TIMP-1 Protects Tight Junctions of Brain Endothelial Cells From MMP-Mediated Degradation.

OBJECTIVE: The blood-brain barrier (BBB) plays a critical role in central nervous system homeostasis, and the integrity of BBB is disrupted in many neurodegenerative diseases. Matrix metalloproteinases (MMPs) degrade the tight junctions (TJs) of endothelial cells and basement membrane components essential to BBB integrity, which leads to increased BBB permeability and allows inflammatory cells and neurotoxic substances to enter the brain. Tissue inhibitors of metalloproteinases (TIMPs), endogenous inhibitors of MMPs, regulate MMP activity, thereby maintaining BBB integrity. METHODS: The disruptive impacts of MMP-3 and MMP-9 on BBB and protective effect of TIMP-1 were investigated in a simplified in vitro model of the BBB, which was generated using rat brain microvascular endothelial cells (RBMEC). The main features of BBB formation, including permeability and the trans-endothelial electrical resistance (TEER), were monitored over time after the addition of MMP-3 and MMP-9 and their complexes with TIMP-1 inhibitor. RESULTS: Our results indicated that MMP-3 and MMP-9 caused a dose-dependent disruption of the BBB, with 1.5 µM MMPs resulting in an over threefold increase in permeability, while TIMP-1 inhibition protected the integrity of the BBB model and recovered TEER and permeability of RBMECs. The disruption and recovery of tight junction proteins of RBMECs after MMP and TIMP treatment were also detected using fluorescent microscopy. CONCLUSION: MMP-9 and MMP-3 disrupt the BBB by degrading tight junctions in endothelial cells, and TIMP-1 could inhibit the disruptive effect of MMP-3 and MMP-9 by showing potential as therapeutic protein against MMP-related diseases where BBB disruption plays a role.

In Vitro and In Silico Studies of Neolignans from Magnolia grandiflora L. Seeds against Human Cannabinoids and Opioid Receptors.

Magnolia grandiflora L. (Magnoliaceae) is a plant of considerable medicinal significance; its flowers and seeds have been used in various traditional remedies. Radioligand binding assays of n-hexane seeds extract showed displacement of radioligand for cannabinoid (CB1 and CB2) and opioid δ (delta), κ (kappa), and µ (mu) receptors. Bioactivity-guided fractionation afforded 4-O-methylhonokiol (1), magnolol (2), and honokiol (3), which showed higher binding to cannabinoid rather than opioid receptors in radioligand binding assays. Compounds 1-3, together with the dihydro analog of 2 (4), displayed selective affinity towards CB2R (Ki values of 0.29, 1.4, 1.94, and 0.99 µM, respectively), compared to CB1R (Ki 3.85, 17.82, 14.55, and 19.08 µM, respectively). An equal mixture of 2 and 3 (1:1 ratio) showed additive displacement activity towards the tested receptors compared to either 2 or 3 alone, which in turn provides an explanation for the strong displacement activity of the n-hexane extract. Due to the unavailability of an NMR or X-ray crystal structure of bound neolignans with the CB1 and CB2 receptors, a docking study was performed to predict ligand-protein interactions at a molecular level and to delineate structure-activity relationships (SAR) of the neolignan analogs with the CB1 and CB2 receptors. The putative binding modes of neolignans 1-3 and previously reported related analogs (4, 4a, 5, 5a, 6, 6a, and 6b) into the active site of the CB1 and CB2 receptors were assessed for the first time via molecular docking and binding free-energy (∆G) calculations. The docking and ∆G results revealed the importance of a hydroxyl moiety in the molecules that forms strong H-bonding with Ser383 and Ser285 within CB1R and CB2R, respectively. The impact of a shift from a hydroxyl to the methoxy group on experimental binding affinity to CB1R versus CB2R was explained through ∆G data and the orientation of the alkyl chain within the CB1R. This comprehensive SAR, influenced by the computational study and the observed in vitro displacement binding affinities, has indicated the potential of magnolia neolignans for developing new CB agonists for potential use as analgesics, anti-inflammatory agents, or anxiolytics.

