In vitro and in vivo correlation of skin and cellular responses to nucleic acid delivery.

Skin, the largest organ in the body, provides a passive physical barrier against infection and contains elements of the innate and adaptive immune systems. Skin consists of various cells, including keratinocytes, fibroblasts, endothelial cells and immune cells. This diversity of cell types could be important to gene therapies because DNA transfection could elicit different responses in different cell types. Previously, we observed the upregulation and activation of cytosolic DNA sensing pathways in several non-tumor and tumor cell types as well in tumors after the electroporation (electrotransfer) of plasmid DNA (pDNA). Based on this research and the innate immunogenicity of skin, we correlated the effects of pDNA electrotransfer to fibroblasts and keratinocytes to mouse skin using reverse transcription real-time PCR (RT-qPCR) and several types of protein quantification. After pDNA electrotransfer, the mRNAs of the putative DNA sensors DEAD (AspGlu-Ala-Asp) box polypeptide 60 (Ddx60), absent in melanoma 2 (Aim2), Z-DNA binding protein 1 (Zbp1), interferon activated gene 202 (Ifi202), and interferon-inducible protein 204 (Ifi204) were upregulated in keratinocytes, while Ddx60, Zbp1 and Ifi204 were upregulated in fibroblasts. Increased levels of the mRNAs and proteins of several cytokines and chemokines were detected and varied based on cell type. Mouse skin experiments in vivo confirmed our in vitro results with increased expression of putative DNA sensor mRNAs and of the mRNAs and proteins of several cytokines and chemokines. Finally, with immunofluorescent staining, we demonstrated that skin keratinocytes, fibroblasts and macrophages contribute to the immune response observed after pDNA electrotransfer.

Physiological role of Kvß2 (AKR6) in murine skeletal muscle growth and regulation.

AIM: Potassium channel accessory subunits (Kvß) play a key role in cardiac electrical activity through ion channel modulation. In this study, we hypothesize that Kvß2 regulates skeletal muscle growth and fibre phenotype via protein-protein interactions. METHODS: Kvß2 knockout mouse model was used for morphometric, immunohistochemical and biochemical analysis to evaluate the role of Kvß2 in skeletal muscle physiology. RESULTS: Deletion of Kvß2 gene in mice (Kvß2 knockout, KO) leads to significant decrease in body weight along with skeletal muscle size. Key hindlimb muscles such as biceps, soleus and gastrocnemius were significantly smaller in size in KO mice compared to that of wild type. Morphometric measurements and histological analysis clearly point that the fibre size is decreased in each of the muscle type in KO compared with wild-type mice. In addition, Kvß2 deletion contributes to fibre-type switching from fast to slow fibre as indicated by more abundant MHCI-expressing fibres in gastrocnemius and soleus muscles, which may underscore the smaller muscle size alongside increase in U3 ubiquitin ligase; NEDD4 expression. Using targeted siRNA knockdown approach, we identified that Kvß2 knockdown does not affect the myoblasts proliferation. However, Pax7 expression was significantly decreased in 4-week-old gastrocnemius muscle, suggesting that cellular reserve for growth may be deficient in KO mice. This is further supported by decreased migratory capacity of C2C12 cells upon siRNA-targeted Kvß2 knockdown. CONCLUSION: Overall, this is the first report identifying that genetic deletion of Kvß2 leads to decreased skeletal muscle size along with isotype switching.

Dietary Inflammatory Index and liver status in subjects with different adiposity levels within the PREDIMED trial.

