Infant blood concentrations of folate markers and catabolites are modified by 5,10-methylenetetrahydrofolate reductase C677T genotype and dietary folate source.

BACKGROUND: Folate intake and polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene may affect folate metabolism in infants. OBJECTIVES: We investigated the association between infant's MTHFR C677T genotype, the dietary folate source, and concentrations of folate markers in the blood. METHODS: We studied 110 breastfed infants (reference) and 182 infants who were randomly assigned to receive infant formulas enriched with either 78 µg folic acid or 81 µg (6S)-5-methyltetrahydrofolate (5-MTHF) per 100 g milk powder for 12 wk. The blood samples were available at the ages of <1 mo (baseline) and 16 wk. MTHFR genotype and concentrations of folate markers and catabolites [i.e., para-aminobenzoylglutamate (pABG)] were analyzed. RESULTS: At baseline, carriers of the TT genotype (vs. CC) had lower mean (SD) concentrations (all in nmol/L) of red blood cell (RBC) folate [1194 (507) vs. 1440 (521), P = 0.033) and plasma pABG [5.7 (4.9) vs. 12.5 (8.1), P < 0.001] but higher plasma 5-MTHF [33.9 (16.8) vs. 24.0 (12.6), P < 0.001]. Irrespective of the genotype, infant formula with 5-MTHF (vs. folic acid) caused a significant increase in RBC folate concentration [1278 (466) vs. 947 (552), P < 0.001]. In breastfed infants, plasma concentrations of 5-MTHF and pABG increased significantly by 7.7 (20.5) and 6.4 (10.5), respectively, from baseline to 16 wk. Infant formula that complies with the present EU legislation for folate intake increased RBC folate and plasma pABG concentrations at 16 wk (P < 0.001) than formula-fed infants. At 16 wk, plasma pABG concentrations remained ∼50% lower in carriers of the TT (vs. the CC) genotype among all feeding groups. CONCLUSIONS: Folate intake from infant formula according to the present EU legislation increased RBC folate and plasma pABG concentrations in infants to a greater extent than breastfeeding, particularly in carriers of the TT genotype. However, this intake did not completely abolish the between-genotype differences in pABG. Whether these differences have any clinical relevance, however, remains unclear. This trial was registered at clinicaltrials.gov as NCT02437721.

Metabolomic signatures distinguish the impact of formula carbohydrates on disease outcome in a preterm piglet model of NEC.

BACKGROUND: Major risk factors for necrotizing enterocolitis (NEC) include premature birth and formula feeding in the context of microbial colonization of the gastrointestinal tract. We previously showed that feeding formula composed of lactose vs. corn syrup solids protects against NEC in preterm pigs; however, the microbial and metabolic effects of these different carbohydrates used in infant formula has not been explored. OBJECTIVE: Our objective was to characterize the effects of lactose- and corn syrup solid-based formulas on the metabolic and microbial profiles of preterm piglets and to determine whether unique metabolomic or microbiome signatures correlate with severity or incidence of NEC. DESIGN/METHODS: Preterm piglets (103 days gestation) were given total parenteral nutrition (2 days) followed by gradual (5 days) advancement of enteral feeding of formulas matched in nutrient content but containing either lactose (LAC), corn syrup solids (CSS), or 1:1 mix (MIX). Gut contents and mucosal samples were collected and analyzed for microbial profiles by sequencing the V4 region of the 16S rRNA gene. Metabolomic profiles of cecal contents and plasma were analyzed by LC/GC mass spectrometry. RESULTS: NEC incidence was 14, 50, and 44% in the LAC, MIX, and CSS groups, respectively. The dominant classes of bacteria were Bacilli, Clostridia, and Gammaproteobacteria. The number of observed OTUs was lowest in colon contents of CSS-fed pigs. CSS-based formula was associated with higher Bacilli and lower Clostridium from clusters XIVa and XI in the colon. NEC was associated with decreased Gammaproteobacteria in the stomach and increased Clostridium sensu stricto in the ileum. Plasma from NEC piglets was enriched with metabolites of purine metabolism, aromatic amino acid metabolism, and bile acids. Markers of glycolysis, e.g., lactate, were increased in the cecal contents of CSS-fed pigs and in plasma of pigs which developed NEC. CONCLUSIONS: Feeding formula containing lactose is not completely protective against NEC, yet selects for greater microbial richness associated with changes in Bacilli and Clostridium and lower NEC incidence. We conclude that feeding preterm piglets a corn syrup solid vs. lactose-based formula increases the incidence of NEC and produces distinct metabolomic signatures despite modest changes in microbiome profiles.

