Term Cord Blood Research Articles

Quantitative Label-Free Mass Spectrometry Identifies Molecular Content and Signaling Differences between Neonatal and Adult Platelets

Observational and randomized studies have associated platelet transfusions with increased morbidity and mortality in preterm neonates. Clinical practice changes have been hindered by a limited understanding of why adult donor-derived platelets might be inappropriate or harmful to infants. Neonatal and adult platelets differ in reactivity and function, but we lack an understanding of underlying molecular differences. As proteins and kinase-driven phosphorylation signaling dictate platelet biology, we aimed to characterize altered protein and phosphoprotein content between neonatal and adult platelets to help explain differences in platelet reactivity and function. We isolated resting platelets from full term cord blood (n=9) and adult peripheral blood (n=7) and used mass spectrometry-based platforms to ascertain protein and phosphoprotein content. We identified 4745 high-confidence proteins in adult and neonatal platelets, including 331 differentially abundant proteins (≥1.5-fold change, p<0.05). Neonatal platelets had increased metabolic and ribosomal proteins, consistent with transcriptional data. Adult platelets were enriched for inflammatory proteins, including complement components, consistent with pro-inflammatory function. We also identified 2115 phospho-proteins (17852 unique phosphopeptides) across all samples. These impacted actin cytoskeletal biology, cell adhesion, and GTPase signaling that regulate platelet functions. Kinase enrichment analysis identified FYN, SRC, ABL1, and AKT1 as kinases most responsible for platelet phosphorylation activities. Granule trafficking and degranulation proteins were among 1183 differentially abundant phosphoproteins in neonatal vs adult samples (p<0.05). For example, 31 different Reticulon 1 (RTN1) phosphopeptides were enriched in adult platelets (p<0.05). RTN1 regulates membrane trafficking and participates in SNARE-mediated exocytosis. Increased RTN1-directed membrane trafficking may promote increased activation and degranulation in adult vs neonatal platelets. Indeed, SNARE complex protein deficiency was previously suggested to mediate hyporeactive degranulation in neonatal platelets. We detected 2 phosphopeptides in adult samples that were absent from any neonatal sample. We reasoned that these could represent developmental stage-specific differences that exist only in adult platelets. One of the adult-specific phosphopeptides was in Rap1GAP2 (phospho-S588). Rap1GAP2 is a highly phosphorylated GTPase-activating protein that inhibits Rap1, a small guanine-nucleotide-binding protein that facilitates integrin activation in platelets. Rap1GAP2 binds synaptotagmin-like protein 1 (Slp1) to regulate dense granule secretion. These findings may link altered developmentally regulated Rap1GAP2 activities with platelet degranulation. A Platelet Cell Adhesion Molecule (PECAM) phosphopeptide (phospho-S726) was also found exclusively in adult platelets. PECAM facilitates inflammation and leukocyte transendothelial migration. PECAM phosphorylation in adult platelets may reflect an enhanced ability to participate in these processes. These findings reveal key differences in protein content and signaling that underlie differential neonatal vs adult platelet reactivity and function, including pro-inflammatory activities and enhanced activation and/or degranulation propensity in adult platelets. RTN1-mediated SNARE complex function and Rap1 signaling activities represent novel and potentially modifiable targets to manipulate platelet activation and/or degranulation. Our results also help provide a biological rationale for the higher morbidity and mortality observed in preterm infants transfused with adult platelets, including the inadvertent introduction of serum complement and other pro-inflammatory molecules within transfusions of donated adult platelets.

Imbalanced Inflammatory Responses in Preterm and Term Cord Blood Monocytes and Expansion of the CD14+CD16+ Subset upon Toll-like Receptor Stimulation

Developmentally regulated features of innate immunity are thought to place preterm and term infants at risk of infection and inflammation-related morbidity. Underlying mechanisms are incompletely understood. Differences in monocyte function including toll-like receptor (TLR) expression and signaling have been discussed. Some studies point to generally impaired TLR signaling, others to differences in individual pathways. In the present study, we assessed mRNA and protein expression of pro- and anti-inflammatory cytokines in preterm and term cord blood (CB) monocytes compared with adult controls stimulated ex vivo with Pam3CSK4, zymosan, polyinosinic:polycytidylic acid, lipopolysaccharide, flagellin, and CpG oligonucleotide, which activate the TLR1/2, TLR2/6, TLR3, TLR4, TLR5, and TLR9 pathways, respectively. In parallel, frequencies of monocyte subsets, stimulus-driven TLR expression, and phosphorylation of TLR-associated signaling molecules were analyzed. Independent of stimulus, pro-inflammatory responses of term CB monocytes equaled adult controls. The same held true for preterm CB monocytes-except for lower IL-1β levels. In contrast, CB monocytes released lower amounts of anti-inflammatory IL-10 and IL-1ra, resulting in higher ratios of pro-inflammatory to anti-inflammatory cytokines. Phosphorylation of p65, p38, and ERK1/2 correlated with adult controls. However, stimulated CB samples stood out with higher frequencies of intermediate monocytes (CD14+CD16+). Both pro-inflammatory net effect and expansion of the intermediate subset were most pronounced upon stimulation with Pam3CSK4 (TLR1/2), zymosan (TR2/6), and lipopolysaccharide (TLR4). Our data demonstrate robust pro-inflammatory and yet attenuated anti-inflammatory responses in preterm and term CB monocytes, along with imbalanced cytokine ratios. Intermediate monocytes, a subset ascribed pro-inflammatory features, might participate in this inflammatory state.

