Post Hoc Analysis of National Institute of Child Health and Human Development Vitamin-D Pregnancy Cohort and The Role of Functional Vitamin-D Deficiency in Pregnancy.

OBJECTIVE: Our objective was to conduct a secondary, post hoc analysis of the National Institute of Child Health and Human Development (NICHD) vitamin D (vitD) pregnancy study by Hollis et al, which reported on the effect of vitD supplementation in pregnant women and determine the potential interaction between intact parathyroid hormone (iPTH) concentrations, vitD status, and various comorbidities associated with pregnancy. Women with low 25-hydroxy vitamin D (25(OH)D) concentrations and high iPTH concentrations during pregnancy, known as functional vitamin-D deficiency (FVDD), were more likely to acquire complications also affecting their neonates. STUDY DESIGN: This post hoc analysis of data collected from a diverse group of pregnant women participating in the NICHD vitD pregnancy study was applied to investigate the applicability of the concept of FVDD in pregnancy (Hemmingway, 2018) in identifying potential risks for certain comorbidities of pregnancy. This analysis defines FVDD as maternal serum 25(OH)D concentrations below 20 ng/mL and iPTH concentrations above 65 pg/mL creating a definitive ratio number, 0.308, to classify mothers as having FVDD prior to delivery (PTD). Statistical analyses were performed using SAS 9.4 (Cary, NC). RESULTS: In total, 281 women (85 African American, 115 Hispanic, and 81 Caucasian) with 25(OH)D and iPTH concentrations measured at monthly visits were included in this analysis. No statistically significant association was found between mothers classified as having FVDD at baseline or 1-month PTD and hypertensive disorders of pregnancy, infection, or admittance to the neonatal intensive care unit. When combining all comorbidities of pregnancy in this cohort, results showed those with FVDD at baseline, 24 weeks' gestation, and 1-month PTD were more likely to experience a comorbidity (p = 0.001; p = 0.001; p = 0.004, respectively). Those with FVDD 1-month PTD were 7.1 times (confidence interval [CI]: 1.71-29.81) more likely to have preterm birth (<37 weeks) than women without FVDD. CONCLUSION: Participants were more likely to have experienced preterm birth if they met the criteria for FVDD. This study supports the importance of FVDD during pregnancy. KEY POINTS: · Functional vitamin D deficiency (FVDD) is defined as the ratio of 25(OH)D divided by iPTH concentration ≤0.308.. · At a minimum, it is recommended that vitamin D status be kept in the healthy range based on current recommendations for pregnant individuals.. · FVDD is a more sensitive predictor of pregnancy risk than 25(OH)D alone.. · FVDD identified those with greater risk of preterm birth in this cohort..

Oxygen and steroids affect the regulatory role of natriuretic peptide receptor-C on surfactant secretion by type II cells.

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.

Sociodemographic factors affecting perceived stress during pregnancy and the association with immune-mediator concentrations.

OBJECTIVES: Determine which sociodemographic factors are most associated with increased maternal perceived stress during pregnancy. Evaluate the association between maternal stress and plasma immune-mediator concentrations (IMCs). METHODS: As part of a prospective, randomized clinical trial, 247 participants completed a Perceived Stress Scale survey (PSS-10) during each trimester of pregnancy. Blood samples were collected from participants and were analyzed for 25-hydroxyvitamin D (25(OH)D) concentration and for several IMCs: interferon-gamma, interleukins (IL-) IL-2, IL-4, IL-5, IL-10, vascular endothelial growth factor, c-reactive protein, and tumor necrosis factor alpha (TNF-α) (R&D Elisa). The potential associations between PSS-10 scores, sociodemographic factors, and IMCs were assessed. RESULTS: In bivariate analysis, participants who were not married and/or had high risk pregnancies were more likely to have increased PSS-10 scores (p<0.05). Increased PSS-10 scores were associated with higher serum concentrations of IL-2 and TNF-α, and decreased concentrations of IL-10 and 25(OH)D. In linear regression analysis, single marital status, high-risk pregnancy, IL-2, and TNF-α were independent predictors of PSS-10 scores. CONCLUSIONS: This study identifies specific sociodemographic factors that are associated with increased perceived stress during pregnancy. This study also provides evidence that increased perceived stress is associated with physiological changes as measured by changes in circulating IL-2, TNF-α, IL-10, and 25(OH)D concentrations.

