Acute respiratory failure and the kinetics of neutrophil recovery in pediatric hematopoietic cell transplantation: a multicenter study.

In this multicenter study, we investigated the kinetics of neutrophil recovery in relation to acuity and survival among 125 children undergoing allogeneic hematopoietic cell transplantation (allo-HCT) who required invasive mechanical ventilation (IMV). Recovery of neutrophils, whether prior to or after initiation of IMV, was associated with a significantly decreased risk of death relative to never achieving neutrophil recovery. A transient increase in acuity (by oxygenation index and vasopressor requirements) occurred among a subset of the patients who achieved neutrophil recovery after initiation of IMV; 61.5% of these patients survived to discharge from the intensive care unit (ICU). Improved survival among patients who subsequently achieved neutrophil recovery on IMV was not limited to those with peri-engraftment respiratory distress syndrome. The presence of a respiratory pathogen did not affect the risk of death while on IMV but was associated with an increased length of IMV (p < 0.01). Among patients undergoing HCT who develop respiratory failure and require advanced therapeutic support, neutrophil recovery at time of IMV and/or presence of a respiratory pathogen should not be used as determining factors when counseling families about survival.

Correction to: Acute respiratory failure and the kinetics of neutrophil recovery in pediatric hematopoietic cell transplantation: a multicenter study.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transgenerational effects of maternal bisphenol: a exposure on offspring metabolic health.

Exposure to the endocrine disruptor bisphenol A (BPA) is ubiquitous and associated with health abnormalities that persist in subsequent generations. However, transgenerational effects of BPA on metabolic health are not widely studied. In a maternal C57BL/6J mice (F0) exposure model using BPA doses that are relevant to human exposure levels (10 µg/kg/day, LowerB; 10 mg/kg/day, UpperB), we showed male- and dose-specific effects on pancreatic islets of the first (F1) and second generation (F2) offspring relative to controls (7% corn oil diet; control). In this study, we determined the transgenerational effects (F3) of BPA on metabolic health and pancreatic islets in our model. Adult F3 LowerB and UpperB male offspring had increased body weight relative to Controls, however glucose tolerance was similar in the three groups. F3 LowerB, but not UpperB, males had reduced ß-cell mass and smaller islets which was associated with increased glucose-stimulated insulin secretion. Similar to F1 and F2 BPA male offspring, staining for markers of T-cells and macrophages (CD3 and F4/80) was increased in pancreas of F3 LowerB and UpperB male offspring, which was associated with changes in cytokine levels. In contrast to F3 BPA males, LowerB and UpperB female offspring had comparable body weight, glucose tolerance and insulin secretion as Controls. Thus, maternal BPA exposure resulted in fewer metabolic defects in F3 than F1 and F2 offspring, and these were sex- and dose-specific.

Exendin-4 increases histone acetylase activity and reverses epigenetic modifications that silence Pdx1 in the intrauterine growth retarded rat.

AIMS/HYPOTHESIS: The abnormal intrauterine milieu of intrauterine growth retardation (IUGR) permanently alters gene expression and function of pancreatic beta cells leading to the development of diabetes in adulthood. Expression of the pancreatic homeobox transcription factor Pdx1 is permanently reduced in IUGR islets suggesting an epigenetic mechanism. Exendin-4 (Ex-4), a long-acting glucagon-like peptide-1 (GLP-1) analogue, given in the newborn period increases Pdx1 expression and prevents the development of diabetes in the IUGR rat. METHODS: IUGR was induced by bilateral uterine artery ligation in fetal life. Ex-4 was given on postnatal days 1-6 of life. Islets were isolated at 1 week and at 3-12 months. Histone modifications, PCAF, USF1 and DNA methyltransferase (Dnmt) 1 binding were assessed by chromatin immunoprecipitation (ChIP) assays and DNA methylation was quantified by pyrosequencing. RESULTS: Phosphorylation of USF1 was markedly increased in IUGR islets in Ex-4 treated animals. This resulted in increased USF1 and PCAF association at the proximal promoter of Pdx1, thereby increasing histone acetyl transferase (HAT) activity. Histone H3 acetylation and trimethylation of H3K4 were permanently increased, whereas Dnmt1 binding and subsequent DNA methylation were prevented at the proximal promoter of Pdx1 in IUGR islets. Normalisation of these epigenetic modifications reversed silencing of Pdx1 in islets of IUGR animals. CONCLUSIONS/INTERPRETATION: These studies demonstrate a novel mechanism whereby a short treatment course of Ex-4 in the newborn period permanently increases HAT activity by recruiting USF1 and PCAF to the proximal promoter of Pdx1 which restores chromatin structure at the Pdx1 promoter and prevents DNA methylation, thus preserving Pdx1 transcription.

