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Anthocyanins, a flavonoid class of polyphenols, are water soluble dark colored natural pigments found in fruits and vegetables. Owing to their wide distribution in plant materials, dietary consumption of anthocyanins is high compared to other flavonoids. Anthocyanins, due to their multifaceted medicinal properties are the active components in many herbal folk medicines. As in vitro and in vivo results, animal models, and clinical trials in various cell lines suggest, anthocyanins possess antioxidant, antidiabetic, antihyperlipidemic, anti-inflammatory, anticarcinogenic, antiulcer, and preventive activities against cardiovascular diseases. Additionally, anthocyanins exhibit chemotherapeutic, cardioprotective, hepatoprotective, and neuroprotective activities. In the diet, anthocyanins are absorbed in the stomach and intestinal cells and rapidly detected in the plasma. These promising properties of anthocyanins may well provide health benefits against chronic diseases., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Brain inflammation, characterized by increased microglia and astrocyte activation, increases during aging and is a key feature of neurodegenerative diseases, such as Alzheimer's disease (AD). In AD, neuronal death and synaptic impairment, induced by amyloid-β (Aβ) peptide, are at least in part mediated by microglia and astrocyte activation. Glial activation results in the sustained production of proinflammatory cytokines and reactive oxygen species, giving rise to a chronic inflammatory process. Astrocytes are the most abundant glial cells in the central nervous system and are involved in the neuroinflammation. Astrocytes can be activated by numerous factors, including free saturated fatty acids, pathogens, lipopolysaccharide, and oxidative stress. Activation of astrocytes produces inflammatory cytokines and the enzyme cyclooxygenase-2, enhancing the production of Aβ. Furthermore, the role of the receptor for advanced glycation end products/nuclear factor-κB (NF-κB) axis in neuroinflammation is in line with the nonenzymatic glycosylation theory of aging, suggesting a central role of the advanced glycation end products in the age-related cognitive and a possible role of nutraceuticals in the prevention of neuroinflammation and AD. However, modulation of P-glycoprotein, rather than antioxidant and anti-inflammatory effects, could be the major mechanism of polyphenolic compounds, including flavonoids. Curcumin, resvertrol, piperine, and other polyphenols have been explored as novel therapeutic and preventive agents for AD. The aim of this review is to critically analyze and discuss the mechanisms involved in neuroinflammation and the possible role of nutraceuticals in the prevention and therapy of AD by targeting neuroinflammation., (© 2017 Elsevier Inc. All rights reserved.)

Oxidative stress is the result of the imbalance between reactive oxygen species (ROS) formation and enzymatic and nonenzymatic antioxidants. Biomarkers of oxidative stress are relevant in the evaluation of the disease status and of the health-enhancing effects of antioxidants. We aim to discuss the major methodological bias of methods used for the evaluation of oxidative stress in humans. There is a lack of consensus concerning the validation, standardization, and reproducibility of methods for the measurement of the following: (1) ROS in leukocytes and platelets by flow cytometry, (2) markers based on ROS-induced modifications of lipids, DNA, and proteins, (3) enzymatic players of redox status, and (4) total antioxidant capacity of human body fluids. It has been suggested that the bias of each method could be overcome by using indexes of oxidative stress that include more than one marker. However, the choice of the markers considered in the global index should be dictated by the aim of the study and its design, as well as by the clinical relevance in the selected subjects. In conclusion, the clinical significance of biomarkers of oxidative stress in humans must come from a critical analysis of the markers that should give an overall index of redox status in particular conditions.

The highly nutritional and ecofriendly Spirulina ( Arthrospira platensis ) has hypolipidemic, hypoglycemic, and antihypertensive properties. Spirulina contains functional compounds, such as phenolics, phycocyanins, and polysaccharides, with antioxidant, anti-inflammatory, and immunostimulating effects. Studies conducted on Spirulina suggest that it is safe in healthy subjects, but attitude to eating probably affects the acceptability of Spirulina containing foods. Although the antioxidant effect of Spirulina is confirmed by the intervention studies, the concerted modulation of antioxidant and inflammatory responses, suggested by in vitro and animal studies, requires more confirmation in humans. Spirulina supplements seem to affect more effectively the innate immunity, promoting the activity of natural killer cells. The effects on cytokines and on lymphocytes' proliferation depend on age, gender, and body weight differences. In this context, ageing and obesity are both associated with chronic low grade inflammation, immune impairment, and intestinal dysbiosis. Microbial-modulating activities have been reported in vitro, suggesting that the association of Spirulina and probiotics could represent a new strategy to improve the growth of beneficial intestinal microbiota. Although Spirulina might represent a functional food with potential beneficial effects on human health, the human interventions used only supplements. Therefore, the effect of food containing Spirulina should be evaluated in the future., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

Andropause or male menopause is defined as androgen decline and onset of hypogonadism in the aging male. Testosterone deficiency in adult male is associated with diabetes mellitus, coronary artery disease, and heart failure. Type 2 diabetic male patients aged above 30 years showed low testosterone levels which is common in diabetic men and had symptoms of hypogonadism. Male sexual dysfunction among diabetic patients can include disorders of libido, ejaculatory problems, and erectile dysfunctions are common among people with diabetes, particularly in older men who had diabetes for years. Older diabetics tend to have both impaired insulin release as well as insulin resistance. There is growing evidence indicating the pathophysiological connections among the mechanisms of oxidative damage by disruption of the oxidative balance, increased levels of enzymatic glycation products in testicular region and glucose transporters, obesity and proinflammatory cytokines in male infertile patients with diabetes. Epidemiological studies suggest that many clinical findings in diabetics are linked to low testosterone levels. This article reviews pathophysiological mechanisms, observational studies, and clinical implications of testosterone therapy in type 2 diabetes mellitus., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Epigallocatechin-3-gallate (EGCG), the main flavonoid of green tea (GT), could play an active role in the prevention of oxidative-stress-related diseases, such as hematologic malignancies. Some effects of EGCG are not imputable to antioxidant activity, but involve modulation of antioxidant enzymes and uric acid (UA) levels. The latter is the major factor responsible of the plasma non-enzymatic antioxidant capacity (NEAC). However, hyperuricemia is a frequent clinical feature caused by tumor lysis syndrome or cyclosporine side effects, both before and after bone marrow transplantation (BMT). Besides this, food-drug interactions could be associated with GT consumption and could have clinical implications. The molecular mechanisms involved in the redox and drug metabolizing/transporting pathways were discussed, with particular reference to the potential role of GT and EGCG in BMT. Moreover, on reviewing data on NEAC, isoprostanes, uric acid, and various enzymes from human studies on GT, its extract, or EGCG, an increase in NEAC, without effect on isoprostanes, and contrasting results on UA and enzymes were observed. Currently, few and contrasting available evidences suggest caution for GT consumption in BMT patients and more studies are needed to better understand the potential impact of EGCG on oxidative stress and metabolizing/transporting systems.

