Winter cholecalciferol supplementation at 55°N has little effect on markers of innate immune defense in healthy children aged 4-8 years: a secondary analysis from a randomized controlled trial.

PURPOSE: We explored the effect of winter cholecalciferol (vitamin D3) supplementation on innate immune markers in healthy Danish children (55°N). METHODS: In the double-blind, placebo-controlled trial, ODIN Junior, 119 healthy, white, 4-8 year-olds were randomized to 0 (placebo), 10 or 20 µg/day of vitamin D3 for 20 weeks (October-March). Cheek mucosal swabs, blood samples, and questionnaires on acute respiratory infections the previous month were collected at baseline and endpoint. Innate immune markers were measured as secondary outcomes including in vivo oral mucosal gene expression of calprotectin (S100A9), lipocalin-2 (LCN2), beta-defensin-4 (DEFB4), interleukin-8 (IL-8), viperin (RSAD2), and the cathelicidin-antimicrobial-peptide (CAMP); ex vivo whole-blood lipopolysaccharide (LPS)-induced cathelicidin, IL-8, and IL-6; and plasma cathelicidin, together with serum 25-hydroxyvitamin D [25(OH)D]. RESULTS: Serum 25(OH)D was 56.7 ± 12.3 nmol/L at baseline and 31.1 ± 7.5, 61.8 ± 10.6, and 75.8 ± 11.5 nmol/L at endpoint after placebo, 10 and 20 µg/day of vitamin D3 (P < 0.0001), respectively. A decreased oral mucosal S100A9 expression with placebo [- 18 (95% CI - 1; - 32)%] was marginally avoided with 20 µg/day [6 (- 13; 28)%] (P = 0.06). Likewise, a decreased LPS-induced IL-8 with placebo [- 438 (95% CI - 693; - 184) ng/L] was marginally avoided with 20 µg/day [- 109 (- 374; 157) ng/L] (P = 0.07). All other immune markers and respiratory infection episodes were unaffected by vitamin D3 supplementation (all P > 0.11). CONCLUSIONS: Winter vitamin D3 supplementation of 10 µg/day did not affect innate immune markers, whereas 20 µg/day tended to maintain the capacity to produce a few markers in healthy children.

Winter Cholecalciferol Supplementation at 55°N Has No Effect on Markers of Cardiometabolic Risk in Healthy Children Aged 4-8 Years.

Background: Low serum 25-hydroxyvitamin D [25(OH)D] has been associated with unfavorable cardiometabolic risk profiles in many observational studies in children, but very few randomized controlled trials have investigated this. Objective: We explored the effect of winter-time cholecalciferol (vitamin D3) supplementation on cardiometabolic risk markers in young, white, 4- to 8-y-old healthy Danish children (55°N) as part of the pan-European ODIN project. Methods: In the ODIN Junior double-blind, placebo-controlled, dose-response trial, 119 children (mean ± SD age: 6.7 ± 1.5 y; 36% male; 82% normal weight) were randomly allocated to 0, 10 or 20 µg/d of vitamin D3 for 20 wk (October-March). Cardiometabolic risk markers including BMI-for-age z score (BMIz), waist circumference, systolic and diastolic blood pressure, serum triglycerides and cholesterol (total, LDL, HDL, and total:HDL), plasma glucose and insulin, and whole-blood glycated hemoglobin were measured at baseline and endpoint as secondary outcomes together with serum 25(OH)D. Intervention effects were evaluated in linear regression models as between-group differences at endpoint adjusted for baseline value of the outcome, and additionally for age, sex, baseline serum 25(OH)D, BMIz, time since breakfast, and breakfast content. Results: Mean ± SD serum 25(OH)D was 56.7 ± 12.3 nmol/L at baseline and differed between groups at endpoint with concentrations of 31.1 ± 7.5, 61.8 ± 10.6, and 75.8 ± 11.5 nmol/L in the 0-, 10-, and 20 µg/d groups, respectively (P < 0.0001). Vitamin D3 supplementation had no effect on any of the cardiometabolic risk markers in analyses adjusted for baseline value of the outcome (all P ≥ 0.05), and additional covariate adjustment did not change the results notably. Conclusions: Preventing the winter decline in serum 25(OH)D with daily vitamin D3 supplementation of 10 or 20 µg had no cardiometabolic effects in healthy 4- to 8-y-old Danish children. This trial was registered at www.clinicaltrials.gov as NCT02145195.

