Pembrolizumab in combination with gemcitabine for patients with HER2-negative advanced breast cancer: GEICAM/2015-04 (PANGEA-Breast) study.

BACKGROUND: We evaluated a new chemoimmunotherapy combination based on the anti-PD1 monoclonal antibody pembrolizumab and the pyrimidine antimetabolite gemcitabine in HER2- advanced breast cancer (ABC) patients previously treated in the advanced setting, in order to explore a potential synergism that could eventually obtain long term benefit in these patients. METHODS: HER2-negative ABC patients received 21-day cycles of pembrolizumab 200 mg (day 1) and gemcitabine (days 1 and 8). A run-in-phase (6 + 6 design) was planned with two dose levels (DL) of gemcitabine (1,250 mg/m2 [DL0]; 1,000 mg/m2 [DL1]) to determine the recommended phase II dose (RP2D). The primary objective was objective response rate (ORR). Tumor infiltrating lymphocytes (TILs) density and PD-L1 expression in tumors and myeloid-derived suppressor cells (MDSCs) levels in peripheral blood were analyzed. RESULTS: Fourteen patients were treated with DL0, resulting in RP2D. Thirty-six patients were evaluated during the first stage of Simon's design. Recruitment was stopped as statistical assumptions were not met. The median age was 52; 21 (58%) patients had triple-negative disease, 28 (78%) visceral involvement, and 27 (75%) ≥ 2 metastatic locations. Progression disease was observed in 29 patients. ORR was 15% (95% CI, 5-32). Eight patients were treated ≥ 6 months before progression. Fourteen patients reported grade ≥ 3 treatment-related adverse events. Due to the small sample size, we did not find any clear association between immune tumor biomarkers and treatment efficacy that could identify a subgroup with higher probability of response or better survival. However, patients that experienced a clinical benefit showed decreased MDSCs levels in peripheral blood along the treatment. CONCLUSION: Pembrolizumab 200 mg and gemcitabine 1,250 mg/m2 were considered as RP2D. The objective of ORR was not met; however, 22% patients were on treatment for ≥ 6 months. ABC patients that could benefit of chemoimmunotherapy strategies must be carefully selected by robust and validated biomarkers. In our heavily pretreated population, TILs, PD-L1 expression and MDSCs levels could not identify a subgroup of patients for whom the combination of gemcitabine and pembrolizumab would induce long term benefit. TRIAL REGISTRATION: ClinicalTrials.gov and EudraCT (NCT03025880 and 2016-001,779-54, respectively). Registration dates: 20/01/2017 and 18/11/2016, respectively.

Untargeted metabolomics for plasma biomarker discovery for early chronic kidney disease diagnosis in pediatric patients using LC-QTOF-MS.

Pediatric chronic kidney disease (CKD) is a clinical syndrome characterized by renal hypofunction occurring due to gradual and irreversible kidney damage that can further progress over time. New biomarkers may help early diagnosis of pediatric patients suffering from CKD and improve the outcome. Untargeted metabolomics based on LC-QTOF-MS has been used to find new biomarkers for the early diagnosis of CKD in plasma from pediatric patients. In order to avoid any bias in the determination of statistically significant entities as a consequence of the data analysis method followed, two different chemometric approaches have been used, Mass Profiler Professional (MPP) software and Matlab R2015a software. Metabolic fingerprints of control and CKD pediatric patients were compared and five metabolites which showed a significant change common to both data analysis procedures were identified. Sphingosine-1-phosphate, n-butyrylcarnitine, cis-4-decenoylcarnitine and an unidentified feature with 126.0930 m/z were found to be increased in plasma from pediatric patients with CKD, whereas bilirubin was significantly decreased. A partial least squares discriminant analysis model built with these 5 entities classified correctly 96% of the samples. In addition, when considering only early CKD patients against controls, a performance of 97% was obtained. Thus, these promising metabolites could be suitable biomarkers for the early diagnosis of pediatric CKD in a clinical setting.

Selection of appropriate Schizosaccharomyces strains for winemaking.