Cannabinoid and Opioid Receptor Affinity and Modulation of Cancer-Related Signaling Pathways of Machaeriols and Machaeridiols from Machaerium Pers.

Machaeriols and machaeridiols are unique hexahydrodibenzopyran-type aralkyl phytocannabinoids isolated from Machaerium Pers. Earlier studies of machaeriol A (1) and B (2) did not show any affinity for cannabinoid receptor 1 (CB1 or CNR1), although they are structural analogs of psychoactive hexahydrocannabinol. This study comprehensively reports on the affinities of isolated Machaerium Pers. compounds, namely machaeriol A-D (1-4) and machaeridiol A-C (5-7), against cannabinoid (CB1 and CB2) and opioid (κ, δ and µ) receptors. Among the isolated compounds, machaeriol D (4) and machaeridiol A-C (5-7) showed some selective binding affinity for the CB2 receptor, using a radioligand binding assay, with Ki values of >1.3, >1.77, >2.18 and >1.1 µM, respectively. On the other hand, none of the compounds showed any binding to the CB1 receptor. Due to recent reports on the anticancer potential of the endocannabinoid system, compounds 1-7 were tested against a battery of luciferase reporter gene vectors that assess the activity of many cancer-related signaling pathways, including Stat3, Smad2/3, AP-1, NF-κB, E2F, Myc, Ets, Notch, FoxO, Wnt, Hedgehog and pTK in HeLa and T98G glioblastoma cells. Complete dose-response curves have been determined for each compound in both of these cell lines, which revealed that machaeridiol 6 displayed activities (IC50 in µM in HeLa and T98G cells) towards Stat3 (4.7, 1.4), Smad2/3 (1.2, 3.0), AP-1 (5.9, 4.2), NF-κB (0.5, 4.0), E2F (5.7, 0.7), Myc (5.3, 2.0), ETS (inactive, 5.9), Notch (5.3, 4.6), Wnt (4.2, inactive) and Hedgehog (inactive, 5.0). Furthermore, a combination study between machaeriol C (3) and machaeridiol B (6) displayed additive effects for E2F, ETS, Wnt and Hedgehog pathways, where these compounds individually were either minimally active or inactive. None of the compounds inhibited luciferase expression driven by the minimal thymidine kinase promoter (pTK), indicating the lack of general cytotoxicity for luciferase enzyme inhibition at the 50 µM concentration in both of these cell lines. The significance of the inhibition of these signaling pathways via machaeridiol 5-7 and their cross-talk potential has been discussed.

Flavonoids from Perovskia atriplicifolia and Their in Vitro Displacement of the Respective Radioligands for Human Opioid and Cannabinoid Receptors.

Bioassay-guided fractionation of the leaves of Perovskia atriplicifolia (Russian sage) resulted in the isolation of four previously known flavonoid derivatives, 5-hydroxy-6,7,3',4'-tetramethoxyflavone (1), 5,7-dihydroxy-6,3',4'-trimethoxyflavone (2), 5-hydroxy-6,7,4'-trimethoxyflavone (3), and 5,7-dihydroxy-6,4'-dimethoxyflavone (4). Compounds 1, 3, and 4 showed displacement of the radioligand for the cloned human δ opioid receptor with Ki values ranging from 3.1 to 26.0 µM. In addition, the binding mode of the compounds in the active site of the δ opioid receptor was investigated through molecular modeling algorithms. This study may have implications in better understanding non-nitrogenous δ opioid receptor ligands.

Novel PCR-based methods enhance characterization of vaginal microbiota in a bacterial vaginosis patient before and after treatment.

Deep characterization, even by next-generation sequencing, of the vaginal microbiota in healthy women or posttreatment bacterial vaginosis patients is limited by the dominance of lactobacilli. To improve detection, we offer two approaches: quantitative PCR (qPCR) using phylogenetic branch-inclusive primers and sequencing of broad-spectrum amplicons generated with oligomers that block amplification of lactobacilli.

