Polymyalgia rheumatica shows metabolomic alterations that are further altered by glucocorticoid treatment: Identification of metabolic correlates of fatigue.

OBJECTIVE: In polymyalgia rheumatica (PMR), glucocorticoids (GCs) relieve pain and stiffness, but fatigue may persist. We aimed to explore the effect of disease, GCs and PMR symptoms in the metabolite signatures of peripheral blood from patients with PMR or the related disease, giant cell arteritis (GCA). METHODS: Nuclear magnetic resonance spectroscopy was performed on serum from 40 patients with untreated PMR, 84 with new-onset confirmed GCA, and 53 with suspected GCA who later were clinically confirmed non-GCA, and 39 age-matched controls. Further samples from PMR patients were taken one and six months into glucocorticoid therapy to explore relationship of metabolites to persistent fatigue. 100 metabolites were identified using Chenomx and statistical analysis performed in SIMCA-P to examine the relationship between metabolic profiles and, disease, GC treatment or symptoms. RESULTS: The metabolite signature of patients with PMR and GCA differed from that of age-matched non-inflammatory controls (R2 > 0.7). There was a smaller separation between patients with clinically confirmed GCA and those with suspected GCA who later were clinically confirmed non-GCA (R2 = 0.135). In PMR, metabolite signatures were further altered with glucocorticoid treatment (R2 = 0.42) but did not return to that seen in controls. Metabolites correlated with CRP, pain, stiffness, and fatigue (R2 ≥ 0.39). CRP, pain, and stiffness declined with treatment and were associated with 3-hydroxybutyrate and acetoacetate, but fatigue did not. Metabolites differentiated patients with high and low fatigue both before and after treatment (R2 > 0.9). Low serum glutamine was predictive of high fatigue at both time points (0.79-fold change). CONCLUSION: PMR and GCA alter the metabolite signature. In PMR, this is further altered by glucocorticoid therapy. Treatment-induced metabolite changes were linked to measures of inflammation (CRP, pain and stiffness), but not to fatigue. Furthermore, metabolite signatures distinguished patients with high or low fatigue.

Trauma Versus Nontrauma Patients Treated by the Danish Helicopter Emergency Medical Service: A Register-Based Study of Patient Characteristics, Hospitalization, and Mortality.

OBJECTIVE: Helicopter emergency medical services (HEMS) are part of many emergency health care systems, ensuring specialized treatment on scene. An accurate use is important for improved resource utilization. The aim of this study was to describe patient characteristics, hospitalization, and mortality in trauma and nontrauma patients seen by the Danish HEMS teams. METHODS: The study is a registry-based study presenting data from the national Danish HEMS database. We included all HEMS missions from October 1, 2014, to April 30, 2018. RESULTS: A total of 13,391 HEMS entries were registered, and 5,524 cases were included in the study. Trauma patients were younger (38 vs. 63 years) and had a lower level of comorbidities (Charlson Comorbidity Index 0: 82% vs. 58%) compared with nontrauma patients. More nontrauma cases were classified with a severity score corresponding to a critical emergency. They also had a higher 30-day mortality compared with trauma patients (26% vs. 11%). CONCLUSION: This national study demonstrated differences in demographics, the severity profile, and mortality among trauma compared with nontrauma patients that could possibly indicate an overuse of HEMS for minor trauma cases. The results might suggest that educational efforts and a review of the current dispatch guidelines could be of importance when trying to improve the use of HEMS.

Common genetic variants are associated with lower serum 25-hydroxyvitamin D concentrations across the year among children at northern latitudes.