Growth and tolerance of formula with lactoferrin in infants through one year of age: double-blind, randomized, controlled trial.

BACKGROUND: Human milk provides necessary macronutrients (protein, carbohydrate, fat) required for infant nutrition. Lactoferrin (Lf), a multifunctional iron-binding protein predominant in human milk, shares similar protein sequence, structure, and bioactivity with bovine Lf (bLf). This large-scale pediatric nutrition study was designed to evaluate growth and tolerance in healthy infants who received study formulas with bLf at concentrations within the range of mature human milk. METHODS: In this multi-center, double-blind, parallel-designed, gender-stratified prospective study 480 infants were randomized to receive a marketed routine cow's milk-based infant formula (Control; n = 155) or one of two investigational formulas with bLf at 0.6 g/L (LF-0.6; n = 165) or 1.0 g/L (LF-1.0; n = 160) from 14-365 days of age. Investigational formulas also had a prebiotic blend of polydextrose (PDX) and galactooligosaccharides (GOS) and adjusted arachidonic acid (ARA). The primary outcome was weight growth rate from 14-120 days of age. Anthropometric measurements were taken at 14, 30, 60, 90, 120, 180, 275, and 365 days of age. Parental recall of formula intake, tolerance, and stool characteristics was collected at each time point. Medically-confirmed adverse events were collected throughout the study period. RESULTS: There were no group differences in growth rate (g/day) from 14-120 days of age; 353 infants completed the study through 365 days of age ( CONTROL: 110; LF-0.6: 127; LF-1.0: 116). Few differences in growth, formula intake, and infant fussiness or gassiness were observed through 365 day of age. Group discontinuation rates and the overall group incidence of medically-confirmed adverse events were not significantly different. From 30 through 180 days of age, group differences in stool consistency (P < 0.005) were detected with softer stools for infants in the LF-0.6 and LF-1.0 groups versus CONTROL. CONCLUSION: Compared to the Control, infants who received investigational formulas with bLf and the prebiotic blend of PDX and GOS experienced a softer stooling pattern similar to that reported in breastfed infants. This study demonstrated routine infant formulas with bLf, a blend of PDX and GOS, and adjusted ARA were safe, well-tolerated, and associated with normal growth when fed to healthy term infants through 365 days of age. TRIAL REGISTRATION: ClinicalTrials.gov NCT01122654 . Registered 10 May 2010.

Delayed initiation but not gradual advancement of enteral formula feeding reduces the incidence of necrotizing enterocolitis (NEC) in preterm pigs.

Enteral formula feeding is a risk factor for necrotizing enterocolitis (NEC) in premature infants, yet studies are conflicting regarding the safest timing for introduction and advancement of feeds. Our aim was to test the effects of early vs. late initiation and abrupt vs. gradual advancement of enteral feeding of an intact vs. hydrolyzed protein formula on NEC incidence and severity in preterm pigs. In Experiment 1, preterm pigs received total parenteral nutrition (TPN) at birth with abrupt initiation of enteral formula feeds (50% full intake) on d of life (DOL) 2 (EA) or 5 (LA) while PN continued. Pigs were also fed formula containing either intact or hydrolyzed protein. In Experiment 2, preterm pigs received TPN at birth with enteral, hydrolyzed-protein formula feeds introduced on DOL 2 either abruptly (EA; 50% full feeds) or gradually (EG; 10-50% full feeds over 5 d) while PN continued. NEC incidence and severity were assessed based on macroscopic and histological scoring. In Experiment 1, NEC incidence (41% vs. 70%, P<0.05) and severity were reduced in LA vs. EA groups and LA was associated with a higher survival rate, daily weight gain and jejunum villus height. Piglets fed hydrolyzed vs. intact protein formula had lower stomach content weights and similar NEC incidence. In Experiment 2, NEC incidence and severity were not different between pigs the EG vs. EA group. Proinflammatory gene expression (IL-1ß, IL-6 and S100A9) in the ileum was lower in both LA and EG vs. EA groups. In conclusion, delayed initiation but not gradual advancement of enteral feeding is protective against NEC in preterm pigs. Feeding hydrolyzed vs. intact protein formula improved gastric transit without affecting the NEC incidence.

