Search

Background Polymorphic alleles of the vitamin D (vitD)-binding protein (VDBP) gene are associated with discriminatory differences in circulating concentrations of 25-hydroxyvitamin D (25-D), the indicator of vitD status (sufficiency defined by the Endocrine Society as ≥75 nmol/L). Within a diverse group of children, we hypothesized that reaching recommended daily allowance (RDA) of vitD intake would have differential impact on vitD status depending on VDBP variability. Methods VDBP alleles (Gc1S, Gc1F, Gc2) in 123 children (1–4 annual visits/child; ages 1–8 years) were compared for relationships with serum 25-D concentrations and daily vitD intake. Results In African-American children, reaching the vitD RDA was associated with significantly higher mean serum 25-D concentrations for the 20% carrying the VDBP 1S allele than for the large majority without this allele (77 vs. 61 nmol/L 25-D; p = 0.038). Children with the Gc1S/1S homozygous genotype (30% Caucasians, 24% Hispanics, 2% African-Americans) who met RDA had 51% (39 nmol/L) greater mean serum 25-D than those below RDA (p

Background For the second aim of the Kellogg Foundation grant, this double-blind RCT investigated the impact of plasma vitamin D metabolite 25-hydroxyvitamin D (25(OH)D) on plasma immune-mediators during pregnancy. We hypothesized that higher 25(OH)D concentrations would associate with reduced pro-inflammatory and increased tolerogenic immune-mediator concentrations. Methods Pregnant women enrolled at 10-14 weeks gestation were randomized to 400 or 4400 IU vitamin D3/day. Data on health, safety, circulating 25(OH)D, and 9 immune-mediators were collected at each trimester. Associations between immune-mediators and 25(OH)D at baseline and at second and third trimesters were examined. Results Baseline TGF-β and second and third trimesters IFN-γ and IL-2 were associated with baseline 25(OH)D. Baseline immune-mediators were associated with immune-mediators at second and third trimesters for all immune-mediators except IL-5 and IL-10. Race was associated with baseline TGF-β, VEGF and IL-10 and with IL-10 at second and third trimesters. Conclusions Both treatment groups had increased 25(OH)D at second and third trimesters, greatest in the 4400 IU group. Though associations between baseline 25(OH)D and baseline TGF-β and second and third trimester IFN-γ and IL-2 were noted, vitamin D supplementation throughout pregnancy did not impact immune-mediators at later trimesters. Supplementing with vitamin D before conception conceivably influences immune-mediator responses during pregnancy. Impact In this vitamin D supplementation clinical trial, baseline (first trimester) but not increasing plasma 25(OH)D concentration impacted select plasma immune-mediator profiles in pregnant women. Baseline 25(OH)D was associated with baseline TGF-β and with IFN-γ and IL-2 at second and third trimesters. Baseline IFN-γ, CRP, TGF-β, TNF-α, VEGF, IL-2, and IL-4 were associated with concentrations at second and third trimesters for respective immune-mediators; however, 25(OH)D concentration at second and third trimesters were not. Some racial differences existed in immune-mediator concentrations at baseline and at second and third trimesters. This study assesses the impact of vitamin D supplementation on multiple immune-mediators in pregnant women of different racial/ethnic groups using longitudinal data from a relatively large randomized controlled trial. This study found that race was associated with baseline TGF-β, VEGF, and IL-10 and with IL-10 at second and third trimesters, a novel finding that sheds light where relationships were less well defined. The results of this study suggest that vitamin D supplementation before conception or early in pregnancy, rather than during pregnancy, may be necessary to significantly impact immune-mediator response. This study sets premise for future clinical trials to evaluate the effect of vitamin D supplementation before conception or prior to pregnancy.

Background Vitamin D has neuroprotective and immunomodulatory properties, and deficiency is associated with worse stroke outcomes. Little is known about effects of hypoxia-ischemia or hypothermia treatment on vitamin D status in neonates with hypoxic-ischemic encephalopathy (HIE). We hypothesized vitamin D metabolism would be dysregulated in neonatal HIE altering specific cytokines involved in Th17 activation, which might be mitigated by hypothermia. Methods We analyzed short term relationships between 25(OH) and 1,25(OH)2 vitamin D, vitamin D binding protein, and cytokines related to Th17 function in serum samples from a multicenter randomized controlled trial of hypothermia 33°C for 48h after HIE birth versus normothermia in 50 infants with moderate to severe HIE. Results Insufficiency of 25(OH) vitamin D was observed after birth in 70% of infants, with further decline over the first 72h, regardless of treatment. 25(OH) vitamin D positively correlated with antiinflammatory cytokine IL-17E in all HIE infants. However, Th17 cytokine suppressor IL-27 was significantly increased by hypothermia, negating the IL-27 correlation with vitamin D observed in normothermic HIE infants. Conclusions Serum 25(OH) vitamin D insufficiency is present in the majority of term HIE neonates and is related to lower circulating anti-inflammatory IL-17E. Hypothermia does not mitigate vitamin D deficiency in HIE.