In a longitudinal study including 642 healthy 8-11-year-old Danish children, we investigated associations between vitamin D dependent SNP and serum 25-hydroxyvitamin D (25(OH)D) concentrations across a school year (August-June). Serum 25(OH)D was measured three times for every child, which approximated measurements in three seasons (autumn, winter, spring). Dietary and supplement intake, physical activity, BMI and parathyroid hormone were likewise measured at each time point. In all, eleven SNP in four vitamin D-related genes: Cytochrome P450 subfamily IIR1 (CYP2R1); 7-dehydrocholesterol reductase/nicotinamide adenine dinucleotide synthetase-1(DHCR7/NADSYN1); group-specific complement (GC); and vitamin D receptor were genotyped. We found minor alleles of CYP2R1 rs10500804, and of GC rs4588 and rs7041 to be associated with lower serum 25(OH)D concentrations across the three seasons (all P<0·01), with estimated 25(OH)D differences of -5·8 to -10·6 nmol/l from major to minor alleles homozygosity. In contrast, minor alleles homozygosity of rs10741657 and rs1562902 in CYP2R1 was associated with higher serum 25(OH)D concentrations compared with major alleles homozygosity (all P<0·001). Interestingly, the association between season and serum 25(OH)D concentrations was modified by GC rs7041 (P interaction=0·044), observed as absence of increase in serum 25(OH)D from winter to spring among children with minor alleles homozygous genotypes compared with the two other genotypes of rs7041 (P<0·001). Our results suggest that common genetic variants are associated with lower serum 25(OH)D concentrations across a school year. Potentially due to modified serum 25(OH)D response to UVB sunlight exposure. Further confirmation and paediatric studies investigating vitamin D-related health outcomes of these genotypic differences are needed.

Seasonal variations in growth and body composition of 8-11-y-old Danish children.

BACKGROUND: Earlier studies on seasonality in growth reported the largest height gains during spring and largest body weight gains during autumn. We examined seasonality in height, body weight, BMI, fat mass index (FMI), and fat-free mass index (FFMI) among contemporary Danish 8-11-y olds. METHODS: A total of 760 children from the OPUS School Meal Study provided >2,200 measurements on height, body weight, and composition between September and June. Average velocities were calculated using change-score analyses based on 3-mo intervals. As a complementary analysis, point velocities derived from estimated growth curves were fitted using semiparametric regression that included covariate adjustment and allowed flexible modeling of the time trend. RESULTS: Average velocities showed the following trends: height was higher than the average (6.10 cm/y) in January-April. Body weight was below the average (4.02 kg/y) in August-January and above in January-May; BMI (average: 0.49 kg/ m(2)/y) and FFMI (average: 0.17 kg/m(2)/y) showed similar trends. In contrast, FMI was above the average (0.38 kg/m(2)/y) in November-March. Similar trends were seen for point velocities. CONCLUSION: Our findings suggest seasonality in growth and body composition of Danish children. We recovered the well-known height velocity peak during spring time, but unlike earlier studies, we found coincident peaks in body weight, BMI, and FFMI velocities.

Vitamin D status and its determinants during autumn in children at northern latitudes: a cross-sectional analysis from the optimal well-being, development and health for Danish children through a healthy New Nordic Diet (OPUS) School Meal Study.

Sufficient summer/autumn vitamin D status appears important to mitigate winter nadirs at northern latitudes. We conducted a cross-sectional study to evaluate autumn vitamin D status and its determinants in 782 Danish 8-11-year-old children (55°N) using baseline data from the Optimal well-being, development and health for Danish children through a healthy New Nordic Diet (OPUS) School Meal Study, a large randomised controlled trial. Blood samples and demographic and behavioural data, including 7-d dietary recordings, objectively measured physical activity, and time spent outdoors during school hours, were collected during September-November. Mean serum 25-hydroxyvitamin D (25(OH)D) was 60·8 (sd 18·7) nmol/l. Serum 25(OH)D levels ≤50 nmol/l were found in 28·4 % of the children and 2·4 % had concentrations <25 nmol/l. Upon multivariate adjustment, increasing age (per year) (ß -2·9; 95 % CI -5·1, -0·7 nmol/l), female sex (ß -3·3; 95 % CI -5·9, -0·7 nmol/l), sampling in October (ß -5·2; 95 % CI -10·1, -0·4 nmol/l) and November (ß -13·3; 95 % CI -17·7, -9·1), and non-white ethnicity (ß -5·7; 95 % CI -11·1, -0·3 nmol/l) were negatively associated with 25(OH)D (all P<0·05). Likewise, immigrant/descendant background was negatively associated with 25(OH)D, particularly in females (ß -16·3; 95 % CI -21·9, -10·7) (P<0·001) (P interaction=0·003). Moderate-to-vigorous physical activity (MVPA) (min/d) (ß 0·06; 95 % CI 0·01, 0·12), outdoor walking during school hours (min/week) (ß 0·4; 95 % CI 0·1, 0·6) and intake of vitamin D-containing supplements ≥3 d/week (ß 8·7; 95 % CI 6·4, 11·0) were positively associated with 25(OH)D (all P<0·05). The high proportion of children with vitamin D status below the recommended sufficiency level of 50 nmol/l raises concern as levels expectedly drop further during winter months. Frequent intake of vitamin D supplements was strongly associated with status. MVPA and outdoor activity during school hours should be investigated further in interventions to improve autumn vitamin D status in children at northern latitudes.