Vitamin B12 status in term pregnant women and the cord blood samples of their newborns: A cross-sectional study.

Background and aim: In India, total vitamin B12 (Vit B12) and its active form (active B12) have not been studied in mother's blood and cord blood. We hypothesized that total and active B12 levels are sufficiently maintained in cord blood despite low levels in mothers. Methods: Two hundred term pregnant mother's blood and cord blood was collected and analyzed for total Vit B12 (radioimmunoassay method) and active B12 levels (enzyme-linked immunosorbent assay). Mean values of constant or continuous variables (hemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), white blood cells (WBC), and Vit B12) were compared in mother's blood and newborn cord blood using Student's t-test and multiple comparisons within the groups were carried out with ANOVA. Spearman's correlation (Vit B12) and multivariable backward regression analyses (height, weight, education, body mass index (BMI) and Hb, PCV, MCV, WBC, and Vit B12 levels) were also performed. Results: Total Vit 12 deficiency was highly prevalent at 89% and active B12 deficiency was 36.7% in mothers. Cord blood showed total Vit B12 deficiency prevalence of 53% and active B12 deficiency being 9.3%. Total Vit B12 (p<0.001) and active B12 (p<0.001) levels were significantly higher in cord blood when compared to mother's blood. In multivariate analysis, higher total and active B12 levels in mother's blood predicted higher levels of total and active B12 levels in cord blood. Conclusion: Our study showed higher prevalence of total and active Vit B12 deficiency in mothers compared to cord blood thus indicating their transfer to fetus irrespective of mother's status. Maternal Vit B12 levels affected cord blood Vit B12 levels.

Maternal and neonatal one-carbon metabolites and the epigenome-wide infant response

Maternal prenatal status, as encapsulated by that to which a mother is exposed through diet and environment, is a key determinant of offspring health and disease. Alterations in DNA methylation (DNAm) may be a mechanism through which suboptimal prenatal conditions confer disease risk later in life. One-carbon metabolism (OCM) is critical to both fetal development and in supplying methyl donors needed for DNAm. Plasma concentrations of one-carbon metabolites across maternal first trimester (M1), maternal term (M3), and infant cord blood (CB) at birth were tested for association with DNAm patterns in CB from the Michigan Mother and Infant Pairs (MMIP) pregnancy cohort. The Illumina Infinium MethylationEPIC BeadChip was used to quantitatively evaluate DNAm across the epigenome. Global and single-site DNAm and metabolite models were adjusted for infant sex, estimated cell type proportions, and batch as covariates. Change in mean metabolite concentration across pregnancy (M1 to M3) was significantly different for S-adenosylhomocysteine (SAH), S-adenosylmethionine (SAM), betaine, and choline. Both M1 SAH and CB SAH were significantly associated with the global distribution of DNAm in CB, with indications of a shift toward less methylation. M3 SAH and CB SAH also displayed significant associations with locus-specific DNAm in infant CB (FDR<0.05). Our findings underscore the role of maternal one-carbon metabolites in shifting the global DNAm pattern in CB and emphasizes the need to closely evaluate how dietary status influences cellular methylation potential and ultimately offspring health.

Placental lipid transport and content in response to maternal overweight and gestational diabetes mellitus in human term placenta

Background and aimsPlacental lipid transport is altered in women with high prepregnancy body mass index (pre-BMI) or gestational diabetes (GDM), which consequently affects foetal growth. However, the interaction of maternal overweight (OW) and GDM on placental lipid metabolism and possible adaptations are less studied. We aimed to examine whether maternal OW or GDM is the main factor disrupting placental lipid processing in human term placenta. Methods and resultsA total of 152 lean (18.5 ≤ pre-BMI ≤ 23.9 kg/m2) and OW (24 ≤ pre-BMI ≤ 27.9 kg/m2) pregnant women with or without GDM with a scheduled delivery by caesarean section were recruited. Maternal venous blood samples were used to measure metabolic parameters during pregnancy. Term placentas and cord blood were collected at delivery to determine placental lipid metabolism and foetal circulating lipid levels. Maternal OW significantly increased the placental mRNA expression of genes involved in lipid metabolism (FAT/CD36, FATP1, FATP4, FATP6, and PPAR-α), elevated placental lipid content (triglyceride, cholesterol), enhanced placental mTORC1-rpS6 and ERK1/2 signalling, increased cord blood insulin levels and birth weight. Neonatal birth weight was positively correlated with maternal pre-BMI, placental ERK1/2 signalling and cord blood insulin. There was an interaction between OW and GDM in regulating key placental fuel transport and storage gene expression (LPL, FATP6, FABP7, PPAR-α, PPAR-β, PPAR-γ, IR-β, GLUT1, SNAT2, SNAT4, and LAT1). ConclusionMaternal OW mainly affects placental lipid metabolism, which may contribute to foetal overgrowth and may impact long-term offspring health. GDM plays a less significant role in affecting placental lipid transfer and other mechanisms may be involved.

