Wine, sirtuins and nephroprotection: not only resveratrol.

Resveratrol (RSV), a red wine component, and red wine itself exert cardio- and nephroprotective effects by modulating the Nitric Oxide system (NO). It has been shown that one of the main actions resulting from NO modulation is sirtuin regulation, especially SIRT-1 regulation. Elucidating both upstream and downstream molecular mechanisms of the SIRT-1 pathway is an open field of investigation that can explain its role not only in long-term processes, such as aging, but also in short-term processes, such as protection against ischemic damage. Our hypothesis suggests the importance of investigating compounds that are routine dietary components and do not necessarily contain RSV. Their nephroprotective activity could involve not only eNOS-dependent, but also NO-dependent but eNOS-independent mechanisms, or other molecular alternative signaling systems.

Results after supracricoid horizontal partial laryngectomy.

OBJECTIVES: This study analyzed oncological and functional results of supracricoid horizontal partial laryngectomy. METHODS: A retrospective study was conducted involving 20 patients with squamous cell carcinoma (SCC) of the larynx who underwent SCPL between 1996 and 2005 in Faculty of Medical Sciences of Santa Casa Hospital of Sao Paulo, Brazil. There were 18 male and 2 female patients with ages ranging from 39 to 74 years (median=58 years), of whom 19 were smokers and 14 alcoholics. The tumors were present in the glottis in 16 cases and supraglottis in 4; 5 were stage I or II and 15 were stage III or IV. We analyzed treatment given when rehabilitation was unsuccessful, oncological results of SCPL, including local and regional recurrences, time to recurrence and treatment given, distal metastases, global survival, survival free of disease, and appearance of second primary tumors. We also calculated the index of functional preservation of the larynx. RESULTS: Rehabilitation of swallowing capabilities and speech was achieved in 18 patients. Removal of the tracheostomy varied between 1 and 9 months. Rehabilitation was unsuccessful in two patients. Three patients required a total laryngectomy, two for unsuccessful rehabilitation and one for recurrence. The preservation of a functional larynx was 85%, with 10% of patients requiring a total laryngectomy after failed rehabilitation. CONCLUSIONS: Supracricoid horizontal partial laryngectomy is an efficient surgical oncology technique that yields good functional results for the treatment of laryngeal cancer.

Immunomodulatory activity of shikimic acid and quercitin in comparison with oseltamivir (Tamiflu) in an in vitro model.

The risk of an avian influenza pandemic has put oseltamivir (Tamiflu) in the spotlight and has given rise to rumors that shikimic acid (SK), which is used for the synthesis of Tamiflu, possesses therapeutic activity. This study was undertaken to determine whether SK, either alone or in combination with quercitin (QT) is able to modulate the release of IL-6 and IL-8 from peripheral blood mononuclear cells (PBMCs). The experiments were conducted comparing the properties of SK, both alone and in combination, with those of Tamiflu. The incubation of PBMCs with 100 nM Tamiflu or SK at two concentrations (10 nM; 100 nM) did not produce any change in IL-6 and IL-8 baseline levels (data expressed as incremental change vs. baseline). On the contrary, incubation with SK and QT at both concentrations (10 and 100 nM) produced a significant increase in the release of IL-8 as compared to other groups (4.19 +/- 0.82, SK-QT 10 nM; 3.83 +/- 1.17 SK-QT 100 nM, P < 0.05 vs. baseline 1.00 +/- 0.10, Tamiflu 100 nM 1.35 +/- 0.16, SK 10 nM 1.68 +/- 0.15 and SK 100 nM 1.80 +/- 0.48). The SK-QT combination also proved to be effective in the upregulation of IL-6 (3.08 +/- 0.46, SK-QT 10 nM; 3.60 +/- 0.74 SK-QT 100 nM, P < 0.05 vs. baseline 1.00 +/- 0.26). According to these findings SK alone is not able to modulate innate immunity in antiviral terms. However, the data show that the SK + QT combination, even at low doses, may be effective for the modulation of innate immunity.

Early epidermal response after a single dose of gamma-rays in organotypic culture of human breast skin.