Accuracy of self-reported intake of signature foods in a school meal intervention study: comparison between control and intervention period.

Bias in self-reported dietary intake is important when evaluating the effect of dietary interventions, particularly for intervention foods. However, few have investigated this in children, and none have investigated the reporting accuracy of fish intake in children using biomarkers. In a Danish school meal study, 8- to 11-year-old children (n 834) were served the New Nordic Diet (NND) for lunch. The present study examined the accuracy of self-reported intake of signature foods (berries, cabbage, root vegetables, legumes, herbs, potatoes, wild plants, mushrooms, nuts and fish) characterising the NND. Children, assisted by parents, self-reported their diet in a Web-based Dietary Assessment Software for Children during the intervention and control (packed lunch) periods. The reported fish intake by children was compared with their ranking according to fasting whole-blood EPA and DHA concentration and weight percentage using the Spearman correlations and cross-classification. Direct observation of school lunch intake (n 193) was used to score the accuracy of food-reporting as matches, intrusions, omissions and faults. The reporting of all lunch foods had higher percentage of matches compared with the reporting of signature foods in both periods, and the accuracy was higher during the control period compared with the intervention period. Both Spearman's rank correlations and linear mixed models demonstrated positive associations between EPA+DHA and reported fish intake. The direct observations showed that both reported and real intake of signature foods did increase during the intervention period. In conclusion, the self-reported data represented a true increase in the intake of signature foods and can be used to examine dietary intervention effects.

The effects of Nordic school meals on concentration and school performance in 8- to 11-year-old children in the OPUS School Meal Study: a cluster-randomised, controlled, cross-over trial.

It is widely assumed that nutrition can improve school performance in children; however, evidence remains limited and inconclusive. In the present study, we investigated whether serving healthy school meals influenced concentration and school performance of 8- to 11-year-old Danish children. The OPUS (Optimal well-being, development and health for Danish children through a healthy New Nordic Diet) School Meal Study was a cluster-randomised, controlled, cross-over trial comparing a healthy school meal programme with the usual packed lunch from home (control) each for 3 months (NCT 01457794). The d2 test of attention, the Learning Rating Scale (LRS) and standard tests on reading and mathematics proficiency were administered at baseline and at the end of each study period. Intervention effects were evaluated using hierarchical mixed models. The school meal intervention did not influence concentration performance (CP; primary outcome, n 693) or processing speed; however, the decrease in error percentage was 0·18 points smaller (P<0·001) in the intervention period than in the control period (medians: baseline 2·03%; intervention 1·46%; control 1·37%). In contrast, the intervention increased reading speed (0·7 sentence, P=0·009) and the number of correct sentences (1·8 sentences, P<0·001), which corresponded to 11 and 25%, respectively, of the effect of one school year. The percentage of correct sentences also improved (P<0·001), indicating that the number correct improved relatively more than reading speed. There was no effect on overall math performance or outcomes from the LRS. In conclusion, school meals did not affect CP, but improved reading performance, which is a complex cognitive activity that involves inference, and increased errors related to impulsivity and inattention. These findings are worth examining in future trials.

Diet-induced changes in iron and n-3 fatty acid status and associations with cognitive performance in 8-11-year-old Danish children: secondary analyses of the Optimal Well-Being, Development and Health for Danish Children through a Healthy New Nordic Diet School Meal Study.

Fe and n-3 long-chain PUFA (n-3 LCPUFA) have both been associated with cognition, but evidence remains inconclusive in well-nourished school-aged children. In the Optimal Well-Being, Development and Health for Danish Children through a Healthy New Nordic Diet (OPUS) School Meal Study, the 3-month intervention increased reading performance, inattention, impulsivity and dietary intake of fish and Fe. This study investigated whether the intervention influenced n-3 LCPUFA and Fe status and, if so, explored how these changes correlated with the changes in cognitive performance. The study was a cluster-randomised cross-over trial comparing school meals with packed lunch (control). At baseline and after each treatment, we measured serum ferritin, whole-blood n-3 LCPUFA and Hb, and performance in reading, mathematics and d2-test of attention. Data were analysed using mixed models (n 726) and principal component analysis of test performances (n 644), which showed two main patterns: 'school performance' and 'reading comprehension'. The latter indicated that children with good reading comprehension were also more inattentive and impulsive (i.e. higher d2-test error%). The intervention improved 'school performance' (P=0·015), 'reading comprehension' (P=0·043) and EPA+DHA status 0·21 (95% CI 0·15, 0·27) w/w % (P<0·001), but it did not affect serum ferritin or Hb. At baseline, having small Fe stores was associated with poorer 'school performance' in girls, but with better 'reading comprehension' in both boys and girls. Both baseline EPA+DHA status and the intervention-induced increase in EPA+DHA status was positively associated with 'school performance', suggesting that n-3 LCPUFA could potentially explain approximately 20 % of the intervention effect. These exploratory associations indicate that increased fish intake might explain some of the increase in reading performance and inattention in the study.