Novel Machaeriol Analogues as Modulators of Cannabinoid Receptors: Structure-Activity Relationships of (+)-Hexahydrocannabinoids and Their Isoform Selectivities.

Machaeriols are an important class of compounds that structurally resemble tetrahydrocannabinol (Δ9-THC), with the major differences being inverted stereochemistry at the ring junction as [6aR, 10aR] and an additional stereocenter at the C9 position of the A-ring due to saturation. A previous study reported that machaeriols did not show any cannabinoid receptor activity, even though these hexahydrodibenzopyran analogues mimic a privileged (+)-tetrahydrocannabinoid scaffold. To unravel structural requisites for modulation of cannabinoid receptors, a simple late-stage divergent approach was undertaken to functionalize the machaeriol scaffold using the Suzuki coupling reaction. Fourteen hexahydro analogues were synthesized and screened against both cannabinoid receptor isoforms, CB1 and CB2. Interestingly, many of the analogues showed a significant binding affinity for both receptors; however, two analogues, 11H and 11J, were identified as possessing CB2 receptor-selective functional activity in the GTPγS assay; they were found to be micromolar-range agonists, with EC50 values of 5.7 and 16 µM, respectively. Furthermore, molecular dynamics simulations between the CB2 receptor and two novel analogues resulted in unique interaction profiles by tightly occupying the active ligand-binding domain of the CB2 receptor and maintaining stable interactions with the critical residues Phe94, Phe281, and Ser285. For the first time, with the aid of structure-activity relationships of (+)-hexahydrocannabinoids, CB2 selective agonists were identified with late-stage diversification using palladium-mediated C-C bond formation. By simply switching to (R)-citronellal as a chiral precursor, enantiomerically pure (-)-hexahydrocannabinoids with better CB1/CB2 receptor isoform selectivity can be obtained using the current synthetic approach.

Physiological and metabolic aspects of very prolonged exercise with particular reference to hill walking.

Hill walking is a popular recreational activity in the developed world, yet it has the potential to impose severe stress simultaneously upon several regulatory systems. Information regarding the physiological strain imposed by prolonged walking outdoors in adverse climatic conditions was reported almost four decades ago and recent research has extended some of this work. These data indicate that once the walker fatigues and starts to slow or stops walking altogether, the rate of heat production falls dramatically. This decrease alone predisposes to the development of hypothermia. These processes, in adverse weather conditions and/or during periods when the level of exertion is low (with low heat production), will be accelerated. Since the majority of walkers pursue this activity in groups, the less fit walkers may be more susceptible to fatigue when exercising at a higher relative intensity compared with their fitter counterparts. The best physiological offset for hypothermia is to maintain heat production by means of exercise, and so fatigue becomes a critical predisposing factor; it is as important to facilitate heat loss, especially during periods of high exertion, as it is to maintain heat production and preserve insulation. This can be partly achieved by clothing adjustments and consideration of the intensity of exercise. Failure to provide adequate energy intake during hill walking activities has been associated with decreased performance (particularly with respect to balance) and impaired thermoregulation. Such impairments may increase susceptibly to both fatigue and injury whilst pursuing this form of activity outdoors. The prolonged low to moderate intensity of activity experienced during a typical hill walk elicits marked changes in the metabolic and hormonal milieu. Available data suggest that during hill walking, even during periods of acute negative energy balance, blood glucose concentrations are maintained. The maintenance of blood glucose concentrations seems to reflect the presence of an alternative fuel source, a hormonally induced increase in fat mobilisation. Such enhancement of fat mobilisation should make it easier to maintain blood glucose by decreasing carbohydrate oxidation and promoting gluconeogenesis, thus sparing glucose utilisation by active muscle. During strenuous hill walking, older age walkers may be particularly prone to dehydration and decreased physical and mental performance, when compared with their younger counterparts. In summary, high rates of energy expenditure and hypohydration are likely to be closely linked to the activity. Periods of adverse weather, low energy intake, lowered fitness or increased age, can all increase the participants' susceptibility to injury, fatigue and hypothermia in the mountainous environment.