Review: Placental programming of postnatal diabetes and impaired insulin action after IUGR.

Being born small due to poor growth before birth increases the risk of developing metabolic disease, including type 2 diabetes, in later life. Inadequate insulin secretion and decreasing insulin sensitivity contribute to this increased diabetes risk. Impaired placental growth, development and function are major causes of impaired fetal growth and development and therefore of IUGR. Restricted placental growth (PR) and function in non-human animals induces similar changes in insulin secretion and sensitivity as in human IUGR, making these valuable tools to investigate the underlying mechanisms and to test interventions to prevent or ameliorate the risk of disease after IUGR. Epigenetic changes induced by an adverse fetal environment are strongly implicated as causes of later impaired insulin action. These have been well-characterised in the PR rat, where impaired insulin secretion is linked to epigenetic changes at the Pdx-1 promotor and reduced expression of this transcription factor. Present research is particularly focussed on developing intervention strategies to prevent or reverse epigenetic changes, and normalise gene expression and insulin action after PR, in order to translate this to treatments to improve outcomes in human IUGR.

Molecular mechanisms for hyperinsulinaemia induced by overproduction of selenium-dependent glutathione peroxidase-1 in mice.

AIMS/HYPOTHESIS: We previously observed hyperglycaemia, hyperinsulinaemia, insulin resistance and obesity in Gpx1-overexpressing mice (OE). Here we determined whether these phenotypes were eliminated by diet restriction, subsequently testing whether hyperinsulinaemia was a primary effect of Gpx1 overexpression and caused by dysregulation of pancreatic duodenal homeobox 1 (PDX1) and uncoupling protein-2 (UCP2) in islets. METHODS: First, 24 male OE and wild-type (WT) mice (2 months old) were given 3 g (diet-restricted) or 5 g (full-fed) feed per day for 4 months to compare their glucose metabolism. Thereafter, several mechanistic experiments were conducted with pancreas and islets of the two genotypes (2 or 6 months old) to assay for beta cell mass, reactive oxygen species (ROS) levels, mitochondrial membrane potential (Deltapsi(m)) and expression profiles of regulatory proteins. A functional assay of islets was also performed. RESULTS: Diet restriction eliminated obesity but not hyperinsulinaemia in OE mice. These mice had greater pancreatic beta cell mass (more than twofold) and pancreatic insulin content (40%) than the WT, along with an enhanced Deltapsi(m) and glucose-stimulated insulin secretion in islets. With diminished ROS production, the OE islets displayed hyperacetylation of H3 and H4 histone in the Pdx1 promoter, elevated PDX1 and decreased UCP2. CONCLUSIONS/INTERPRETATION: Overproduction of the major antioxidant enzyme, glutathione peroxidase 1, caused seemingly beneficial changes in pancreatic PDX1 and UCP2, but eventually led to chronic hyperinsulinaemia by dysregulating islet insulin production and secretion.

Hormonal and metabolic defects in a prader-willi syndrome mouse model with neonatal failure to thrive.

Prader-Willi syndrome (PWS) has a biphasic clinical phenotype with failure to thrive in the neonatal period followed by hyperphagia and severe obesity commencing in childhood among other endocrinological and neurobehavioral abnormalities. The syndrome results from loss of function of several clustered, paternally expressed genes in chromosome 15q11-q13. PWS is assumed to result from a hypothalamic defect, but the pathophysiological basis of the disorder is unknown. We hypothesize that a fetal developmental abnormality in PWS leads to the neonatal phenotype, whereas the adult phenotype results from a failure in compensatory mechanisms. To address this hypothesis and better characterize the neonatal failure to thrive phenotype during postnatal life, we studied a transgenic deletion PWS (TgPWS) mouse model that shares similarities with the first stage of the human syndrome. TgPWS mice have fetal and neonatal growth retardation associated with profoundly reduced insulin and glucagon levels. Consistent with growth retardation, TgPWS mice have deregulated liver expression of IGF system components, as revealed by quantitative gene expression studies. Lethality in TgPWS mice appears to result from severe hypoglycemia after postnatal d 2 after depletion of liver glycogen stores. Consistent with hypoglycemia, TgPWS mice appear to have increased fat oxidation. Ghrelin levels increase in TgPWS reciprocally with the falling glucose levels, suggesting that the rise in ghrelin reported in PWS patients may be secondary to a perceived energy deficiency. Together, the data reveal defects in endocrine pancreatic function as well as glucose and hepatic energy metabolism that may underlie the neonatal phenotype of PWS.