BACKGROUND & AIMS: To assess the possible association between a validated Dietary Inflammatory Index (DII) and specific dietary components with suitable non-invasive markers of liver status in overweight and obese subjects within the PREDIMED study. METHODS: A cross-sectional study encompassing 794 randomized overweight and obese participants (mean ± SD age: 67.0 ± 5.0 y, 55% females) from the PREDIMED (PREvención con DIeta MEDiterránea) trial was conducted. DII is a validated tool evaluating the effect of diet on six inflammatory biomarkers (IL-1b, IL-4, IL-6, IL-10, TNF-α and C-reactive protein). Furthermore, a validated 137-item food-frequency-questionnaire was used to obtain the information about the food intake. In addition, anthropometric measurements and several non-invasive markers of liver status were assessed and the Fatty Liver Index (FLI) score was calculated. RESULTS: A higher DII and lower adherence to Mediterranean diet (MeDiet) were associated with a higher degree of liver damage (FLI > 60) in obese as compared to overweight participants. Furthermore, the DII score was positively associated with relevant non-invasive liver markers (ALT, AST, GGT and FLI) and directly affected FLI values. Interestingly, a positive correlation was observed between liver damage (>50th percentile FLI) and nutrients and foods linked to a pro-inflammatory dietary pattern. CONCLUSIONS: This study reinforced the concept that obesity is associated with liver damage and revealed that the consumption of a pro-inflammatory dietary pattern might contribute to obesity and fatty liver disease features. These data suggest that a well-designed precision diet including putative anti-inflammatory components could specifically prevent and ameliorate non-alcoholic fatty liver manifestations in addition to obesity.

Association between Siesta (Daytime Sleep), Dietary Patterns and the Presence of Metabolic Syndrome in Elderly Living in Mediterranean Area (Medis Study): The Moderating Effect of Gender.

OBJECTIVES: Several lifestyle parameters including diet, physical activity and sleep were associated in isolation with the presence of Metabolic Syndrome (MetS) in adults, to date there is a paucity of studies which evaluated their combined role aging populations and especially with respect to gender. Therefore, the aim of the present study was to provide a global consideration of the lifestyle factors associated with MetS among elderly individuals. DESIGN: Cross-sectional observational study. SETTING: 21 Mediterranean islands and the rural Mani region (Peloponnesus) of Greece. PARTICIPANTS: during 2005-2015, 2749 older (aged 65-100 years) from were voluntarily enrolled in the study. MEASUREMENTS: Dietary habits, energy intake, physical activity status, socio-demographic characteristics, lifestyle parameters (sleeping and smoking habits) and clinical profile aspects were derived through standard procedures. The presence of MetS was defined using the definition provided by NCEP ATP III (revised) and cluster analysis was used to identify overall dietary habit patterns. RESULTS: The overall prevalence of MetS in the study sample was 36.2%, but occurred more frequently in females (40.0% vs. 31.8%, respectively, p=0.03). Individuals with MetS were more likely to sleep during the day (89.4% vs. 76.8% respectively, p=0.039) and frequent 'siesta' was positively linked to the odds of MetS presence in females (Odds Ratio (OR) =3.43, 95% Confidence Intervals (CI): 1.08-10.9), but not for men (p=0.999). The lower carbohydrate (i.e., 45.2% of total daily energy, 120±16gr/day) dietary cluster was inversely associated with the odds for MetS presence, but only for men (OR=0.094, 95%CI: 0.010-0.883). CONCLUSIONS: Lifestyle parameters including sleep and diet quality are strongly associated with the presence of MetS in elderly cohort, but different their level of influence appears to be different, depending on gender. Further research is needed to better consider the role of lifestyle characteristics in the management of MetS in clinical practice.

Macrophages and Mitochondria: A Critical Interplay Between Metabolism, Signaling, and the Functional Activity.

Macrophages are phagocytic cells that participate in a broad range of cellular functions and they are key regulators of innate immune responses and inflammation. Mitochondria are highly dynamic endosymbiotic organelles that play key roles in cellular metabolism and apoptosis. Mounting evidence suggests that mitochondria are involved in the interplay between metabolism and innate immune responses. The ability of these organelles to alter the metabolic profile of a cell, thereby allowing an appropriate response to each situation, is crucial for the correct establishment of immune responses. Furthermore, mitochondria act as scaffolds for many proteins involved in immune signaling pathways and as such they are able to modulate the function of these proteins. Finally, mitochondria release molecules, such as reactive oxygen species, which directly regulate the immune response. In summary, mitochondria can be considered as core components in the regulation of innate immune signaling. Here we discuss the intricate relationship between mitochondria, metabolism, intracellular signaling, and innate immune responses in macrophages.