Autophagy dysfunction is a common feature in neurodegenerative disorders characterized by accumulation of toxic protein aggregates. Increasing evidence has demonstrated that activation of TFEB (transcription factor EB), a master regulator of autophagy and lysosomal biogenesis, can ameliorate neurotoxicity and rescue neurodegeneration in animal models. Currently known TFEB activators are mainly inhibitors of MTOR (mechanistic target of rapamycin [serine/threonine kinase]), which, as a master regulator of cell growth and metabolism, is involved in a wide range of biological functions. Thus, the identification of TFEB modulators acting without inhibiting the MTOR pathway would be preferred and probably less deleterious to cells. In this study, a synthesized curcumin derivative termed C1 is identified as a novel MTOR-independent activator of TFEB. Compound C1 specifically binds to TFEB at the N terminus and promotes TFEB nuclear translocation without inhibiting MTOR activity. By activating TFEB, C1 enhances autophagy and lysosome biogenesis in vitro and in vivo. Collectively, compound C1 is an orally effective activator of TFEB and is a potential therapeutic agent for the treatment of neurodegenerative diseases.

Systemic biomarkers of oxidative stress can be relevant for assessment of psoriasis severity, for prediction of the outcome of therapy and of the development of comorbidities. In this review we aimed to evaluate the relationship between plasma total antioxidant capacity (TAC) and peroxidation biomarkers, as well as their association with dyslipidemia and systemic inflammation in psoriasis. The review of 59 case-control comparisons (from 41 studies) and 17 interventions (from 13 studies) suggests that peroxidation markers are more sensitive than TAC in the evaluation of oxidative stress in psoriasis. Although few studies investigated the effect of treatment on oxidative stress, it seems that biological drugs could be the better choice in the treatment of psoriasis. However, considering the limitations of TAC and plasma peroxidation markers, this review suggests that new methods should be developed in order to evaluate systemic oxidative stress in psoriasis.

Total Antioxidant Capacity (TAC) is a biomarker often used in order to investigate oxidative stress in many pathological conditions. Saliva and urine can be collected noninvasively and represent attractive diagnostic fluids for detecting biomarkers of various pathological conditions. The reviewed case-control and intervention studies that measured salivary or urinary TAC revealed that diseases, antioxidant foods, or supplements and age, gender, and lifestyle factors influenced salivary or urinary TAC. Salivary and urinary TAC were particularly affected by oral or renal status, respectively, as well as by infection; therefore these factors must be taken into account in both case-control and intervention studies. Furthermore, some considerations on sample collection and normalization strategies could be made. In particular, unstimulated saliva could be the better approach to measure salivary TAC, whereas 24 h or spontaneous urine collection should be chosen on the basis of the study outcome and of the creatinine clearance. Finally, the uric acid-independent TAC could be the better approach to evaluate red-ox status of body, in particular after nutritional interventions and in diseases associated with hyperuricaemia.

Despite tea increased plasma nonenzymatic antioxidant capacity, the European Food Safety Administration (EFSA) denied claims related to tea and its protection from oxidative damage. Furthermore, the Supplement Information Expert Committee (DSI EC) expressed some doubts on the safety of green tea extract (GTE). We performed a pilot study in order to evaluate the effect of a single dose of two capsules of a GTE supplement (200 mg × 2) on the peroxidation of leukocytes index ratio (PLIR) in relation to uric acid (UA) and ferric reducing antioxidant potential (FRAP), as well as the sample size to reach statistical significance. GTE induced a prooxidant effect on leukocytes, whereas FRAP did not change, in agreement with the EFSA and the DSI EC conclusions. Besides, our results confirm the primary role of UA in the antioxidant defences. The ratio based calculation of the PLIR reduced the sample size to reach statistical significance, compared to the resistance to an exogenous oxidative stress and to the functional capacity of oxidative burst. Therefore, PLIR could be a sensitive marker of redox status., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

For the first time, we investigated the relationship between postprandial dysmetabolism and the Peroxidation of Leukocytes Index Ratio (PLIR), a test that measures the resistance of leukocytes to exogenous oxidative stress and their functional capacity of oxidative burst upon activation. Following a blind, placebo controlled, randomized, crossover design, ten healthy subjects ingested, in two different occasions, a high fat and high carbohydrates meal with Snello cookie (HFHCM-S) or with control cookies (HFHCM-C). Snello cookie, a functional food covered by dark chocolate and containing glucomannan, inulin, fructooligosaccharides, and Bacillus coagulans strain GanedenBC30, significantly improved postprandial metabolic stress (insulin, glucose, and triglycerides) and reduced the postprandial increase of uric acid. HFHCM-S improved PLIR of lymphocytes, but not of monocytes and granulocytes. Both meals increased granulocytes' count and reduced the lipoperoxidation induced by both exogenous free radicals and reactive oxygen species (ROS) produced by oxidative burst. Our results suggest that the healthy status of the subjects could be a limitation of this pilot study for PLIR evaluation on cells that produce ROS by oxidative burst. In conclusion, the relationship between PLIR and postprandial dysmetabolism requires further investigations.

