Jason A. Gersting

Circulating Concentrations of Chemokines in Cord Blood, Neonates, and Adults

Chemokines are critical for the movement of leukocytes. Chemotaxis is deficient in neonates, particularly those delivered prematurely, and this likely contributes to their increased vulnerability to sepsis. The concentrations of circulating chemokines in neonates have not been reported, nor is it known whether low chemokine concentrations contribute to their defective chemotaxis. We hypothesized that serum concentrations of chemokines 1) would be lower in preterm than term neonates, and 2) would be lower in preterm and term neonates than adults. Samples were obtained from preterm and term neonates with normal neutrophil and eosinophil counts, umbilical cord blood samples from pregnancies without clinical evidence of intra-amniotic infection, and healthy adult volunteers. The concentrations of epithelial neutrophil activating peptide-78, growth-related oncogene-α, eotaxin, RANTES (regulated upon activation, normal T cell expressed and secreted), and macrophage inflammatory protein-1α were measured using specific ELISA. Serum concentrations from preterm infants were either similar to or higher than those measured in term neonates and adults. We conclude that the chemotactic defect observed in premature neonates is not the result of diminished circulating concentrations of any of the specific chemokines we measured.

Development of ER-α and ER-β expression in the developing ovine brain and pituitary

Fetal neuroendocrine development in late gestation is critical for maintenance of fetal homeostasis, growth, and readiness for birth. We designed the present study to identify the regional patterns of expression of the two main isoforms of the estrogen receptor, ER-alpha and ER-beta, in the developing ovine fetal brain. Fetal (80, 100, 120, 130, and 145 days gestation), neonatal (1 and 7 days), and adult sheep were euthanized and the following tissues were collected: pituitary, hypothalamus, hippocampus, cerebral cortex, and brainstem. Both ERs are expressed in the ovine brain as early as 80 days gestation, and the expression of both receptors appears to be developmentally regulated. We conclude that both forms of the estrogen receptor are expressed in fetal brain and pituitary throughout the latter half of gestation.

Inhibition of Brain Prostaglandin Endoperoxide Synthase-2 Prevents the Preparturient Increase in Fetal Adrenocorticotropin Secretion in the Sheep Fetus

Maturation of the fetal hypothalamus-pituitary-adrenal axis is critical for the timely somatic development of the fetus and readiness for birth. Recently, we proposed that prostaglandin generation within the fetal central nervous system is critical for the modulation of hypotension-induced fetal ACTH secretion. The present study was designed to test the hypothesis that the preparturient increase in fetal ACTH secretion is dependent upon fetal central nervous system prostaglandin synthesis mediated by the activity of prostaglandin endoperoxide synthase (PGHS)-2 (cyclooxygenase-2) in the fetal brain. We performed two studies in chronically catheterized fetal sheep. In the first study, we infused nimesulide or vehicle intracerebroventricularly (i.c.v) into singleton fetal sheep and collected blood samples until spontaneous parturition. Nimesulide significantly delayed parturition, and inhibited fetal ACTH and proopiomelanocortin secretion but did not prevent the preparturient increase in fetal plasma cortisol concentration. In the second study, we used twin fetuses. One fetus received intracerebroventricular nimesulide and the other intracerebroventricular vehicle. Nimesulide reduced brain tissue concentrations of prostaglandin estradiol, while not affecting plasma prostaglandin E(2) concentrations, demonstrating an action restricted to the fetal brain. Nimesulide reduced PGHS-2 mRNA and increased PGHS-2 protein, while not altering PGHS-1 mRNA or protein in most brain regions, suggesting an effect of the inhibitor on PGHS-2 turnover and relative specificity for PGHS-2 in vivo. We conclude that the preparturient increase in fetal ACTH and proopiomelanocortin is dependent upon the activity of PGHS-2 in the fetal brain. However, we also conclude that the timing of parturition is not solely dependent upon ACTH in this species.