This paper describes the selection of Schizosaccharomyces yeasts with adequate oenological suitability and high capacity for the degradation of malic acid. Despite the almost non-existent number of commercial strains, the use of this yeast genus has recently been recommended by the International Organisation of Vine and Wine (OIV, in French). Thus, in the present study, a large number of Schizosaccharomyces strains were isolated using a selective differential medium. Initially, classic parameters of oenological interest for the use of fermentative strains of Saccharomyces cerevisiae (the most frequently used type of yeast) were assessed. Only five strains of moderate acetic acid production lower than 0.4 g/L were obtained at the end of fermentation. Other, more specific features of this yeast genus' physiology were also studied, including urease activity and the production of pyruvic acid and glycerol. Finally, oenological suitability was determined by comparing selected strains with other Schizosaccharomyces reference and S cerevisiae control strains. Schizosaccharomyces strains produced 80% less urea content, four times higher pyruvic acid levels and 1 g higher glycerol contents than the Saccharomyces reference strains. The results confirmed that it is possible to perform selective processes on microorganisms from the genus Schizosaccharomyces using methodology developed in this work to obtain strains of industrial interest.

Non-linear oscillatory rheological properties of a generic continuum foam model: comparison with experiments and shear-banding predictions.

The occurrence of shear bands in a complex fluid is generally understood as resulting from a structural evolution of the material under shear, which leads (from a theoretical perspective) to a non-monotonic stationary flow curve related to the coexistence of different states of the material under shear. In this paper we present a scenario for shear-banding in a particular class of complex fluids, namely foams and concentrated emulsions, which differs from other scenarios in two important ways. First, the appearance of shear bands is shown to be possible both without any intrinsic physical evolution of the material (e.g. via a parameter coupled to the flow such as concentration or entanglements) and without any finite critical shear rate below which the flow does not remain stationary and homogeneous. Secondly, the appearance of shear bands depends on the initial conditions, i.e. the preparation of the material. In other words, it is history dependent. This behaviour relies on the tensorial character of the underlying model (2D or 3D) and is triggered by an initially inhomogeneous strain distribution in the material. The shear rate displays a discontinuity at the band boundary whose amplitude is history dependent and thus depends on the sample preparation.

An optimized MNK1b aptamer, apMNKQ2, and its potential use as a therapeutic agent in breast cancer.

Breast cancer is the most commonly diagnosed and leading cause of cancer death among women worldwide. Mitogen-activated protein kinase-interacting kinases (MNKs) promote the expression of several oncogenic proteins and are overexpressed in several types of cancer. In human cells, there are four isoforms of MNKs. The truncated isoform MNK1b, first described in our laboratory, has a higher basal activity and is constitutively active. Aptamers are emerging in recent years as potential therapeutic agents that show significant advantages over drugs of other nature. We have previously obtained and characterized a highly specific aptamer against MNK1b, named apMNK2F, with a dissociation constant in the nanomolar range, which produces significant inhibition of proliferation, migration, and colony formation in breast cancer cells. Furthermore, its sequence analysis predicted two G-quadruplex structures. In this work, we show the optimization process of the aptamer to reduce its size, improving its stability. The obtained aptamer, named apMNKQ2, is able to inhibit proliferation, colony formation, migration, and invasion in breast cancer cells. In murine models of breast cancer, apMNKQ2 has demonstrated its efficacy in reducing tumor volume and the number of metastases. In conclusion, apMNKQ2 could be used as an anti-tumor drug in the future.

Mediterranean diet and the incidence of cardiovascular disease: a Spanish cohort.

BACKGROUND AND AIM: The Mediterranean diet is considered a model for healthy eating. However, prospective evidence in Mediterranean countries evaluating the relationship between this dietary pattern and non-fatal cardiovascular events is scarce. The aim of the present study was to evaluate the association between the adherence to the Mediterranean diet and the incidence of fatal and non-fatal cardiovascular events among initially healthy middle-aged adults from the Mediterranean area. METHODS AND RESULTS: We followed-up 13,609 participants (60 percent women, mean age: 38 years) initially free of cardiovascular disease (CVD) during 4.9 years. Participants were part of a prospective cohort study of university graduates from all regions of Spain. Baseline diet was assessed using a validated 136-item food-frequency questionnaire. A 9-point score was used to appraise adherence to the Mediterranean diet. Incident clinical events were confirmed by a review of medical records. We observed 100 incident cases of CVD. In multivariate analyses, participants with the highest adherence to the Mediterranean diet (score>6) exhibited a lower cardiovascular risk (hazard ratio=0.41, 95% confidence interval [CI]: 0.18-0.95) compared to those with the lowest score (<3). For each 2-point increment in the score, the adjusted hazard ratios were 0.80 (95% CI: 0.62-1.02) for total CVD and 0.74 (0.55-0.99) for coronary heart disease. CONCLUSIONS: There is an inverse association between adherence to the Mediterranean diet and the incidence of fatal and non-fatal CVD in initially healthy middle-aged adults.