Effects of dietary almond- and olive oil-based docosahexaenoic acid- and vitamin E-enriched beverage supplementation on athletic performance and oxidative stress markers.

Functional beverages based on almonds and olive oil and enriched with α-tocopherol and docosahexaenoic acid (DHA) could be useful in modulating oxidative stress and enhancing physical performance in sportsmen. The aim of this work was to evaluate the effects of supplementation with functional beverages on physical performance, plasma and erythrocyte fatty acids' and polyphenol handling, oxidative and nitrative damage, and antioxidant and mitochondrial gene expression in young and senior athletes. Athletes performed maximal exercise tests before and after one month of dietary supplementation and blood samples were taken immediately before and one hour after each test. The beverages did not alter performance parameters during maximal exercise. Supplementation increased polyunsaturated and reduced saturated plasma fatty acids while increasing the DHA erythrocyte content; it maintained basal plasma and blood polyphenol levels, but increased the blood cell polyphenol concentration in senior athletes. Supplementation protects against oxidative damage although it enhances nitrative damage in young athletes. The beverages enhance the gene expression of antioxidant enzymes in peripheral blood mononuclear cells after exercise in young athletes.

Effects of total dietary polyphenols on plasma nitric oxide and blood pressure in a high cardiovascular risk cohort. The PREDIMED randomized trial.

BACKGROUND AND AIM: Hypertension is one of the main cardiovascular risk factors in the elderly. The aims of this work were to evaluate if a one-year intervention with two Mediterranean diets (Med-diet) could decrease blood pressure (BP) due to a high polyphenol consumption, and if the decrease in BP was mediated by plasma nitric oxide (NO) production. METHODS AND RESULTS: An intervention substudy of 200 participants at high cardiovascular risk was carried out within the PREDIMED trial. They were randomly assigned to a low-fat control diet or to two Med-diets, one supplemented with extra virgin olive oil (Med-EVOO) and the other with nuts (Med-nuts). Anthropometrics and clinical parameters were measured at baseline and after one year of intervention, as well as BP, plasma NO and total polyphenol excretion (TPE) in urine samples. Systolic and diastolic BP decreased significantly after a one-year dietary intervention with Med-EVOO and Med-nuts. These changes were associated with a significant increase in TPE and plasma NO. Additionally, a significant positive correlation was observed between changes in urinary TPE, a biomarker of TP intake, and in plasma NO (Beta = 4.84; 95% CI: 0.57-9.10). CONCLUSIONS: TPE in spot urine sample was positively correlated with plasma NO in Med-diets supplemented with either EVOO or nuts. The statistically significant increases in plasma NO were associated with a reduction in systolic and diastolic BP levels, adding to the growing evidence that polyphenols might protect the cardiovascular system by improving the endothelial function and enhancing endothelial synthesis of NO.

Docosahexaenoic acid supplementation promotes erythrocyte antioxidant defense and reduces protein nitrosative damage in male athletes.

The aim of this study was to determine the influence of long-term docosahexaenoic acid (DHA) dietary supplementation on the erythrocyte fatty acid profile and oxidative balance in soccer players after training and acute exercise. Fifteen volunteer male athletes (age 20.0 ± 0.5 years) were randomly assigned to a placebo group that consumed an almond-based beverage (n = 6), or to an experimental group that consumed the same beverage enriched with DHA (n = 9) for 8 weeks. Blood samples were taken in resting conditions at the beginning and after 8 weeks of nutritional intervention and training in resting and in post-exercise conditions. Oxidative damage markers (malonyldialdehyde, carbonyl and nitrotyrosine indexes) and the activity and protein level of antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase and peroxidase) were assessed. The results showed that training increased antioxidant enzyme activities in erythrocytes. The experimental beverage increased DHA from 34.0 ± 3.6 to 43.0 ± 3.6 nmol/10(9) erythrocytes. DHA supplementation increased the catalytic activity of superoxide dismutase from 1.48 ± 0.40 to 10.5 ± 0.35 pkat/10(9) erythrocytes, and brought about a reduction in peroxidative damage induced by training or exercise. In conclusion, dietary supplementation with DHA changed the erythrocyte membrane composition, provided antioxidant defense and reduced protein peroxidative damage in the red blood cells of professional athletes after an 8-week training season and acute exercise.

