INTRODUCTION:Human milk oligosaccharides (HMOs) arise from tightly regulated glycosylation pathways and exert pleiotropic effects on infant immune, microbial, and neurodevelopmental trajectories. Vitamin D influences gene transcription and cellular metabolism, yet its potential role in mammary glycosylation remains unexplored. We examined whether maternal vitamin D status during lactation is associated with HMO composition and whether effects vary by maternal secretor phenotype. METHODS:We performed a post hoc analysis of two clinical studies: the NICHD vitamin D lactation randomized controlled trial (2005-2012) and the Lactation Immune Trial pilot (n = 88). Serum 25-hydroxyvitamin D [25(OH)D, vitamin D status] and 19 HMOs concentrations were measured at 1 and 4 months postpartum. Secretor status was defined by presence of 2'-fucosyllactose. Statistical analyses were conducted using SAS 9.4 with p < 0.05. RESULTS:The cohort comprised 41% White, 7% Black, and 52% Hispanic women. Black and Hispanic mothers exhibited lower 25(OH)D concentrations than White mothers (p < 0.001), and Hispanic mothers were more likely to be secretors (p = 0.006). In secretors, higher 25(OH)D was significantly associated with increased fucosylated and complex HMOs, including lacto-N-fucopentaose II, lacto-N-hexaose, and fucosyl-disialyl-lacto-N-hexaose I at 1 month, with sustained association for the latter at 4 months. In non-secretors, higher 25(OH)D was associated with lower 6'-sialyllactose at 4 months. Distinct HMO patterns were observed by secretor phenotype. CONCLUSION:Vitamin D status is associated with differential HMO profiles during early lactation, supporting a potential role for vitamin D-mediated regulation of mammary glycosylation that is modified by secretor status.

Objective Evaluate the association between vitamin D (vitD) status and Corona Virus-19 (COVID-19) infection in adults aged 50 years and older. Design Adults ≥50 undergoing COVID-19 testing from July 2020 to December 2021, without prior vaccination, consented to blood analysis. SARS-CoV-2 PCR confirmed current COVID-19 infection. VitD status was assessed via 25(OH)D concentration (LCMS/MS, ZRT Labs, Portland, OR). Sociodemographic data were collected at enrollment. Statistical analyses (SAS 9.4) examined associations between sociodemographics, COVID-19, and vitD status. Multivariate logistic regression analyzed factors linked to COVID-19 or vitD status. Results Of 131 participants, 46.6% were ≥65 years old, 71.0% married, 19.9% Black American, 36.6% male, 38.9% Medicaid/Medicare/self-pay, and 42.8% BMI≥30. VitD status and Black American (P=0.0001) significantly associated with COVID-19 infection (P=0.0001). Black American (P=0.0003), males (P=0.003), and BMI (P=0.007) were inversely associated with 25(OH)D concentration. In a multiple logistic regression model predicting COVID-19 infection, only vitamin D status remained significant after controlling for certain sociodemographic and clinical factors (P<0.0001, OR 0.92, 95% CI 0.89–0.95).Of the 44 COVID-positive participants, 35 (79.6%) were hospitalized and 19 (43.2%) were admitted to the Intensive Care Unit (ICU). Hospitalization due to COVID-19 was associated with age ≥65 years old (P=0.02; OR 12.0, 95% CI 1.34–106.79), male (P=0.02, OR 10.7, 95% CI 1.20–94.73), and 25(OH)D <40 ng/mL (P=0.0006, OR 42.5, 95% CI 3.90–461.01). In multivariate analysis, the association between vitamin D status and the risk of COVID-related hospitalization remained significant and inversely associated (P=0.03, OR 0.88, 95% CI 0.78–0.99).In unadjusted analysis, COVID pneumonia was associated with male sex (P=0.049; OR 4.6, 95% CI 1.06–20.16) and 25(OH)D <40 ng/mL (P=0.006, OR 18.8, 95% CI 1.9–184.10). Participants with COVID infection and 25(OH)D <20 ng/mL were 2.1 times more likely to be admitted to ICU/death (P=0.03). In unadjusted analysis, ICU admission and/or death were linked to age ≥65 years (P=0.0002, OR 16.9, 95% CI 3.63–78.56), Medicaid/Medicare/self-pay insurance status (P=0.004, OR 0.1, 0.04–0.56), and 25(OH)D <20 (P=0.03, OR 3.9, 1.09–13.66) and <40 ng/mL (P=0.03); however, only age ≥65 remained significant in multivariate analysis (P=0.04, OR 6.7, CI 1.05–43.0). Conclusions Lower 25(OH)D concentration was a significant predictor and/or contributor to COVID-19 infection, suggesting the importance of maintaining adequate vitamin D status in reducing infection risk and mitigating severe outcomes.