Adherence inhibition of Cronobacter sakazakii to intestinal epithelial cells by lactoferrin.

Cronobacter sakazakii is now recognized as an opportunistic pathogen and has been implicated in rare but severe cases of necrotizing enterocolitis, meningitis, and sepsis in neonates. The first step in bacterial pathogenesis requires that the organism adheres to host cells surfaces; therefore, agents that inhibit adherence might be useful for preventing infections. Lactoferrin, an iron binding protein found in milk, has been shown to inhibit bacterial adherence by direct interaction and disruption of bacterial surfaces. Therefore, the goal of this research was to assess the ability of two different types of bovine lactoferrin, alone and in combination with a 1:1 blend of galactooligosaccharides and polydextrose, to inhibit adherence of C. sakazakii to a HEp-2 human cell line. Results showed that the adherence of C. sakazakii was significantly reduced at a minimum lactoferrin concentration of 10 mg/ml. However, in combination with the oligosaccharide blend, no synergistic effect was observed in adherence inhibition. These results suggest that lactoferrin might interact with C. sakazakii and directly inhibit adhesion to tissue culture cells.

Behavioral effects of bovine lactoferrin administration during postnatal development of rats.

We tested the hypothesis that rats consuming bovine lactoferrin (bLf) during postnatal development would show better performance of stressful tasks during adolescence. In the first study, we orally administered bLf (750 mg/kg) once daily between postnatal days 16-34. Rats then underwent a battery of behavioral tests: open field (forced exploration of risky environment), light-dark emergence (voluntary exploration of risky environment), baited holeboard (working and reference memory), food neophobia (preference for familiar versus novel food), forced swim (test for antidepressant efficacy), and shuttle-box escape (learning to escape footshock). bLf-supplemented rats showed less exploration of the risky environment, greater preference for the familiar food odor, and faster escape responses. The effect of bLf on forced-swim behavior depended on sex: immobility increased for males and decreased for females. In the next study, we replaced the forced-swim test with an escape-swim test in which rats learned to use a visual cue to locate an escape platform, and we tested the dose response of bLf on this and the shuttle-box escape test, with subjects receiving vehicle or bLf at 500, 1,000, or 2,000 mg/kg. Under this modified testing battery, improvement of escape from footshock was not observed at any dose. However, males, but not females, showed a significant dose-dependent effect of bLf on acquisition of the water-escape task. On average, males receiving a higher dose mastered the task 20-25 % sooner than rats receiving a lower dose or vehicle. These results offer preliminary evidence that bLf supplementation during development can improve subsequent cognitive performance during stress.

Lack of effect of bovine lactoferrin in respiratory syncytial virus replication and clinical disease severity in the mouse model.

BACKGROUND: Lactoferrin (LF) is a glycoprotein present in human milk with known antimicrobial effects. In vitro, LF has demonstrated antiviral activity against respiratory syncytial virus (RSV). We sought to assess the effect of bovine (b)LF in RSV replication, lung inflammation and function, cytokine profiles and clinical disease in an in vivo murine model. METHODS: Female BALB/c mice were inoculated with 10(7)PFU RSV A2 or 10% EMEM. bLF or placebo (DPBS) were administered once or twice daily by oral gavage or intraperitoneal (IP) injection at doses ranging from 2 to 10mg/animal/day, from 48h before until 96h post-RSV inoculation. Bronchoalveolar lavage (BAL), whole lung and serum samples were harvested on day 5 post-inoculation to asses RSV loads, lung inflammation and cytokine concentrations. Weight loss, airway obstruction and disease severity were assessed daily in all groups. RESULTS: On day 5 post-inoculation BAL RSV loads, lung inflammation and serum innate, Th1, Th2 and Th17 cytokine concentrations showed no differences between RSV infected mice treated with bLF and RSV infected but untreated mice independent of bLF dosing and administration route (p>0.05). In addition, all bLF groups showed similar weight loss, degree of airway obstruction, and disease severity scores on days 1-5 post-inoculation which was comparable to infected untreated mice (p>0.05) but higher than uninfected controls. CONCLUSIONS: Administration of oral or IP bLF at different doses did not demonstrate antiviral activity or significant effects on disease severity in the RSV mouse model. Whether these observations could be extrapolated to infants at risk for RSV infection needs to be further explored.