Eight strategies to engage industry in biosecurity.

A significant portion of the domestic bio-research base-and the one most likely to provide translational research-is not engaged in biodefense. Despite the fact that more than one-third of all life science researchers are employed in commercial bio-research, fewer than 3% of the 1,500 U.S. bio-technology companies are involved in biosecurity initiatives. The bio-tech industry has largely not aligned itself to play an integral role in biosecurity, but there are a few policy changes that could dramatically alter this balance. These include engaging and motivating the bio-technology middle class, seeding secondary markets, focusing on system solutions, providing reagents and standards, aligning communications, and prioritizing translational research. By reaching out, policymakers can span the current chasm between the bio-industry and government, build a stable biodefense industrial base, establish solid working relationships, and secure better services and products. The rewards would be significant for government and industry alike.

In vitro opioid receptor affinity and in vivo behavioral studies of Nelumbo nucifera flower.

ETHNOPHARMACOLOGICAL RELEVANCE: Nelumbo nucifera Geartn., known as sacred lotus, has been used traditionally in South East Asia as a traditional medicine for various CNS disorders including stress, fever, depression, insomnia, and cognitive conditions. AIM OF THE STUDY: To investigate the in vitro cannabinoid and opioid receptor binding affinities, and in vivo behavioral actions of Nelumbo flower extracts and to isolate the potential compounds to treat CNS associated disorders. MATERIALS AND METHODS: The white and pink flowers of N. nucifera were extracted with 95% EtOH, followed by acid-base partitioning using CHCl3 to give acidic and basic partitions. These partitions were subjected to Centrifugal Preparative TLC (CPTLC) to yield benzyltetrahydroisoquinoline (BTIQ) alkaloids and long chain fatty acids, identified by physical and spectroscopic methods. In addition, EtOH extracts and partitions were analyzed for chemical markers by UHPLC/MS and GC/MS. In vitro neuropharmacological effects were evaluated by cannabinoid (CB1 and CB2) and opioid [delta (δ), kappa (ĸ), and mu (µ)] competitive radioligand binding and GTPγS functional assays. The in vivo behavioral effect was studied through the use of the mouse tetrad assay at 10, 30, 75 and 100mg/kg/ip doses that revealed the effect on locomotion, catalepsy, body temperature, and nociception of acidic and basic CHCl3 partitions, fractions, and compounds. RESULTS: Three aporphines, nuciferine (1), N-nor-nuciferine (2), asimilobine (3), and five BTIQs, armepavine (4), O-methylcoclaurine (5), N-methylcoclaurine (6), coclaurine (7), neferine (10), and a mixture of linoleic and palmitic acids (LA and PA), were identified and evaluated for cannabinoid and opioid receptor displacement activities. Compounds 5-7 showed binding affinities for the ĸ opioid receptor with equilibrium dissociation constant (Ki) values of 3.5 ± 0.3, 0.9 ± 0.1, 2.2 ± 0.2 µM, respectively. Compound 10 displayed affinities for δ-and µ- opioid receptors with Ki values of 0.7 ± 0.1 and 1.8 ± 0.2 µM, respectively, and was determined to be a weak δ agonist by GTPγS functional assay. The mixture of LA and PA (1:1) showed an affinity for δ opioid receptor with a Ki value of 9.2 ± 1.1 µM. The acidic and basic CHCl3 partitions, compounds 1 and 7, and 5-7 mixture were subjected to the tetrad assay, of which the acidic partition displayed decreased locomotion and increased catalepsy, antinociception, and hypothermia in animal at doses of 75-100 mg/kg/ip, and also showed clonic-tonic seizures upon touch at 100mg/kg. CONCLUSION: Bioassay-guided isolation revealed compounds 5-7, 10, and the mixture of LA and PA displayed various degrees of opioid receptor radioligand displacement affinities. The in vivo tetrad assay of acidic CHCl3 partition, enriched with aporphines 1 and 2, displayed actions on all four points of behavioral parameters. It can be concluded that the in vivo mild canabimimetic-type effect observed for the CHCl3 partition is likely mediated through other CNS mechanisms since the extracts, partitions, and isolated compounds had no affinity for the in vitro CB1 and CB2 receptors. This work, along with traditional use and the reported bioactivities of the BTIQ alkaloids, suggested further studies on N. nucifera are needed to understand the roles that the extracts and/or individual compounds might contribute to the behavioral effects.