OBJECTIVE:To evaluate the levels of inflammatory cytokines and symptom survey scores in patients diagnosed with Menière's disease or vestibular migraine from a single center by a single neurotologist compared to control subjects with no history of dizziness or migraine. STUDY DESIGN:Cross-sectional pilot study. SETTING:Single-center tertiary referral center in Charleston, SC. PATIENTS:Patients were recruited from the neurotology clinic at a tertiary referral center. Patients with definite Menière's disease or definite vestibular migraine as defined by the Barany consensus criteria were eligible. Control subjects presented to clinic without dizziness, vertigo, fluctuating hearing loss, or a history of migraine. MAIN OUTCOME MEASURES:Questionnaire scores include DHI, SF-20, CFQ, PHQ-9, PSWQ, GAD-7, NVI, DCS, VM-PATHI, and MD-POSI. Circulating and in vitro levels of cytokines include ENA-78, GROα, IFN-α2a, IFN-γ, IL-10, IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-8, MCP-1, MCP-2, MDC, MIP-1α, MIP-1β, and TNF-α. Cytokine levels were compared with effect size analysis. RESULTS:There were 20 Menière's disease, 20 vestibular migraine, and 10 control patients enrolled in this study. Episode frequencies ranged from three per week to two to five per year in the MD group and daily to 1 every 2 to 3 months in the VM group. When patient-derived PBMC samples were compared to vestibular migraine, TNF-α ( d = -0.427 [-0.879, 0.025]) and IFN-γ ( d = -0.818 [-1.313, -0.323]) were found to be higher in Menière's disease, whereas ENA-78 ( d = -0.652 [-1.361, 0.056]) was found to be lower. No differences were found when cytokines were measured following stimulation with LPS. CONCLUSIONS:This pilot study suggests Menière's disease patients may have higher levels of TNF-α and IFN-γ and lower levels of ENA-78 than vestibular migraine patients when measured following in vitro release from patient-derived PBMC. Increased sample size, optimized blood draw timing, and more specific PBMC stimulation may help us further elucidate inflammatory pathways implicated in these disorders.

Introduction: Vitamin D (VitD) has been shown to impact neurodevelopment. Studies have shown that higher 25-hydroxy-vitamin D (25(OH)D) concentrations (the indicator of vitD status) may be associated with better neurodevelopmental outcomes, although current data are conflicting. This study examined the relationship between total circulating 25(OH)D concentrations and neurodevelopmental outcomes in 3–5-year-old (3–5 yo) children. Methods: In this study, pregnant women were randomized to receive 400 (standard dose), 2000, or 4000 IU vitD3/day. Offspring then underwent the Brigance Screen at 3–5 yo. The 25(OH)D concentration was measured at birth and 3–5 yo. Relationships between Brigance scores and 25(OH)D and Brigance scores and vitamin D binding protein (VDBP) genotype were examined. Results: Higher 25(OH)D at the time of testing was associated with better overall performance on neurodevelopmental testing as measured by the Brigance quotient (B = 0.208, p = 0.049). Scores were then broken down into sub-scores. Children born to mothers in the 2000 IU/day group scored higher on the Brigance language component of the assessment versus the standard dose group (B = 4.667, p = 0.044). The group of children who had the Gc1f-1s or Gc1f-2 genotypes scored higher on the Brigance academic component (B = 9.993, p < 0.001) and lower on the Brigance language component versus the 1f1f genotype (B = −9.313, p < 0.001). Children with the Gc1s-1s, Gc1s-2, or Gc2-2 genotypes also scored lower than the Gc1f-1f genotype (B = −6.757, p = 0.003). Conclusion: These results suggest that higher 25(OH)D concentrations early in life and higher doses of maternal vitamin D supplementation during pregnancy may have a positive association with neurodevelopmental outcomes. This study also suggests that the VDBP genotype is associated with neurodevelopment and differentially affects various fields of neurodevelopment.