BACKGROUND: Skin reaction is the most common side-effect of radiation therapy. Radiation-induced dermal fibrosis has been characterized histologically, but little is known about the epidermis overlying fibronecrotic lesions. OBJECTIVES: To characterize the epidermal response 24 h after a single clinically relevant dose of gamma-rays in cultured human breast skin. METHODS: Biopsies obtained from cosmetic surgery (n = 7) were placed epidermis upwards in a Transwell system, and were exposed to a single dose of gamma-irradiation (2 Gy). A parallel set of nonirradiated skin fragments was incubated under the same conditions. Both irradiated and nonirradiated fragments were harvested 24 h after irradiation and processed for light microscopy and molecular biology analysis. A quantitative analysis of cell proliferation was performed after 5-bromo-2'-deoxyuridine incorporation. Cytokeratin 10 (CK10) and desmocollin 1 (Dsc1) expression was evaluated by immunofluorescence. Dsc1 and transforming growth factor (TGF)-beta1 gene expression was measured by reverse transcriptase-polymerase chain reaction analysis. RESULTS: The mean percentage inhibition of epidermal proliferation in irradiated samples was 53.7% (P < 0.01, paired Student's t-test). The inhibition of cell proliferation was significant in five of seven samples (P < 0.05, unpaired Student's t-test). Normal cell architecture was found in irradiated samples. Throughout the epithelial compartment, the distribution patterns of CK10 and Dsc1 were comparable in nonirradiated and irradiated fragments. Condensation of CK10 filaments suggested a cytoskeletal rearrangement in irradiated samples. Dsc1 and TGF-beta1 mRNA levels were, respectively, reduced and unmodified 24 h after irradiation. CONCLUSIONS: A perturbation of epidermal homeostasis occurs as early as 24 h after a single dose of gamma-rays. Our immunofluorescence observations indicate that keratinocyte terminal differentiation is not yet affected at the protein level 24 h after exposure to gamma-rays. The lack of an inverse relationship between TGF-beta1 gene expression and epidermal proliferation, together with decreased Dsc1 gene expression, may represent the early molecular basis for the development of the late effects of radiotherapy observed many months/years after radiotherapy. Our findings set the stage for further investigation of the best time to begin topical treatment at the start of radiation therapy.

Evaluation of residual immunoreactivity in red and white wines clarified with gluten or gluten derivatives.

Gluten or hydrolyzed gluten could be a suitable alternative to animal proteins in the wine clarification process, but their residues could represent a risk for individuals suffering from coeliac disease or allergic to cereal proteins. The aim of this study was to investigate the presence of gluten in wines treated with gluten or its hydrolysate in the clarification process and to assess its antigenicity in commercial products. The presence of residual immunoreactive gluten was evaluated by electrophoresis (SDS-PAGE) and immunoblotting. Data obtained in several red and white wine samples showed that no residue was detectable in any of the red wines. In white wines, gluten reduced the protein content less completely, but most samples showed no immunoreactivity after the wine had been treated with gluten or its derivatives, either alone or combined with bentonite, silica gel or tannins. The use of gluten derivatives coupled with bentonite was the most effective method of removing immunoreactive protein in white wines. In conclusion, the use of gluten derivatives in wine clarification seems to exclude a risk for subjects susceptible to coeliac disease or gluten allergy. However, it is recommended that wine producers continuously monitor the clarification process in order to protect the most sensitive individuals.

Assessment of residual immunoreactivity in red or white wines clarified with pea or lupin extracts.

Vegetable proteins could be a suitable alternative to animal proteins in the clarification of wine, but their residues could represent a risk for subjects with food allergy or intolerance. The aim of this study was to investigate the presence of specific immunoreactivity in red and white wines treated, as must or wine, with vegetable proteins in the clarification process. The proteins considered were prepared from lupins and peas, which are not included among the allergens listed in annex Illbis of Directive 2003/89/EC. The presence of residual immunoreactivity to specific rabbit anti-lupin and anti-pea polyclonal antibodies in treated wines was assessed by electrophoresis (SDS-PAGE) and immunoblotting. Residual protein was not detectable in red wines clarified with lupin, pea or a mixture of pea and lupin proteins or in white wines clarified with pea proteins. A small number of musts treated with lupin or pea proteins and white wines treated with lupin proteins yielded equivocal results, probably because of the presence of interfering material (e.g., sugar-rich proteins from grape and yeast). The use of bentonite as a secondary clarifying agent is therefore recommended since its combination with vegetable proteins is particularly effective in removing overall protein immunoreactivity.