Transforming growth factor-α (TGFα) is a cytokine secreted by stimulated alveolar macrophages (AM) in vitro and after in vivo particulate or hyperoxia exposure and has been implicated in the processes of postnatal lung development. It is unknown if AM TGFα secretion changes during normal postnatal lung development. After sacrifice of New Zealand white rabbits on postnatal d 0–2, 5–7, 9–10, 14, 21, and 28 and > 4 mo (adult), AM were isolated by discontinuous density centrifugation and placed in culture in the presence or absence of concanavalin A (ConA) for 24 h. Media were collected, and the concentration and isoforms of TGFα in AM media samples were determined by an epidermal growth factor/TGFα radioreceptor assay and Western immunodetection, respectively. TGFα was present in media of AM from the 1.06 and 1.08 g/dL Percoll densities, but not in the 1.10 g/dL density. Statistically significant differences in TGFα secretion by unstimulated and ConA-stimulated AM at the various ages were not detected until d 14 (p < 0.02). Western blot analysis of unstimulated AM media samples from d 0–7 rabbits demonstrated the presence of TGFα isoforms at 46, 30, and 14.3 kD. At later postnatal ages (≥ d 9), a single 14.3-kD isoform was present. In contrast, analysis of ConA-stimulated AM media samples showed TGFα isoforms at 46, 30, and 14.3 kD for all ages; however, the 6-kD mature isoform was present only in juvenile (d 28) and adult media. These results demonstrate an age-dependent effect on AM TGFα secretion and biochemical isoforms.

Early Stooling as an Indicator of Intestinal Motility/Maturity and Its Relation To Feeding Intolerance

Pediatric, OB/Gyn and Family Medicine Residents' Knowledge about Breastfeeding and Human Milk Composition at a University Hospital

It is known that nourishment with human milk can minimize morbidities associated with high-risk neonates and infants. Nationwide, 54% of all mothers choose to initiate breastfeeding; at our institution that number is 40%. Infants born to adolescent mothers are one of these high-risk groups, yet little is known about the prevalence of breastfeeding, motivators, or effects of intervention in this age group. A cross-sectional survey was undertaken to determine the baseline attitudes towards breastfeeding among 29 nulliparous females, aged 13-19 years, with the goal of elucidating those factors most associated with a decision to breastfeed. The research instrument included 39 statements scored on a 5-point Likert scale, followed by 18 open and closed ended questions administered by the PI. Respondents were primarily single, Afro-American students, 38% insured by Medicaid and 24% uninsured. 45% of respondents would choose artificial milk to nourish their infants, 28% were undecided, 14% would choose both human and artificial milks, and 14% would breastfeed. Data were then examined to detect who or what most influenced this decision, the role of exposure to breastfeeding, concerns regarding quality and/or quantity of breast milk, and breastfeeding's social acceptability. Likert scale questions were divided into the following positive vs. negative categories: self image, process perceptions, emotional perceptions, and outside influences. Negative attitudes were identified in responses to the following statements: “My boyfried/husband wants me to breastfeed.” “Breastfeeding makes your breasts sag.”“I have heard from someone that breastfeeding hurts.” “No one else could feed my baby.” “I would have to eat differently.” “My mother wants me to bottle feed.” 62% of respondents agreed or strongly agreed that they did not know enough about breastfeeding. We conclude that our population of teenagers suffer from misinformation about breastfeeding and have had little exposure to it. Our data also suggest that breastfeeding prevalence may be increased in this population through education and exposure to positive role models.