Obesity is associated with many pathological conditions. Tumor Necrosis Factor-α (TNF-α) is one of the key mediators of inflammation involved in the obesity-related insulin resistance development. We aim to review the human evidence useful to clarify the relationship between inflammation and body weight, with particular reference to TNF-α. Genetic polymorphisms and epigenetic factors, such as diet, could affect TNF-α activity. TNF-α is associated with obesity, but also with anorexia and cachexia. Despite the role of TNF-α in obesity-related diseases, anti-TNF-α antibody therapy is associated with an increase in adiposity. In conclusion the reviewed results suggest that inflammation is more likely a consequence rather than a cause of obesity., (Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

The health benefits of plant food-based diets could be related to both integrated antioxidant and antiinflammatory mechanisms exerted by a wide array of phytochemicals present in fruit, vegetables, herbs and spices. Therefore, there is mounting interest in identifying foods, food extracts and phytochemical formulations from plant sources which are able to efficiently modulate oxidative and inflammatory stress to prevent diet-related diseases. This paper reviews available evidence about the effect of supplementation with selected fruits, vegetables, herbs, spices and their extracts or galenic formulation on combined markers of redox and inflammatory status in humans., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: Transcription factors (TFs) act downstream of the major signalling pathways functioning as master regulators of cell fate. Their activity is tightly regulated at the transcriptional, post-transcriptional and post-translational level. Proteins modifying TF activity are not easily identified by experimental high-throughput methods., Results: We developed a computational strategy, called Differential Multi-Information (DMI), to infer post-translational modulators of a transcription factor from a compendium of gene expression profiles (GEPs). DMI is built on the hypothesis that the modulator of a TF (i.e. kinase/phosphatases), when expressed in the cell, will cause the TF target genes to be co-expressed. On the contrary, when the modulator is not expressed, the TF will be inactive resulting in a loss of co-regulation across its target genes. DMI detects the occurrence of changes in target gene co-regulation for each candidate modulator, using a measure called Multi-Information. We validated the DMI approach on a compendium of 5,372 GEPs showing its predictive ability in correctly identifying kinases regulating the activity of 14 different transcription factors., Conclusions: DMI can be used in combination with experimental approaches as high-throughput screening to efficiently improve both pathway and target discovery. An on-line web-tool enabling the user to use DMI to identify post-transcriptional modulators of a transcription factor of interest che be found at http://dmi.tigem.it.

Previous evidence suggested an active role of cocoa products and flavanols in modulating platelet aggregation. However, cocoa flavanols are characterized by a low bioavailability that can deeply affect their presence in biological fluids and raise questions on their biological effect in humans. We performed a systematic search on Medline, Embase, Cochrane and ProQuest databases, until April 2015, on the effect of cocoa products on platelet aggregation in human intervention studies. We identified 13 interventions, of which only five involved repeated administration. Different effects were observed on the basis of the platelet aggregation test used, whereas neither a longer duration of treatment nor a higher dose was associated with a higher inhibition of platelet aggregation. In conclusion, the reviewed results suggest that consumption of cocoa products in bolus administration positively affects platelet aggregation in both healthy subjects and diseased patients. On the other hand, more evidence is required in order to assess the effect of long-term cocoa product ingestion and to identify the bioactive components involved.

Introduction: Red wine consumption is considered to be protective against oxidative stress. Diet strongly influences non-alcoholic fatty liver disease, which is associated with oxidative stress and is considered the hepatic manifestation of the metabolic syndrome., Methods: We reviewed the available evidence that investigated the effects of red wine on the postprandial-induced metabolic and oxidative stress in humans., Results: After red wine consumption with meal, despite the improvement in non-enzymatic antioxidant capacity and lipoperoxidation markers, the influence of confounding factors such as uric acid should be taken into account. Both uric acid and triglycerides increases, induced by ethanol, could cause liver damage. On the other hand, further researches are required in order to understand the meaning of the induction of antioxidant enzymes by red wine and red wine polyphenols in the context of non-alcoholic fatty liver disease., Conclusion: In conclusion, inconsistent and contrasting findings exist regarding the potential benefits of red wine consumption against postprandial stress.

The consumption of flavonoid-rich foods could have beneficial effects on health. However, different classes of flavonoids have different effect on disease risk and the relationship between flavonoid intake and risk of disease appeared to be non-linear. Furthermore, contrarily to vitamins, there are no symptoms of deficiency for flavonoids; therefore, our body treats them like other xenobiotics. Therefore, a therapeutic index should be determined. Despite flavonoids are at the pharma-nutrition interface, drugs and foods are subject to different regulatory frameworks and there is no recommended daily allowance (RDA) for flavonoids. Relatively little is known about the efficacy, safety and underlying mechanisms of these bioactive compounds, especially when taken in concert with drugs. Flavonoids could act both as drugs and pro-drugs with pharmacological and toxicological promiscuity. Due to the low bioavailability, the gastrointestinal tract could be the primary target of flavonoids and metabolites. Different effects have been observed after acute and chronic consumption and bioavailability and bioactivity have high inter-individual variability. Furthermore, the difficulties in the design and in the interpretation of human intervention studies make difficult the establishment of a therapeutic index for flavonoids. Probably the concept of 'personalized nutrition' previously proposed could be the better approach. However, despite more studies are needed in order to establish a therapeutic index for each flavonoid subclasses, at the moment RDA of total flavonoids could be between 250-400 mg/d, respecting the seasonality of food sources., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

The view of the lysosome as the terminal end of cellular catabolic pathways has been challenged by recent studies showing a central role of this organelle in the control of cell function. Here we show that a lysosomal Ca2+ signalling mechanism controls the activities of the phosphatase calcineurin and of its substrate ​TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy. Lysosomal Ca2+ release through ​mucolipin 1 (​MCOLN1) activates calcineurin, which binds and dephosphorylates ​TFEB, thus promoting its nuclear translocation. Genetic and pharmacological inhibition of calcineurin suppressed ​TFEB activity during starvation and physical exercise, while calcineurin overexpression and constitutive activation had the opposite effect. Induction of autophagy and lysosomal biogenesis through ​TFEB required ​MCOLN1-mediated calcineurin activation. These data link lysosomal calcium signalling to both calcineurin regulation and autophagy induction and identify the lysosome as a hub for the signalling pathways that regulate cellular homeostasis.

Statins and dietary modifications are the cornerstone of hypercholesterolemia management. Although it is well known that possible adverse effect of statins can occur due to drug-drug interactions, food-drug interactions are a commonly overlooked aspect. In particular, flavonoids could interfere with statins' bioavailability through different mechanisms, such as competition with cytochrome P450 (CYP) enzymes, esterases, uridine diphosphate glucuronosyltransferases and transporters (P-glycoprotein, multi-drug resistance-associated proteins, organic anion transporting polypeptides, breast cancer-resistance protein and monocarboxylate transporters). Transporters are characterized by low substrate specificity and flavonoid- rich foods could interfere with the bioavailability of all statins at this level. On the other hand, in addition to being substrates of drug metabolism/ transport systems, flavonoids are also able to modulate gene expression of enzymes and transporters. Therefore, long-term transcriptional induction may increase the clearance of statins, despite flavonoids act as competitive inhibitors after bolus consumption. In humans, major interactions were observed between grapefruit juice and statins that are substrates of P-glycoprotein/CYP3A, but other fruit juices also affect the bioavailability of statins that are not metabolised by CYP. Even if flavonoids could play a role in the prevention of hypercholesterolemia, the question whether there's a helpful or dangerous association between flavonoid-rich foods and statins, due to the interactions between flavonoid-rich foods and statins and the potential associated adverse effects of statins, remain unanswered. Therefore, the anamnesis of patients must include detailed information about their eating habits and the present review suggests monitoring and reporting any possible case of interaction between a prescribed statin and food.