Maternal 11-Ketoandrostenedione Rises Through Normal Pregnancy and Is the Dominant 11-Oxygenated Androgen in Cord Blood.

Adrenal-derived 11-oxygenated androgens (11oAs) are known important contributors to human physiology and disease but have not been studied in pregnancy. We characterize 11oAs in normal human pregnancy and neonatal period and assess the ratios between 11oAs and compare with ratios of other steroids that undergo placental metabolism. Prospective cohort study, 2010-2018. Academic institution. Pairs of pregnant women and newborns (n = 120) were studied. Inclusion criteria were maternal age between 18 and 42 years old, spontaneous singleton pregnancies, and intention to deliver at University of Michigan. Maternal venous blood was collected during first trimester and at term. Neonatal cord blood was collected following delivery. Steroids were measured via liquid chromatography-tandem mass spectrometry. Levels of 11β-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), 11β-hydroxytestosterone, and 11-ketotestoterone (11KT) in maternal first trimester, maternal term, and neonatal cord blood were compared. 11OHA4-to-11KA4 ratios were correlated with cortisol-to-cortisone ratios. Dominant 11oAs in pregnancy and the cord blood are 11OHA4 and 11KA4, compared to 11OHA4 and 11KT in adult men and nonpregnant women. We found a rise in 11oA concentrations, particularly 11KA4, from first to third trimester. In cord blood, the concentration of 11KA4 exceeded those of both 11OHA4 and 11KT, reflecting placental 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) and 17β-hydroxysteroid dehydrogenase (17βHSD2) activities, respectively. 11OHA4-to-11KA4 ratios are concordant with cortisol-to-cortisone ratios across all maternal and fetal compartments, reflecting placental 11βHSD2 activity. Placental 17βHSD2 activity defends the fetus against the androgen 11KT. Our normative values may be used in future studies of 11oAs in complicated pregnancies.

Noval Insights into the Role of Platelet Angiogenic Growth Factors on the Regulation of Normal Vascular Development

The role of platelets as angiogenic regulators has been the subject of intense research over the last decade, and has led to the recognition of platelets as the predominant source of key angiogenic growth factors (AGFs) in the circulation. Through the regulated delivery of these factors, platelets contribute to wound healing, maintenance of vascular integrity, and tumor angiogenesis. Prior studies have shown that thrombocytopenia in adult mice leads to hemorrhages primarily from structurally abnormal new blood vessels, both in in vivo angiogenesis assays and inside tumors. The rapid growth during fetal and neonatal life is associated with a developmental stage-specific need for accelerated angiogenesis and vascular development. However, whether and how platelets participate in normal fetal/neonatal vascular development is unknown, except for their role in blood/lymphatic separation. We hypothesized that platelets would be important for normal fetal/neonatal vascular development, and that their AGF profile would be different from that of adult platelets. As a first step, we quantified five key AGFs (VEGF-A, bFGF, ANG-1, PDGF-BB and TGF-b) by ELISA in platelet lysates and platelet-poor plasma from term cord blood samples (CB; n=12) and from the peripheral blood of healthy adult volunteers (PB; n=8). These studies demonstrated that CB platelets, just like PB platelets, are the major source of AGFs in the circulation, carrying more than 80% of the levels of these AGFs present in the blood. However, the angiogenic profiles were different in platelets of different developmental origins. Fetal/neonatal platelets contained ~70% more bFGF and VEGF-A than adult platelets (p<0.01 for both), but had significantly lower levels of ANG-1, PDGF-BB and TGF-β1 (p<0.05 vs. adult platelets). VEGF-A and bFGF are essential for the proliferation and differentiation of endothelial cells, while ANG-1, PDGFs and TGF-b concert mural cell coverage, basement membrane formation and vascular maturation, thus stabilizing and “strengthening” the vasculature. Based on these characteristics, we hypothesized that neonatal and adult platelets would have different biological effects. To test this, we first assessed the effects of CB- vs. PB-derived platelet lysates on human umbilical vascular endothelial cells (HUVEC), in a tube formation assay. In these experiments, CB platelets more potently stimulated angiogenesis, quantified as number of capillary branch points at 4, 5, and 6 hours, than PB platelets. Subsequently, we compared the proliferative effects of CB vs. PB platelet lysates on human primary CB-derived endothelial progenitor cells (EPCs) and amniotic membrane-derived mesenchymal stromal cells (MSCs). Consistent with the effects on HUVEC cells, CB platelet lysates induced approximately 50% more proliferation of EPCs, compared to PB platelet lysates (p<0.001). In contrast, PB platelet lysates were approximately twice more potent in promoting MSCs growth (p<0.001 vs. CB platelets). Since EPCs give rise to the vascular endothelium and MSCs differentiate into mural cells, these results strongly suggested that the AGF content in fetal/neonatal platelets promotes rapid angiogenesis at the expense of a stable vasculature, matching the developmental needs of rapidly growing fetuses and neonates. Finally, since birth is associated with an abrupt change in oxygen concentration (from the profoundly hypoxic intrauterine to the normoxic extrauterine environment), we assessed whether oxygen concentration would modulate the VEGF content in CB-derived cultured MKs (and thus platelets). Consistent with this hypothesis, CB MKs cultured in 5% oxygen released significantly more VEGF in the medium compared to MKs cultured in 20% oxygen (p<0.05). Our findings strongly suggest that the platelet AGF profile changes from neonatal to adult life to help regulate vascular development at different stages of life, and suggest that it might also change in response to environmental factors such as oxygen concentration. Whether a change in platelet angiogenic profile associated with the sudden increase in oxygen concentration during preterm birth contributes to diseases of prematurity characterized by disrupted vascular development (i.e. retinopathy of prematurity) requires further investigation. DisclosuresSola-Visner:Sysmex Inc.: Research Funding.