In vitro assessment of the ability of probiotics, blueberry and food carbohydrates to prevent S. pyogenes adhesion on pharyngeal epithelium and modulate immune responses.

Group A streptococci (GAS) cause 20-30% of pediatric pharyngitis episodes, which are a major cause of ambulatory care visits. Therefore, a strategy to prevent GAS dissemination in children could significantly benefit public healthcare. Contextually, we assessed the possibility of employing alternative food-grade strategies to be used with the oral probiotic L. helveticus MIMLh5 for the prevention of pharyngeal infections. First, we demonstrated through an antagonism-by-exclusion assay that guaran may potentially prevent S. pyogenes adhesion on pharyngeal cells. Subsequently, we showed that an anthocyanin-rich fraction extracted from wild blueberry (BbE) exerts anti-inflammatory effects on the human macrophage cell line U937. Finally, we showed that BbE reduces interferon-ß expression in MIMLh5-stimulated murine dendritic cells, resulting in a reduction in the pro-inflammatory cytokines IL-12 and TNF-α. In conclusion, this proof-of-concept study indicates that different food-grade strategies may be concomitantly adopted to potentially prevent GAS colonization and modulate local immune defences.

Reduced ex vivo stimulated IL-6 response in infants randomized to fish oil from 9 to 18 months, especially among PPARG2 and COX2 wild types.

We investigated whether n-3 LCPUFA affected immune function in late infancy and explored effect-modification by single nucleotide polymorphisms (SNPs) and links to intestinal microbiota. Infants (n=105) were randomized to fish oil (FO, 1.2g/d n-3 LCPUFA) or sunflower oil (SO)-supplements from age 9-18 months. Immune function was assessed by ex vivo cytokine production in stimulated blood and plasma immunoglobulin E (IgE). We genotyped functional SNPs in PPARG2 and COX2 and analyzed fecal microbiota by 16S-rRNA terminal restriction fragment length polymorphism. FO compared to SO reduced Lactobacillus paracasei-stimulated IL-6 at 18 months (P=0.03, n=104). This effect was most pronounced among infants wild-type for PPARG2-Pro12Ala and/or COX2-T8473C (P<0.05). Predominant bacterial fragments were associated with 18 months IgE in all infants (P=0.004) (bp100) and with IL-6 production among infants weaned before 9 months (P=0.047) (bp102). Thus, FO reduced IL-6 in a genotype-modified manner. The microbiota was partly linked to IL-6 and IgE, not directly to FO.

Postnatal amniotic fluid intake reduces gut inflammatory responses and necrotizing enterocolitis in preterm neonates.

Preterm neonates are susceptible to gastrointestinal disorders such as necrotizing enterocolitis (NEC). Maternal milk and colostrum protects against NEC via growth promoting, immunomodulatory, and antimicrobial factors. The fetal enteral diet amniotic fluid (AF), contains similar components, and we hypothesized that postnatal AF administration reduces inflammatory responses and NEC in preterm neonates. Preterm pigs (92% gestation) were delivered by caesarean section and fed parental nutrition (2 days) followed by enteral (2 days) porcine colostrum (COLOS, n = 7), infant formula (FORM, n = 13), or AF supplied before and after introduction of formula (AF, n = 10) in experiment 1, and supplied only during the enteral feeding period in experiment 2 (FORM, n = 16; AF, n = 14). The NEC score was reduced in both AF and COLOS pigs, relative to FORM, when AF was provided prior to full enteral feeding (9.9 and 7.7 compared with 17.3, P < 0.05). There was no effect of AF when provided only during enteral feeding. AF pigs showed decreased bacterial abundance in colon and intestinal inflammation-related genes (e.g., TNF-α, IL-1α, IL-6, NOS) were downregulated, relative to FORM pigs with NEC. Anti-inflammatory properties of AF were supported by delayed maturation and decreased TNF-α production in murine dendritic cells, as well as increased proliferation and migration, and downregulation of IL-6 expression in intestinal cells (IEC-6, IPEC-J2). Like colostrum, AF may reduce NEC development in preterm neonates by suppressing the proinflammatory responses to enteral formula feeding and gut colonization when provided before the onset of NEC.