Flavonoids, through a modulation of immune function, have been suggested to be involved in the role played by plant foods in disease prevention. We performed a systematic search in the MEDLINE database to review the effect of flavonoid-rich foods and flavonoids supplements on immune function. A total of 58 studies, were identified as suitable: 41 addressed in vivo proinflammatory cytokines and 15 measured ex vivo markers of immune function. According to our findings and on the basis of single food items, the number of studies in humans is limited and, for galenic supplements, only quercetin has been investigated. More evidences are needed to clarify the role of flavonoids as modulator of immune function in humans.

Delays in processing are frequent because of problems associated with transporting the samples to the laboratory. Thus, we aimed to evaluate the effect of sample storage on the Peroxidation of Leukocytes Index Ratio (PLIR). Differences between PLIR values of lymphocytes (PLIR-L), monocytes (PLIR-M) and granulocytes (PLIR-G) were observed in fresh samples. Sample storage affected the evaluation of PLIR. In particular, PLIR-L was lower in stored samples compared to fresh samples. In conclusion, our results suggest that fresh samples are recommended for assessing the PLIR.

The evaluation of oxidative burst is particularly relevant in many pathological and subclinical conditions. Flow cytometry provides quick and accurate measures of the reactive oxygen species production by leukocytes in most situations. However, spurious results, related to probes' efflux may be observed in several instances. Many factors affect the evaluation of the oxidative burst with fluorescent probes that require intracellular deacetylation and could be substrate of the multidrug resistance proteins (MDR). After discussing the implications of the efflux of fluorophores in the normalization strategies in flow cytometry assays, we have pointed out the possible interference of flavonoids with fluorescet probes' staining and signal. We have also reviewed the results from human intervention studies regarding the evaluation of oxidative burst with these probes. In vitro, at concentrations close to post-ingestion circulating levels, some flavonoids and their metabolites could interfere with probes' staining and fluorescence signal through different mechanisms, such as the inhibition of esterases, the modulation of the MDR-mediate efflux of probe and the inhibition of the oxidation of probe. These effects may explain the contrasting results obtained by human intervention studies. Finally, also inflammatory state or the use of drugs substrate of MDR proteins could affect the evaluation of the oxidative burst with intracellular probes., (© 2014 International Society for Advancement of Cytometry.)

The metabolic syndrome can be prevented by the Mediterranean diet, characterized by fiber, omega-3 polyunsaturated fatty acids and polyphenols. However, the composition of the Mediterranean diet, which can be viewed as a natural multiple supplement, is poorly controlled, and its beneficial effects poorly predictable. The metabolic syndrome is associated with intestinal dysbiosis and the gut microbioma seems to be the main target and player in the interactions occurring between probiotics, prebiotics, omega 3 polyunsaturated fatty acids, and polyphenols. From the reviewed evidence, it is reasonable to manage growth and metabolism of gut microflora with specific prebiotics and polyphenols. Even though the healthy properties of functional foods and nutraceuticals still need to be fully elucidated, available data suggest that well-designed supplements, containing the better ratio of omega-3 polyunsaturated fatty acids and antioxidants, specific probiotic strains, and selected polyphenols and prebiotics, could be useful in metabolic syndrome prevention and treatment.

Intracellular probes used for oxidative burst evaluation could be a substrate of the multidrug resistance proteins (MDR), which may cause misinterpretation of experimental data. We aimed to study the effect of the culture condition and of the MDR-interfering antioxidant quercetin on free radical measurement in serum depleted HCT-8 cells and chick embryo hepatocytes. Serum depletion and/or quercetin affected the traffic of 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and calcein. The comparative results obtained with 2',7'-dichlorofluorescein (DCF) and with the plasma-membrane probe C11-BODIPY 581/591 [4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid], suggest caution in the use of intracellular probes to evaluate oxidative stress in vitro., (Copyright © 2014 Elsevier B.V. All rights reserved.)

High-fat meals (HFM) induce metabolic stress, leading to the activation of protective mechanisms, including inflammation and endogenous antioxidant defences. In the present study, we investigated the effects of antioxidant-rich fruit juice drinks on the endogenous antioxidant response induced by HFM. In a double-blind, cross-over design (10 d washout), fourteen overweight volunteers were randomly assigned to one of the following interventions: HFM+500 ml placebo beverage (HFM-PB, free from fruit); HFM+500 ml antioxidant beverage 1 (HFM-AB1; apple, grape, blueberry and pomegranate juices and grape skin, grape seed and green tea extracts); HFM+500 ml antioxidant beverage 2 (HFM-AB2; pineapple, black currant and plum juices). HFM-PB consumption increased the plasma levels of thiols (SH) (4 h, P< 0·001) and uric acid (UA) (2 h, P< 0·01) and total radical-trapping antioxidant parameter (TRAP) (4 h, P< 0·01). Following the consumption of drinks, UA production was significantly reduced with respect to placebo beverage consumption 8 h after HFM-AB2 consumption (P< 0·05). SH levels were reduced 0·5 (P< 0·05), 1 (P< 0·05) and 2 h (P< 0·01) after HFM-AB1 consumption and 2, 4 and 8 h (P< 0·05) after HFM-AB2 consumption. Plasma TRAP (2 h, P< 0·001) and urinary ferric reducing antioxidant power (0-8 h, P< 0·01) were increased by HFM-AB1 consumption, the drink with the highest in vitro antioxidant capacity, but not by HFM-AB2 consumption. In urine, UA levels were significantly increased from basal levels after the consumption of HFM-PB and HFM-AB2. However, neither of the beverages increased the urinary excretion of UA with respect to the placebo beverage. In conclusion, the increase in UA and SH levels induced by HFM as part of an endogenous antioxidant response to postprandial stress can be prevented by the concomitant ingestion of antioxidant-rich fruit juice drinks.