Vitamin D status is associated with cardiometabolic markers in 8-11-year-old children, independently of body fat and physical activity.

Vitamin D status has been associated with cardiometabolic markers even in children, but the associations may be confounded by fat mass and physical activity behaviour. This study investigated associations between vitamin D status and cardiometabolic risk profile, as well as the impact of fat mass and physical activity in Danish 8-11-year-old children, using baseline data from 782 children participating in the Optimal well-being, development and health for Danish children through a healthy New Nordic Diet (OPUS) School Meal Study. We assessed vitamin D status as serum 25-hydroxyvitamin D (25(OH)D) and measured blood pressure, fasting plasma glucose, homoeostasis model of assessment-insulin resistance, plasma lipids, inflammatory markers, anthropometry and fat mass by dual-energy X-ray absorptiometry, and physical activity by 7 d accelerometry during August-November. Mean serum 25(OH)D was 60·8 (sd 18·7) nmol/l. Each 10 mmol/l 25(OH)D increase was associated with lower diastolic blood pressure (-0·3 mmHg, 95 % CI -0·6, -0·0) (P=0·02), total cholesterol (-0·07 mmol/l, 95 % CI -0·10, -0·05), LDL-cholesterol (-0·05 mmol/l, 95 % CI -0·08, -0·03), TAG (-0·02 mmol/l, 95 % CI -0·03, -0·01) (P≤0·001 for all lipids) and lower metabolic syndrome (MetS) score (P=0·01). Adjustment for fat mass index did not change the associations, but the association with blood pressure became borderline significant after adjustment for physical activity (P=0·06). In conclusion, vitamin D status was negatively associated with blood pressure, plasma lipids and a MetS score in Danish school children with low prevalence of vitamin D deficiency, and apart from blood pressure the associations were independent of body fat and physical activity. The potential underlying cause-effect relationship and possible long-term implications should be investigated in randomised controlled trials.

Provision of healthy school meals does not affect the metabolic syndrome score in 8-11-year-old children, but reduces cardiometabolic risk markers despite increasing waist circumference.

An increasing number of children are exhibiting features of the metabolic syndrome (MetS) including abdominal fatness, hypertension, adverse lipid profile and insulin resistance. Healthy eating practices during school hours may improve the cardiometabolic profile, but there is a lack of evidence. In the present study, the effect of provision of school meals rich in fish, vegetables and fibre on a MetS score (primary outcome) and on individual cardiometabolic markers and body composition (secondary outcomes) was investigated in 834 Danish school children. The study was carried out as a cluster-randomised, controlled, non-blinded, cross-over trial at nine schools. Children aged 8-11 years received freshly prepared school lunch and snacks or usual packed lunch from home (control) each for 3 months. Dietary intake, physical activity, cardiometabolic markers and body composition were measured at baseline and after each dietary period. The school meals did not affect the MetS score (P= 1.00). However, it was found that mean arterial pressure was reduced by 0.4 (95% CI 0.0, 0.8) mmHg (P= 0.04), fasting total cholesterol concentrations by 0.05 (95% CI 0.02, 0.08) mmol/l (P= 0.001), HDL-cholesterol concentrations by 0.02 (95% CI 0.00, 0.03) mmol/l, TAG concentrations by 0.02 (95% CI 0.00, 0.04) mmol/l (both P< 0.05), and homeostasis model of assessment-insulin resistance by 0.10 (95% CI 0.04, 0.16) points (P= 0.001) compared with the control diet in the intention-to-treat analyses. Waist circumference increased 0.5 (95% CI 0.3, 0.7) cm (P< 0.001), but BMI z-score remained unaffected. Complete-case analyses and analyses adjusted for household educational level, pubertal status and physical activity confirmed the results. In conclusion, the school meals did not affect the MetS score in 8-11-year-olds, as small improvements in blood pressure, TAG concentrations and insulin resistance were counterbalanced by slight undesired effects on waist circumference and HDL-cholesterol concentrations.