Dietary factors associated with subclinical inflammation among girls.

BACKGROUND/OBJECTIVES: Dietary patterns and biomarkers of inflammation have been scarcely associated. The aim was to assess dietary factors associated with subclinical inflammation among girls. SUBJECTS/METHODS: Fasting blood samples were collected from 12- to 17-year old girls (n=219) to measure adiponectin, leptin, tumor necrosis factor-alpha, plasminogen activator inhibitor 1 (PAI-1), interleukin-6 (IL-6) and high-sensitivity C-reactive protein (hs-CRP) levels. Body mass index (BMI) and waist-to-height ratio (WHtR), and food intake were also measured. Western (WDP) and Mediterranean (MDP) dietary patterns were identified. RESULTS: BMI and WHtR were associated with adiponectin, leptin and hs-CRP (the last, only associated with BMI). Intakes of ß-carotene equivalents and vitamin C were associated with adiponectin; saturated fatty acids (SFA), vitamin A, manganese and selenium with leptin; linoleic acid with PAI-1; and oleic acid and vitamin E with IL-6. Selenium was inversely associated with adiponectin, whereas magnesium was positively associated with IL-6. MDP was associated with higher plasma concentrations of adiponectin (ß=0.174, P<0.05); after adjustment for BMI, associations were not significant (ß=0.144, P=0.076). WDP was negatively associated with adiponectin (ß=-0.177, P<0.05) and positively with IL-6 (ß=0.183, P<0.05). CONCLUSIONS: Subclinical inflammation is detectable with increasing BMI and also WHtR. Measures of adiposity (BMI and WHtR) are significant predictors of adiponectin, leptin and hs-CRP. Dietary patterns per se have a small role in affecting inflammatory markers among adolescents.

Inflammatory markers and metabolic syndrome among adolescents.

BACKGROUND/OBJECTIVES: The alarming increase in obesity worldwide is of concern, owing to the associations of obesity with metabolic syndrome (MetS), which has been associated with a proinflammatory state characterized by elevated plasma concentrations of several markers of inflammation. The aim of this study was to assess levels of inflammatory markers and their association with MetS among adolescents. SUBJECTS/METHODS: A random sample of adolescents (n=362, 143 boys and 219 girls, 12-17 years) was interviewed, anthropometrically measured and provided a fasting blood sample. Circulating levels of adiponectin, leptin, tumour necrosis factor-alpha, plasminogen activator inhibitor 1 (PAI-1), interleukin-6 and high-sensitivity C-reactive protein were measured. The association between inflammatory markers and sex, age, body mass index (BMI) status, MetS, physical activity and blood pressure was also calculated. RESULTS: Adiponectin levels are inversely associated and leptin levels are directly associated with MetS and BMI, but directly with gender (females show higher levels than boys), and PAI-1 levels are directly associated with MetS, among adolescents. CONCLUSIONS: Leptin, adiponectin and PAI-1 may be used as biomarkers to predict MetS among adolescents.

Scuba diving activates vascular antioxidant system.