Maternal and Infant Immune Repertoire Sequencing Analysis Identifies Distinct Immunoglobulin and T Cell Receptor Development in Term and Preterm Infants

Preterm labor (PTL) is the leading cause of neonatal morbidity and mortality worldwide. While many studies have focused on investigating the maternal immune responses that cause PTL, fetal immune cell activation has recently been raised as an important contributor to the pathogenesis of PTL. Here, we analyzed lymphocyte receptor repertoires in maternal and cord blood from 14 term and 10 preterm deliveries and hypothesized that the high incidence of infection in patients with PTL may result in specific changes in the TCR and BCR repertoires. We analyzed T-cell receptor beta chain (TCR-β) and immunoglobulin heavy chain (IgH) diversity, CDR3 lengths, clonal sharing, and preferential usage of variable (V), diversity (D), and joining (J) gene segments. Additionally, we studied the rates of somatic hypermutation (SHM) in IgH. Overall, the cord blood IgH repertoires had significantly (padj < 0.05) lower rates of SHM and both TCR-β and IgH repertoires had shorter CDR3s compared to maternal blood. When comparing term and PPROM cord blood samples, we found that CDR3 lengths correlated with gestational age, with the shorter CDR3s in preterm neonates suggesting a ‘less developed’ repertoire. Preterm cord blood displayed preferential usage of a number of V genes and J genes. Furthermore, the term maternal repertoires displayed significant preferential usage of TRBV7-8 compared to preterm maternal repertoires. In preterm pregnancies, we observed significantly higher prevalence of convergent clones between mother/baby pairs than in term pregnancies. Together, our results suggest the repertoire of preterm infants displays a combination of immature features yet preferential use of particular genes and convergence with maternal TCR-β clones compared to term infants. While the higher TCR-β diversity might reflect less clonal expansion, the higher clonal convergence between mothers and infants in PTL could represent mother and fetus both responding to a shared stimulus like an infection. These data provide a detailed analysis of the maternal-fetal immune repertoire in term and preterm patients, and contribute to a better understanding of neonate immune repertoire development and potential changes associated with preterm labor.

Placental HTRA1 Protease Cleaves Alpha-1-Antitrypsin and Generates Neonatal NET-Inhibitory Factor