Thermosensitive liposomes (TSL) are triggered nanoparticles that release the encapsulated drug in response to hyperthermia. Combined with localized hyperthermia, TSL enabled loco-regional drug delivery to tumors with reduced systemic toxicities. More recent TSL formulations are based on intravascular triggered release, where drug release occurs within the microvasculature. Thus, this delivery strategy does not require enhanced permeability and retention (EPR). Compared to traditional nanoparticle drug delivery systems based on EPR with passive or active tumor targeting (typically <5%ID/g tumor), TSL can achieve superior tumor drug uptake (>10%ID/g tumor). Numerous TSL formulations have been combined with various drugs and hyperthermia devices in preclinical and clinical studies over the last four decades. Here, we review how the properties of TSL dictate delivery and discuss the advantages of rapid drug release from TSL. We show the benefits of selecting a drug with rapid extraction by tissue, and with quick cellular uptake. Furthermore, the optimal characteristics of hyperthermia devices are reviewed, and impact of tumor biology and cancer cell characteristics are discussed. Thus, this review provides guidelines on how to improve drug delivery with TSL by optimizing the combination of TSL, drug, and hyperthermia method. Many of the concepts discussed are applicable to a variety of other triggered drug delivery systems.

Pregnancy is a unique time when amplified sex steroid concentrations promote an escalation in vitamin D binding protein (DBP) synthesis, associated with increased total vitamin D and metabolites, including 25-hydroxyvitamin D (25(OH)D). Free 25(OH)D concentration increases disproportionately to total 25(OH)D during pregnancy, likely an adaptation to supply the woman and fetus with readily available 25(OH)D. Highlighting the importance of the calcium metabolic stress during pregnancy, the interactional relationship between serum 25(OH)D and PTH has been evaluated. Maternal total 25(OH)D and total 25(OH)D/iPTH are measures of vitamin D status and biomarkers for potential pregnancy complications. It has been proposed that free 25(OH)D and free 25(OH)D/iPTH could be better indicators of vitamin D status and predictors of pregnancy complications such as gestational diabetes (GDM), hypertensive disorders of pregnancy, and preterm delivery. This study aims to determine if free 25(OH)D and its association with PTH are more accurate predictors of comorbidities of pregnancy than total 25(OH)D and its association with PTH. In this post hoc analysis of the Kellogg Pregnancy Study, a double-blind randomized placebo-controlled trial, participants included 297 women with singleton pregnancies: 191 participants were randomized into a group receiving a daily prenatal (400 IU vitamin D3) while 196 received a prenatal plus extra supplementation (4400 IU vitamin D3). Blood and urine samples were collected monthly. 297 participants' serum total 25(OH)D concentrations were measured using radioimmunoassay at baseline (visit 1) and 5-7 months' gestation (visit 6-7). 93 participants' serum free 25(OH)D and PTH concentrations were measured using ELISA and immunoradiometric assay, respectively, at visit 1 and 6-7; 66 participants had paired samples and were included in this analysis. Data were analyzed using SAS 9.4, Cary, N.C. or SPSS v28, IBM Corporation, Armonk, N.Y. Results were considered significant with a p < 0.05. A significant relationship exists between the ratio of total 25(OH)D/iPTH and free 25(OH)D/iPTH grouped by total 25(OH)D ≥ 30 ng/mL and < 30 ng/mL as an indicator of maternal vitamin D status. There was a statistically significant relationship between lower mean free 25(OH)D/iPTH and the development of GDM at visit 1 (p = 0.0003) and at visit 6-7 (p = 0.001) while total 25(OH)D/iPTH and GDM were significantly related only at visit 1 (p = 0.029). In this exploratory cohort, neither free 25(OH)D/iPTH nor total 25(OH)D/iPTH were significantly associated with increased incidence of preterm delivery, hypertensive disorders, or combined comorbidities of pregnancy. An univariate logistic regression evaluating the outcome of gestational diabetes while independently controlling for independent factors showed the ratio of free 25(OH)D/iPTH was more closely associated with gestational diabetes than the ratio of total 25(OH)D/iPTH, although neither were significant. This proof-of-concept analysis suggests that the ratio of free 25(OH)D/iPTH is associated with the development of gestational diabetes throughout pregnancy while total 25(OH)D/iPTH is only associated with the outcome early in pregnancy. Further investigation is warranted to explore this relationship between calcium metabolic stress during pregnancy with a larger cohort to improve validity,reproducibility, and relevance to other pregnancy comorbidities.