The aim was to study the effects of scuba diving immersion on plasma antioxidant defenses, nitric oxide production, endothelin-1 and vascular endothelial growth factor levels. 9 male divers performed an immersion at 50 m depth for a total time of 35 min. Blood samples were obtained before diving at rest, immediately after diving, and 3 h after the diving session. Leukocyte counts, plasma 8oxoHG, malondialdehyde and nitrite levels significantly increased after recovery. Activities of lactate dehydrogenase, creatine kinase, catalase and superoxide significantly increased immediately after diving and these activities remained high after recovery. Plasma myeloperoxidase activity and protein levels and extracellular superoxide dismutase protein levels increased after 3 h. Endothelin-1 concentration significantly decreased after diving and after recovery. Vascular endothelial growth factor concentration significantly increased after diving when compared to pre-diving values, returning to initial values after recovery. Scuba diving at great depth activated the plasma antioxidant system against the oxidative stress induced by elevated pO2 oxygen associated with hyperbaria. The decrease in endothelin-1 levels and the increase in nitric oxide synthesis could be factors that contribute to post-diving vasodilation. Diving increases vascular endothelial growth factor plasma levels which can contribute to the stimulation of tissue resistance to diving-derived oxidative damage.

Body mass index, life-style, and healthy status in free living elderly people in Menorca Island.

OBJECTIVES: To assess the BMI, life-style, and healthy status, and explore relationships between these parameters, among Menorca's free living elderly people. METHODS: A cross-sectional survey carried out in Menorca Island in 2009. A random sample (n=450) of the elderly population (≥65 years) was interviewed. Anthropometric measurements and a general questionnaire incorporating questions related to socio-demographic and life-style factors and health status were used. RESULTS: Approximately five per cent of elders were underweight and 60% overweight or obese. Underweight were positively and overweight and obesity negatively affected by age. The prevalence of central obesity, according to the WC cut-off points, was 66.8% in men and 85.1% in women. Low education, socioeconomic status and physical activity were risk factors for malnutrition and overweight/obesity. A possible cognitive impairment was found among elderly persons with BMI<22 kg/m2. A J-shaped association between BMI and hypertension, hypercholesterolemia, heart failure and other CV diseases, a U-shaped relation between BMI and diabetes mellitus, arthritis, and chronic bronchitis, and an inverted J-shape between BMI and gastric ulcer, osteoporosis and bone fractures, cancer, and prostatitis (in men) were found. CONCLUSIONS: Both low and high BMI are associated with a wide range of prevalent conditions and diseases in Menorca elderly men and women.

Vitamin C supplementation influences the antioxidant response and nitric oxide handling of erythrocytes and lymphocytes to diving apnea.

OBJECTIVE: We have investigated the influence of vitamin C diet supplementation on the antioxidant response and nitrite levels in lymphocytes and erythrocytes during diving apnea. SUBJECTS: Seven male professional apnea divers participated in a double blind crossover study. Divers were randomly assigned to either vitamin C supplemented or placebo groups. The subjects did not take any other supplements than the ones provided for this study. INTERVENTION: One group was supplemented with vitamin C capsules (1 g per day) for 7 days while the other group took a placebo composed of lactose. The usual dietary habits of participants were assessed using a self-reported 7-days 24-h recall before the day of the study. Blood samples were taken under basal conditions, immediately after diving apnea for 4 h and after 1 h of recovery. RESULTS: Catalase activity increased in erythrocytes (23%) and superoxide dismutase increased in lymphocytes (35%) during the recovery only in the placebo group. Lymphocyte ascorbate levels increased in the supplemented group after diving (85%) and maintained high at recovery. Plasma nitrite levels increased about twofold in both groups during the recovery. Erythrocyte nitrite levels increased after diving (50%) and about twofold during the recovery in the supplemented group. Nitrite levels and iNOS levels in lymphocytes were higher in the placebo group than in the supplemented during the recovery. Erythrocyte carbonyl derivates were unchanged in all situations. CONCLUSIONS: Vitamin C supplementation influenced the antioxidant response and NO handling in erythrocytes and lymphocytes to the oxidative stress induced by hypoxia-reoxygenation.

Differential response of plasma and immune cell's vitamin E levels to physical activity and antioxidant vitamin supplementation.