N-terminal pro-B-type natriuretic peptide levels pre-transcatheter aortic valve implantation and relationship with long-term outcomes.

Background: Blood levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP) has been suggested as a future guidance tool for the selection of patients for aortic valve replacement. This study aimed to examine how levels of NT-proBNP pre-transcatheter aortic valve implantation (TAVI) is associated with one-year rates of heart failure (HF) admission and mortality following TAVI. Methods: With Danish nationwide registries, we identified all patients undergoing TAVI from 2014 to 2021 who had at least one recorded NT-pro-BNP measurement within one year before TAVI. Patients were compared by quartiles of pre-TAVI NT-proBNP: quartile 4 (high NT-proBNP group) vs quartile 1-3 (low NT-proBNP group). Comparisons of all-cause mortality and HF-admissions were conducted using Kaplan-Meier analysis, cumulative incidence, and Cox analysis, as appropriate. Results: We identified 1,140 patients undergoing first-time TAVI with a recorded NT-pro-BNP; 846 (74.2 %) with a low NT-proBNP (<420 pmol/L) (55.0 % male, median age 81 year) and 294 (25.8 %) with a high NT-proBNP (≥420 pmol/L) (53.1 % male, median age 82 year). A high versus low NT-proBNP was associated with increased one-year cumulative incidence of HF-admissions (9.1 % vs. 23.1 %, adjusted HR 2.00 [95 % CI, 1.40-2.85]) and all-cause mortality (6.0 % vs. 14.6 %, adjusted HR 1.95 [95 % CI: 1.24-3.07]). A high NT-proBNP was associated with higher rates of outcomes irrespective of previously known atrial fibrillation, HF, chronic kidney disease, and hypertension. Conclusion: In patients undergoing TAVI, a baseline NT-proBNP ≥ 420 pmol/L was associated with increased one-year rates of HF-admission and mortality post-TAVI and may be utilized to identify a high-risk population.

Performing [18F]MFBG Long-Axial-Field-of-View PET/CT Without Sedation or General Anesthesia for Imaging of Children with Neuroblastoma.

Meta-[123I]iodobenzylguanidine ([123I]MIBG) scintigraphy with SPECT/CT is the standard of care for diagnosing and monitoring neuroblastoma. Replacing [123I]MIBG with the new PET tracer meta-[18F]fluorobenzylguanidine ([18F]MFBG) and further improving sensitivity and reducing noise in a new long-axial-field-of-view (LAFOV) PET/CT scanner enable increased image quality and a faster acquisition time, allowing examinations to be performed without sedation or general anesthesia (GA). Focusing on feasibility, we present our first experience with [18F]MFBG LAFOV PET/CT and compare it with [123I]MIBG scintigraphy plus SPECT/CT for imaging in neuroblastoma in children. Methods: A pilot of our prospective, single-center study recruited children with neuroblastoma who were referred for [123I]MIBG scintigraphy with SPECT/CT. Within 1 wk of [123I]MIBG scintigraphy and SPECT/low-dose CT, [18F]MFBG LAFOV PET/ultra-low-dose CT was performed 1 h after injection (1.5-3 MBq/kg) without sedation or GA, in contrast to the 24-h postinjection interval needed for scanning with [123I]MIBG, the 2- to 2.5-h acquisition time, and the GA often needed in children less than 6 y old. Based on the spirocyclic iodonium-ylide precursor, [18F]MFBG was produced in a fully automated good manufacturing practice-compliant procedure. We present the feasibility of the study. Results: In the first paired scans of the first 10 children included (5 at diagnosis, 2 during treatment, 2 during surveillance, and 1 at relapse), [18F]MFBG PET/CT scan showed a higher number of radiotracer-avid lesions in 80% of the cases and an equal number of lesions in 20% of the cases. The SIOPEN score was higher in 50% of the cases, and the Curie score was higher in 70% of the cases. In particular, intraspinal, retroperitoneal lymph node, and bone marrow involvement was diagnosed with much higher precision. None of the children (median age, 1.6 y; range, 0.1-7.9 y) had sedation or GA during the PET procedure, whereas 80% had GA during [123I]MIBG scintigraphy with SPECT/CT. A PET acquisition time of only 2 min without motion artifacts was the data requirement of the 10-min acquisition time for reconstruction to provide a clinically useful image. Conclusion: This pilot study demonstrates the feasibility of performing [18F]MFBG LAFOV PET/CT for imaging of neuroblastoma. Further, an increased number of radiotracer-avid lesions, an increased SIOPEN score, and an increased Curie score were seen on [18F]MFBG LAFOV PET/CT compared with [123I]MIBG scintigraphy with SPECT/CT, and GA and sedation was avoided in all patients. Thus, with a 1-d protocol, a significantly shorter scan time, a higher sensitivity, and the avoidance of GA and sedation, [18F]MFBG LAFOV PET/CT shows promise for future staging and response assessment and may also have a clinical impact on therapeutic decision-making for children with neuroblastoma.