The oleic acid/alpha-lactalbumin complex HAMLET (human alpha-lactalbumin made lethal to tumors) is cytotoxic to various cancerous cell lines and is assembled from alpha-lactalbumin (ALA) and free oleic acid (OA). HAMLET is also cytotoxic to normal immature intestinal cells. It remains unclear if HAMLET, experimentally assembled with OA and heat, can spontaneously assemble in frozen human milk over time. To approach this issue, we used a set of timed proteolytic experiments to evaluate the digestibility of HAMLET and native ALA. The purity of HAMLET in human milk was confirmed by ultra high performance liquid chromatography coupled to tandem mass spectrometry and western blot to resolve the ALA and OA components. Timed proteolytic experiments were used to identify HAMLET in whole milk samples. Structural characterization of HAMLET was performed by Fournier transformed infrared spectroscopy and indicated a transformation of secondary structure with increased alpha-helical character of ALA upon binding to OA.

Atrial natriuretic peptide (ANP) and its receptors natriuretic peptide receptor (NPR)-A and NPR-C are all highly expressed in alveolar epithelial type II cells (AEC2s) in the late-gestation ovine fetal lung and are dramatically decreased postnatally. However, of all the components, NPR-C stimulation inhibits ANP-mediated surfactant secretion. Since alveolar oxygen increases dramatically after birth, and steroids are administered to mothers antenatally to enhance surfactant lung maturity, we investigated the effects of O2 concentration and steroids on NPR-C-mediated surfactant secretion in AEC2s. NPR-C expression was highest at 5% O2 while being suppressed by 21% O2, in cultured mouse lung epithelial cells (MLE-15s) and/or human primary AEC2s. Surfactant protein-B (SP-B) was significantly elevated in media from both in vitro and ex vivo culture at 13% O2 versus 21% O2 in the presence of ANP or terbutaline (TER). Both ANP and C-ANP (an NPR-C agonist) attenuated TER-induced SP-B secretion; this effect was reversed by dexamethasone (DEX) pretreatment in AEC2s and by transfection with NPR-C siRNA in MLE-15 cells. DEX markedly reduced AEC2 NPR-C expression, and pregnant ewes treated with betamethasone showed reduced ANP in fetal sheep lung fluid. These data suggest that elevated O2 downregulates AEC2 NPR-C and that steroid-mediated NPR-C downregulation in neonatal lungs may provide a novel mechanism for their effect on perinatal surfactant production.

Objective Thermosensitive liposomes (TSL) and other triggered drug delivery systems (DDS) are promising therapeutic strategies for targeted drug delivery. However, successful designs with candidate drugs depend on many variables, including nanoparticle formulation, drug properties, and cancer cell properties. We developed a computational model based on experimental data to predict the potential efficacies of drugs when used with triggered DDS, such as TSL. Methods A computer model based on the Krogh cylinder was developed to predict uptake and cell survival with four anthracyclines when delivered by intravascular triggered DDS (e.g., TSL): doxorubicin (DOX), idarubicin (IDA), pirarubicin (PIR), and aclarubicin (ACLA). We simulated three tumor types derived from SVR angiosarcoma, LLC lung cancer, or SCC-1 oral carcinoma cells. In vitro cellular drug uptake and cytotoxicity data were obtained experimentally and incorporated into the model. Results For all three cell lines, ACLA and IDA had the fastest cell uptake, with slower uptake for DOX and PIR. Cytotoxicity was highest for IDA and lowest for ACLA. The computer model predicted the highest tumor drug uptake for ACLA and IDA, resulting from their rapid cell uptake. Overall, IDA was most effective and produced the lowest tumor survival fraction, with DOX being the second best. Perivascular drug penetration was reduced for drugs with rapid cell uptake, potentially limiting delivery to cancer cells distant from the vasculature. Conclusion Combining simple in vitro experiments with a computer model could provide a powerful screening tool to evaluate the potential efficacy of candidate investigative drugs preceding TSL encapsulation and in vivo studies.