Dysfunctional lactate metabolism in human alveolar type II cells from idiopathic pulmonary fibrosis lung explant tissue.

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.

Effects of vitamin D supplementation on circulating concentrations of growth factors and immune-mediators in healthy women during pregnancy.

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-ß 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-ß, 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-ß 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-ß and with IFN-γ and IL-2 at second and third trimesters. Baseline IFN-γ, CRP, TGF-ß, 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-ß, 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.

Vitamin D binding protein polymorphisms significantly impact vitamin D status in children.

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 < 0.0001). CONCLUSIONS: VDBP genetic variability was a significant factor affecting childhood vitD status when following RDA guidelines. This study may inform public health policy of uniformity in recommended childhood vitD dosage, especially regarding racially/ethnically associated disparities.

Transforming growth factor ß1 (TGFß1) regulates CD44V6 expression and activity through extracellular signal-regulated kinase (ERK)-induced EGR1 in pulmonary fibrogenic fibroblasts.

The appearance of myofibroblasts is generally thought to be the underlying cause of the fibrotic changes that underlie idiopathic pulmonary fibrosis. However, the cellular/molecular mechanisms that account for the fibroblast-myofibroblast differentiation/activation in idiopathic pulmonary fibrosis remain poorly understood. We investigated the functional role of hyaluronan receptor CD44V6 (CD44 containing variable exon 6 (v6)) for differentiation of lung fibroblast to myofibroblast phenotype. Increased hyaluronan synthesis and CD44 expression have been detected in numerous fibrotic organs. Previously, we found that the TGFß1/CD44V6 pathway is important in lung myofibroblast collagen-1 and α-smooth-muscle actin synthesis. Because increased EGR1 (early growth response-1) expression has been shown to appear very early and nearly coincident with the expression of CD44V6 found after TGFß1 treatment, we investigated the mechanism(s) of regulation of CD44V6 expression in lung fibroblasts by TGFß1. TGFß1-mediated CD44V6 up-regulation was initiated through EGR1 via ERK-regulated transcriptional activation. We showed that TGFß1-induced CD44V6 expression is through EGR1-mediated AP-1 (activator protein-1) activity and that the EGR1- and AP-1-binding sites in the CD44v6 promoter account for its responsiveness to TGFß1 in lung fibroblasts. We also identified a positive-feedback loop in which ERK/EGR1 signaling promotes CD44V6 splicing and found that CD44V6 then sustains ERK signaling, which is important for AP-1 activity in lung fibroblasts. Furthermore, we identified that HAS2-produced hyaluronan is required for CD44V6 and TGFßRI co-localization and subsequent CD44V6/ERK1/EGR1 signaling. These results demonstrate a novel positive-feedback loop that links the myofibroblast phenotype to TGFß1-stimulated CD44V6/ERK/EGR1 signaling.

Succination is Increased on Select Proteins in the Brainstem of the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) Knockout Mouse, a Model of Leigh Syndrome.

Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus. Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to vestibular nucleus pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltage-dependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys(77) and Cys(48) were identified as endogenous sites of succination in voltage-dependent anion channels 2. Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model.

The commonly used nonionic surfactant Span 80 has RXRα transactivation activity, which likely increases the obesogenic potential of oil dispersants and food emulsifiers.