Longitudinal analysis of vaginal microbiome dynamics in women with recurrent bacterial vaginosis: recognition of the conversion process.

Bacterial vaginosis (BV) affects ∼ 30% of women of reproductive age, has a high rate of recurrence, and is associated with miscarriage, preterm birth, and increased risk of acquiring other sexually transmitted infections, including HIV-1. Little is known of the daily changes in the vaginal bacterial composition as it progresses from treatment to recurrence, or whether any of these might be useful in its prediction or an understanding of its causes. We used phylogenetic branch-inclusive quantitative PCR (PB-qPCR) and Lactobacillus blocked/unblocked qPCR (Lb-qPCR) to characterize longitudinal changes in the vaginal microbiota in sequential vaginal self-swabs from five women with recurrent BV, from diagnosis through remission to recurrence. Both patients with acute BV samples dominated by G. vaginalis recurred during the study with similar profiles, whereas the three patients with acute BV samples dominated by other anaerobes did not recur or recurred to an intermediate Nugent score. L. iners dominated remission phases, with intermittent days of abnormal microbial profiles typically associated with menses. The exception was a newly discovered phenomenon, a sustained period of abnormal profiles, termed conversion, which preceded symptomatic acute BV. Species known to have antagonistic activity towards Lactobacillus were detected in pre-conversion samples, possibly contributing to the decline in Lactobacillus. Lb-qPCR scores define two categories of response in the initial post-treatment visit samples; scores <5 may correspond with poor response to treatment or rapid recurrence, whereas scores >8 may predict delayed or no recurrence. Amsel criteria or Nugent scores did not have this potential predictive capability. Larger studies are warranted to evaluate the prognostic potential of detecting conversion and poor Lb-qPCR scores at the post-treatment visit of recurrent BV patients.

How we will produce the evidence-based EURRECA toolkit to support nutrition and food policy.

BACKGROUND: There is considerable variation in the recommended micronutrient intakes used by countries within Europe, partly due to different methodologies and concepts used to determine requirements and different approaches used to express the recommendations. As populations become more mobile and multi-national, and more traditional foods become available internationally, harmonised recommendations based on up to date science are needed. This was recognised by the European Commission's (EC) Directorate-General (DG) Research in their 2005 call for proposals for a Network of Excellence (NoE) on 'nutrient status and requirements of specific vulnerable population groups'. EURopean micronutrient RECommendations Aligned (EURRECA), which has 34 partners representing 17 European countries, started on its 5-year EC-funded programme in January 2007. The programme of work was developed over 2 years prior to submitting an application to the EC. The Network's first Integrating Meeting (IM) held in Lisbon in April 2007, and subsequent consultations, has allowed further refinement of the programme. AIM: This paper presents the rationale for the EURRECA Network's roadmap, which starts by establishing the status quo for devising micronutrient recommendations. The Network has the opportunity to identify previous barriers and then explore 'evidence-based' solutions that have not been available before to the traditional panels of experts. The network aims to produce the EURRECA 'toolkit' to help address and, in some cases, overcome these barriers so that it can be used by those developing recommendations. RESULTS: The status quo has been largely determined by two recent initiatives; the Dietary Reference Intake (DRI) reports from the USA and Canada and suggestions for approaches to international harmonisation of nutrient-based dietary standards from the United Nations University (UNU). In Europe, the European Food Safety Authority (EFSA) has been asked by the EC's Directorate-General for Health and Consumer Protection to produce values for micronutrient recommendations. Therefore, EURRECA will draw on the uniqueness of its consortium to produce the sustainable EURRECA toolkit, which will help make such a task more effective and efficient. Part of this uniqueness is the involvement in EURRECA of small and medium-sized enterprises (SMEs), consumer organisations, nutrition societies and other stakeholders as well as many scientific experts. The EURRECA toolkit will contain harmonised best practice guidance for a more robust science base for setting micronutrient recommendations. Hence, in the future, the evidence base for deriving nutrient recommendations will have greater breadth and depth and will be more transparent. CONCLUSIONS: The EURRECA Network will contribute to the broader field of food and nutrition policy by encouraging and enabling the alignment of nutrient recommendations. It will do this through the development of a scientific toolkit by its partners and other stakeholders across Europe. This will facilitate and improve the formulation of micronutrient recommendations, based on transparently evaluated and quantified scientific evidence. The Network aims to be sustainable beyond its EC funding period.