Human milk fortifiers (HMF) are commonly added to human milk to increase caloric density and nutrient delivery to preterm infants. TGFβ2 is present in human milk, has immunoregulatory properties, and is involved in growth, differentiation, and repair of neonatal gut epithelia. The effect of HMF on TGFβ2 concentration is unknown. We sought to measure the concentration of TGFβ2 in whole milk following the addition of a commercially available powdered HMF. Human milk samples (N=23) were collected and stored at -20°C. For analysis, milk samples were thawed and divided into two aliquots. HMF was added at a concentration of 0.0384 g/mL (or one 0.96 g packet per 25 mL) to the 2nd aliquot of each pair. Controls included:(1) HMF at a concentration of 0.0384 g/mL in calibrator diluent (CD) and (2) HMF 0.0384 g/mL in CD with recombinant TGFβ2 at 2000 and 500 pg/mL. The concentration of the 25 kD acid-activated TGFβ2 isoform was measured using an ELISA (R&D Systems). The mean±SD concentration of TGFβ2 in unfortified milk was 5554±5345 (range 143-17640) pg/mL human milk. The mean±SD concentration of TGFβ2 in fortified milk was 7135±4774 (range 1641-17390) pg/mL. The mean relative increase in milk TGFβ2 concentration after the addition of HMF was 3.4 times that of unfortified milk. The mean TGFβ2 concentration in HMF control was 2518 pg/mL. HMF doubled the concentration of TGFTGFβ2 standards. In summary, with the addition of HMF to whole milk samples, TGFβ2 concentration decreased 2-to-10% in 4 samples but increased 1-to- 3174% in 19 samples. It appears that HMF is an additional source of TGFβ2 when added to human milk. What effect this alteration in human milk TGFβ2 concentration has on neonatal immune and gut function remains unknown.

The Effect of Antenatal Steroids on Transforming Growth Factor β-2(TGFβ 2 ) Concentrations in Human Milk ♦ 588

Human milk fortifier (HMF) is routinely added to human milk feedings to augment nutrient and caloric delivery to premature infants. We previously reported that the TGFα immunoreactive concentration in the fat, but not the aqueous compartment, of human milk is decreased with the addition of HMF[Ped Res 1997; 41(4):515A]. TGFα is detectable in HMF when reconstituted in sterile water (concentration 2 pg/mL), but not in commercially available formulas. It is unknown if HMF alters the biochemical properties, including the TGFα isoform profile, of human milk. To address this question, the TGFα isoform profiles of whole milk and its fractions were compared with the same fractions with a commercially available powered HMF (Mead-Johnson). Fifteen whole milk samples were collected from 12 mothers on postpartum days 4 through 90; gestational age of the infants ranged from 26-40 weeks. These samples were divided into 2 aliquots of 25 mL; 1 packet of HMF (0.96 g) was added to the first aliquot. Two cc of whole milk were retained for later analysis. The remainder of each sample was centrifuged at 1000 X g for 20 min at 4°C to achieve separation of the fat and aqueous fractions. Sodium taurocholate, a bile salt, was added 1:1 (v/v) to the fat for emulsion. Western blot analysis of the samples was performed with a monoclonal TGFα antibody (0.5 μg/mL; RDI) and a polyclonal secondary antibody (1:20,000; Pierce) using 15% SDS-PAGE electrophoresis- semi-dry transfer method. Isoform bands were visualized by enhanced chemilluminescence(Amersham). TGFα isoforms were detected in whole milk and its separated fractions at 6.5, 12-16, 22, 26, and 30 kD. HMF alone and HMF with sodium taurocholate had a single detectable band at 18 kD. While whole and aqueous milk samples with HMF present also showed the 18 kD band, the fat fraction samples with HMF did not have the 18 kD band. It appears that HMF differentially alters the biochemical profile of human milk with regard to TGFα concentration and the immunodetection of TGFα isoforms. What effect this alteration in human milk biochemistry has on neonatal gut function remains unknown.

Transforming growth factor-alpha (TGFα) is a polypeptide that plays an important role in the growth and differentiation of a variety of ectodermal and mesodermal cells both during fetal and postnatal life. Epidermal growth factor (EGF), a growth factor that acts similarly to TGFα through binding of the EGF receptor, is associated with fetal growth and maturation: EGF cord blood concentrations correlate significantly with birth weight and placental weight (Biol Neonate, 1992:61). It is unknown if a similar relationship exists between cord blood TGFα concentration and advancing gestational age. We sought to determine if such an association exists. Eighty newborns ranging in age from 26 to 42 weeks gestation were enrolled in the study, and clinical data collected prospectively. At delivery, a cord blood sample was collected from each placenta and processed within 4 hours for serum separation. Serum samples were then frozen at -80°C until later analysis. Each sample was analyzed for TGFα concentration (pg/mL) by a commercially available RIA (RDI; sensitivity 1 pg/100 μL; cross-reactivity with EGF of 0%). The concentration of cord serum TGFα was expressed as the mean ± S.D. There was a strong association between TGFα cord serum concentration and gestational age (p