Astragalus root and elderberry fruit extracts enhance the IFN-ß stimulatory effects of Lactobacillus acidophilus in murine-derived dendritic cells.

Many foods and food components boost the immune system, but little data are available regarding the mechanisms by which they do. Bacterial strains have disparate effects in stimulating the immune system. In dendritic cells, the gram-negative bacteria Escherichia coli upregulates proinflammatory cytokines, whereas gram-positive Lactobacillus acidophilus induces a robust interferon (IFN)-ß response. The immune-modulating effects of astragalus root and elderberry fruit extracts were examined in bone marrow-derived murine dendritic cells that were stimulated with L. acidophilus or E. coli. IFN-ß and other cytokines were measured by ELISA and RT-PCR. Endocytosis of fluorescence-labeled dextran and L. acidophilus in the presence of elderberry fruit or astragalus root extract was evaluated in dendritic cells. Our results show that both extracts enhanced L. acidophilus-induced IFN-ß production and slightly decreased the proinflammatory response to E. coli. The enhanced IFN-ß production was associated with upregulation of toll-like receptor 3 and to a varying degree, the cytokines IL-12, IL-6, IL-1ß and TNF-α. Both extracts increased endocytosis in immature dendritic cells, and only slightly influenced the viability of the cells. In conclusion, astragalus root and elderberry fruit extracts increase the IFN-ß inducing activity of L. acidophilus in dendritic cells, suggesting that they may exert antiviral and immune-enhancing activity.

Comparative analysis of a large panel of non-starch polysaccharides reveals structures with selective regulatory properties in dendritic cells.

SCOPE: Structural-based recognition of foreign molecules is essential for activation of dendritic cells (DCs) that play a key role in regulation of gut mucosal immunity. Orally ingested non-starch polysaccharides (NSP) are ascribed many health-promoting properties, but currently we lack insight into the impact of structure and size for their capacity to affect immune responses. METHODS AND RESULTS: This study addresses the importance of chemical structure, size, origin and presence of contaminants for the capacity of both dietary and non-food NSP to modulate DC. Of 28 NSP products, ß-glucans of microbial and plant origin and the galactomannan guar gum were found to modulate the DC cytokine pattern induced by the Toll-like receptor 4-ligand LPS giving rise to reduced IL-12p70 and increased IL-10 levels, whereas IL-6 production was unaffected. A large proportion of the tested NSP were able to down-regulate LPS-induced IL-12p70 production. The most potent NSP induced up-regulation of CD86 on DC independently of LPS stimulation. Cereal-based ß-glucans showed less potency than ß-glucans of microbial origin, but proper molecular weight composition and preparation may improve effectiveness. CONCLUSIONS: Collectively, this comparative study revealed that some plant-derived NSP besides those of microbial origin exert modulation of the DC phenotype, with the exact structure being important for the activity.

Bovine colostrum is superior to enriched formulas in stimulating intestinal function and necrotising enterocolitis resistance in preterm pigs.

Milk contains immunomodulatory compounds that may be important to protect the immature intestine in preterm neonates from harmful inflammatory reactions involved in disorders like necrotising enterocolitis (NEC). We hypothesised that bovine colostrum and milk formulas enriched with sialic acids (SL), gangliosides (Gang) or osteopontin (OPN) would improve gastrointestinal function and NEC resistance in preterm neonates. Forty-seven caesarean-delivered preterm pigs were given total parenteral nutrition for 2 d followed by 1·5 d of enteral feeding. In Expt 1, a control formula was compared with an OPN-enriched formula (n 13), while Expt 2 compared a control formula with bovine colostrum or formulas enriched with Gang or SL (n 4-6). OPN enrichment decreased NEC severity relative to control formula (P < 0·01), without any significant effects on NEC incidence, digestive enzyme activities and hexose absorption. Neither SL- nor Gang-enriched formulas improved NEC resistance or digestive functions, while all the intestinal functional parameters were significantly improved in pigs fed bovine colostrum, relative to formula. The effects in vivo were supported in vitro by bacteria- and dose-dependent modulation by colostrum whey of the cytokine response from bacteria-stimulated murine bone marrow-derived dendritic cells (DC). In conclusion, OPN had only moderate NEC-protective effects, while formulas enriched with Gang or SL were ineffective. The observed modulation of DC cytokine response by bovine colostrum whey in vitro may be due to a synergistic action of various milk bioactives, and it may explain its beneficial effects on NEC development and intestinal function in a piglet model of preterm infants.