Intrauterine growth retardation leads to the development of type 2 diabetes in the rat.

Intrauterine growth retardation has been linked to the development of type 2 diabetes in later life. The mechanisms underlying this phenomenon are unknown. We have developed a model of uteroplacental insufficiency, a common cause of intrauterine growth retardation, in the rat. Bilateral uterine artery ligation was performed on day 19 of gestation (term is 22 days) in the pregnant rat; sham-operated pregnant rats served as controls. Birth weights of intrauterine growth-retarded (IUGR) animals were significantly lower than those of controls until approximately 7 weeks of age, when IUGR rats caught up to controls. Between 7 and 10 weeks of age, the growth of IUGR rats accelerated and surpassed that of controls, and by 26 weeks of age, IUGR rats were obese (P < 0.05 vs. controls). No significant differences were observed in blood glucose and plasma insulin levels at 1 week of age. However, between 7 and 10 weeks of age, IUGR rats developed mild fasting hyperglycemia and hyperinsulinemia (P < 0.05 vs. controls). At age 26 weeks, IUGR animals had markedly elevated levels of glucose (P < 0.05 vs. controls). IUGR animals were glucose-intolerant and insulin-resistant at an early age. First-phase insulin secretion in response to glucose was also impaired early in life in IUGR rats, before the onset of hyperglycemia. There were no significant differences in beta-cell mass, islet size, or pancreatic weight between IUGR and control animals at 1 and 7 weeks of age. However, in 15-week-old IUGR rats, the relative beta-cell mass was 50% that of controls, and by 26 weeks of age, beta-cell mass was less than one-third that of controls (P < 0.05). The data presented here support the hypothesis that an abnormal intrauterine milieu can induce permanent changes in glucose homeostasis after birth and lead to type 2 diabetes in adulthood.

Workshop to identify critical windows of exposure for children's health: cardiovascular and endocrine work group summary.

The work group on cardiovascular and endocrine effects was asked to review the current state of knowledge about children's windows of vulnerability to developmental toxicants and to recommend how that information may be used to improve risk assessment and public health. We considered differences between structural defects, where periods of vulnerability are rather well defined, and functional defects, where periods of vulnerability are quite elusive.

Placental expression of glucose transporter proteins 1 and 3 in growth-restricted fetal rats.

OBJECTIVE: The purpose of this study was to determine the effect of intrauterine growth restriction on the placental expression of glucose transporter proteins. STUDY DESIGN: Intrauterine growth restriction was induced by bilateral uterine artery ligation in the pregnant rat at a gestational age of 19 days (term is 21.5 days). Maternal rats were killed and fetuses were delivered by hysterotomy on gestational days 20 and 21. Control fetuses from mothers that had been subjected to a sham operation were studied simultaneously. Glucose transporter protein 1 and glucose transporter protein 3 messenger ribonucleic acid was quantified by reverse transcriptase-polymerase chain reaction amplification. Glucose transporter protein 1 and glucose transporter protein 3 densities in placental membranes were also assessed by Western blotting and by immunohistochemical analysis. RESULTS: Glucose transporter protein 1 messenger ribonucleic acid, expressed as a multiple of the matched sham control value, was unchanged on both days 20 and 21 of gestation. Glucose transporter protein 3 messenger ribonucleic acid was also unchanged. Western blotting demonstrated no change in expression of glucose transporter protein 1 or glucose transporter protein 3 on either day 20 or 21 of gestation. Immunohistochemical staining patterns for glucose transporter protein 1 and glucose transporter protein 3 on the syncytiotrophoblastic membranes were similar between the growth-restricted group and the sham control group. CONCLUSION: Placental expression of glucose transporter proteins in the pregnant rat is unchanged with uteroplacental insufficiency.

Localization and quantification of glucose transporters in liver of growth-retarded fetal and neonatal rats.