Human milk contains transforming growth factor-beta isoforms(TGFβ1 and β2). TGFβ affects growth and differentiation of neonatal intestinal epithelia, and modulates restitution of the gut epithilium following injury. A differential distribution of some bioactive factors in milk fat and aqueous fractions has been shown, which could affect bioavailability. It is unknown if TGF-β2 shares similar distribution properties. We sought to determine if there is a differential concentration of TGFβ2 in human milk aqueous and fat compartments. Eighteen milk samples were collected from 15 lactating mothers on postpartum day 2 to 27; gestational age of their infants at delivery ranged from 24 to 40 weeks. Whole milk was centrifuged at 2500g × 20 min at 4°C to achieve separation and isolation of its fat, aqueous and cellular components. Sodium taurcholate(STC) was added to the fat for emulsion. The concentration of acid-activated TGFβ2 in the aqueous and fat components of human milk was measured by ELISA (RD sensitivity 55 pg/ml sample; cross-reactivity with other TGFβ species/receptors is not significant; standard curve unaffected by STC). TGFβ2 was detected in all aqueous samples (6214 ± 5083 pg/mL; range 1619 to 20686); and all fat samples (6987 ± 5344 pg/mL; range 1736 to 20576). Similac Special Care formula and Enfamil Premature formula, also tested, did not have detectable levels of TGFβ2. In conclusion TGFβ2 is found in high concentrations in both the aqueous and fat compartments of human milk. These findings may be of significance for differential distribution and bioavailability of TGFβ2 in the newborn gut. Results of this study are important to future research, as fat is often discarded during human milk investigations.

Human milk contains many cytokines, including transforming growth factor-beta isoforms (TGFβ1 and β2). TGFβ appears to control growth and differentiation of neonatal intestinal epithelia, and to act in the repair of the gut following injury. It is thought that the bioavailability of these factors can be affected by their compartmentalization in milk. While an increase of certain bioactive factors within the fat compartment of milk has been shown, it is unknown if TGFβ1 shares similar distribution properties. We sought to determine if there is a differential concentration of TGFβ1 in the human milk fat layer compared with whole milk and its aqueous fraction. Thirty-six whole milk samples and an additional 13 milk fat samples were collected from 35 lactating mothers on postpartum day 3 to 6 mo.; gestational age of their infants at delivery ranged from 32 to 42 weeks. Whole milk was centrifuged at 1000g× 20 min at 4°C to achieve separation and isolation of its fat, aqueous and cellular components. Tris buffered saline (TBS; 200 μL) with 10μL Tween-20 was added to the fat for emulsion. The concentration of acid-activated TGFβ1 in whole milk, and its aqueous and fat components was measured by ELISA (Promega; sensitivity 25 pg/ml sample; cross-reactivity with other TGFβ species of

The newborn GI tract undergoes extensive maturational changes in the weeks following birth. Activation of the epidermal growth factor receptor (EGFr) following its binding of EGF or TGFα is thought to play a major role in that process. Both growth factors are present in human milk. While milk-derived EGF has been shown to induce gut epithelial proliferation in bothin vitro and in vivo models, the role of TGFα is less clear. We devised an in vitro model to compare the effect of recombinant, human milk- and HMM-derived TGFα on small intestinal cell proliferation. A fetal small intestinal cell line with epithelial characteristics (FhS-74; American Tissue Type Co.) was grown to confluence in the presence of Hybri-Care medium with EGF (30 ng/ml medium). The cells were removed from flasks following Trypsan-EDTA exposure, washed in EGF-free medium, then placed in culture in EGF-free medium at a concentration of 104 cells/well (60% confluence; 96-well plates). After 24 hr, either recombinant TGFα (0.01-50 ng/mL media), human milk supernatant (0.5 -1 ng TGFα/mL) or HMM media (20-100 pg TGFα/mL) was added to separate wells (5 wells/condition) in the presence or absence of 1° monoclonal TGFα antibody (RDI; 1 μg/mL media) for 24 hr. Cell proliferation was then measured (CellTiter 96-AQ, Promega) and expressed as% above control. A positive dose-response effect on cell proliferation by all three agents that paralleled TGFα concentration was observed. This effect was decreased in the presence of 1° TGFα antibody: 22 ± 7.1% decline of TGFα-stimulated proliferation; 25 ± 3.1% decline of HM-stimulated growth; and 27.6 ± 3.2% decline of HMM media-stimulated growth at all concentrations tested. With removal of the 1° antibody, cell proliferation returned to control levels. Binding of the TGFα present in human milk and HMM media led to a consistent decrease in in vitro gut epithelial proliferation without affecting cell viability. The proliferative effect of human milk on the gut appears to be due, in part, to TGFα.