Effects of Enterally Administering Granulocyte Colony-Stimulating Factor to Suckling Mice

Gastrointestinal (GI) tract development is influenced by multiple growth factors, some of which are delivered directly to the GI lumen, as they are swallowed constituents of amniotic fluid, colostrum, and milk. Granulocyte colony-stimulating factor (G-CSF), traditionally known as a granulocytopoietic growth factor, is an example of one such factor. However, it is not clear whether the large amounts of G-CSF that are normally swallowed by the fetus and neonate have systemic effects on circulating neutrophils or local effects in the developing intestine. To assess this, we administered either active or heat-denatured (control) recombinant human G-CSF to 5- to 7-d-old C57BL/6 × 129SvJ mice. Pups received either a low dose (3 ng) that was calculated to approximate the amount of G-CSF swallowed in utero from amniotic fluid or an isovolemic high dose 100 times larger (300 ng). Oral dosing was performed daily for either 3 or 7 d, after which pups were killed and measurements were made on the blood and the GI tract. Absolute blood neutrophil counts and immature to total neutrophil ratios did not differ from controls in any of the test groups. However, intestinal villus area, perimeter, length, crypt depth, and proliferating cell nuclear antigen index increased significantly among those that were treated with active G-CSF. Thus, in suckling mice, enterally administered G-CSF had no effect on the concentration of circulating neutrophils but had trophic effects on the intestine. We speculate that the G-CSF present in amniotic fluid, colostrum, and milk acts as a topical intestinal growth factor and has little or no granulocytopoietic action.

G-CSF and Erythropoietin Stability in Amniotic Fluid during Simulated in vitro Digestion Conditions

Objective: To determine the stability of granulocyte colony-stimulating factor (G-CSF) and erythropoietin (Epo) in human amniotic fluid and recombinant G-CSF (Neupogen) and Epo (Epogen) in simulated amniotic fluid to digestions at pH concentrations of 3.2, 4.5, and 5.8 to assess their bioavailability to the neonate. Design: A simulated amniotic fluid containing Neupogen and Epogen was subjected to in vitro conditions that mimicked preprandial and postprandial neonatal intestinal digestion. Human amniotic fluid was tested using identical digestion conditions as well as human amniotic fluid to which Epogen and Neupogen had been added. Main Outcome Measures: The percentages of G-CSF/Epo and Neupogen/Epogen remaining after 1 and 2 hours of simulated digestions were compared with those at time zero, and concentrations at 2 hours were compared with those at 1 hour and time zero. Results: In simulated amniotic fluid at pH 3.2, significant degradation of G-CSF was observed at 1 hour (p = 0.03). No differences were observed at 1 or 2 hours for either pH 4.5 (p = 0.30 and 0.11, respectively) or pH 5.8 (p = 0.20 and 0.49, respectively). Human amniotic fluid exhibited significant degradation pH 3.2 (p = 0.04) and pH 4.5 (p < 0.05) at 1 hour; no difference was noted at pH 5.8 at 1 hour (p = 0.34). When additional Neupogen was added to human amniotic fluid, significant degradation was observed at pH 3.2 (p < 0.05) and pH 4.5 (p = 0.03) at 1 hour; no difference was noted at 1 hour at pH 5.8 (p = 0.11). In simulated amniotic fluid at pH 3.2, significant degradation of Epo occurred at 1 hour (p < 0.05). There were no differences at 1 hour for pH 4.5 (p = 0.50) or pH 5.8 (p = 0.17). Human amniotic fluid exhibited significant degradation at pH 3.2 (p < 0.05) and pH 4.5 (p < 0.05) at 1 hour; no difference was noted at 1 hour at pH 5.8 (p = 0.34). When additional Epogen was added to human amniotic fluid, significant degradation was observed at pH 3.2 (p = 0.001) and pH 4.5 (p = 0.003); no difference was noted at 1 hour at pH 5.8 (p = 0.31). Conclusions: G-CSF/Epo in human amniotic fluid and Neupogen/Epogen in simulated amniotic fluid are preserved to varying degrees during simulated digestion conditions. The degree of degradation of both cytokines was time- and pH-dependent. Measurable quantities of G-CSF and Epo are biologically available when swallowed by the fetus or a preterm neonate.