Evidence for resveratrol-induced preservation of brain mitochondria functions after hypoxia-reoxygenation.

We have previously shown, as have other authors, that trans-resveratrol (E-resveratrol, 3,4,5-trihydroxy-E-stilbene) reduces reactive oxygen species (ROS) generation of mitochondria freshly isolated from healthy rat brains and that it also counteracts the effect of uncouplers (CCCP) on mitochondrial respiration and oxidative phosphorylation. Two main mechanisms have been shown: firstly, a scavenger effect toward O2- and secondly inhibition of complex III ROS generation. We now report on the effects of resveratrol in a pathological model that mimics the ischemia followed by the reperfusion process which may occur in the human brain. Isolated brain mitochondria were submitted first to hypoxia then to reoxygenation. The aim of this study was to determine the extent of mitochondrial damage induced by this experimental model, to demonstrate which mitochondrial functions were altered and to quantify the extent to which they were prevented by resveratrol. Resveratrol was either added to mitochondria freshly isolated from healthy rat brains or was injected by subcutaneous chronically implanted pumps (0.5, 2 and 10 mg/kg/day for 7 days). The rats were then sacrificed and mitochondria were extracted from brains. To evaluate the respective effects of hypoxia and reoxygenation on mitochondrial functions and the relevant effects of resveratrol, this drug was added (first protocol) either before the complete process (i.e., hypoxia and reoxygenation), or after anoxia before reoxygenation. We found that resveratrol prevented alterations of mitochondrial functions. This substance partly counteracted the decrease in respiratory control and the increase in ROS generation. It fully inhibited the alteration of membrane fluidity and the mitochondrial step of the apoptotic process (evidenced by cytochrome c release and membrane potential collapse). The effects of resveratrol were concentration-dependent (in vitro) or dose-dependent (ex vivo, second protocol). They were not significantly different when the drug was added before or after hypoxia, which suggests that in this model, reoxygenation was the most deleterious process and the stage at which resveratrol was most effective.

Evaluation by SDS-Page and immunoblotting of residual antigenicity in gluten-treated wine: a preliminary study.

Hydrolyzed gluten could be a suitable alternative to animal proteins in the wine clarification process, but the residual proteins could constitute a risk for subjects suffering from celiac disease or allergy to cereals. The aim of this study was to investigate possible traces of gluten in treated wine and to assess its antigenicity in commercial products. The presence of gluten in treated wine was evaluated by an electrophoretic method [sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] and its immunoreactivity was evaluated by immunoblotting. No traces of protein were found in untreated samples. A small quantity of protein was detected in treated wine but this produced no significant immunochemical reaction. In an experimental clarification process, a protein fraction was detectable in untreated samples and in the first stages of the clarification process. However, there was no significant gluten-associated immunochemical reaction in clarified wine samples, confirming strong binding between the clarifying agent and the phenolic fraction. In conclusion, the clarifying process strongly reduced the amount of protein material, at least in red wines. Under the most restrictive tests of the presence of gluten in the product, the predictable residue of gluten in wine was safe for celiac subjects. For allergic subjects the data are less conclusive because there is no known limit for allergic reactions, but clear labeling of the method of treating the wine should also protect this group of consumers.

Inhibitory activity of the white wine compounds, tyrosol and caffeic acid, on lipopolysaccharide-induced tumor necrosis factor-alpha release in human peripheral blood mononuclear cells.

