of clinicopathologic characteristics (i.e., portal vein embolization, extended hepatectomy, and positive margin). The incidence of severe complications was 46% for the BC types 2 and 3 patients and 51% for the BC type 4 patients (p = 0.1). Moreover, the 90-day mortality was 13% for the BC types 2 and 3 patients and 12% for the BC type 4 patients (p = 0.57). Lymph-node metastasis (N1; hazard-ratio [HR], 1.62), positive margins (R1; HR, 1.36), perineural invasion (HR, 1.53), and poor grade of differentiation (HR, 1.25) were predictors of survival (all p ≤0.004), but BC type was not associated with prognosis. Among the N0 and R0 patients, the 5-year overall survival was 43% for the patients with BC types 2 and 3 pCCA and 41% for those with BC type 4 pCCA (p = 0.60). Conclusions In this analysis of a large Western multi-institutional cohort, resection was shown to be an acceptable curative treatment option for selected patients with BC type 4 pCCA although a more technically challenging surgical approach was required.

Colon epithelium is exposed to oxidative stress conditions via exogenous and endogenous routes and a number of colon diseases are associated with the imbalance of the cellular redox system. The aim of the present work was to study the intracellular antioxidant activity of red grape polyphenolic extracts on superficial enterocytes experimentally exposed to prooxidant conditions. In situ confocal microscopy observation on CM-H2DCFDA charged explants exposed to H2O2 (5, 10, and 15min) was performed. First, the ability of the experimental set up to detect intracellular ROS scavenging activity by intracellularly absorbed antioxidants was demonstrated by a dose-response inhibition of the H2O2 induced intracellular probe fluorescence following pre-incubation with Trolox, synthetic analog of vitamin E. Then, the explants were incubated with grape skin polyphenolic extracts for 1 h producing a significant decrease of the H2O2 induced fluorescence. This effect was more pronounced following 15 min H2O2 exposure with respect to 5 min and it was also more evident for extracts obtained from mature grapes, which showed an increased ORAC value and qualitative peculiarities in the polyphenolic composition. Results demonstrated that red grape polyphenolic extracts are able to exert an intracellular antioxidant activity on superficial colonocytes, inducing a protection action against pro-oxidant condition. Extracts obtained from mature grapes showed an increased protective activity

Under physiopathological conditions reactive oxygen species can be over produced evoking an intracellular state of oxidative stress. Dietary polyphenols are known to have potent antioxidant activity by various mechanisms. While a number of data is available on the antioxidant behavior of single polyphenolic compounds, little is known about possible interactions among them, which could influence the “quality” of their intracellular antioxidant activity when present in mixture. This study was aimed to assess the intracellular antioxidant activity of two red wine polyphenols, resveratrol and quercetin, both single and in mixture. HeLa cells charged with the ROS-sensitive cell permeant dye, CM-H2DCFDA, were used as model. Spectrofluorimetric and confocal microscopy analysis was performed. In the range of concentration tested (5-50 µM) quercetin alone exhibited a dose-dependent intracellular antioxidant activity against an oxidative stress induced by H2O2 used as pro-oxidant agent. On the other hand resveratrol showed a pro-oxidant intracellular activity. When the cells were exposed to equimolar mixtures of the two compounds, an intracellular interaction among them was highlighted, with a non-predictable behavior on the basis of the analysis conducted on the single compounds. In conclusion results demonstrated that antioxidant properties of polyphenolic compounds depend not only on the nature of the compound itself, but also on the type of their interaction in a cellular system.

Objective: Red wine polyphenols have long been associated to a reduced morbidity and mortality from atherosclerotic disease in part by reducing inflammation, however the effects of different polyphenolic compounds are not clear. The aim of this study was to evaluate the anti-inflammatory and anti-oxidant effects of polyphenolic extracts from two red wines typical of Southern Italy, and to dissect the specific contribute of hydroxycinnamic acids, flavonols and stilbenes and to investigate underlying mechanisms of action in endothelial cells. Methodology and Results: Through multiple assays, we showed that both Primitivo and Negroamaro Wine Polyphenolic Extracts significantly inhibited monocyte adhesion to endothelium, endothelial expression of adhesion molecules, monocyte chemoattractant protein-1 and macrophage colony-stimulating factor as well as intracellular oxidative stress and the activation of NF-?B and AP-1, without any reduction in cell viability. We also showed that all tested polyphenols exhibited a strong antioxidant action in stimulated human endothelial cells but not all significantly exhibited anti-inflammatory activity which in turn was related to the inhibition of NF-?B and AP-1 activation. Conclusions: The present study provides new evidences on specifc anti-inflammatory and anti-atherosclerotic role of red wine polyphenols including hydroxycinnamic acids, flavonols and stilbenes.