Obesity has reached pandemic proportions, and there is mounting evidence that environmental exposures to endocrine disrupting chemicals known as "obesogens" may contribute to obesity and associated medical conditions. The Deepwater Horizon (DWH) oil spill resulted in a massive environmental release of crude oil and remediation efforts applied large quantities of Corexit dispersants to the oil spill. The Corexit-enhanced Water Accommodated Fraction (CWAF) of DWH crude oil contains PPARγ transactivation activity, which is attributed to dioctyl sodium sulfosuccinate (DOSS), a probable obesogen. In addition to its use in oil dispersants, DOSS is commonly used as a stool softener and food additive. Because PPARγ functions as a heterodimer with RXRα to transcriptionally regulate adipogenesis we investigated the potential of CWAF to transactivate RXRα and herein demonstrated that the Corexit component Span 80 has RXRα transactivation activity. Span 80 bound to RXRα in the low micromolar range and promoted adipocyte differentiation of 3T3-L1 preadipocytes. Further, the combination of DOSS and Span 80 increased 3T3-L1 adipocyte differentiation substantially more than treatment with either chemical individually, likely increasing the obesogenic potential of Corexit dispersants. From a public health standpoint, the use of DOSS and Span 80 as food additives heightens concerns regarding their use and mandates further investigations.

Lactate as substrate for mitochondrial respiration in alveolar epithelial type II cells.

Because of the many energy-demanding functions they perform and their physical location in the lung, alveolar epithelial type II (ATII) cells have a rapid cellular metabolism and the potential to influence substrate availability and bioenergetics both locally in the lung and throughout the body. A thorough understanding of ATII cell metabolic function in the healthy lung is necessary for determining how metabolic changes may contribute to pulmonary disease pathogenesis; however, lung metabolism is poorly understood at the cellular level. Here, we examine lactate utilization by primary ATII cells and the ATII model cell line, MLE-15, and link lactate consumption directly to mitochondrial ATP generation. ATII cells cultured in lactate undergo mitochondrial respiration at near-maximal levels, two times the rates of those grown in glucose, and oxygen consumption under these conditions is directly linked to mitochondrial ATP generation. When both lactate and glucose are available as metabolic substrate, the presence of lactate alters glucose metabolism in ATII to favor reduced glycolytic function in a dose-dependent manner, suggesting that lactate is used in addition to glucose when both substrates are available. Lactate use by ATII mitochondria is dependent on monocarboxylate transporter (MCT)-mediated import, and ATII cells express MCT1, the isoform that mediates lactate import by cells in other lactate-consuming tissues. The balance of lactate production and consumption may play an important role in the maintenance of healthy lung homeostasis, whereas disruption of lactate consumption by factors that impair mitochondrial metabolism, such as hypoxia, may contribute to lactic acid build-up in disease.

Alveolar type II cells maintain bioenergetic homeostasis in hypoxia through metabolic and molecular adaptation.

Although many lung diseases are associated with hypoxia, alveolar type II epithelial (ATII) cell impairment, and pulmonary surfactant dysfunction, the effects of O(2) limitation on metabolic pathways necessary to maintain cellular energy in ATII cells have not been studied extensively. This report presents results of targeted assays aimed at identifying specific metabolic processes that contribute to energy homeostasis using primary ATII cells and a model ATII cell line, mouse lung epithelial 15 (MLE-15), cultured in normoxic and hypoxic conditions. MLEs cultured in normoxia demonstrated a robust O(2) consumption rate (OCR) coupled to ATP generation and limited extracellular lactate production, indicating reliance on oxidative phosphorylation for ATP production. Pharmacological uncoupling of respiration increased OCR in normoxic cultures to 175% of basal levels, indicating significant spare respiratory capacity. However, when exposed to hypoxia for 20 h, basal O(2) consumption fell to 60% of normoxic rates, and cells maintained only ∼50% of normoxic spare respiratory capacity, indicating suppression of mitochondrial function, although intracellular ATP levels remained at near normoxic levels. Moreover, while hypoxic exposure stimulated glycogen synthesis and storage in MLE-15, glycolytic rate (as measured by lactate generation) was not significantly increased in the cells, despite enhanced expression of several enzymes related to glycolysis. These results were largely recapitulated in murine primary ATII, demonstrating MLE-15 suitability for modeling ATII metabolism. The ability of ATII cells to maintain ATP levels in hypoxia without enhancing glycolysis suggests that these cells are exceptionally efficient at conserving ATP to maintain bioenergetic homeostasis under O(2) limitation.