A method for estimating Dekkera/Brettanomyces populations in wines.

AIMS: The formation of ethylphenols in wines, a consequence of Dekkera/Brettanomyces metabolism, can affect their quality. The main aims of this work were to further our knowledge of Dekkera/Brettanomyces with respect to ethylphenol production, and to develop a methodology for detecting this spoilage yeast and for estimating its population size in wines using differential-selective media and high performance liquid chromatography (HPLC). METHODS AND RESULTS: This work examines the reduction of p-coumaric acid and the formation of 4-vinylphenol and 4-ethylphenol (recorded by HPLC-DAD) in a prepared medium because of the activities of different yeast species and populations. A regression model was constructed for estimating the population of Dekkera/Brettanomyces at the beginning of fermentation via the conversion of hydroxycinnamic acids into ethylphenols. CONCLUSIONS: The proposed methodology allows the populations of Dekkera/Brettanomyces at the beginning of fermentation to be estimated in problem wines. Moreover, it avoids false positives because of yeasts resistant to the effects of the selective elements of the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This may help prevent the appearance of organoleptic anomalies in wines at the winery level.

LC-QQQ-MS routine analysis method for new biomarker quantification in plasma aimed at early chronic kidney disease diagnosis.

Pediatric chronic kidney disease (CKD) is currently assessed using glomerular filtration rate (GFR), which is calculated from different equations based on serum creatinine concentration. However, serum creatinine is affected by several factors and is not reliable enough for the diagnosis of CKD, especially at early stages. Recent targeted and untargeted metabolomics studies found 7 new potential biomarkers that could be useful for early pediatric chronic kidney disease diagnosis. Thus, a new LC-QQQ-MS analytical method has been developed and validated aimed at routine analysis of these 7 potential biomarkers: NG,NG'-dimethyl-l-arginine di(p-hydroxyazobenzene-p'-sulfonate) (SDMA), S-adenosyl-l-methionine (SAM), n-butyryl-l-carnitine (nC4), iso-butyryl-l-carnitine (iC4), citrulline (CIT), creatinine (CNN) and d-erytro-sphingosine-1-phosphate (S1P), in addition to creatinine, the classical biomarker for CKD diagnosis. Then, this analytical method has been used for the quantification of plasma samples from a heterogeneous group of CKD pediatric patients and a control pediatric population. Data analysis of these results showed that it is possible to differentiate between CKD and control populations based on the metabolite concentration in plasma.

Evaluation of the effect of polymorphism on G-quadruplex-ligand interaction by means of spectroscopic and chromatographic techniques.

Guanine-rich sequences may fold into highly ordered structures known as G-quadruplexes. Apart from the monomeric G-quadruplex, these sequences may form multimeric structures that are not usually considered when studying interaction with ligands. This work studies the interaction of a ligand, crystal violet, with three guanine-rich DNA sequences with the capacity to form multimeric structures. These sequences correspond to short stretches found near the promoter regions of c-kit and SMARCA4 genes. Instrumental techniques (circular dichroism, molecular fluorescence, size-exclusion chromatography and electrospray ionization mass spectrometry) and multivariate data analysis were used for this purpose. The polymorphism of G-quadruplexes was characterized prior to the interaction studies. The ligand was shown to interact preferentially with the monomeric G-quadruplex; the binding stoichiometry was 1:1 and the binding constant was in the order of 105M-1 for all three sequences. The results highlight the importance of DNA treatment prior to interaction studies.

Plasma biomarker discovery for early chronic kidney disease diagnosis based on chemometric approaches using LC-QTOF targeted metabolomics data.