There is a growing interest in producing food plants with increased amounts of antioxidants because of their potential health benefits. Polyphenolic secondary metabolites, such as flavonoids and stilbenes, have been investigated for their significant antioxidant activity, important both for plant physiology and human nutrition. With the aim to verify whether the synthesis of resveratrol in tomato fruit affected the redox status of transformed tomato tissues, in the present work we took advantage of the availability of two different transgenic tomato lines in which a grape stilbene synthase gene was expressed under a constitutive or a tissue specific promoter, respectively. The induction of resveratrol synthesis in tomato affected the redox status of transformed tomato fruit. In particular, ascorbate and glutathione pool increased, significantly and proportionally to the amount of the resveratrol synthesised in transformed tissues. Noteworthy, the increase in ascorbate and glutathione pool paralleled with a significant increase in the activities of the main enzymes involved in redox homeostasis, i.e. catalase, ascorbate peroxidase as well the enzymes responsive for ascorbate recycling. Furthermore, lipoxygenase activity and levels of polyunsaturated fatty acids hydroperoxides, were reduced in fruit tissues from transgenic tomato lines. The synthesis of resveratrol, together with the global redox status had an impact on the total antioxidant activity of transgenic fruits. Again the total antioxidant capability increased proportionally to the amount of synthesised resveratrol with the most remarkable increase recorded from the lipophilic-resveratrol containing-fraction of transgenic fruits. Overall, our results pointed a higher nutritional value of resveratrol synthesizing tomato fruits.

The metabolic engineering of plants offers the opportunity to change the content of specific phytonutrients in plant - derived foods. The plant polyphenol trans-resveratrol (3, 5, 4'-trihydroxystilbene), mainly found in grape, peanut, and other few plants, displays a wide range of biological effects. Numerous in vitro studies have described various biological effects of resveratrol. In order to provide more information regarding absorption, metabolism, and bioavailability of resveratrol, different research approaches have been performed. In recent years, the induction of resveratrol synthesis in plants which normally do not accumulate such polyphenol has been successfully achieved by molecular engineering. In this context, the ectopic production of resveratrol could have positive effects on the enhancement of the nutritional value of several widely consumed fruits and vegetables. This chapter focuses on the latest findings regarding on resveratrol bio-production in tomato (Solanum lycopersicum) fruits.

Red wine is a treasured source of polyphenols, such as stilbenes, flavonols and hydroxycinnamic acids, depending on different factors, such as grape cultivar and wine-making practices. The red wine polyphenols (RWPs) have long been associated to reduced mortality and morbidity from cardiovascular diseases and cancer. Since matrix metalloproteinase (MMP) activity is involved in extracellular matrix degradation, a crucial step both in inflammation and cell migration, aim of this study was to examine the effects of specific classes of polyphenols from Primitivo and Negroamaro grapes and wines produced in the Apulian region (Southern Italy), on the release and activity of MMP-9 in human monocytoid cells. U937 monocitoid cells were pre-treated with increasing concentrations of polyphenolic extracts (PE) of Primitivo and Negramaro grape and wine, before stimulation with 30 nM phorbol myristate acetate (PMA) for 24 h. The release and activity of MMP-9 in culture medium was tested by ELISA and zymography, respectively. Both Primitivo and Negroamaro PE exhibited a concentration-dependent inhibition of MMP-9 release and activity, without any reduction in cell viability. The inhibitory effect was higher than that produced by individual chemically synthesized polyphenols.. In this study, the PE from Primitivo and Negramaro grapes and red wines were characterized, and shown to significantly and synergistically inhibit the monocyte-derived release and activity of extracellular matrix proteases. These inhibitory effects could explain, at least in part, the anti-inflammatory and anti-cancerogenic properties of RWP.

Phenolic coumpounds present in grape berries are extracted from the skin, seeds and flesh during the winemaking process. These substances have a potentially positive effect, on human health, thus giving to red wine "bioactive properties" that an contribute to decrease the incidence of atherosclerosis, cancer, neurodegenerative diseases. Grape variety, quality, climate, geographical origin and phythopatologies can affect the quality and quantity of phenolic compounds that accumulate in the cells. Viticulture and vinification practice, which varies in different countries, determine the concentration of phenolic compounds in wine. During the vinemaking process numerous parameters, such as temperature, presence of seeds and berry skins and addition of enzymes, have been reported to affect the extraction of phenolic compounds during grape must fermentation. Conversely, poor knowledge is available on the effects of different yeast strains on the final concentration of polyphenols in red wine. Indeed, yeasts enzymatic activity are likely to influence the extraction of phenolic substances from grape tissues. With the aim to evaluate the existence of a potential correlation between polyphenols content, antioxidant capacity of wine and yeast starter cultures, three selected autochthonous strains of Saccharomyces cerevisiae, were used to inoculate large scale must fermentation. At the end of the process, the experimental wines obtained were evaluated for the content of different class of polyphenols and antioxidant capacity. The statistical analysis of obtained results revealed a significant influence of the autochthonous strain on the concentration of several class of polyphenols and total antioxidant capacity in the produced wines. The different strain influence on the analysed parameters assumes a technological significance and it strongly contribute in determining the final quality of the wine by influencing "functional parameters" important for the preservation of human health.