BACKGROUND: Neutrophil extracellular traps (NET) are extracellular lattices of decondensed chromatin associated with anti-microbial proteins and degradative enzymes released by polymorphonuclear leukocytes (PMN) to trap and kill invading microbes. Dysregulated NET formation, however, contributes to inflammatory tissue damage. We have identified a novel NET-inhibitory peptide, neonatal NET-Inhibitory Factor (nNIF), present in the fetal circulation. nNIF is formed as a carboxy-terminus cleavage fragment of alpha-1 antitrypsin (AAT), an abundant, circulating protease inhibitor with homologs in human and mouse blood. However, the exact mechanisms by which nNIF is generated in fetal and neonatal blood remains unknown.OBJECTIVE: High temperature requirement A 1 (HTRA1) is expressed in the placenta during fetal development and inhibits AAT. We hypothesized that placentally expressed HTRA1, a serine protease, regulates the formation of NET-inhibitory peptides, such as nNIF, through cleavage of AAT.DESIGN/METHODS: Term and preterm placenta were lysed and probed for HTRA1 expression. HTRA1 and AAT plasma expression from term and preterm infants and adults were determined by ELISA. Recombinant, bioactive HTRA1 or placenta-eluted HTRA1 were incubated with AAT and the generation of carboxy-terminus fragments of AAT was assessed using western blotting and mass spectrometry. Fragments of AAT generated by HTRA1 were incubated with LPS-stimulated PMNs and NET formation was examined qualitatively using live cell imaging and quantitatively using a high throughput fluorescence assay. The effect of the HTRA-AAT cleavage fragment on reactive oxygen species generation, neutrophil chemotaxis, phagocytosis, and bacterial killing was measured using flow cytometry, a modified Boyden chamber asssay, neutrophil labeled Escherichia coli uptake assay, and a bacterial killing assay with a pathogenic strain of Escherichia coli, respectively. Finally, NET formation was evaluated qualitatively and quantitatively in murine PMNs isolated from neonatal WT and HTRA1-/- pups between 1-3, 4-6 and 7-10 days after birth to determine when PMNs become NET-competent.RESULTS: Term and preterm infant placentas express HTRA1, and we detected significantly (p<0.05) higher levels of HTRA1 in plasma from term (465.1±71.8 µg/mL) and preterm (385.9±71.3 µg/mL) infant cord blood compared to adults (58.6±11.6 µg/mL). Recombinant, bioactive HTRA1 and placenta-derived HTRA1 incubated with AAT generate a 4kD AAT fragment based on western blot and mass spectrometry similar to the nNIF fragment found in cord blood from term and preterm infants. Pre-incubation of this fragment with LPS-stimulated PMNs significantly inhibits NET formation (p<0.05). The cleavage fragment from HTRA1-AAT, however, has no effect on reactive oxygen species generation, chemotaxis, or phagocytosis. However, incubation of this fragment with LPS-stimulated PMNs significantly (p<0.05) reduces NET-associated bacterial killing by 62% compared to a scrambled HTRA-AAT control peptide. In addition, the HTRA1-AAT fragment significantly (p<0.05) reduces nuclear decondensation by 93% compared to LPS-stimulated PMN, suggesting this fragment inhibits PAD4 activation similar to other NIFs previously examined. Neonatal murine plasma contains a 4kD AAT fragment which inhibits NET formation by adult mouse PMNs, indicating that nNIF generation is conserved in mice. Neonatal PMNs stimulated with LPS exhibit delayed NET formation following birth with PMNs becoming NET-competent by day 8 of life. However, neonatal PMNs from pups born from HTRA1-/- deficient mice generate significantly (p<0.05) more NETs between day 4-6 of life compared to WT controls, suggesting that HTRA1 regulates NET formation through nNIF production.CONCLUSIONS: Placental HTRA1 interacts with AAT to generate a carboxy-terminus cleavage fragment of AAT with identical NET-inhibitory properties to nNIF. Our data strongly indicate that placental HTRA1 generates nNIF in the fetal circulation. We speculate that nNIF participates in the required tolerance to new microbial antigens encountered during the transition to extrauterine life. DisclosuresNo relevant conflicts of interest to declare.

Antimicrobial peptide LL-37 and recombinant human mannose-binding lectin express distinct age- and pathogen-specific antimicrobial activity in human newborn cord blood in vitro

Background: There is a need to prevent and treat infection in newborns. One approach is administration of antimicrobial proteins and peptides (APPs) such as LL-37, a membrane-active cathelicidin antimicrobial peptide, and mannose-binding lectin (MBL), a pattern-recognition protein that binds to microbial surface polysaccharides resulting in opsonization and complement activation. Low plasma/serum levels of LL-37 and of MBL have been correlated with infection and exogenous administration of these agents may enhance host defense. Methods: The antimicrobial activity of LL-37 (15 µg/ml) or rMBL (0.5, 2 and 10 µg/ml) was tested in hirudin-anticoagulated preterm and term human cord blood (N = 12–14) against Staphylococcus aureus (SA) USA 300 (2x10 4 CFU/ml), Staphylococcus epidermis (SE) 1457 (2x10 4 CFU/ml) and Candida albicans (CA) SC5314 (1x10 4 CFU/ml). After incubation (1, 45, or 180 min), CFUs were enumerated by plating blood onto agar plates. Supernatants were collected for measurement of MBL via ELISA. Results: Preterm cord blood demonstrated impaired endogenous killing capacity against SA and SE compared to term blood. Addition of LL-37 strongly enhanced antimicrobial/antifungal activity vs SA, SE and CA in term blood and SE and CA in preterm blood. By contrast, rMBL showed modest fungistatic activity vs CA in a sub-analysis of term newborns with high basal MBL levels. Baseline MBL levels varied within preterm and term cohorts with no correlation to gestational age. In summary, exogenous LL-37 demonstrated significant antimicrobial activity against SA, SE and CA in term and SE and CA in preterm human blood tested in vitro. rMBL demonstrated modest antifungal activity in term cord blood of individuals with high baseline MBL levels. Conclusions: To the extent that our in vitro results predict the effects of APPs in vivo, development of APPs for prevention and treatment of infection should take into account host age as well as the target pathogen.