It has been suggested that some fruit-based drinks (FBD) may delay the onset of postprandial stress, which is involved in the pathogenesis of many diseases. The majority of the studies, which have investigated the effects of FBD on postprandial stress, involved a placebo that was a drink with the same content in sugars or carbohydrates of the FBD, but without the bioactive antioxidant compounds. These studies were aimed more at evaluating the effect of the antioxidants rather than the effect of the FBD as a whole. Only 4 studies compared the effect of FBD with water as control and did not support the hypothesis that FBD could inhibit postprandial dysmetabolism, as well as the studies that compared the effect of orange juice and cola. Overall, the results suggest a complex relationship between postprandial dysmetabolism, inflammation, and oxidative stress. Furthermore, the inflammatory and oxidative stress markers need further analytical validation and normal ranges should be established in order to reach a firm conclusion. Finally, caution should be taken in the interpretation of the effect of FBD in postprandial studies and the reviewed results suggest that dietary recommendations should aim to limit rather than increase sugar-sweetened beverages consumption.

Postprandial stress induced by acute consumption of meals with a high fat content results in an increase of markers of cardiometabolic risk. Repeated acute dietary stress may induce a persistent low-grade inflammation, playing a role in the pathogenesis of functional gut diseases. This may cause an impairment of the complex immune response of the gastrointestinal mucosa, which results in a breakdown of oral tolerance. We investigated the effect of ingestion of a fruit-juice drink (FJD) composed by multiple fruit juice and extracts, green tea extracts and vitamin C on postprandial stress induced by a High Fat Meal (HFM) in healthy overweight subjects. Following a double blind, placebo controlled, cross-over design, 15 healthy overweight subjects were randomized to a HFM providing 1334 Kcal (55% fat, 30% carbohydrates and 15% proteins) in combination with 500 mL of a placebo drink (HFM-P) or a fruit-juice drink (HFM-FJD). Ingestion of HFM-P led to an increase in circulating levels of cholesterol, triglycerides, glucose, insulin, TNF-α and IL-6. Ingestion of HFM-FJD significantly reduced plasma levels of cholesterol and triglycerides, decreasing inflammatory response mediated by TNF-α and IL-6. Ingestion of a fruit-juice drink reduce markers of postprandial stress induced by a HFM.

Background: High-fat meals may lead to hypotension, oxidative stress and increases of lipopolysaccharide (LPS). Contrasting results have been reported after treatment of isolated tissues with hydrogen peroxide and LPS, whereas the effects of peroxyl radicals, involved in the propagation reaction of lipoperoxidation, have not been investigated previously., Aim: In the present study, we aimed to evaluate the effects of peroxyl radicals on the contractile responses in isolated rabbit aorta and guinea pig atria., Methods and Results: We treated isolated guinea pig atria, rabbit aorta strips and rings with 2,2'-Azobis (2- amidinopropane) dihydrochloride (AAPH). AAPH did not affect isoprenaline-induced contraction in guinea pig atria, whilst it dose-dependently reduced the contractile responses induced in rabbit aorta strips by cumulative doses of adrenaline (ADR) and induced an endothelium-independent relaxation of noradrenaline (NA)-contracted aorta rings. The effects of KCl-induced and BaCl2-induced contractions were small. Furthermore, alkalinization with NH4Cl of NA-contracted aorta rings significantly reduced the vasodilatatory activity of AAPH., Conclusion: The present study suggests that peroxyl radicals induce acute functional alterations on vascular contraction through intracellular pH regulation. This finding could be related to the documented after meal increase in oxidative burst and endotoxin and the related hypotension.

Epidemiological or in vitro evidence suggests a potential role for flavonoids as anti-inflammatory agents; we investigated the effect of flavonoids-rich foods or supplements on tumor necrosis factor- alpha (TNF-α) and interleukin-6 (IL-6) in long-term placebo-controlled human intervention trials. From 110 human intervention studies selected (MEDLINE, EMBASE, CHORANE, and FSTA databases), 32 long-term placebo-controlled trials were suitable for meta-analysis. After sensitivity analysis, seven studies imputed of bias were excluded and 25 studies were analyzed (TNF-α, n = 2404; IL-6, n = 2174). Levels of TNF-α decreased after flavonoid consumption in the fixed model only (mean difference (MD) (95% CI): -0.098 (-0.188, -0.009), p = 0.032), but metaregression results showed that neither higher dose, nor a longer duration of intervention were associated with a greater effect size. Subgroup analysis did not reveal any significant effect for quercetin and soy, but other sources (red wine, pomegranate, and tea extracts) showed a significant effect size both in fixed (MD (95% CI): TNF-α -0.449 (-0.619, -0.280), p < 0.001; IL-6 -0.346 (-0.612, -0.079), p = 0.011) and random (MD (95% CI): TNF-α -0.783 (-1.476, -0.090), p = 0.027; IL-6, -0.556 (-1.062, -0.050), p = 0.031) effect models. High-quality placebo-controlled trials are needed in order to identify flavonoids as the active ingredients., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and Aim: A complex relationship between immune system and metabolic pathway exists and can induce oxidative stress. The objective of this study was to design a new methodology allowing the measurement of oxidative status of leukocytes., Methods and Results: We developed a flow cytometry technique, based on C11-BODIPY 581/591 staining, to evaluate peroxidation in leukocytes. We defined the Peroxidation of Leukocytes Index Ratio (PLIR) as the ratio between the damage after AAPH-induced and PMA-induced peroxidation, using Trolox as standard antioxidant. Sensitivity of the method was assessed by correlating results with plasma antioxidant capacity (TRAP and FRAP), levels of endogenous antioxidants (uric acid and sulfhydryls) and markers of metabolic status (cholesterol, triglycerides, glucose and insulin). PLIR measures the ratio between the resistance to exogenous and endogenous ROS injury, independently from baseline level of oxidation, which was directly correlated with plasma cholesterol on lymphocytes (0.738, p=0.029), monocytes (0.691, p=0.047) and neutrophils (0.690, p=0.047). PLIR of lymphocytes was inversely correlated with uric acid (-0.810, p=0.009) and FRAP (-0.738, p=0.029) levels. On the other hand, PLIR of monocytes was directly correlated with the total scavenger antioxidant capacity attributable to nutritional antioxidants (0.738, p=0.029), calculated as the difference between TRAP and the contribution of uric acid and sulfhydryls to its value., Conclusions: This study reports a feasible and reproducible new flow cytometry assay for assessing the leukocytes redox status. PLIR discriminates between reducing and scavenger activities and is able to appreciate the potentially dangerous effect of uric acid on innate immune response., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Purpose: Postprandial metabolic stress as a consequence of ingestion of high-energy meals is recognized as an important risk factor for cardiovascular disease. The objective of this study was to evaluate the inflammatory and antioxidant response of the body to the acute ingestion of a high-fat meal (HFM)., Methods: Fifteen healthy overweight subjects were recruited for the study. After HFM consumption, plasma glucose, insulin, uric acid (UA), triglycerides (TG), total cholesterol (TC), thiols (SH), inflammatory cytokines (IL-6 and TNF-α) and dietary antioxidants were measured at 0, 0, 5, 1, 2, 4, 6 and 8 h points from ingestion., Results: The ingestion of HFM induced significant increases in both TG and TC, with peaks at 4 h (p < 0.001) and 8 h (p < 0.01), respectively. IL-6 and TNF-α significantly increased postprandially, reaching maximum concentrations 8 h after meal consumption (p < 0.001). Whereas plasma concentrations of vitamins and carotenoids were not changed by HFM, SH and UA increased, peaking 2-4 h postingestion (p < 0.001 and 0.01, respectively). Increments of SH and UA were positively correlated with AUC for TG (Pearson coefficient 0.888, p < 0.001 and 0.923, p < 0.001, respectively)., Conclusions: Present results indicate that as a consequence of an excess of dietary fat, the body responds through an inflammatory reaction, which is accompanied by an increment of endogenous antioxidant defenses, mediated by UA and SH, but not by vitamins C and E and carotenoids. Although further studies are needed, results of the current investigation represent novel findings on endogenous strategies of redox defense from fat overloads.