Current micronutrient recommendations in Europe: towards understanding their differences and similarities.

BACKGROUND: Nowadays most countries in Europe have established their own nutrient recommendations to assess the adequacy of dietary intakes and to plan desirable dietary intakes. As yet there is no standard approach for deriving nutrient recommendations, they may vary from country to country. This results in different national recommendations causing confusion for policy-makers, health professionals, industry, and consumers within Europe. EURRECA (EURopean micronutrient RECommendations Aligned) is a network of excellence funded by the European Commission (EC), and established to identify and address the problem of differences between countries in micronutrient recommendations. The objective of this paper is to give an overview of the available micronutrient recommendations in Europe, and to provide information on their origin, concepts and definitions. Furthermore this paper aims to illustrate the diversity in European recommendations on vitamin A and vitamin D, and to explore differences and commonalities in approaches that could possibly explain variations observed. METHODS: A questionnaire was developed to get information on the process of establishing micronutrient recommendations. These questionnaires were sent to key informants in the field of micronutrient recommendations to cover all European countries/regions. Also the latest reports on nutrient recommendations in Europe were collected. Standardisation procedures were defined to enable comparison of the recommendations. Recommendations for vitamin A and vitamin D were compared per sex at the ages 3, 9 months and 5, 10, 15, 25, 50 and 70 years. Information extracted from the questionnaires and reports was compared focusing on: (1) The concept of recommendation (recommended daily allowance (RDA), adequate intake (AI) or acceptable range), (2) The year of publication of the report (proxy for available evidence), (3) Population groups defined, (4) Other methodological issues such as selected criteria of adequacy, the type of evidence used, and assumptions made. RESULTS: Twenty-two countries, the World Health Organization (WHO)/the Food and Agriculture Organization of the United Nations (FAO) and the EC have their own reports on nutrient recommendations. Thirteen countries based their micronutrient recommendations on those from other countries or organisations. Five countries, WHO/FAO and the EC defined their own recommendations. The DACH-countries (Germany, Austria and Switzerland) as well as the Nordic countries (Norway, Sweden, Finland, Denmark and Iceland) cooperated in setting recommendations. Greece and Portugal use the EC and the WHO/FAO recommendations, respectively and Slovenia adopted the recommendations from the DACH-countries. Rather than by concepts, definitions, and defined population groups, variability appears to emerge from differences in criteria for adequacy, assumptions made and type of evidence used to establish micronutrient recommendations. DISCUSSION: The large variation in current micronutrient recommendations for population groups as illustrated for vitamin A and vitamin D strengthens the need for guidance on setting evidence based, up-to-date European recommendations. Differences in endpoints, type of evidence used to set recommendations, experts' opinions and assumptions are all likely to contribute to the identified variation. So far, background information was not sufficient transparent to disentangle the relative contribution of these different aspects. CONCLUSION: EURRECA has an excellent opportunity to develop tools to improve transparency on the approaches used in setting micronutrient recommendations, including the selection of criteria for adequacy, weighing of evidence, and interpretation of data.