Antimicrobial peptide LL-37 and recombinant human mannose-binding lectin express distinct age- and pathogen-specific antimicrobial activity in human newborn cord blood in vitro.

Background: There is a need to prevent and treat infection in newborns. One approach is administration of antimicrobial proteins and peptides (APPs) such as LL-37, a membrane-active cathelicidin antimicrobial peptide, and mannose-binding lectin (MBL), a pattern-recognition protein that binds to microbial surface polysaccharides resulting in opsonization and complement activation. Low plasma/serum levels of LL-37 and of MBL have been correlated with infection and exogenous administration of these agents may enhance host defense. Methods: The antimicrobial activity of LL-37 (15 µg/ml) or rMBL (0.5, 2 and 10 µg/ml) was tested in hirudin-anticoagulated preterm and term human cord blood (N = 12-14) against Staphylococcus aureus (SA) USA 300 (2x10 4 CFU/ml), Staphylococcus epidermis (SE) 1457 (2x10 4 CFU/ml) and Candida albicans (CA) SC5314 (1x10 4 CFU/ml). After incubation (1, 45, or 180 min), CFUs were enumerated by plating blood onto agar plates. Supernatants were collected for measurement of MBL via ELISA. Results: Preterm cord blood demonstrated impaired endogenous killing capacity against SA and SE compared to term blood. Addition of LL-37 strongly enhanced antimicrobial/antifungal activity vs SA, SE and CA in term blood and SE and CA in preterm blood. By contrast, rMBL showed modest fungistatic activity vs CA in a sub-analysis of term newborns with high basal MBL levels. Baseline MBL levels varied within preterm and term cohorts with no correlation to gestational age. In summary, exogenous LL-37 demonstrated significant antimicrobial activity against SA, SE and CA in term and SE and CA in preterm human blood tested in vitro. rMBL demonstrated modest antifungal activity in term cord blood of individuals with high baseline MBL levels. Conclusions: To the extent that our in vitro results predict the effects of APPs in vivo, development of APPs for prevention and treatment of infection should take into account host age as well as the target pathogen.

Characterization of CD31 expression on murine and human neonatal T lymphocytes during development and activation

Abstract CD31, expressed by the majority of the neonatal T cell pool, is involved in modulation of T-cell receptor (TCR) signalling by increasing the threshold for T cell activation. Therefore, CD31 could impact neonatal tolerance or immune responses during infection. Cells were harvested from murine neonates at various days post-birth, human late preterm and term cord blood and adult peripheral blood. The number of CD31 expressing CD4+ and CD8a+ T cells increase in the neonatal mouse over the first week of life, which is consistent with previous work demonstrating an increase in CD31 expression across human gestation. Human samples were activated in vitro over a five-day period to simulate acute inflammation similar to chorioamnionitis or early onset sepsis. The frequency of human CD31+ CD4+ and CD8a+ T cells decrease upon activation, with infants having a lower frequency of CD31+ cells compared to adults. In order to further evaluate the regulation of CD31 during acute infection, murine neonates at 3 and 7 days of age were infected with influenza. There was an increased number of naïve and activated CD31+ T lymphocytes at the site of infection at 6 and 9 days post-infection in the 3-day old neonates (p&amp;lt;0.05), as compared to 7-day old. However, the opposite is true in the periphery; mice infected at 7-days of age have an increased number of activated CD31+ T lymphocytes in the spleen at 9 days post-infection (p&amp;lt;0.05). Trafficking differences of CD31+T lymphocytes to the site of infection suggest a differential regulation in younger mice which could contribute to the dampened adaptive immune response in neonates. Further murine and human mechanistic studies which examine CD31+ cytotoxic T lymphocyte function at the site of infection are needed.

Characterization of CD31 expression on murine and human neonatal T lymphocytes during development and activation.

BackgroundCD31, expressed by the majority of the neonatal T cell pool, is involved in modulation of T-cell Receptor signalling by increasing the threshold for T cell activation. Therefore, CD31 could modulate neonatal tolerance and adaptive immune responses.MethodsLymphocytes were harvested from murine neonates at different ages, human late preterm and term cord blood, and adult peripheral blood. Human samples were activated over a five-day period to simulate acute inflammation. Mice were infected with influenza; lungs and spleens were harvested at days 6 and 9 post-infection and analyzed by flow cytometry.ResultsCD31 expressing neonatal murine CD4+ and CD8a+ T cells increase over the first week of life. Upon in vitro stimulation, human infants’ CD4+ and CD8a+ T cells shed CD31 faster in comparison to adults. In the context of acute infection, mice infected at 3-days old have an increased number of naive and activated CD31+ T lymphocytes at the site of infection at day 6 and 9 post-infection, as compared to 7-days old; however, the opposite is true in the periphery.ConclusionDifferences in trafficking of CD31+ Cytotoxic T Lymphocytes (CTLs) during acute influenza infection could modulate tolerance and contribute to a dampened adaptive immune response in neonates.