Resveratrol, a plant phenolic compound, is accumulated in grape and red wine, but it is not widely distributed in other food crops. Our previous works showed that resveratrol biosynthesis can be induced in tomato plant (Lycopersicum esculentum Mill.) via the heterologous expression of a grape (Vitis vinifera L.) stilbene synthase cDNA under the control of a constitutive (CaMV 35S) or a fruit specific (TomLoxB) promoter. Resveratrol synthesising fruits showed an increase in the levels of ascorbate and glutathione, the soluble antioxidants of primary metabolism and the total antioxidant activity. These results paralleled with a higher capability of transgenic fruit to counteract the pro-inflammatory effects of phorbol ester in mocyte-macrophages, via the inhibition of induced cyclooxygenase-2, and indicated the potential of our approach to improve the nutritional value of tomato. Since the levels of resveratrol detected in transgenic fruit were limited to a maximum of 50 ug/g fresh weight, we started a breeding programme to increase the content of this metabolite. We took advantage of the ability of an Arabidopsis myb-type transcription factor (AtMYB12) to strongly activate the expression of several phenylpropanoid biosynthetic genes that act upstream of stilbene synthase, suggesting that AtMYB12 can provide high rates of metabolic flux for resveratrol biosynthesis. To further increase the level of resveratrol, we have introduced in the breeding programme a spontaneous flavanone 3-hydroxylase tomato mutant. The presence of this mutation reduces the amount flavonols and should therefore enhance substrate availability to stilbene synthase. Preliminary data show that a combination of biosynthetic and regulatory genes together with the availability of natural tomato varieties is a powerful strategy for plant metabolic engineering.

With the rapid advances in biomedical research, there has been a growing interest in unravelling the extraordinary properties of some commonly used natural products. Natural compounds can be extracted for commercial use from different natural sources. Natural secondary metabolites generally contain a mixture of different molecules. For use as "nutraceutical", it may be important to control the composition of metabolite mixtures, as different molecules exhibit different bioactive properties. Furthermore, industry requires compounds that can have stable activity (pigmentation, antioxidant activity) in a range of food and pharmaceutical systems. A viable cell culture system for production of natural compounds has the potential advantage of facilitating selective production of stable bioactive molecules and circumventing problems of uncertain or seasonal supply. The improvement of the natural compounds uptake is another important challenge. The utilization of micro/nano-capsules offers several advantages respect to the delivery of free compounds because it can increase the uptake of the selected compounds through the gastrointestinal barrier protecting them from adverse environmental parameters. The use of micro/nano-capsules to store and deliver a wide variety of natural compounds is of great interest due to their potential applications in areas such as medicine, catalysis, cosmetics as well as food-biotechnology. Resveratrol, a plant phenolic compound, is found in grapes and red wine but is not widely distributed in other common food sources. Aims of the present study are the production of health promoting polyphenols from V. vinifera cell cultures, and the improvement of their delivery through encapsulation into nano/micro-carriers with tailored structure according to polyphenol chemical features.

Resveratrol, a plant phenolic compound, is found in grapes and red wine, but is not widely distributed in other common food sources. The pathway for resveratrol biosynthesis is well characterized. Metabolic engineering of this compound has been achieved in tomato plants (Lycopersicon esculentum Mill.) in order to improve their nutritional value. Tomato plants synthesizing resveratrol were obtained via the heterologous expression of a grape (Vitis vinifera L.) cDNA encoding for the enzyme stilbene synthase (StSy), under the control of the fruit-specific promoter TomLoxB or the constitutive CaMV 35S promoter. The resulting 35S plants accumulate resveratrol in all the tissues, but in the highest amount in the fruits. While, as expected, LoxS transgenic plants only accumulate it in the fruits, in particular in the skin of the mature fruits. Quantitative analyses carried out on LoxS fruits were compared with those of 35S tomato lines. The LoxS fruits contained levels of trans -resveratrol that were 20-fold lower than those reported for the 35S line. The total antioxidant capability, ascorbate and gluthatione contents were also evaluated. A significant increase in all these antioxidant paramenters was found in the LoxS and 35S lines. These results could explain the higher capability that extract from transgenic fruits have to counteract the pro-inflammatory effects of phorbol ester in monocyte- macrophages via the inhibition of induced cyclo-oxygenase-2 enzyme.