Risk loci involved in giant cell arteritis susceptibility: a genome-wide association study.

BACKGROUND: Giant cell arteritis is an age-related vasculitis that mainly affects the aorta and its branches in individuals aged 50 years and older. Current options for diagnosis and treatment are scarce, highlighting the need to better understand its underlying pathogenesis. Genome-wide association studies (GWAS) have emerged as a powerful tool for unravelling the pathogenic mechanisms involved in complex diseases. We aimed to characterise the genetic basis of giant cell arteritis by performing the largest GWAS of this vasculitis to date and to assess the functional consequences and clinical implications of identified risk loci. METHODS: We collected and meta-analysed genomic data from patients with giant cell arteritis and healthy controls of European ancestry from ten cohorts across Europe and North America. Eligible patients required confirmation of giant cell arteritis diagnosis by positive temporal artery biopsy, positive temporal artery doppler ultrasonography, or imaging techniques confirming large-vessel vasculitis. We assessed the functional consequences of loci associated with giant cell arteritis using cell enrichment analysis, fine-mapping, and causal gene prioritisation. We also performed a drug repurposing analysis and developed a polygenic risk score to explore the clinical implications of our findings. FINDINGS: We included a total of 3498 patients with giant cell arteritis and 15 550 controls. We identified three novel loci associated with risk of giant cell arteritis. Two loci, MFGE8 (rs8029053; p=4·96 × 10-8; OR 1·19 [95% CI 1·12-1·26]) and VTN (rs704; p=2·75 × 10-9; OR 0·84 [0·79-0·89]), were related to angiogenesis pathways and the third locus, CCDC25 (rs11782624; p=1·28 × 10-8; OR 1·18 [1·12-1·25]), was related to neutrophil extracellular traps (NETs). We also found an association between this vasculitis and HLA region and PLG. Variants associated with giant cell arteritis seemed to fulfil a specific regulatory role in crucial immune cell types. Furthermore, we identified several drugs that could represent promising candidates for treatment of this disease. The polygenic risk score model was able to identify individuals at increased risk of developing giant cell arteritis (90th percentile OR 2·87 [95% CI 2·15-3·82]; p=1·73 × 10-13). INTERPRETATION: We have found several additional loci associated with giant cell arteritis, highlighting the crucial role of angiogenesis in disease susceptibility. Our study represents a step forward in the translation of genomic findings to clinical practice in giant cell arteritis, proposing new treatments and a method to measure genetic predisposition to this vasculitis. FUNDING: Institute of Health Carlos III, Spanish Ministry of Science and Innovation, UK Medical Research Council, and National Institute for Health and Care Research.

Design of the OPUS School Meal Study: a randomised controlled trial assessing the impact of serving school meals based on the New Nordic Diet.

INTRODUCTION: Danish children consume too much sugar and not enough whole grain, fish, fruit, and vegetables. The Nordic region is rich in such foods with a strong health-promoting potential. We lack randomised controlled trials that investigate the developmental and health impact of serving school meals based on Nordic foods. AIM: This paper describes the rationale, design, study population, and potential implications of the Optimal well-being, development and health for Danish children through a healthy New Nordic Diet (OPUS) School Meal Study. METHODS: In a cluster-randomised cross-over design, 1021 children from 3rd and 4th grades (8-11 years old) at nine Danish municipal schools were invited to participate. Classes were assigned to two 3-month periods with free school meals based on the New Nordic Diet (NND) or their usual packed lunch (control). Dietary intake, nutrient status, physical activity, cardiorespiratory fitness, sleep, growth, body composition, early metabolic and cardiovascular risk markers, illness, absence from school, wellbeing, cognitive function, social and cultural features, food acceptance, waste, and cost were assessed. RESULTS: In total, 834 children (82% of those invited) participated. Although their parents were slightly better educated than the background population, children from various socioeconomic backgrounds were included. The proportion of overweight and obese children (14%) resembled that of earlier examinations of Danish school children. Drop out was 8.3%. CONCLUSIONS: A high inclusion rate and low drop out rate was achieved. This study will be the first to determine whether school meals based on the NND improve children's diet, health, growth, cognitive performance, and early disease risk markers.