Fish oil in combination with high or low intakes of linoleic acid lowers plasma triacylglycerols but does not affect other cardiovascular risk markers in healthy men.

Both (n-3) long-chain PUFA (LCPUFA) and linoleic acid [LA, 18:2(n-6)] improve cardiovascular disease (CVD) risk factors, but a high-LA intake may weaken the effect of (n-3) LCPUFA. In a controlled, double-blind, 2 x 2-factorial 8-wk intervention, we investigated whether fish oil combined with a high- or low-LA intake affects overall CVD risk profile. Healthy men (n = 64) were randomized to 5 mL/d fish oil capsules (FO) [mean intake 3.1 g/d (n-3) LCPUFA] or olive oil capsules (control) and to oils and spreads with either a high (S/B) or a low (R/K) LA content, resulting in a 7.3 g/d higher LA intake in the S/B groups than in the R/K groups. Diet, (n-3) LCPUFA in peripheral blood mononuclear cells, blood pressure (BP), heart rate (HR), and plasma CVD risk markers were measured before and after the intervention. FO lowered fasting plasma triacylglycerol (TAG) (P < 0.001) by 51% and 19% in the FO+R/K-group and FO+S/B-group, respectively, which was also reflected in postprandial TAG measured after the intervention (P < 0.01). Although a fat x FO interaction was found for monocyte chemoattractant protein-1, neither the FO nor fat intervention affected fasting plasma cholesterol, glucose, insulin, fibrinogen, C-reactive protein, interleukin-6, vascular cell adhesion molecule-1, P-selectin, oxidized LDL, cluster of differentiation antigen 40 ligand (CD40L), adiponectin, or fasting or postprandial BP or HR after adjustment for body weight changes. In conclusion, neither fish oil supplementation nor the LA intake had immediate pronounced effects on the overall CVD risk profile in healthy men, but fish oil lowered plasma TAG in healthy subjects with initially low concentrations.

The effects of fish oil and high or low linoleic acid intake on fatty acid composition of human peripheral blood mononuclear cells.

Dietary intake of 18: 2n-6 and 18: 3n-3 may affect endogenous production and incorporation of n-3 long-chain PUFA (LCPUFA) from fish oils (FO). This double-blinded controlled 2 x 2-factorial 8-week intervention investigates the effects of high and low 18: 2n-6 intake in combination with FO-supplementation on tissue fatty acid composition. Healthy young men (n 64) were randomized to capsules with FO or olive oil (control) (4.4 (2.0-5.6) ml/d) and to either sunflower oil and margarine (S/B) or rapeseed oil and a butter spread (R/K) to provide a high or a low 18: 2n-6 intake. Diet was measured by 4-d weighed dietary records at baseline, during and 8 weeks after the intervention and tissue incorporation as fatty acid composition of peripheral blood mononuclear cells (PBMC). The fat intervention gave a mean difference in the 18: 2n-6 intake of 7.3 g/d (95 % CI 4.6, 10.0) and a similar 18: 3n-3 intake in the groups. The R/K groups had a 0.2 % fatty acid (FA%) (95 % CI 0.0, 0.4, P = 0.02) higher content of 22: 5n-3 in the PBMC, a tendency of slightly higher 20: 5n-3 (P = 0.06), but no more 22: 6n-3 (P = 0.83) than the S/B groups. FO effectively raised the PBMC content of all n-3 LCPUFA (P < 0.001). The fat intervention did not markedly influence the effect of FO; the mean PBMC content of n-3 LCPUFA was 10.3 (sem 0.3) FA% in the FO+S/B group and 10.6 (sem 0.2) FA% in the FO+R/K group. In conclusion, increasing the 18: 2n-6 intake did not have any pronounced effect on incorporation of n-3 LCPUFA in PBMC, either alone or with simultaneous FO supplementation.