OBJECTIVE: To assess the differential response of plasma, lymphocyte and neutrophil vitamin E levels to high-intensity physical activity and to vitamin C and E supplementation. SUBJECTS: In all, 14 male trained amateur runners (32-36 y old) were randomly divided in two groups (supplemented and placebo), and participated in a half marathon race. The subjects did not take any other supplements than the ones provided for this study. INTERVENTION: Vitamin C (152 mg/day) and E (50 mg/day) supplementation was administrated to athletes for a month, using a new almond-based isotonic and energetic beverage (supplemented group). The usual dietary habits of participants were assessed using a self-reported 7-day 24-h recall before the day of the study. To avoid the beverage influence, nonenriched vitamin C and E almond-based isotonic and energetic beverage was given to the placebo group. After 1 month, subjects participated in a half marathon race (21 km run). Vitamin E concentration was determined in plasma, neutrophils and lymphocytes before and immediately after the race, and 3 h after finishing the race. RESULTS: Daily energy intake and caloric profile of supplemented and placebo group were not different except for vitamin C and E supplementation. Vitamin supplementation and exercise had no effect on vitamins E levels in plasma. The exercise significantly (P<0.05) increased the lymphocyte vitamin E concentration both in the placebo (+119%) and supplemented groups (+128%), and neutrophil vitamin E content in the supplemented group (+88%). These levels remained significantly (P<0.05) high after the short recovery. After exercise, vitamin E levels in lymphocytes and neutrophils of supplemented subjects were practically twice the levels before exercise, whereas neutrophil vitamin E content of the placebo group was close to those in plasma. CONCLUSION: After endurance exercise, lymphocytes increased their vitamin E content in the supplemented and placebo subjects whereas this trend in neutrophils was just observed in the supplemented group. The determination of vitamin E content in lymphocytes and neutrophils after exercise is a useful tool to assess the functional status of vitamin E.

[Clinical and neuroimaging findings in a family with CADASIL associated to C475T mutation]. / Hallazgos clínicos y de neuroimagen en una familia con CADASIL asociada a la mutación C475T.

INTRODUCTION: The term CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) refers to an autosomal dominant hereditary arteriopathy of the brain that is characterised by headache, recurring strokes and progressive cognitive deterioration. We report the case of another family with CADASIL and emphasise the importance of a genetic study in its diagnosis. CASE REPORT: A 62-year-old female patient with repeating lacunar strokes, subcortical dementia and a family history of dementia and strokes. Neuroimaging studies conducted on the patient and her siblings showed signs of leukoencephalopathy and lacunar infarctions. The ultrastructural study of the biopsy performed on a sample of the patient's skin, which included five dermal vessels, did not show any electron-dense deposits. The genetic study revealed the presence of mutation C475T in exon 4 of NOTCH3. CONCLUSIONS: The possible presence of CADASIL must be suspected in patients with symptoms of cerebrovascular disease or dementia who present characteristic alterations in the magnetic resonance brain scan, especially when there is a compatible family history. The first choice diagnostic procedure must be a genetic study.

Olive oil and the Mediterranean diet: beyond the rhetoric.

OBJECTIVE: The purpose of this study was to analyze the association of food, nutrient and energy intakes with olive oil consumption in Spain. DESIGN: Cross-sectional study by face-to-face interview. SETTING: Population-based random sample derived from the Catalan Nutrition Survey. SUBJECTS: In total, 1600 individuals between 18 and 60 y of age. INTERVENTION: Two 24-h recalls were administered to measure food and nutrient intakes. Food consumption and nutrient intakes were analyzed comparing the highest and lowest quartiles of olive oil consumption. RESULTS: Those with the highest consumption of olive oil (greater than 13.5% of total calories, fourth quartile) consumed less cereal, baked goods, whole milk, sausages, candy, fruit juice and soft drinks, but more fish, eggs, vegetables and added fats, as compared to those with the lowest olive oil consumption (less than 6.8% of total calories, first quartile). The group with the greatest olive oil consumption also demonstrated high total fat intake, although saturated fats showed a lower percentage of total energy intake. Vitamin intake was more adequate in those with the highest consumption of olive oil. CONCLUSION: Olive oil is a key contributor to the healthy aspects attributed to the Mediterranean diet, and as such, nutritional objectives in Mediterranean countries should address reducing saturated fats, without modifying quantities of olive oil.