Adherence inhibition of Cronobacter sakazakii to intestinal epithelial cells by prebiotic oligosaccharides.

Cronobacter sakazakii is an opportunistic pathogen that has been implicated in meningitis, NEC, and sepsis in neonates. Colonization and subsequent infection and invasion of C. sakazakii require that the organism adheres to host cell surfaces. Agents that inhibit or block attachment of the pathogen to epithelial cells could be useful in reducing infections. The goal of this research was to assess the ability of prebiotic galactooligosaccharides (GOS) and polydextrose (PDX) to inhibit adherence of C. sakazakii 4603 to a HEp-2 human cell line. Adherence experiments were performed in the presence or absence of prebiotics using HEp-2 cells grown to confluency on glass coverslips. Prebiotics and bacteria were added and incubated for 3 h. Coverslips were washed, and adherence was determined by cultural and microscopic methods. When measured microscopically or by cultural methods, significant reductions in adherence (56 and 71%, respectively) of C. sakazakii were observed in the presence of GOS (16 mg/ml). Adherence inhibition also occurred (48%) when a GOS-PDX blend (8 mg/ml each) was tested, although PDX by itself had less effect. Similar results were also observed for Caco-2 cells and also for another strain of C. sakazakii (29004). These results suggest that GOS and PDX, alone and in combination, may have an anti-adhesive effect on C. sakazakii and directly inhibit the adherence to gastrointestinal epithelial cells.

Vitamin D receptor expression by the lung micro-environment is required for maximal induction of lung inflammation.

Mice lacking the vitamin D receptor (VDR) are resistant to airway inflammation. Pathogenic immune cells capable of transferring experimental airway inflammation to wildtype (WT) mice are present and primed in the VDR KO mice. Furthermore, the VDR KO immune cells homed to the WT lung in sufficient numbers to induce symptoms of asthma. Conversely, WT splenocytes, Th2 cells and hematopoetic cells induced some symptoms of experimental asthma when transferred to VDR KO mice, but the severity was less than that seen in the WT controls. Interestingly, experimentally induced vitamin D deficiency failed to mirror the VDR KO phenotype suggesting there might be a difference between absence of the ligand and VDR deficiency. Lipopolysaccharide (LPS) induced inflammation in the lungs of VDR KO mice was also less than in WT mice. Together the data suggest that vitamin D and the VDR are important regulators of inflammation in the lung and that in the absence of the VDR the lung environment, independent of immune cells, is less responsive to environmental challenges.

Vitamin D status, 1,25-dihydroxyvitamin D3, and the immune system.

Vitamin D is an important immune system regulator. The active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], has been shown to inhibit the development of autoimmune diseases, including inflammatory bowel disease (IBD). Paradoxically, other immune system-mediated diseases (experimental asthma) and immunity to infectious organisms were unaffected by 1,25(OH)2D3 treatment. There are similar paradoxical effects of vitamin D deficiency on various immune system functions. Vitamin D and vitamin D receptor (VDR) deficiency resulted in accelerated IBD. Experimental asthma was unaffected by 1,25(OH)2D3 treatment and was less severe among VDR-deficient mice. Vitamin D is a selective regulator of the immune system, and the outcome of 1,25(OH)2D3 treatment, vitamin D deficiency, or VDR deficiency depends on the nature of the immune response (eg, infectious disease, asthma, or autoimmune disease). An additional factor that determines the effect of vitamin D status on immune function is dietary calcium. Dietary calcium has independent effects on IBD severity. Vitamin D-deficient mice on low-calcium diets developed the most severe IBD, and 1,25(OH)2D3 treatment of mice on low-calcium diets improved IBD symptoms. However, the best results for IBD were observed when the calcium concentration was high and 1,25(OH)2D3 was administered. Both the type of immune response and the calcium status of the host determine the effects of vitamin D status and 1,25(OH)2D3 on immunity.