MicroRNAs regulating toll like receptor inflammatory pathways in preterm and term placenta and cord blood

MicroRNAs regulating toll like receptor inflammatory pathways in preterm and term placenta and cord blood

Males are from Mars, and females are from Venus: sex-specific fetal brain gene expression signatures in a mouse model of maternal diet-induced obesity

Maternal obesity is associated with adverse neurodevelopmental outcomes in children, including autism spectrum disorders, developmental delay, and attention-deficit hyperactivity disorder. The underlying mechanisms remain unclear. We previously identified second-trimester amniotic fluid and term cord blood gene expression patterns suggesting dysregulated brain development in fetuses of obese compared with lean women. We sought to investigate the biological significance of these findings in a mouse model of maternal diet-induced obesity. We evaluated sex-specific differences in fetal growth, brain gene expression signatures, and associated pathways. Female C57BL/6J mice were fed a 60% high-fat diet or 10% fat control diet for 12-14 weeks prior to mating. During pregnancy, obese dams continued on the high-fat diet or transitioned to the control diet. Lean dams stayed on the control diet. On embryonic day 17.5, embryos were weighed and fetal brains were snap frozen. RNA was extracted from male and female forebrains (10 per diet group per sex) and hybridized to whole-genome expression arrays. Significantly differentially expressed genes were identified using a Welch's t test withthe Benjamini-Hochberg correction. Functional analyses were performed using ingenuity pathways analysis and gene set enrichment analysis. Embryos of dams on the high-fat diet were significantly smaller than controls, with males more severely affected than females (P= .01). Maternal obesity and maternal obesity with dietary change in pregnancy resulted in significantly more dysregulated genes in male vs female fetal brains (386 vs 66, P < .001). Maternal obesity with and without dietary change in pregnancy was associated with unique brain gene expression signatures for each sex, with an overlap of only 1 gene. Changing obese dams to a control diet in pregnancy resulted in more differentially expressed genes in the fetal brain than maternal obesity alone. Functional analyses identified common dysregulated pathways in both sexes, but maternal obesity and maternal dietary change affected different aspects of brain development in males compared with females. Maternal obesity is associated with sex-specific differences in fetal size and fetal brain gene expression signatures. Male fetal growth and brain gene expression may be more sensitive toenvironmental influences during pregnancy. Maternal diet during pregnancy has a significant impact on the embryonic brain transcriptome. It is important toconsider both fetal sex and maternal diet when evaluating the effects ofmaternal obesity on fetal neurodevelopment.

Gender-Specific Effects on Gestational Length and Birth Weight by Early Pregnancy BPA Exposure.

Effects of prenatal exposure to bisphenol A (BPA) on gestational and birth outcomes are controversial. The aim of the study was to evaluate the relationship between prenatal exposure to BPA and birth and gestational outcomes. design, setting, participants, and outcome: Levels of unconjugated (uBPA) and BPA glucuronide in 80 matching samples of pregnant women during the first trimester of pregnancy and at delivery and matching term cord blood obtained from a prospective study conducted at the University of Michigan Hospitals were determined using a methodology validated in the National Institutes of Environmental Health Sciences funded Round Robin study and related to pregnancy outcomes. Highest levels of uBPA were found in maternal term samples followed by first trimester maternal (M1) samples and cord blood. A 2-fold increase in M1 uBPA was associated with 55-g less birth weight when male and female pregnancies were combined and 183-g less birth weight with only female pregnancies. A 2-fold increase in maternal term uBPA was associated with an increased gestational length of 0.7 days for all pregnancies and 1.1 days for only female pregnancies. Higher uBPA exposure levels during first trimester and term are associated with sex-specific reduction in birth weight and increase in gestational length, respectively. Race, parity, and employment have an effect on BPA exposure. Because low birth weight is associated with adverse health outcomes, effect of early pregnancy BPA levels on reducing birth weight highlights the risk posed by developmental exposure to BPA.

Cytokine Levels in Late Pregnancy: Are Female Infants Better Protected Against Inflammation?

Inflammatory responses have been implicated in several forms of infant deaths (sudden expected deaths and stillbirths) and the initiation of pre-term births. In this study, we examined matched samples of term maternal blood, cord blood, and amniotic fluid obtained from 24 elective cesarean deliveries for both pro- and anti-inflammatory cytokines thought to be important in maintaining a balanced response leading to successful pregnancy outcome. These included interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-10, and IL-1 receptor antagonist (IL-1ra). Amniotic fluid levels for each of the cytokines examined were significantly higher than those for cord blood or maternal plasma. While pro-inflammatory cytokines were higher in amniotic fluid associated with male fetuses compared with females, the major significant difference was higher levels of IL-1ra in amniotic fluid associated with female fetuses. Our study supports similar findings for cytokines during mid-trimester, which noted that amniotic fluid levels were higher than those in maternal blood. Our study suggests that maternal decidua secretes additional IL-ra in the presence of a female conceptus which improves the likelihood of a good outcome compared to pregnancies with male fetuses.