It is unknown if vitamin D (vitD) sufficiency in breastfeeding mothers can lead to physiological outcomes for their children that are discernible from infant vitD sufficiency per se. In a 3-month, randomized vitD supplementation study of mothers and their exclusively breastfeeding infants, the effects of maternal vitD sufficiency were determined on infant plasma concentrations of 25-hydroxyvitamin D (i.e., vitD status) and 11 cytokines. An inverse correlation was seen between maternal vitD status and infant plasma TNF concentration (r = −0.27; p < 0.05). Infant whole blood was also subjected to in vitro antigenic stimulation. TNF, IFNγ, IL-4, IL-13, and TGFβ1 responses by infant leukocytes were significantly higher if mothers were vitD sufficient but were not as closely correlated to infants’ own vitD status. Conversely, IL-10 and IL-12 responses after antigenic challenge were more correlated to infant vitD status. These data are consistent with vitD-mediated changes in breast milk composition providing immunological signaling to breastfeeding infants and indicate differential physiological effects of direct-infant versus maternal vitD supplementation. Thus, consistent with many previous studies that focused on the importance of vitD sufficiency during pregnancy, maintenance of maternal sufficiency likely continues to affect the health of breastfed infants.

Human milk, the best enteral selection for a preterm infant, becomes altered during freezing and soluble free fatty acid is generated over time. Free fatty acids may form complexes, such as the oleic acid-bound protein called HAMLET (human α-lactalbumin made lethal to tumor cells). We determined the in vitro biological activity of preterm human milk protein-oleic complexes (HAMLET-like complexes) and tested the hypothesis that laboratory-synthesized HAMLET exhibits cytotoxicity in human immature epithelial intestinal cell culture. Thirty-four milk samples from 15 mothers of hospitalized preterm infants were donated over time. Milk fractions were tested repeatedly for FHs 74 Int and HIEC-6 fetal cell cytotoxicity, using a sensitive viability assay. Protein and fatty acid identities were confirmed by Western blot, high performance liquid chromatography, and mass spectrometry. Cytotoxicity of intestinal cells exposed to milk increased respective to milk storage time (p < 0.001) and was associated with free oleic acid (p = 0.009). Synthesized HAMLET was cytotoxic in cultures of both lines. Preterm milk samples killed most cells in culture after an average 54 days in frozen storage (95% C.I. 34–72 days). After prolonged storage time, preterm milk and HAMLET showed a degree of cytotoxicity to immature intestinal cells in culture.

BACKGROUND:Idiopathic Pulmonary Fibrosis (IPF) is the most common and progressive form of the interstitial lung diseases, leading most patients to require lung transplants to survive. Despite the relatively well-defined role of the fibroblast in the progression of IPF, it is the alveolar type II epithelial cell (AEC2) that is now considered the initiation site of damage, driver of disease, and the most efficacious therapeutic target for long-term resolution. Based on our previous studies, we hypothesize that altered lactate metabolism in AEC2 plays a pivotal role in IPF development and progression, affecting key cellular and molecular interactions within the pulmonary microenvironment.METHODS:AEC2s isolated from human patient specimens of non-fibrotic and IPF lungs were used for metabolic measurements, lactate dehydrogenase (LDH) analyses and siRNA-mediated knockdown experiments.RESULTS:AEC2s isolated from human IPF lung explant tissues had lower rates of oxidative metabolism and were more glycolytic lactate-producing cells than were AEC2 from control, non-fibrotic lung explant tissues. Consistent with this shift in metabolism, patient-derived IPF AEC2s exhibited LDH tetramers that have higher ratios of LDHA:LDHB (i.e., favoring pyruvate to lactate conversion) than control AEC2s. Experimental manipulation of LDHA subunit expression in IPF AEC2s restored the bioenergetic profile characteristic of AEC2 from non-fibrotic lungs.CONCLUSIONS:These results are consistent with the concept that altered lactate metabolism may be an underlying feature of AEC2 dysfunction in IPF and may be a novel and important target for therapeutic treatment.