Vitamin D receptor-deficient mice fail to develop experimental allergic asthma.

The active metabolite of vitamin D (1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))) is known to modulate the immune response in Th1 cell-directed diseases. To investigate the role of vitamin D in Th2 cell-directed diseases, experimental allergic asthma was induced in vitamin D receptor (VDR) knockout and in wild-type (WT) mice. As expected, WT mice developed symptoms of airway inflammation with an influx of eosinophils, elevated Th2 cytokine levels, mucous production, and airway hyperresponsiveness. The administration of 1,25(OH)(2)D(3) had no effect on asthma severity. The only discernable effect of 1,25(OH)(2)D(3) on experimental allergic asthma in WT mice was an increased expression of two Th2-related genes (soluble CD23 and GATA-3) in lungs of BALB/c mice exposed to Ag through the nasal route only. By contrast, asthma-induced VDR knockout mice failed to develop airway inflammation, eosinophilia, or airway hyperresponsiveness, despite high IgE concentrations and elevated Th2 cytokines. The data suggest that although 1,25(OH)(2)D(3) induced these Th2-type genes, the treatment failed to have any affect on experimental asthma severity. However, VDR-deficient mice failed to develop experimental allergic asthma, suggesting an important role for the vitamin D endocrine system in the generation of Th2-driven inflammation in the lung.

The targets of vitamin D depend on the differentiation and activation status of CD4 positive T cells.

Vitamin D is a potent immune system regulator. The active form of vitamin D (1,25(OH)(2)D(3)) suppresses the development of animal models of human autoimmune diseases. 1,25(OH)(2)D(3) decreased the proliferation of all T helper (h) cells and decreased the production of IFN-gamma, IL-2, and IL-5. In Th2 cells 1,25(OH)(2)D(3) increased the production of IL-4. Quiescent CD4+ T cells express vitamin D receptors but only at a low level, which increased five-fold following activation. 1,25(OH)(2)D(3) treatment of Th0 cells, but not Th1 or Th2 cells, induced the expression of the transcription factor GATA-3. Microarray technology identified over 102 targets of 1,25(OH)(2)D(3) in CD4+ T cells. Of the 102 genes, 57 genes were down-regulated and 45 were up-regulated by 1,25(OH)(2)D(3) treatment of the CD4+ T cells. Two of the identified genes are regulators of NFkB. Other genes of interest included the IL-2Rbeta gene and IgE binding factor. Th2 and Th0 cells produced more IgE binding factor after treatment with 1,25(OH)(2)D(3) while Th1 cell IgE binding factor expression was unaffected by 1,25(OH)(2)D(3) addition. It is unclear why some of the genes identified are expressed in CD4+ T cells and furthermore why 1,25(OH)(2)D(3) regulates the expression of these genes. Clearly CD4+ T cells can be direct targets of vitamin D. The targets of vitamin D in CD4+ T cells depend on the state of activation and differentiation status of the cells.

Transformation of Bacillus subtilis in chocolate milk: evidence for low frequency of establishment of cells transformed under non-selective conditions.

Transformation of naturally competent Bacillus subtilis with plasmid was carried out in chocolate milk without antibiotics. Transformed cells were enumerated during the entire growth phase in chocolate milk. When DNA was added to aliquots of a batch culture after different times of incubation, transformation events were detected at all different growth stages. When DNA was added to a batch culture together with the inoculum, transformed cells were detected at the onset of exponential growth. However, apparently no or only limited growth of these transformed cells was observed. To clarify, whether the limitation of growth was due to suppression by non-transformed cells, different proportions of B. subtilis cells either carrying or not carrying the plasmid were mixed and inoculated into chocolate milk without antibiotic. Our results indicate that suppression appears to be of minor importance. Instead, plasmid-bearing cells appear to suffer from a prolonged lag-phase. However, the failure to exhibit significant growth of cells which had taken up the plasmid in chocolate milk appears to be due to failure of these cells to establish themselves as permanently transformed under non-selective conditions.