Factors Affecting Postpartum Bone Mineral Density in a Clinical Trial of Vitamin D Supplementation.

Background: Few studies evaluate the effects of vitamin D status and supplementation on maternal bone mineral density (BMD) during lactation and further lack inclusion of diverse racial/ethnic groups, body mass index (BMI), or physical activity. Objective: Determine the effects of vitamin D treatment/status, feeding type, BMI, race/ethnicity, and physical activity on postpartum women's BMD to 7 months. Methods: Women with singleton pregnancies beginning 4-6 weeks' postpartum were randomized into two treatment groups (400 or 6400 IU vitamin D/day). Participant hip, spine, femoral neck, and whole-body BMD using Dual-energy X-ray absorptiometry (DXA Hologic), serum 25-hydroxyvitamin D [25(OH)D] (RIA; Diasorin), BMI, and physical activity were measured at 1, 4, and 7 months postpartum. A general linear mixed modeling approach was undertaken to assess the effects of vitamin D status [both serum 25(OH)D concentrations and treatment groups], feeding type, race/ethnicity, BMI, and physical activity on BMD in postpartum women. Results: During the 6-month study period, lactating women had 1-3% BMD loss in all regions compared with 1-3% gain in nonlactating women. Higher maternal BMI was associated with less bone loss in femoral neck and hip regions. Black American women had less BMD loss than White/Caucasian or Hispanic lactating women in spine and hip regions. Exclusively breastfeeding women in the 6400 IU vitamin D group had less femoral neck BMD loss than the 400 IU group at 4 months sustained to 7 months. Physical activity was associated with higher hip BMD. Conclusion: While there was BMD loss during lactation to 7 months, the loss rate was less than previously reported, with notable racial/ethnic variation. Breastfeeding was associated with loss in BMD compared with formula-feeding women who gained BMD. Higher BMI and physical activity independently appeared to protect hip BMD, whereas higher vitamin D supplementation appeared protective against femoral neck BMD loss.

Persistence of LPS-induced lung inflammation in surfactant protein-C-deficient mice.

Pulmonary surfactant protein-C (SP-C) gene-targeted mice (Sftpc(-/-)) develop progressive lung inflammation and remodeling. We hypothesized that SP-C deficiency reduces the ability to suppress repetitive inflammatory injury. Sftpc(+/+) and Sftpc(-/-) mice given three doses of bacterial LPS developed airway and airspace inflammation, which was more intense in the Sftpc(-/-) mice at 3 and 5 days after the final dose. Compared with Sftpc(+/+)mice, inflammatory injury persisted in the lungs of Sftpc(-/-) mice 30 days after the final LPS challenge. Sftpc(-/-) mice showed LPS-induced airway goblet cell hyperplasia with increased detection of Sam pointed Ets domain and FoxA3 transcription factors. Sftpc(-/-) type II alveolar epithelial cells had increased cytokine expression after LPS exposure relative to Sftpc(+/+) cells, indicating that type II cell dysfunction contributes to inflammatory sensitivity. Microarray analyses of isolated type II cells identified a pattern of enhanced expression of inflammatory genes consistent with an intrinsic low-level inflammation resulting from SP-C deficiency. SP-C-containing clinical surfactant extract (Survanta) or SP-C/phospholipid vesicles blocked LPS signaling through the LPS receptor (Toll-like receptor [TLR] 4/CD14/MD2) in human embryonic kidney 293T cells, indicating that SP-C blocks LPS-induced cytokine production by a TLR4-dependent mechanism. Phospholipid vesicles alone did not modify the TLR4 response. In vivo deficiency of SP-C leads to inflammation, increased cytokine production by type II cells, and persistent inflammation after repetitive LPS stimulation.

Genetic replacement of surfactant protein-C reduces respiratory syncytial virus induced lung injury.