Vitamin D (VitD) affects immune function across the lifespan, which includes pregnancy and lactation. We hypothesized that the response of fully breastfeeding (BrF) infants to HBV would differ as a function of maternal and/or infant's vitD status as measured by circulating 25-hydroxyD (25-D) concentration. Plasma 25-D concentration and HBV titers were measured in a subset of mothers and exclusively BrF infants (n = 56 pairs) participating in a lactation vitD supplementation clinical trial. Mothers were randomized to receive either 400 vs. 6400 IU vitD3/day and infants 400 IU/day or placebo (if mother was in 6400 IU group). An additional 14 infants were exclusively formula-fed (FF). 25-D concentration (RIA) and infant anti-HBV IgG titers (ELISA) after 3 vaccinations (7 months of age) were measured. The associations between maternal vitD treatment group, circulating 25-D, and HBV IgG titers were explored using t-tests, ANOVA and linear correlations. After 3 vaccinations, all infants in this study were considered to be highly immune to HBV (plasma anti-HBV surface IgG titer >100 IU/mL). However, we found that mean anti-HBV IgG titers in exclusively BrF infants were significantly lower if mother was vitD sufficient (2200 vs 4500 IU/ml; P = 0.017), with inverse correlation between infant IgG titers and maternal vitD status (r = −0.31; P = 0.02). We also found that these infant anti-HBV titers were not significantly correlated with their own vitD status (P = 0.18). Results also showed that mean titers in exclusively FF infants were not correlated with vitD status and were nearly identical to those of BrF infants of vitD-insufficient mothers. Though still considered immune after 3 vaccinations, HBV IgG titers of BrF infants differed at 7 months of age by maternal vitD treatment and not on the basis of infant vitD status. These findings suggest that effects of vitD on breastmilk composition results in regulation of an infant's immune response perhaps by induction of immunotolerance, whether through blunting of a massive immune response to HBV antigens or affecting a more rapid switch from active plasma cells to memory B cells. A currently ongoing lactation pilot study continues to collect samples to confirm and expand these findings. NIH/NCATS, SC Translational Research Institute, MUSC Pediatrics.

Background: The safety of higher dose vitamin D (vitD) supplementation in women who change from exclusive or full breastfeeding to combination feeding or who continue supplementation after cessation of breastfeeding is unknown. Objective: Compare vitD supplementation safety of 6,400 to 400 IU/day and 2,400 IU/day using specific laboratory parameters in postpartum women and their infants through 7 months postpartum by feeding type. Design: In this randomized controlled trial, mothers (exclusively breastfeeding or formula-feeding) were randomized at 4-6 weeks' postpartum to 400, 2,400, or 6,400 IU vitD3 (cholecalciferol)/day for 6 months. Breastfeeding infants in 400 IU group received oral 400 IU vitD3/day; infants in 2,400 and 6,400 IU groups received placebo. Maternal safety parameters (serum vitD, 25-hydroxy-vitamin D [25(OH)D; calcidiol], calcium, phosphorus, intact PTH; urinary calcium/creatinine ratios; and feeding type/changes) were measured monthly; infant parameters were measured at months 1, 4, and 7. Sufficiency was defined as 25(OH)D >50 nmol/L. Feeding type was defined as exclusive/full, combination, or formula-feeding. Data were analyzed using SAS 9.4. Results: Four hundred nineteen mother-infant pairs were randomized into the three treatment groups and followed: 346 breastfeeding and 73 formula-feeding pairs. A dose of 6400 IU/day safely and significantly increased maternal vitD and 25(OH)D from baseline in all mothers regardless of feeding type (p < 0.0001) and was superior to the 400 and 2,400 IU groups in achieving vitD sufficiency with no other differences in safety parameters by treatment or feeding type. Infants in the 2,400 IU group were more likely vitD-deficient than the other groups; otherwise, there were no infant safety parameter differences. Conclusions: While 6,400 IU/day was more effective than 400 or 2,400 IU/day in achieving maternal vitD sufficiency in all feeding groups, the groups did not differ on other safety parameters. Similarly, infant safety parameters did not differ by treatment group or feeding status. Clinical Trial Registration: FDA IND Number: 66,346; ClinicalTrials.gov Number: NCT00412074.

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 D 3 /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.