Diet supplementation with vitamin E, vitamin C and beta-carotene cocktail enhances basal neutrophil antioxidant enzymes in athletes.

Exercise increases oxygen consumption and causes a disturbance of intracellular pro-oxidant-antioxidant homeostasis. Few data are available as to the cumulative effects of exercise on the antioxidant defenses of the neutrophil. We studied the effects of 90 days' supplementation with placebo or an antioxidant cocktail of vitamin E (500 mg/day) and beta-carotene (30 mg/day) and the last 15 days also with vitamin C (1 g/day) on sportsmen's basal neutrophil antioxidant defenses. We analyzed the activities of catalase, glutathione peroxidase, glutathione reductase and the activities and levels of superoxide dismutase, glutathione and glutathione disulfide in neutrophils purified from antecubital vein blood of sportsmen before and after diet supplementation. Plasma vitamin E, beta-carotene and vitamin C concentrations in the antioxidant-supplemented group were approximately 1.6, 10, and 1.2 times higher respectively than those of the placebo group. The antioxidant-supplemented group presented a significantly higher glutathione versus glutathione disulfide ratio in neutrophils (about 20%) than the placebo one. Antioxidant supplementation enhances the antioxidant enzyme activity of superoxide dismutase and catalase in neutrophils.

Vitamins in Spanish food patterns: the eVe Study.

OBJECTIVE: To describe vitamin intakes in Spanish food patterns, identify groups at risk for inadequacy and determine conditioning factors that may influence this situation. DESIGN: Pooled-analysis of eight cross-sectional regional nutrition surveys. SUBJECTS: Ten thousand two hundred and eight free-living subjects (4728 men, 5480 women) aged 25-60 years. Respondents of population nutritional surveys carried out in eight Spanish regions (Alicante, Andalucia, Balearic Islands, Canary Islands, Catalunya, Galicia, Madrid and Basque Country) from 1990 to 1998. The samples were pooled together and weighted to build a national random sample. METHODS: Dietary assessment by means of repeated 24-hour recall using photograph models to estimate portion size. Adjusted data for intra-individual variation were used to estimate the prevalence of inadequate intake. A Diet Quality Score (DQS) was computed considering the risk for inadequate intake for folate, vitamin C, vitamin A and vitamin E. DQS scores vary between 0 (good) and 4 (very poor). Influence of lifestyle (smoking, alcohol consumption and physical activity) was considered as well. RESULTS: Inadequate intakes (<2/3 Recommended Dietary Intake) were estimated in more than 10% of the sample for riboflavin (in men), folate (in women), vitamin C, vitamin A, vitamin D and vitamin E. More than 35% of the sample had diets classified as poor quality or very poor quality. Factors identified to have an influence on a poor-quality diet were old age, low education level and low socio-economical level. A sedentary lifestyle, smoking, usual consumption of alcohol and being overweight were conditioning factors for a poor-quality diet as well. CONCLUSION: Results from The eVe Study suggest that a high proportion of the Spanish population has inadequate intakes for at least one nutrient and nearly 50% should adjust their usual food pattern towards a more nutrient-dense, healthier diet.

The presenilin 1 C92S mutation increases abeta 42 production.

Although wild-type human presenilin 1 (PS1) rescues the C. elegans egg-laying (egl) phenotype that is caused by a loss of function mutation in the C. elegans presenilin homologue sel12, most familial Alzheimer's disease (FAD)-linked PS1 mutants only partially rescue this phenotype. To investigate the effects of the loss of function sel12 mutation on Abeta production in mammalian cells, we analyzed Abeta production in transfected H4 neuroglioma cells expressing the PS1 homologue of the sel12 C60S mutant, PS1 C92S. This analysis revealed that PS1 C92S increased Abeta42 levels in a similar fashion to other pathogenic Alzheimer's disease (AD) PS1 mutations. Significantly, the PS1 C92S mutation has recently been identified as the pathogenic mutation in an Italian family with FAD. Thus, placing a mutation that results in loss of function in C. elegans into a context whereby its effect on mammalian cells can be evaluated suggests that all FAD-linked PS1 mutants result in increased Abeta42 production through a partial loss of function mechanism.