Chronic kidney disease (CKD) is a progressive pathological condition in which renal function deteriorates in time. The first diagnosis of CKD is often carried out in general care attention by general practitioners by means of serum creatinine (CNN) levels. However, it lacks sensitivity and thus, there is a need for new robust biomarkers to allow the detection of kidney damage particularly in early stages. Multivariate data analysis of plasma concentrations obtained from LC-QTOF targeted metabolomics method may reveal metabolites suspicious of being either up-regulated or down-regulated from urea cycle, arginine methylation and arginine-creatine metabolic pathways in CKD pediatrics and controls. The results show that citrulline (CIT), symmetric dimethylarginine (SDMA) and S-adenosylmethionine (SAM) are interesting biomarkers to support diagnosis by CNN: early CKD samples and controls were classified with an increase in classification accuracy of 18% when using these 4 metabolites compared to CNN alone. These metabolites together allow classification of the samples into a definite stage of the disease with an accuracy of 74%, being the 90% of the misclassifications one level above or below the CKD stage set by the nephrologists. Finally, sex-related, age-related and treatment-related effects were studied, to evaluate whether changes in metabolite concentration could be attributable to these factors, and to correct them in case a new equation is developed with these potential biomarkers for the diagnosis and monitoring of pediatric CKD.

Closed tracheal suction systems versus open tracheal suction systems for mechanically ventilated adult patients.

BACKGROUND: Ventilator-associated pneumonia is a common complication in ventilated patients. Endotracheal suctioning is a procedure that may constitute a risk factor for ventilator-associated pneumonia. It can be performed with an open system or with a closed system. In view of suggested advantages being reported for the closed system, a systematic review comparing both techniques was warranted. OBJECTIVES: To compare the closed tracheal suction system and the open tracheal suction system in adults receiving mechanical ventilation for more than 24 hours. SEARCH STRATEGY: We searched CENTRAL (The Cochrane Library 2006, Issue 1) MEDLINE, CINAHL, EMBASE and LILACS from their inception to July 2006. We handsearched the bibliographies of relevant identified studies, and contacted authors and manufacturers. SELECTION CRITERIA: The review included randomized controlled trials comparing closed and open tracheal suction systems in adult patients who were ventilated for more than 24 hours. DATA COLLECTION AND ANALYSIS: We included the relevant trials fitting the selection criteria. We assessed methodological quality using method of randomization, concealment of allocation, blinding of outcome assessment and completeness of follow up. Effect measures used for pooled analyses were relative risk (RR) for dichotomous data and weighted mean differences (WMD) for continuous data. We assessed heterogeneity prior to meta-analysis. MAIN RESULTS: Of the 51 potentially eligible references, the review included 16 trials (1684 patients), many with methodological weaknesses. The two tracheal suction systems showed no differences in risk of ventilator-associated pneumonia (11 trials; RR 0.88; 95% CI 0.70 to 1.12), mortality (five trials; RR 1.02; 95% CI 0.84 to 1.23) or length of stay in intensive care units (two trials; WMD 0.44; 95% CI -0.92 to 1.80). The closed tracheal suction system produced higher bacterial colonization rates (five trials; RR 1.49; 95% CI 1.09 to 2.03). AUTHORS' CONCLUSIONS: Results from 16 trials showed that suctioning with either closed or open tracheal suction systems did not have an effect on the risk of ventilator-associated pneumonia or mortality. More studies of high methodological quality are required, particularly to clarify the benefits and hazards of the closed tracheal suction system for different modes of ventilation and in different types of patients.

[Cyclosporin A causes oxidative stress and mitochondrial dysfunction in renal tubular cells]. / La ciclosporina A origina estrés oxidativo y disfunción mitocondrial en células tubulares renales.

Reactive oxygen species (ROS) have been implicated in cyclosporin A (CsA) nephrotoxicity. As mitochondria are one of the main sources of ROS in cells, we evaluated the role of CsA in mitochondrial structure and function in LLC-PK1 cells. We incubated cells with CsA 1 microM for 24 hours and studies were performed with flow citometry and confocal microscopy. We studied mitochondrial NAD(P)H content, superoxide anion (O2.-) production (MitoSOX Red), oxidation of cardiolipin of inner mitochondrial membrane (NAO) and mitochondrial membrane potential (DIOC2(3)). Also we analyzed the intracellular ROS synthesis (H2DCF-DA) and reduced glutation (GSH) of cells. Our results showed that CsA decreased NAD(P)H and membrane potential, and increased O2.- in mitochondria. CsA also provoked oxidation of cardiolipin. Furthermore, CsA increased intracellular ROS production and decreased GSH content. These results suggest that CsA has crucial effects in mitochondria. CsA modified mitochondrial physiology through the decrease of antioxidant mitochondrial compounds as NAD(P)H and the dissipation of mitochondrial membrane potential and increase of oxidants as O2.-. Also, CsA alters lipidic structure of inner mitochondrial membrane through the oxidation of cardiolipin. These effects trigger a chain of events that favour intracellular synthesis of ROS and depletion of GSH that can compromise cellular viability. Nephrotoxic cellular effects of CsA can be explained, at least in part, through its influence on mitochondrial functionalism.