Public health aspects of food fortification: a question of balance.

Micronutrient malnutrition is widespread throughout the world, with important health and economic consequences. Tools to address this situation include food fortification, supplementation and dietary diversification, each having different and complementary roles. Fortification (mandatory and voluntary) has been practised over several decades in Western countries as well as in developing countries. Iodised salt was introduced in the USA in 1924 to reduce severe I deficiency. In 1938 voluntary enrichment of flours and breads with niacin and Fe was initiated to reduce the incidence of pellagra and Fe-deficiency anaemia respectively. Micronutrient intakes in European countries appear to be generally adequate for most nutrients. However, a number of population subgroups are at higher risk of suboptimal intakes (below the lower reference nutrient intake) for some micronutrients, e.g. folate, Fe, Zn and Ca in children, adolescents and young women. Dietary surveys indicate that fortified foods play a role in mitigating such risks for several important nutrients. The number of foods suited to fortification are considerably limited by several factors, including technological properties (notably moisture, pH and O2 permeability), leading to unacceptable taste and appearance, as well as cost and consumer expectations. In countries in which voluntary fortification is widely practised micronutrient intakes are considerably below tolerable upper intake levels. Concerns about safety are addressed in relation to the potentially increased level or proportion of fortified foods (e.g. following potential EU legislation), for nutrients with relatively low tolerable upper intake levels and where the potential benefit and risks are in different subpopulations (e.g. folic acid). Recent models for assessing these issues are discussed.

Dietary intake and nutritional status of children and adolescents in Europe.

The objective of this project was to collect and evaluate data on nutrient intake and status across Europe and to ascertain whether any trends could be identified. Surveys of dietary intake and status were collected from across Europe by literature search and personal contact with country experts. Surveys that satisfied a defined set of criteria -- published, based on individual intakes, post-1987, adequate information provided to enable its quality to be assessed, small age bands, data for sexes separated above 12 years, sample size over 25 and subjects representative of the population -- were selected for further analysis. In a small number of cases, where no other data for a country were available or where status data were given, exceptions were made. Seventy-nine surveys from 23 countries were included, and from them data on energy, protein, fats, carbohydrates, alcohol, vitamins, minerals and trace elements were collected and tabulated. Data on energy, protein, total fat and carbohydrate were given in a large number of surveys, but information was very limited for some micronutrients. No surveys gave information on fluid intake and insufficient gave data on food patterns to be of value to this project. A variety of collection methods were used, there was no consistency in the ages of children surveyed or the age cut-off points, but most surveys gave data for males and females separately at all ages. Just under half of the surveys were nationally representative and most of the remainder were regional. Only a small number of local surveys could be included. Apart from anthropometric measurements, status data were collected in only seven countries. Males had higher energy intakes than females, energy intake increased with age but levelled off in adolescent girls. Intakes of other nutrients generally related to energy intakes. Some north-south geographical trends were noted in fat and carbohydrate intakes, but these were not apparent for other nutrients. Some other trends between countries were noted, but there were also wide variations within countries. A number of validation studies have shown that misreporting is a major problem in dietary surveys of children and adolescents and so all the dietary data collected for this project should be interpreted and evaluated with caution. In addition, dietary studies rely on food composition tables for the conversion of food intake data to estimated nutrient intakes and each country uses a different set of food composition data which differ in definitions, analytical methods, units and modes of expression. This can make comparisons between countries difficult and inaccurate. Methods of measuring food intake are not standardised across Europe and intake data are generally poor, so there are uncertainties over the true nutrient intakes of children and adolescents across Europe. There are insufficient data on status to be able to be able to draw any conclusions about the nutritional quality of the diets of European children and adolescents.