Alzheimer's disease presenilin-1 exon 9 deletion and L250S mutations sensitize SH-SY5Y neuroblastoma cells to hyperosmotic stress-induced apoptosis.

Mutations in the presenilin-1 (PS1) and presenilin-2 (PS2) genes account for the majority of early-onset familial Alzheimer's disease cases. Recent studies suggest that presenilin gene mutations predispose cells to apoptosis by mechanisms involving altered calcium homeostasis and oxidative damage. In the present study, we determined whether PS1 mutations also sensitize cells to hyperosmotic stress-induced apoptosis. For this, we established SH-SY5Y neuroblastoma cell lines stably transfected with wild-type PS1 or either the PS1 exon 9 deletion (deltaE9) or PS1 L250S mutants. Cultured cells were exposed to an overnight (17 h) serum deprivation, followed by a 30 min treatment with either 20 mM glucose, 10 nM insulin-like growth factor-1 or 20 mM glucose + 10 nM insulin-like growth factor-1. Cells were then cultured for a further 3, 6 or 24 h and stained for apoptotic condensed nuclei using propidium iodide. Confirmation that cells were undergoing an active apoptotic process was achieved by labelling of DNA strand breaks using the terminal dUTP nick end labelling (TUNEL) technique. We also determined cell viability using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Propidium iodide staining revealed that all cell lines and controls showed an increased number of apoptotic cells appearing with condensed nuclei at 24 h compared with 6 h and 3 h. High glucose-induced hyperosmotic stress resulted in significantly more apoptotic cells in the PS1 deltaE9 and PS1 L250S mutation cell lines at 24 h, compared with the wild-type PS1 lines (P < 0.001, ANOVA for both comparisons). Mean values (+/-S.D.) for the percentage number of apoptotic cells at 24 h following high glucose treatment were 16.1 +/- 3.5%, 26.7 +/- 5.5% and 31.0 +/- 5.7% for the wild-type PS1, PS1 deltaE9 and PS1 L250S lines, respectively. The pro-apoptotic effects of high glucose treatment were reversed by 10 nM insulin-like growth factor-1, although to a lesser extent in the mutation cell lines (5.8 +/- 2.4%, 15.2 +/- 7.3% and 13.2 +/- 2.0% for the wild-type PS1, PS1 deltaE9 (P < 0.01 for comparison with wild-type PS1) and PS1 L250S (P < 0.01 for comparison with wild-type PS1) transfected lines, respectively. TUNEL labelling of cells at 24 h following treatment gave essentially the same results pattern as obtained using propidium iodide. The percentage number of apoptotic cells with DNA strand breaks (means +/- S.D.) following high glucose treatment was 15.4 +/- 2.6% for the wild-type PS1, 26.8 +/- 3.2% for the PS1 deltaE9 (P < 0.001 for comparison with wild-type PS1) and 29.7 +/- 6.1% for the PS1 L250S transfected lines (P < 0.001 for comparison with wild-type PS1). The PS1 deltaE9 and PS1 L250S transfected lines also showed a higher number of apoptotic cells with DNA strand breaks at 24 h following high glucose plus insulin-like growth factor-1 treatment (11.4 +/- 2.0% and 14.3 +/- 2.8%, respectively), compared with values for the wild-type PS1 lines (8.5 +/- 2.4%). These differences were significant (P < 0.01) for the comparison of wild-type PS1 and PS1 L250S, but not PS1 deltaE9 lines. The mutation-related increases in number of apoptotic cells at 24 h following high glucose treatment were not accompanied by significant differences in cell viability at this time-point. Our results indicate that PS1 mutations predispose to hyperosmotic stress-induced apoptosis and that the anti-apoptotic effects of insulin-like growth factor-1 are compromised by these mutations. Perturbations of insulin-like growth factor-1 signalling may be involved in PS1 mutation-related apoptotic neuronal cell death in Alzheimer's disease.