TLR2 and TLR9 synergistically control herpes simplex virus infection in the brain.

Viruses are recognized by the innate immune system through pattern recognition receptors (PRRs). For instance, HSV virions and genomic DNA are recognized by TLR2 and TLR9, respectively. Although several viruses and viral components have been shown to stimulate cells through TLRs, only very few studies have defined essential roles for single TLRs in innate immune defense in vivo. This could suggest that PRRs act in concert to mount the first line of defense against virus infections. To test this hypothesis we have examined the host response of C57BL/6, TLR2(-/-), TLR9(-/-), and TLR2/9(-/-) mice toward HSV-2 infection. After a systemic infection, the cytokine serum response was markedly reduced in the double knockout mice, but only partly affected in either strain of the single knockout mice. This was supported by in vitro data showing that HSV-induced cytokine expression relayed on TLR2 and TLR9 in a cytokine- and cell type-dependent manner. With respect to the cellular response to infection, we found that recruitment but not activation of NK cells was impaired in TLR2/9(-/-) mice. Importantly, the viral load in the brain, but not liver, was significantly higher in the brain of TLR2/9(-/-) mice whereas the viral loads in organs of single knockout mice were statistically indistinguishable from C57BL/6 mice. In the brain we found that TNF-alpha and the IFN-stimulated gene CXCL9 were expressed during infection and were dependent on either TLR2 or TLR9. Thus, TLR2 and TLR9 synergistically stimulate innate antiviral activities, thereby protecting against HSV infection in the brain.

Simultaneous determination of ochratoxin A, mycophenolic acid and fumonisin B(2) in meat products.

Here we present a method for simultaneous determination of the fungal metabolites mycophenolic acid, ochratoxin A (OTA) and fumonisin B(2) (FB(2)) in meat products. Extraction was performed with water-acetonitrile, followed by acetone-induced precipitation of salts and proteins. Purification and identification of analytes was performed by mixed-mode reversed-phase anion-exchange chromatography in direct ion-exchange mode, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection. Quantification was based on isotope dilution with fully (13)C-labelled FB(2) and OTA, and matrix-spiked calibration curves. Fermented sausages inoculated with an OTA- and FB(2)-producing strain of Aspergillus niger were analysed, but no analytes were detected. Analysis of 22 retail products showed one Parma meat with a very high level of OTA contamination (56-158 microg/kg) that clearly exceeded the Italian regulatory limit of 1 microg/kg. This sample and uninfected control samples were subsequently reanalysed, and the high OTA content was verified by two other techniques: (i) LC-time-of-flight MS confirmed the accurate mass as well as chlorine isotope pattern; and (ii) sample methylation in methanol-BF(3) and subsequent LC-MS/MS provided indirect confirmation by detection of the OTA methyl ester. In the contaminated Parma ham, the high OTA level most likely originated from growth of Penicillium nordicum on the meat.

Proteome analysis of Aspergillus niger: lactate added in starch-containing medium can increase production of the mycotoxin fumonisin B2 by modifying acetyl-CoA metabolism.