BACKGROUND: Individuals with deficiencies of pulmonary surfactant protein C (SP-C) develop interstitial lung disease (ILD) that is exacerbated by viral infections including respiratory syncytial virus (RSV). SP-C gene targeted mice (Sftpc -/-) lack SP-C, develop an ILD-like disease and are susceptible to infection with RSV. METHODS: In order to determine requirements for correction of RSV induced injury we have generated compound transgenic mice where SP-C expression can be induced on the Sftpc -/- background (SP-C/Sftpc -/-) by the administration of doxycycline (dox). The pattern of induced SP-C expression was determined by immunohistochemistry and processing by Western blot analysis. Tissue and cellular inflammation was measured following RSV infection and the RSV-induced cytokine response of isolated Sftpc +/+ and -/- type II cells determined. RESULTS: After 5 days of dox administration transgene SP-C mRNA expression was detected by RT-PCR in the lungs of two independent lines of bitransgenic SP-C/Sftpc -/- mice (lines 55.3 and 54.2). ProSP-C was expressed in the lung, and mature SP-C was detected by Western blot analysis of the lavage fluid from both lines of SP-C/Sftpc -/- mice. Induced SP-C expression was localized to alveolar type II cells by immunostaining with an antibody to proSP-C. Line 55.3 SP-C/Sftpc -/- mice were maintained on or off dox for 7 days and infected with 2.6x107 RSV pfu. On day 3 post RSV infection total inflammatory cell counts were reduced in the lavage of dox treated 55.3 SP-C/Sftpc -/- mice (p = 0.004). The percentage of neutrophils was reduced (p = 0.05). The viral titers of lung homogenates from dox treated 55.3 SP-C/Sftpc -/- mice were decreased relative to 55.3 SP-C/Sftpc -/- mice without dox (p = 0.01). The cytokine response of Sftpc -/- type II cells to RSV was increased over that of Sftpc +/+ cells. CONCLUSIONS: Transgenic restoration of SP-C reduced inflammation and improved viral clearance in the lungs of SP-C deficient mice. The loss of SP-C in alveolar type II cells compromises their response to infection. These findings show that the restoration of SP-C in Sftpc -/- mice in response to RSV infection is a useful model to determine parameters for therapeutic intervention.

Association of maternal vitamin D and placenta growth factor with the diagnosis of early onset severe preeclampsia.

OBJECTIVE: Decreased maternal 25-hydroxyvitamin D (25-OH-D) and placenta growth factor (PlGF) have both been associated with the diagnosis of early onset severe preeclampsia (EOSPE). This investigation aimed to define the association of these biomarkers with EOSPE. STUDY DESIGN: Patients with EOSPE (n = 40) and healthy controls (n = 40) were recruited and information on demographics, outcomes, and plasma was collected at diagnosis of EOSPE or gestational age-matched controls. 25-OH-D was assessed by radioimmunoassay and reported in nanogram per milliliter. PlGF was assessed by enzyme-linked immunosorbent assay and reported in picogram per milliliter. Kruskal-Wallis test was used to compare biomarkers between groups. Multivariable logistic regression was used to determine associations between 25-OH-D or PlGF and the diagnosis of EOSPE. RESULTS: In EOSPE, both 25-OH-D and PlGF were decreased significantly compared with controls. After controlling for age, race, body mass index, and gestational age at sample collection, both 25-OH-D (adjusted odds ratio 0.14 [0.05, 0.36]) and PlGF (adjusted odds ratio 0.03 [0.01, 0.24] were significantly associated with diagnosis of ESOPE (p < 0.001 for both markers). CONCLUSION: PlGF and 25-OH-D are both associated with the diagnosis of EOSPE. These biomarkers may be helpful in development of novel rapid diagnostic tests for preeclampsia.

HAMLET in human milk is resistant to digestion and carries essential free long chain polyunsaturated fatty acids and oleic acid.

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.

Predicting comorbidities of pregnancy: A comparison between total and free 25(OH)D and their associations with parathyroid hormone.

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.

Vitamin D and Child Neurodevelopment-A Post Hoc Analysis.

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.

Free Fatty Acid and α-Lactalbumin-Oleic Acid Complexes in Preterm Human Milk Are Cytotoxic to Fetal Intestinal Cells in vitro.

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.