Development of prostaglandin endoperoxide synthase expression in the ovine fetal central nervous system and pituitary

In this study, we tested the hypothesis that prostaglandin endoperoxide synthase-1 and -2 (PGHS-1 and PGHS-2) are expressed throughout the latter half of gestation in ovine fetal brain and pituitary. Hypothalamus, pituitary, hippocampus, brainstem, cortex and cerebellum were collected from fetal sheep at 80, 100, 120, 130, 145days of gestational age (DGA), 1 and 7days postpartum lambs, and from adult ewes (n=4-5 per group). mRNA and protein were isolated from each region, and expression of prostaglandin synthase-1 (PGHS-1) and -2 (PGHS-2) were evaluated using real-time RT-PCR and western blot. PGHS-1 and -2 were detected in every brain region at every age tested. Both enzymes were measured in highest abundance in hippocampus and cerebral cortex, and lowest in brainstem and pituitary. PGHS-1 and -2 mRNAs were upregulated in hypothalamus and pituitary after 100 DGA. The hippocampus exhibited decreases in PGHS-1 and increases in PGHS-2 mRNA after 80 DGA. Brainstem PGHS-1 and -2 and cortex PGHS-2 exhibited robust increases in mRNA postpartum, while cerebellar PGHS-1 and -2 mRNAs were upregulated at 120 DGA. Tissue concentrations of PGE(2) correlated with PGHS-2 mRNA, but not to other variables. We conclude that the regulation of expression of these enzymes is region-specific, suggesting that the activity of these enzymes is likely to be critical for brain development in the late-gestation ovine fetus.

Bioavailability of granulocyte colony-stimulating factor administered enterally to suckling mice

The developing fetal and neonatal gastrointestinal (GI) tract is influenced by many growth factors, including epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), and erythropoietin (Epo). Granulocyte colony-stimulating factor (G-CSF), typically regarded as a hematopoietic growth factor, might also be included because it exists in high concentrations in amniotic fluid, colostrum, and human milk, and because granulocyte CSF receptors (G-CSF-R) are abundantly expressed on the villous enterocytes of the developing intestine. As a first step toward understanding whether the effects of G-CSF on the GI tract were local or systemic, we sought to determine whether recombinant human G-CSF (rhG-CSF) administered enterally to suckling mice, is absorbed into the circulation, and if so, whether the G-CSF-R is essential for this absorption. We enterally administered rhG-CSF to suckling mice, selecting a daily dose based on the amount of G-CSF normally swallowed by the fetus and neonate (3 ng), or in other mice, a dose of G-CSF 100 times larger (300 ng). Pups were tested at either 5-7 days of age, or at 14-16 days of age. C57BL/6 x 129SvJ mice were used. Some mice had a targeted null mutation in the G-CSF-R gene, producing a non-functional G-CSF-R protein. At intervals following the enteral G-CSF dosing, G-CSF concentrations in plasma were measured by specific ELISA. The bioavailability of G-CSF was invariably <1%, regardless of the dose of rhG-CSF given, the age of the pups, or whether they had a functional G-CSF-R. After enteral administration of rhG-CSF to suckling mice, only minimal quantities of G-CSF are absorbed into the circulation, and the G-CSF-R is not essential for this absorption.

Messenger RNA Expression of Granulocyte Colony-Stimulating Factor Receptor Isoforms in the Fetus

Granulocyte colony-stimulating factor (G-CSF) is a growth factor known to support the proliferation, differentiation, and survival of cells of the neutrophil lineage. G-CSF affects these cells after binding to its specific receptor, G-CSF-R, which exists in seven isoforms. While information exists about the distribution of these isoforms in hematopoietic cells and placenta, G-CSF-R isoforms on non-hematopoietic fetal tissues have not been described. Using RT-PCR, we analyzed a variety of human fetal tissues ranging from 6 to 18 weeks gestation. Isoforms I and III were present in all tissues, and the expression of isoform III varied with gestational age. The remaining isoforms were variably expressed in relation to tissue type and gestational age. Thus in the human fetus, G-CSF-R isoform I is the predominant form expressed on non-hematopoietic and hematopoietic tissues.