Background: Inherited retinal dystrophies, including Retinitis Pigmentosa and Leber Congenital Amaurosis among others, are a group of genetically heterogeneous disorders that lead to variable degrees of visual deficits. They can be caused by mutations in over 100 genes and there is evidence for the presence of as yet unidentified genes in a significant proportion of patients. We aimed at identifying a novel gene for an autosomal recessive form of early onset severe retinal dystrophy in a patient carrying no previously described mutations in known genes., Methods: An integrated strategy including homozygosity mapping and whole exome sequencing was used to identify the responsible mutation. Functional tests were performed in the medaka fish (Oryzias latipes) model organism to gain further insight into the pathogenic role of the ADAMTS18 gene in eye and central nervous system (CNS) dysfunction., Results: This study identified, in the analyzed patient, a homozygous missense mutation in the ADAMTS18 gene, which was recently linked to Knobloch syndrome, a rare developmental disorder that affects the eye and the occipital skull. In vivo gene knockdown performed in medaka fish confirmed both that the mutation has a pathogenic role and that the inactivation of this gene has a deleterious effect on photoreceptor cell function., Conclusion: This study reveals that mutations in the ADAMTS18 gene can cause a broad phenotypic spectrum of eye disorders and contribute to shed further light on the complexity of retinal diseases.

The involvement of both oxidative stress and hyperlipaemia in atherosclerosis development is well established. Oxidative burst is an innate immune response to infection, the latter being associated also with marked changes in lipid and lipoprotein metabolism, aimed to neutralize endotoxin toxic effects. On the other hand, lipid overload may increase lipopolysaccharide circulating levels and oxidative stress. Whilst these changes may be beneficial from the perspective of host defense, if they become chronic, they likely increase the risk of atherosclerosis. In particular, oxidation of lipoproteins, resulting from an imbalance of the pro- and antioxidant equilibrium, is involved in the pathologic process of atherosclerosis, changing cellular functions. Lipid oxidation, induced by leukocytes derived reactive oxygen species, can amplify foam cell formation through oxidized low density lipoproteins LDL (oxLDL) formation and uptake. The main enzymes, operating during oxidative burst, involved in LDL oxidation are NADPH oxidase and myeloperoxidase. In vitro studies have shown that oxLDL are able to induce many proatherogenic processes, including modulation of oxidative burst. OxLDL may also induce maturation of dendritic cells and regulate the shift from classical (M1) to alternative (M2) macrophage activation and from T helper 1 to T helper 2 response, suggesting that these could act as a bridge between innate and adaptative immunity, both involved in plaque development. Understanding the relationship between oxLDL and leukocyte oxidative burst helps to explain the involvement of innate immune responses in the early phases of atherosclerosis. The present review focuses on this interplay.

We propose a new data analysis approach for reactive oxygen species detection using Dihydrorhodamine 123 in blood monocytes and neutrophils. This approach, based on data transformation using lymphocytes as internal standard, allows to appreciate free radical production by monocytes also without Phorbol 12-myristate 13-acetate (PMA) activation. In addition, this method is sensitive to differences in healthy subjects due to sub-pathological conditions, such as hypercholesterolemia., (Copyright © 2012 Elsevier B.V. All rights reserved.)

An emerging role of IL-17 in the inflammatory response associated with pathogenesis of neurodegeneration has been recently suggested. However, though diet represents a key factor in the modulation of inflammatory processes, evidence is not currently available on the nutritional regulation of IL-17 in humans. In a double blind, randomized, placebo controlled, crossover study, we investigated the effect of High Fat Meal (HFM) on IL-17 circulating levels in presence of a placebo (HFM-P) or with a Fruit Juice Drink (HFM-FJD) composed of pineapple, blackcurrant and plum in fourteen healthy overweight humans. Fasting in the morning subjects ingested a test meal providing 1344 Kcal. Ingestion of HFM-P induced an inflammatory response mediated by TNF-α (p < 0.001), IL-6 (p < 0.001) and IL-17 (p < 0.01). Plasma IL-17 concentration significantly increased at 1 h (+2.6 ± 1.1 pg/ml), remaining high at 4 h (+2.98 ± 1.2 pg/ml), 6 h (+2.38 ± 0.6 pg/ml) and 8 h (+2.8 ± 0.9 pg/ml) (ANOVA for time-course p=0.009). When the HFM was consumed in the presence of the FJD a marked inhibition of IL-17 response to the HFM was observed (ANOVA between treatment p=0.037). We provided, for the first time, evidence on the role of diet in modulating IL-17 production in healthy overweight subjects.