BACKGROUND: Aspergillus niger is a filamentous fungus found in the environment, on foods and feeds and is used as host for production of organic acids, enzymes and proteins. The mycotoxin fumonisin B2 was recently found to be produced by A. niger and hence very little is known about production and regulation of this metabolite. Proteome analysis was used with the purpose to reveal how fumonisin B2 production by A. niger is influenced by starch and lactate in the medium. RESULTS: Fumonisin B2 production by A. niger was significantly increased when lactate and starch were combined in the medium. Production of a few other A. niger secondary metabolites was affected similarly by lactate and starch (fumonisin B4, orlandin, desmethylkotanin and pyranonigrin A), while production of others was not (ochratoxin A, ochratoxin alpha, malformin A, malformin C, kotanin, aurasperone B and tensidol B). The proteome of A. niger was clearly different during growth on media containing 3% starch, 3% starch + 3% lactate or 3% lactate. The identity of 59 spots was obtained, mainly those showing higher or lower expression levels on medium with starch and lactate. Many of them were enzymes in primary metabolism and other processes that affect the intracellular level of acetyl-CoA or NADPH. This included enzymes in the pentose phosphate pathway, pyruvate metabolism, the tricarboxylic acid cycle, ammonium assimilation, fatty acid biosynthesis and oxidative stress protection. CONCLUSIONS: Lactate added in a medium containing nitrate and starch can increase fumonisin B2 production by A. niger as well as production of some other secondary metabolites. Changes in the balance of intracellular metabolites towards a higher level of carbon passing through acetyl-CoA and a high capacity to regenerate NADPH during growth on medium with starch and lactate were found to be the likely cause of this effect. The results lead to the hypothesis that fumonisin production by A. niger is regulated by acetyl-CoA.

Comorbid insomnia, psychological symptoms and widespread pain among patients suffering from musculoskeletal pain in general practice: a cross-sectional study.

OBJECTIVE: The aim of this study was to examine the association between musculoskeletal (MSK) pain, insomnia, anxiety and depressive symptoms in patients from general practice. DESIGN: This is a cross-sectional study. SETTING: This study was conducted in general practice in Denmark. PARTICIPANTS: A consecutive sample of 390 general practice patients (aged 12 years or older) were included; 183 patients with MSK pain and 207 patients without MSK pain. To be included in the MSK pain group, participants with MSK pain were required to report MSK pain at least weekly during the preceding month, which had a negative impact on daily activities. PRIMARY AND SECONDARY OUTCOMES MEASURES: The primary outcome was insomnia evaluated by the Athens Insomnia Scale. The secondary outcomes were psychological symptoms assessed by the Hospital Anxiety and Depression Scale. RESULTS: Patients with MSK pain had a significantly higher prevalence of insomnia (difference 25.5%, p<0.0001), anxiety (difference 24.3%, p<0.0001) and depressive symptoms (difference 11%, p<0.0001) compared with patients without MSK pain. Furthermore, patients with MSK pain and comorbid insomnia had significantly higher levels of anxiety and symptoms of depression compared with patients with MSK pain without insomnia (p<0.0001). These relationships remained robust when controlling for age, sex and body mass index in linear regression. CONCLUSION: One in two patients in general practice report MSK pain. Comorbid MSK pain and insomnia are common and are associated with a higher prevalence of anxiety and depression. This highlights the importance of establishing the presence of insomnia and affective disorders as potentially modifiable factors during treatment of MSK pain in general practice.

Type I interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through Toll-like receptor 9, the mitochondrial antiviral signaling protein pathway, and novel recognition systems.

Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes simplex virus (HSV) infection. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent alpha/beta IFN (IFN-alpha/beta) response is derived from several cell types and induced independently of TLR9. In conventional DCs, the IFN response occurred independently of viral replication but was dependent on viral entry. Moreover, using a HSV-1 UL15 mutant, which fails to package viral DNA into the virion, we found that entry-dependent IFN induction also required the presence of viral genomic DNA. In macrophages and fibroblasts, where the virus was able to replicate, HSV-induced IFN-alpha/beta production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms of pathogen recognition are active, which operate in cell-type- and time-dependent manners to trigger expression of type I IFN and coordinate the antiviral response.

Determination of mycophenolic acid in meat products using mixed mode reversed phase-anion exchange clean-up and liquid chromatography-high-resolution mass spectrometry.

A method for determination of mycophenolic acid (MPA) in dry-cured ham, fermented sausage and liver pâté is described. MPA was extracted from meat with bicarbonate-acetonitrile, further cleaned-up by mixed mode reversed phase-anion exchange and detected using a LC-MS system with electrospray ionisation-time-of-flight detection. The limit of detection was 4 microg/kg in sausage and 6 microg/kg in ham and pâté. The method was successfully used for quantification of MPA in dry-cured ham and liver pâté artificially inoculated with Penicillium brevicompactum. Levels ranged from 190 microg/kg in centre to 11 mg/kg in surface of ham and from 150 microg/kg in bottom to 14 mg/kg in surface of pâté.