Viability qPCR, a new tool for Legionella risk management.

BACKGROUND: Viability quantitative Polymerase Chain Reaction (v-qPCR) is a recent analytical approach for only detecting live microorganisms by DNA amplification-based methods This approach is based on the use of a reagent that irreversibly fixes dead cells DNA. In this study, we evaluate the utility of v-qPCR versus culture method for Legionellosis risk management. METHODS: The present study was performed using 116 real samples. Water samples were simultaneously analysed by culture, v-qPCR and qPCR methods. Results were compared by means of a non-parametric test. RESULTS: In 11.6% of samples using both methods (culture method and v-qPCR) results were positive, in 50.0% of samples both methods gave rise to negative results. As expected, equivalence between methods was not observed in all cases, as in 32.1% of samples positive results were obtained by v-qPCR and all of them gave rise to negative results by culture. Only in 6.3% of samples, with very low Legionella levels, was culture positive and v-qPCR negative. In 3.5% of samples, overgrowth of other bacteria did not allow performing the culture. When comparing both methods, significant differences between culture and v-qPCR were in the samples belonging to the cooling towers-evaporative condensers group. The v-qPCR method detected greater presence and obtained higher concentrations of Legionella spp. (p<0.001). Otherwise, no significant differences between methods were found in the rest of the groups. CONCLUSIONS: The v-qPCR method can be used as a quick tool to evaluate Legionellosis risk, especially in cooling towers-evaporative condensers, where this technique can detect higher levels than culture. The combined interpretation of PCR results along with the ratio of live cells is proposed as a tool for understanding the sample context and estimating the Legionellosis risk potential according to 4 levels of hierarchy.

Use of Schizosaccharomyces strains for wine fermentation-Effect on the wine composition and food safety.

Schizosaccharomyces was initially considered as a spoilage yeast because of the production of undesirable metabolites such as acetic acid, hydrogen sulfide, or acetaldehyde, but it currently seems to be of great value in enology.o ced Nevertheless, Schizosaccharomyces can reduce all of the malic acid in must, leading to malolactic fermentation. Malolactic fermentation is a highly complicated process in enology and leads to a higher concentration of biogenic amines, so the use of Schizosaccharomyces pombe can be an excellent tool for assuring wine safety. Schizosaccharomyces also has much more potential than only reducing the malic acid content, such as increasing the level of pyruvic acid and thus the vinylphenolic pyranoanthocyanin content. Until now, few commercial strains have been available and little research on the selection of appropriate yeast strains with such potential has been conducted. In this study, selected and wild Sc. pombe strains were used along with a Saccharomyces cerevisiae strain to ferment red grape must. The results showed significant differences in several parameters including non-volatile and volatile compounds, anthocyanins, biogenic amines and sensory parameters.

Optimized symbolic dynamics approach for the analysis of the respiratory pattern.

Traditional time domain techniques of data analysis are often not sufficient to characterize the complex dynamics of respiration. In this paper, the respiratory pattern variability is analyzed using symbolic dynamics. A group of 20 patients on weaning trials from mechanical ventilation are studied at two different pressure support ventilation levels, in order to obtain respiratory volume signals with different variability. Time series of inspiratory time, expiratory time, breathing duration, fractional inspiratory time, tidal volume and mean inspiratory flow are analyzed. Two different symbol alphabets, with three and four symbols, are considered to characterize the respiratory pattern variability. Assessment of the method is made using the 40 respiratory volume signals classified using clinical criteria into two classes: low variability (LV) or high variability (HV). A discriminant analysis using single indexes from symbolic dynamics has been able to classify the respiratory volume signals with an out-of-sample accuracy of 100%.

Classification of patients undergoing weaning from mechanical ventilation using the coherence between heart rate variability and respiratory flow signal.