Usher syndrome (USH) is a clinically and genetically heterogeneous disorder characterized by visual and hearing impairments. Clinically, it is subdivided into three subclasses with nine genes identified so far. In the present study, we investigated whether the currently available Next Generation Sequencing (NGS) technologies are already suitable for molecular diagnostics of USH. We analyzed a total of 12 patients, most of which were negative for previously described mutations in known USH genes upon primer extension-based microarray genotyping. We enriched the NGS template either by whole exome capture or by Long-PCR of the known USH genes. The main NGS sequencing platforms were used: SOLiD for whole exome sequencing, Illumina (Genome Analyzer II) and Roche 454 (GS FLX) for the Long-PCR sequencing. Long-PCR targeting was more efficient with up to 94% of USH gene regions displaying an overall coverage higher than 25×, whereas whole exome sequencing yielded a similar coverage for only 50% of those regions. Overall this integrated analysis led to the identification of 11 novel sequence variations in USH genes (2 homozygous and 9 heterozygous) out of 18 detected. However, at least two cases were not genetically solved. Our result highlights the current limitations in the diagnostic use of NGS for USH patients. The limit for whole exome sequencing is linked to the need of a strong coverage and to the correct interpretation of sequence variations with a non obvious, pathogenic role, whereas the targeted approach suffers from the high genetic heterogeneity of USH that may be also caused by the presence of additional causative genes yet to be identified.

Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease., Competing Interests: The authors have declared that no competing interests exist.

Background: MicroRNAs (miRNAs) are key regulators of biological processes. To define miRNA function in the eye, it is essential to determine a high-resolution profile of their spatial and temporal distribution., Results: In this report, we present the first comprehensive survey of miRNA expression in ocular tissues, using both microarray and RNA in situ hybridization (ISH) procedures. We initially determined the expression profiles of miRNAs in the retina, lens, cornea and retinal pigment epithelium of the adult mouse eye by microarray. Each tissue exhibited notably distinct miRNA enrichment patterns and cluster analysis identified groups of miRNAs that showed predominant expression in specific ocular tissues or combinations of them. Next, we performed RNA ISH for over 220 miRNAs, including those showing the highest expression levels by microarray, and generated a high-resolution expression atlas of miRNAs in the developing and adult wild-type mouse eye, which is accessible in the form of a publicly available web database. We found that 122 miRNAs displayed restricted expression domains in the eye at different developmental stages, with the majority of them expressed in one or more cell layers of the neural retina., Conclusions: This analysis revealed miRNAs with differential expression in ocular tissues and provided a detailed atlas of their tissue-specific distribution during development of the murine eye. The combination of the two approaches offers a valuable resource to decipher the contributions of specific miRNAs and miRNA clusters to the development of distinct ocular structures.

5'-Deoxy-5'-methylthioadenosine phosphorylase II from Sulfolobus solfataricus (SsMTAPII) and purine nucleoside phosphorylase from Pyrococcus furiosus (PfPNP) are hyperthermophilic purine nucleoside phosphorylases stabilized by intrasubunit disulfide bonds. In their C-terminus, both enzymes harbour a CXC motif analogous to the CXXC motif present at the active site of eukaryotic protein disulfide isomerase. By monitoring the refolding of SsMTAPII, PfPNP and their mutants lacking the C-terminal cysteine pair after guanidine-induced unfolding, we demonstrated that the CXC motif is required for the folding of these enzymes. Moreover, two synthesized CXC-containing peptides with the same amino acid sequences present in the C-terminal regions of SsMTAPII and PfPNP were found to act as in vitro catalysts of oxidative folding. These small peptides are involved in the folding of partially refolded SsMTAPII, PfPNP and their CXC-lacking mutants, with a concomitant recovery of their catalytic activity, thus indicating that the CXC motif is necessary to obtain complete reversibility from the unfolded state of the two proteins. The two CXC-containing peptides are also able to reactivate scrambled RNaseA. The data obtained in the present study represent the first example of how the CXC motif improves both stability and folding in hyperthermophilic proteins with disulfide bonds.

We report the biochemical and structural characterization of the purine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus (SsIAG-NH). SsIAG-NH is a homodimer of 70kDa specific for adenosine, guanosine and inosine. SsIAG-NH is highly thermophilic and is characterized by extreme thermodynamic stability (T(m), 107 degrees C), kinetic stability and remarkable resistance to guanidinium chloride-induced unfolding. A disulfide bond that, on the basis of SDS-PAGE is positioned intersubunits, plays an important role in thermal stability. SsIAG-NH shares 43% sequence identity with the homologous pyrimidine-specific nucleoside hydrolase from S. solfataricus (SsCU-NH). The comparative sequence alignment of SsIAG-NH, SsCU-NH, purine non-specific nucleoside hydrolase from Crithidia fasciculata and purine-specific nucleoside hydrolase from Trypanosoma vivax shows that, only few changes in the base pocket are responsible for different substrate specificity of two S. solfataricus enzymes. The structure of SsIAG-NH predicted by homology modeling allows us to infer the role of specific residues in substrate specificity and thermostability.

We report on a female patient with severe infantile spasms, profound global developmental arrest, hypsarrhythmia and severe mental retardation, associated with a de novo apparently balanced X;autosome translocation. Her neurological phenotype resembles that of X-linked infantile spasms (ISSX). Molecular study showed that the translocation disrupts a transcript involved in GTPases signalling, IQSEC2, mapped to the Xp11.22 region. Several genes involved in intracellular signalling pathways via Ras-homologous small GTPase have been implicated in X-linked neurological disorders. Expression studies revealed that the murine homolog of this transcript, Iqsec2, is highly expressed in the nervous system from the early stages of development. These data suggest that IQSEC2 could be considered a candidate gene for X-linked neurological disorders.

Embryonic stem (ES) cells are pluripotent cell lines with the capacity of self-renewal and the ability to differentiate into specific cell types. We performed the first genome-wide analysis of the mouse ES cell transcriptome using approximately 250,000 gene trap sequence tags deposited in public databases. We unveiled >8000 novel transcripts, mostly non-coding, and >1000 novel alternative and often tissue-specific exons of known genes. Experimental verification of the expression of these genes and exons by RT-PCR yielded a 70% validation rate. A novel non-coding transcript within the set studied showed a highly specific pattern of expression by in situ hybridization. Our analysis also shows that the genome presents gene trapping hotspots, which correspond to 383 known and 87 novel genes. These "hypertrapped" genes show minimal overlap with previously published expression profiles of ES cells; however, we prove by real-time PCR that they are highly expressed in this cell type, thus potentially contributing to the phenotype of ES cells. Although gene trapping was initially devised as an insertional mutagenesis technique, our study demonstrates its impact on the discovery of a substantial and unprecedented portion of the transcriptome.