The objective of this study was to assess whether tyrosol and caffeic acid are able to inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha release. TNF is one of the most important cytokines involved in inflammatory reactions. The results show that both tyrosol and caffeic acid are able to inhibit LPS-induced TNF-alpha release from human monocytes, even at low doses. Their mechanisms of action are discussed and we conclude that high doses of the two compounds are not required to achieve effective inhibition of inflammatory reactions due to TNF-alpha release.

Cardioprotection with white wine.

The cardioprotective effects of red wine have been attributed to several polyphenolic antioxidants including resveratrol and proanthocyanidins. The goal of the present study was to determine whether white wines could also provide cardioprotection. Three different white wines (white wine #1, #2 and #3) were chosen for this study. Ethanol-free extracts of the wines were prepared by vacuum evaporation. Rats weighing approximately 200 g were given either 50 mg/kg or 100 mg/kg of each wine extract for 3 weeks. The rats were anesthetized and sacrificed and their hearts were excised for the preparation of isolated working rat heart. All hearts were subjected to 30 min of global ischemia followed by 2 h of reperfusion. Cardiac function including heart rate, left ventricular developed pressure (LVDP), maximum first derivative of developed pressure (LVdp/dtmax), left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEP), aortic flow (AF) and coronary flow (CF) were continuously monitored and myocardial infarct size was measured at the end of the experiments. The results of our study demonstrated that among the three different white wines, only white wine #2 conferred cardioprotection as evidenced by improved postischemic ventricular recovery compared with controls. The same white wine at a dose of 50 mg/kg also showed improvement in postischemic contractile recovery but the differences compared with controls were not significant. The amount of malondialdehyde production from these hearts was lower than that found in control hearts, indicating reduced formation of reactive oxygen species in white wine #2-treated rats. In vitro studies using a chemiluminescence technique revealed that white wine #2 scavenged both superoxide anions and hydroxyl radicals. The results of our study demonstrate that white wine #2 provided cardioprotection and the cardioprotective effect of the wine can be attributed, at least in part, to its ability to function as an in vivo antioxidant.

Effect of some white wine phenols in preventing inflammatory cytokine release.

Some well-known antioxidant phenols present in extravirgin olive oil have also been found in white wine. Both tyrosol and caffeic acid are phenols that are present not only in extravirgin olive oil, but also in wine, especially white wine. Their antioxidant properties are well known, but their biological effects have not yet been elucidated. In a previous study we found that these substances were able to inhibit tumor necrosis factor alpha release. The present study was carried out to assess whether these compounds are able to inhibit other inflammatory cytokines, such as interleukin-1 beta and interleukin-6. The results show that low concentrations of these phenols, which can be found in the bloodstream after intake of moderate quantities of white wine, exert significant inhibitory activity on the release of several inflammatory cytokines.

Nanomolar level of resveratrol (trans-3,5,4'-trihydroxystilbene) is required, but is not sufficient, to inhibit the growth of human monocytoid tumor cells through an apoptotic-like mechanism.

The chemopreventive activity of resveratrol, a stilbene found in grapes and wine, was evaluated in a human monocytic leukemia cell line at the same concentration (100 nM to 1 microM) as that found in the blood-stream after moderate wine intake. As early as at 4 h after intake, resveratrol exhibited antiproliferative and cytotoxic activity. At the same time, some apoptotic-like phenomena were detected such as cell membrane perturbation (phosphatidylserine-annexin V binding), apolipoprotein (APO)-1/FAS (CD95) expression and mitochondrial (delta psi) depolarization. The anticancer drug camptothecin, used as a positive control, did not significantly increase APO-1/FAS (CD95) levels, while only a modest increase in APO-1/FAS-CD95 ligand (CD95-L) was detected. At 12 h, however, resveratrol at concentrations of 100 nM and 1 microM did not exhibit the same antiproliferative activity and increased cell proliferation was correlated to a significant increase in FAS-L expression. We conclude that treatment with low doses of resveratrol, such as those found after moderate wine intake, is not sufficient to stop human leukemia cell line proliferation and that cell resistance, marked by high FAS-L (CD95-L) expression, could be mediated by low (delta psi) mitochondria-released antiapoptotic factors such as BCL-2. It is also suggested that the synergistic action of other wine components with resveratrol might, at least partially, explain its chemopreventive activity.