Exclusively breastfeeding (BrF) infants are at risk for vitD deficiency if they or their mothers are not supplemented. However, it is unknown if maternal or direct infant vitD supplementation could have differential effects on the BrF child. We hypothesized that maternal vitD status may affect concentrations of pro-inflammatory cytokines in infants. Exclusively BrF infants and mothers were enrolled in a clinical study in which infant vitD sufficiency was achieved by either direct supplementation with 400 IU/d vitD3 or mothers were supplemented with 6400 IU/d. Infant and maternal plasma 25-hydroxyvitamin D (25-D) values were determined by radioimmunoassay at enrollment (1-mo infant) and completion (5-mo). Concentrations of 10 pro-inflammatory cytokines in infant plasma were determined by electrochemiluminescence. ANOVA, t-tests and linear correlations were used to calculate relationships between vitD and circulating cytokines in 49 infant-mother pairs at study enrollment and 39 pairs at completion. Plasma concentrations of 10 pro-inflammatory cytokines measured were very low (pg/ml range). Neither infant nor maternal vitD status (sufficiency defined as plasma 25-D > 30 ng/ml) were associated with 9 measured cytokines in infant blood (IFN-γ, IL-1b, –2, –4, –6, –8, –10, –12, –13). In contrast, concentrations of tumor necrosis factor (TNF)-α in infant plasma were inversely correlated to maternal vitD status (r = −0.37; P = 0.02 at study completion). At study enrollment, BrF infants of vitD-sufficient mothers had mean 20% lower plasma TNF-α concentration than infants of vitD-insufficient mothers (P = 0.02), regardless of the infant's own vitD status. VitD sufficiency in infants per se was associated with mean 5% lower plasma TNF-α concentration, non-significantly different to that of vitD-deficient infants. These results indicate that vitD-mediated changes in breastmilk composition can affect the immune system of BrF infants. Circulating concentrations of the major pro-inflammatory cytokine TNF-α were significantly lower in infants with vitD-sufficient mothers regardless of the infant's own vitD status. Studies are currently investigating vitD-associated effects on responses of BrF infants' immune cells to antigenic challenge. NIH/NCATS, SC Translational Research Institute, MUSC Pediatrics.

Maternal vitD deficiency as defined by circulating 25(OH)D concentration is linked with certain adverse pregnancy outcomes (e.g., preterm birth) and childhood outcomes (e.g., asthma), with the effect seemingly more pronounced if deficiency occurs earlier in pregnancy. OBJ: Assess the long-term effect of maternal and neonatal vitD status on later risk of childhood allergy, wheezing and/or asthma to 4 yrs. It was hypothesized that deficiency earlier in pregnancy would have a significant effect on risk that would continue during pregnancy. In this follow-up post hoc analysis of women and their offspring enrolled in 1 of 2 pregnancy vitD supplementation trials (NICHD, n = 348 and Kellogg Foundation, n = 298), women were randomized to either 400, 2000 or 4000 IU vitD/day (NICHD) at 12–16 wks' or 400 or 4400 IU/day at 10–14 wks' (Kellogg). Baseline then monthly 25(OH)D concentration as the primary outcome in both studies and as the indicator of vitD status was measured by RIA until delivery. Neonatal vitD status was measured in cord blood. Follow-up data on the offspring were available through 4 yrs using an EMR with ICD-9 and 10 codes for eczema, wheezing and/or asthma. Student's t-test was used to analyze differences in mean 25(OH)D and eczema, wheezing, and asthma. Chi-square analyses were used to test for differences in incidence of 25(OH)D below 20, 30, and 40 and eczema, wheezing, and asthma. In NICHD Pregnancy, 326/348 (93.7%) offspring had EMR data available: 48 (14.7%) had eczema; 32 (9.8%) had wheezing; and 48 (14.7%) had asthma. In Kellogg Pregnancy, 205/298 (68.8%) had EMR data available; 36 (17.6%) had eczema; 14 (6.8%) had wheezing; and 10 (4.9%) had asthma. Maternal baseline 25(OH)D < 30 ng/mL was associated with eczema (P = 0.024) and asthma (P = 0.035) by age 4 yrs. Neonatal 25(OH)D was inversely associated with eczema (P = 0.01) and asthma by age 4 (P = 0.0012). When dichotomized, neonates with 25(OH)D < 20 ng/mL had a significantly higher risk of eczema (P = 0.02) and asthma (P = 0.004) and those below 40 ng/mL had a higher risk of eczema (P = 0.03). In this combined cohort of pregnant women and their offspring, both maternal and neonatal vitD status were associated with later allergy, wheezing and asthma risk. Efforts to improve maternal vitD status may have later significant consequences on childhood health outcomes. NIH/NICHD/NCATS.