Helicobacter pylori (Hp) infection is associated with gastric inflammation and ulceration. The pathways of tissue damage in Hp-infected subjects are complex, but evidence indicates that T cell-derived cytokines enhance the synthesis of matrix metalloproteinases (MMP) that contribute to mucosal ulceration and epithelial damage. In this study, we have examined the role of the T cell cytokine IL-21 in Hp-infected gastric mucosa and evaluated whether IL-21 regulates MMP production by gastric epithelial cells. We show that IL-21 is constitutively expressed in gastric mucosa and is more abundant in biopsy specimens and purified mucosal CD3(+) T cells from Hp-infected patients compared with normal patients and disease controls. We also demonstrate that IL-21R is expressed by primary gastric epithelial cells, as well as by the gastric epithelial cell lines AGS and MKN28. Consistently, AGS cells respond to IL-21 by increasing production of MMP-2 and MMP-9, but not MMP-1, MMP-3, MMP-7, or tissue inhibitors of MMP. Analysis of signaling pathways leading to MMP production reveals that IL-21 enhances NF-kappaB but not MAPK activation, and inhibition of NF-kappaB activation reduces IL-21-induced MMP-2 and MMP-9 production. Finally, we show that treatment of Hp-infected gastric explants with anti-IL-21 reduces epithelial cell-derived MMP-2 and MMP-9 production. These data indicate that IL-21 is overexpressed in Hp-infected gastric mucosa where it could contribute to increased epithelial gelatinase production.

Recent studies have shown that probiotics are beneficial in T-cell-mediated inflammatory diseases. The molecular mechanism by which probiotics work remains elusive, but accumulating evidence indicates that probiotics can modulate immune cell responses. Since T cells express receptors for bacterial products or components, we examined whether different strains of lactobacilli directly regulate the functions of human T cells. CD4(+) T cells were isolated from blood and intestinal lamina propria (LP) of normal individuals and patients with inflammatory bowel disease (IBD). Mononuclear cells were also isolated from Peyer's patches. Cells were activated with anti-CD3/CD2/CD28 in the presence or absence of Lactobacillus paracasei subsp. paracasei B21060, L. paracasei subsp. paracasei F19, or L. casei subsp. casei DG. Cell proliferation and death, Foxp3, intracellular pH, and cytokine production were evaluated by flow cytometry. We showed that L. paracasei subsp. paracasei B21060 but neither L. paracasei subsp. paracasei F19 nor L. casei subsp. casei DG inhibited blood CD4(+) T-cell growth. This effect was associated with no change in cell survival, expression of Foxp3, or production of gamma interferon, interleukin-4 (IL-4), IL-5, and IL-10. L. paracasei subsp. paracasei B21060-mediated blockade of CD4(+) T-cell proliferation required a viable bacterium and was associated with decreased MCT-1 expression and low intracellular pH. L. paracasei subsp. paracasei B21060 also inhibited the growth of Peyer's patch mononuclear cells, normal lymphocytes, and IBD CD4(+) LP lymphocytes without affecting cytokine production. The data show that L. paracasei subsp. paracasei B21060 blocks T-cell growth, thus suggesting a mechanism by which these probiotics could interfere with T-cell-driven immune responses.

Purpose: MicroRNAs (miRNAs) are a class of small, endogenous RNAs that negatively regulate gene expression post-transcriptionally by binding to target sites in the 3' untranslated region (UTR) of messenger RNAs. Although they have been found to regulate developmental and physiological processes in several organs and tissues, their role in the eye transcriptome is completely unknown. This study was conducted to gain understanding of their eye-related function in mammals, by looking for miRNAs significantly expressed in the mouse eye by means of high-resolution expression analysis., Methods: The spatiotemporal localization of miRNAs was analyzed in the murine embryonic and postnatal eye by RNA in situ hybridization (ISH) using LNA-modified oligonucleotide probes., Results: Seven miRNAs were expressed in the eye with diverse and partially overlapping patterns, which may reflect their role in controlling cell differentiation of the retina as well as of other ocular structures. Most eye-expressed miRNAs overlap with or are in the near vicinity of transcripts derived predominantly from eye cDNA libraries. We found that these transcripts share very similar cellular distribution with their corresponding miRNAs, suggesting that miRNAs may share common expression regulatory elements with their host genes., Conclusions: The data provide a detailed characterization of expression of eye-enriched miRNAs. Knowledge of the spatiotemporal distribution of miRNAs is an essential step toward the identification of their targets and eventually the elucidation of their biological role in eye development and function.

High expression of IL-21 and/or IL-21R has been described in T cell-mediated inflammatory diseases characterized by defects of counterregulatory mechanisms. CD4(+)CD25(+) regulatory T cells (Treg) are a T cell subset involved in the control of the immune responses. A diminished ability of these cells to inhibit T cell activation has been documented in immune-inflammatory diseases, raising the possibility that inflammatory stimuli can block the regulatory properties of Treg. We therefore examined whether IL-21 controls CD4(+)CD25(+) T cell function. We demonstrate in this study that IL-21 markedly enhances the proliferation of human CD4(+)CD25(-) T cells and counteracts the suppressive activities of CD4(+)CD25(+) T cells on CD4(+)CD25(-) T cells without affecting the percentage of Foxp3(+) cells or survival of Treg. Additionally, CD4(+)CD25(+) T cells induced in the presence of IL-21 maintain the ability to suppress alloresponses. Notably, IL-21 enhances the growth of CD8(+)CD25(-) T cells but does not revert the CD4(+)CD25(+) T cell-mediated suppression of this cell type, indicating that IL-21 makes CD4(+) T cells resistant to suppression rather than inhibiting CD4(+)CD25(+) T cell activity. Finally, we show that IL-2, IL-7, and IL-15, but not IL-21, reverse the anergic phenotype of CD4(+)CD25(+) T cells. Data indicate that IL-21 renders human CD4(+)CD25(-) T cells resistant to Treg-mediated suppression and suggest a novel mechanism by which IL-21 could augment T cell-activated responses in human immune-inflammatory diseases.