Human leukocyte antigen (HLA)-G during pregnancy part I: Correlations between maternal soluble HLA-G at midterm, at term, and umbilical cord blood soluble HLA-G at term

Human leukocyte antigen (HLA)-G is a class Ib molecule with restricted tissue distribution expressed on trophoblast cells and has been proposed to have immunomodulatory functions during pregnancy. Soluble HLA-G1 (sHLA-G1) can be generated by the shedding of membrane-bound HLA-G molecules; however, three soluble isoforms also exist (HLA-G5 to -G6). During pregnancy, it is unknown whether there is a correlation between sHLA-G levels in maternal and fetal blood. In 246 pregnancies, we have measured the levels of sHLA-G1/-G5 in maternal blood plasma samples from gestational week 20 (GW20) and at term, as well as in umbilical cord blood samples. Soluble HLA-G levels declined by 38.4% in maternal blood from GW20 to term, and sHLA-G levels were significantly lower in maternal blood at term than in GW20 (P<0.001). At term, the sHLA-G levels were significantly higher in maternal blood than in umbilical blood (P<0.001). HLA-G levels in maternal blood in GW20 and at term, and in maternal blood at term and umbilical cord blood, were correlated (P<0.001 and P<0.01, respectively). This is the first large study simultaneously measuring sHLA-G in both maternal and umbilical cord blood. The finding that sHLA-G levels are significantly lower in fetal compared with maternal blood at term documents for the first time that sHLA-G is not freely transferred over the placental barrier. Soluble HLA-G levels in maternal and fetal blood were found to be correlated, which may be due to shared genetic factors of importance for production of sHLA-G in the mother and child, or it may support the theory that sHLA-G in the pregnant woman and the fetus is partly derived from a “shared organ”, the placenta.

Application of a Novel High Yield Volume-Reduction Method for the Cryopreservation of 950 UC Blood Units

Umbilical cord blood represents an increasingly useful source of hematopoietic stem cells for Bone Marrow Transplantation (BMT), although currently, low cell numbers generally limit its use to pediatric patients. The aim of this study was to evaluate the routine use of a recently published volume reduction protocol for long term cord blood banking as was applied in a large number of cord blood samples. 950 cord blood samples with mean volume collected 78.81 ± 23.56 ml, Nucleated Cell (NC) 0.91 ± 0.44 × 109 NC, Monocytes (MN) 0.49 ± 0.23 × 109 MN and CD34+ HPCs (Haematopoietic Progenitors Stem Cells) 2.4 ± 2.08 × 106 CD34+ HPCs counts, have been collected respectively. We found a significant correlation between leukocyte counts and CD34+ HPCs with the volume collected (r=0.71, P<0.0001). We found a highly statistically significant correlation between leukocyte cell count and CD34+ HPCs cell number, (r=0.66, P<0.0001). After volume reduction the respective UCB units contained 1.06 ± 0.54 × 109 NC and 3.4 ± 3.07 × 106 CD34+ HPC , 17.88 ± 9.38 × 109 Red Blood Cells (RBC), while the viability and the volume were 92 ± 5.9 % and 6.43 ± 0.99 ml respectively.

P025 Cytokine profile of healthy preterm and term cord blood and peripheral blood of septic newborns

Introduction Regardless of pregnancy, birth triggers a stress-induced pro-inflammatory response with an overall increase in cytokine production in the infant. Term labor may affect inflammatory cytokine concentrations in the mother, fetus and neonate. Preterm infants often experience a heightened pro-inflammatory response from the stress of birth and due to the immaturity of the endocrine system at preterm birth, the infant may be unable to maintain this inflammatory response under control, which may predispose it to sepsis. Our aim is to evaluate serum concentrations of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ, IL-12p70, TNF-α and IL-17A in healthy preterm and term newborns and in newborns diagnosed with sepsis. Methods Cytokines were measured by cytometric bead array in cord sera from healthy preterm Results The concentrations of pro-inflammatory cytokines IL-8, IL-6, IL-1β and IL-17A significantly depended on the mode of delivery, being higher in vaginal delivery than that in elective cesarean section. On the other hand, septic neonates presented significantly increased levels of all inflammatory cytokines, particularly IL-6 and the anti-inflammatory cytokine IL-10. Regarding the Th1 cytokines, IL-2 and IL-4, low concentrations were detected in all groups, and INF-g was not detected in all samples tested. Equivalent levels of IL-17A were detected in Groups 2 and 3, and in septic newborns. Conclusion Term and preterm labor itself might constitute an inflammatory-like process, and the results of this study strengthen the suggestion that the mode of delivery influences cytokine levels. The results with septic newborns indicate that IL-6 and IL-10 are good indicators of sepsis. Financial Support: FAPESP (2009/54246-5, 2009/52515-9, 2009/54400-5).