Weaning from mechanical ventilation is still one of the most challenging problems in intensive care. Unnecessary delays in discontinuation and weaning trials that are undertaken too early are both undesirable. This study investigated the contribution of spectral signals of heart rate variability (HRV) and respiratory flow, and their coherence to classifying patients on weaning process from mechanical ventilation. A total of 121 candidates for weaning, undergoing spontaneous breathing tests, were analyzed: 73 were successfully weaned (GSucc), 33 failed to maintain spontaneous breathing so were reconnected (GFail), and 15 were extubated after the test but reintubated within 48 h (GRein). The power spectral density and magnitude squared coherence (MSC) of HRV and respiratory flow signals were estimated. Dimensionality reduction was performed using principal component analysis (PCA) and sequential floating feature selection. The patients were classified using a fuzzy K-nearest neighbour method. PCA of the MSC gave the best classification with the highest accuracy of 92% classifying GSucc versus GFail patients, and 86% classifying GSucc versus GRein patients. PCA of the respiratory flow signal gave the best classification between GFail and GRein patients (79% accuracy). These classifiers showed a good balance between sensitivity and specificity. Besides, the spectral coherence between HRV and the respiratory flow signal, in patients on weaning trial process, can contribute to the extubation decision.

Carbohydrate quality, weight change and incident obesity in a Mediterranean cohort: the SUN Project.

BACKGROUND/OBJECTIVES: To evaluate the association between the carbohydrate quality index (CQI) and weight change or incident overweight/obesity (body mass index ⩾25 kg/m(2)) in the 'Seguimiento Universidad de Navarra (SUN)' cohort. SUBJECTS/METHODS: A total of 8741 participants who were initially free of overweight/obesity were followed up for a median of 7.9 years. We evaluated at baseline the CQI according to four criteria: dietary fiber intake, glycemic index, whole grains/total grains ratio and solid carbohydrates/total carbohydrates ratio. Subjects were classified into quintiles according to CQI. Weight was recorded at baseline and updated every 2 years during follow-up. RESULTS: Increasing CQI of diet was not significantly associated with lower weight gain, although participants in the highest quintile had the lowest average crude weight gain (+211 g/year). We observed 1862 incident cases of overweight/obesity during follow-up. CQI was significantly associated (P for trend 0.006) with a lower risk of overweight/obesity; adjusted odds ratio for the fourth and fifth quintiles were 0.81 (95% confidence interval (CI): 0.66-0.99) and 0.74 (95% CI: 0.60-0.92), respectively. CONCLUSIONS: In this Mediterranean cohort, CQI showed a significant inverse association with the incidence of overweight/obesity, which highlights that carbohydrate intake guidelines related to obesity prevention should be focused on improving the CQI of the diet.

A flavonoid-rich diet increases nitric oxide production in rat aorta.

1. Red wine intake is associated with a low risk of cardiovascular disease. This effect has been partly attributed to the action of polyphenolic compounds, which decrease the oxidation of plasma low density lipoproteins. Moreover, nitric oxide ((*)NO) is a vasodilator and polyphenolic compounds induce endothelium-dependent vasorelaxation in vitro. 2. Here we studied whether a diet rich in dealcoholated red wine (DRW) increases acetylcholine-induced vasorelaxation and whether ingestion of DRW-, quercetin- or catechin-rich diets modifies the (*)NO-cyclic guanosine-3',5'-monophosphate (cyclic GMP) pathway and superoxide anion (O2(.-)) release in aorta in a resting state in rats fed semi-purified diets containing either 35% (v w(-1)) DRW, 0.3% (w w(-1)) quercetin or 0.3% (w w(-1)) catechin for 10 days. 3. (*)NO-mediated vasorelaxation induced by acetylcholine was greater in rats fed the DRW-rich diet than in those that received the control diet. 4. Expression of endothelial (*)NO synthase (eNOS) was similar in the four dietary groups. The aortic rings of rats fed either the DRW-, quercetin-, or catechin-rich diets showed higher NOS activity, (*)NO production and cyclic GMP content than those of rats fed the control diet. No changes were observed in O2(.-) production. 5. In summary, diets rich in either DRW, quercetin or catechin induced endothelium-dependent vasorelaxation in rat aorta in a resting state through the enhancement of (*)NO production, without modifying O2(.-) generation, thus the bioavailability of (*)NO was increased. The increase in the (*)NO-cyclic GMP pathway explains the beneficial effect of flavonoids at vascular level.