Resveratrol, a polyphenol found in wine, reduces ischemia reperfusion injury in rat kidneys.

Reactive oxygen species have been implicated in the pathophysiology of renal ischemia reperfusion injury. Antioxidants including polyphenolics have been found to protect renal cells from the cellular injury induced by ischemia and reperfusion. Resveratrol, a stilbene polyphenol found in grapes and red wine, has recently been found to protect isolated rat heart from ischemia reperfusion injury. This study was sought to determine if resveratrol could also protect renal cells from ischemic injury. Male Wistar rats were treated with control, resveratrol (0.23 microg/kg), vehicle used to solubilize resveratrol, and resveratrol plus L-NAME (15 mg/kg body wt), a nitric oxide blocker. Our results demonstrated that resveratrol administration reduced the mortality of ischemic rats from 50% to 10% and renal damage was reduced as indicated by histologic examination and serum creatinine level. The short-term administration of resveratrol also inhibited renal lipid peroxidation induced by ischemia and reperfusion both in cortex and in medulla. Electron paramagnetic resonance detected an increased formation of nitric oxide in the resveratrol-treated kidney that was reduced to the baseline value after treating the rats with L-NAME in addition to resveratrol. The results suggest that resveratrol reduced the renal ischemia reperfusion injury through a nitric oxide-dependent mechanism.

Anti-apoptotic effect of trans-resveratrol on paclitaxel-induced apoptosis in the human neuroblastoma SH-SY5Y cell line.

Paclitaxel, an anticancer drug, induces apoptosis in human neuroblastoma cell line SH-SY5Y. The addition of trans-resveratrol, a natural antioxidant present in grapes and red wine, to SH-SY5Y cultures exposed to paclitaxel significantly reduces cellular death. The neuroprotective action of trans-resveratrol is due neither to its antioxidant capacity nor to interference with the polymerization of tubulin induced by paclitaxel. However, trans-resveratrol is able to inhibit the activation of caspase 7 and degradation of poly-(ADP-ribose)-polymerase which occur in SH-SY5Y exposed to paclitaxel. Resveratrol, therefore, exerts its anti-apoptotic effect by modulating the signal pathways that commit these neuronal-like cells to apoptosis.

Resveratrol inhibits TNF alpha-induced endothelial cell activation.

Resveratrol, a phytoalexin found in grapes and wine, has been shown to have anti-inflammatory properties. Since endothelium is activated during inflammation by some cytokines released by macrophages and many other cells, we tested whether resveratrol could modulate endothelial cell activation. We studied the effect of resveratrol treatment in vitro on the expression of vascular cell adhesion molecule-1 by tumour necrosis factor alpha-stimulated human umbilical vein endothelial cells. In addition, we studied the effect of resveratrol treatment in vivo (in a murine experimental model) on the modulation of tumour necrosis factor alpha-induced vascular permeability. Resveratrol, used at the concentrations present in human plasma following moderate wine consumption, was demonstrated to be an inhibitor of the adhesion molecule expression by tumour necrosis factor alpha-stimulated endothelial cells. In addition, resveratrol significantly prevented the cytokine-induced vascular leakage. Our results (both in vitro and in vivo) may explain some aspects of the anti-inflammatory effects of resveratrol.

Reactive oxygen metabolites and prooxidant status in children with Down's syndrome.

Children with Down's syndrome suffer many diseases among which cardiovascular diseases, increased susceptibility to infections, leukemia, endocrine alterations, immune defects, nutritional disturbance and mental retardation have clinical relevance. It has been suggested that the pathogenesis of Down's syndrome involves reactive oxygen species arising from a mutation in gene encoding, which disproportionately elevates superoxide dismutase activity. Reactive oxygen species and total antioxidant capacity were evaluated using two new spectrophotometric methods in a selected group of 40 children with Down's syndrome and in 20 apparently healthy children used as controls. Reactive oxygen species were significantly higher (p <0.05) in children with Down's syndrome than in controls: 452 (+/- 72) U.Carr vs. 270 (+/- 66) U.Carr respectively. Total antioxidant capacity was significantly higher (p <0.05) in controls than in children with Down's syndrome: 380 (+/- 52) micromol hypochlorous acid (HCLO)/ml vs. 281 (+/- 33) micromol HCLO/ml, respectively. In fact, thiol groups (sulfhydryl) were significantly higher (p <0.05) in controls than in children with Down's syndrome: 644 (+/- 78) micromol/l vs. 462 (+/- 54) micromol/l, respectively Our data show how to simply measure chemical indices of oxidative status in serum samples from children with Down's syndrome. We determined the plasmatic activities of reactive oxygen metabolites and oxidative defense molecules. Accumulated macromolecular damage may be one of the causes of some of the abnormalities that are considered part of the syndrome. Therefore, children with Down's syndrome have to cope with a significant prooxidant environment. Oxidative stress causes alterations such as atherosclerosis, early aging, immunological default and neurologic disorders in Down's syndrome patients. The new test available for measuring reactive oxygen species in serum proved to be reliable and useful as an early marker of tissue damage.

Does resveratrol induce pharmacological preconditioning?

Resveratrol is a grape component with complex pharmacology related to its antioxidant activity. Little is known about the direct effects of resveratrol on the myocardium. We tested whether resveratrol administration before ischemia could attenuate ischemic/reperfusion damage. We examined how resveratrol affects high-energy phosphate metabolism (31P-nuclear magnetic resonance) and contractility of isolated Langendorff perfused rat hearts subjected to 20 min no-flow ischemia and 30 min reperfusion. During 10 min resveratrol infusion (10 microM) before ischemia, basal phosphorylation potential dropped by 40% (p < 0.05 vs. preinfusion value) without affecting contractility. The level of effluent adenosine was increased by 68%, parallel to a 50% increase in coronary flow. Resveratrol significantly improved postischemic recovery of rate-pressure product (62 +/- 5.2 vs. 23 +/- 8.1% of controls; p < 0.05). The metabolic pattern following resveratrol infusion was similar to that produced by ischemic preconditioning, suggesting that an increase in adenosine availability is involved in cardioprotection.

Myocardial protection with red wine extract.

Cardioprotective action of red wine was studied by preperfusing isolated rat hearts with ethanol-free red wine extract for 15 min before subjecting them to 30 min of global ischemia followed by 2 h of reperfusion. Four other group of rats were studied under identical conditions, of which one served as control; one was treated with 10 microM trans-resveratrol (RVT), one of the major antioxidants found in red wines; another, with 0.07% ethanol; and another, with 0.07% ethanol plus 10 microM RVT. The results of our study demonstrated that both red wine extract and RVT were equally cardioprotective, as evidenced by their abilities to improve postischemic ventricular functions including developed pressure and aortic flow. Developed pressure values at 60 min after reperfusion were 81.8 +/- 1.2 and 68.8 +/- 4.1 mm Hg for the red wine extract and RVT groups, respectively, versus 49.7 +/- 2.7 mm Hg for the control group. These compounds also reduced myocardial infarct size compared with the control hearts (20.1 +/- 0.5% and 10.5 +/- 0.3% for red wine extract and RVT groups, respectively, vs. 29.9 +/- 3.1% for the control group). The ethanol-treated group displayed slightly better functional recovery, which deteriorated sharply toward the end of the reperfusion period, and the extent of infarction was comparable to that of the control group (31.5 +/- 0.9%). In the ethanol plus RVT group, postischemic contractile function was significantly better than control, and infarct size also was reduced to 20.9 +/- 0.7%. The amount of malonaldehyde formation in the postischemic myocardium was reduced by red wine extract and RVT, indicating a reduction of oxidative stress developed in the ischemic reperfused myocardium. In vitro studies revealed that red wine extract is a potent antioxidant as evidenced by its ability to scavenge peroxyl radical in vitro. Taken together, the results of our study indicate that red wines are cardioprotective by their ability to function as an in vivo antioxidant.