To determine whether altered transport of glucose into the hepatocyte may be an important factor contributing to abnormal hepatic glucose metabolism in the intrauterine growth-retarded (IUGR) fetus and newborn, we measured glucose transport (glucose uptake, GLUT protein, and mRNA) and localization of GLUT protein in liver of control (sham operated) and IUGR fetal (day 20) and postnatal (1, 4, 14, and 21 days) rats. GLUT-1 and -2 proteins were localized to the hepatocyte. Glucose uptake and GLUT-1 protein and mRNA levels were increased in IUGR fetal and neonatal liver. GLUT-2 protein and mRNA levels were low in IUGR and control fetal liver. After birth, GLUT-2 abundance did not differ from controls. Run-on experiments showed that the rate of transcription of GLUT-1 and -2 did not differ between IUGR and control rats. However, the transcription rate of GLUT-1 decreased with age, and the GLUT-2 transcription rate increased with age. These studies indicate that the metabolic and physiological factors that cause IUGR also alter glucose transporter expression in fetal liver.

Effect of uteroplacental insufficiency upon brain neuropeptide Y and corticotropin-releasing factor gene expression and concentrations.

Various hypothalamic functions such as feeding behavior, energy expenditure, body weight gain, level of anxiety, and sexual maturation are mediated by a balance between the concentrations of neuropeptide Y (NPY) and corticotropin-releasing factor (CRF). To test the hypothesis that maternal uteroplacental insufficiency alters the offspring's brain NPY and/or CRF levels, we examined the effect of maternal uterine artery ligation with intrauterine growth restriction (IUGR) (p < 0.05) upon fetal (20 d) and postnatal (4, 14, and 21 d) brain NPY and CRF synthesis, concentrations, and regional distribution. An age-related increase in NPY (0.8 kb) and CRF (1.4 kb) mRNA levels with peak amounts at the 14-d postnatal age (p < 0.05) was observed. IUGR was associated with a 75% increase in fetal brain NPY mRNA levels (p < 0.05) with no change in NPY peptide, CRF mRNA and peptide amounts. Although the increase in NPY mRNA levels persisted postnatally (p < 0.05) at d 4 and 21, CRF mRNA amounts were 2.5-fold higher only in the 4-d IUGR (p < 0.05). Paralleling the mRNA changes, an age-related increase in RIA of NPY and CRF peptide concentrations was noted (p < 0.05). IUGR caused postnatal brain NPY and CRF peptide changes similar to corresponding mRNA levels (p < 0.05), despite normal postnatal circulating glucose, insulin, corticosterone, and leptin concentrations. The age-specific intergroup differences in the NPY and CRF peptide immunoreactivity appeared predominantly in the hypothalamic region. We conclude that maternal uteroplacental insufficiency causing IUGR leads to a pretranslational imbalance in the immediate (4 d) postnatal brain NPY and CRF peptide concentrations, thereby altering the developmental pattern. This alteration in NPY and CRF peptide concentrations, despite normalization of the metabolic milieu was associated with a persistent diminution in body weight. The IUGR-associated pretranslational increase in NPY and not CRF peptide levels at d 21, may herald changes in feeding behavior during the postsuckling phase.

Modulation of glucose transport in fetal rat lung: a sexual dimorphism.

Male fetuses exhibit delayed lung maturation and surfactant production in comparison with female fetuses. This delay may be related to sex hormone effects: estrogen enhances and androgens delay lung development. The uptake of glucose, an important precursor for surfactant synthesis, may be differently affected by estrogen and androgens. In these studies we determined the effects of these two hormones on glucose transport (glucose uptake, glucose transporter [Glut] 1 protein, and mRNA) and hexokinase activity in lung tissue of fetal rats. On Day 20 of gestation (term = 21.5 d) lung tissue was harvested from female and male fetal rats, minced into explants, and cultured for 24 h. Basal glucose uptake, measured in the absence of sex hormones, was 37% higher (P < 0.05) in female compared with male lungs. Explants were washed and cultured for an additional 3 h or 24 h in either estradiol or dihydrotestosterone (DHT) at 0, 1, 10, or 100 nM. Twenty-four-hour treatment with estradiol in both male and female explants increase 2-deoxyglucose uptake, Glut 1 protein, and mRNA levels (P < 0.05). However, explants from male fetuses were not as responsive to estradiol treatment as were those from females (P < 0.05). Treatment for 24 h with DHT decreased 2-deoxyglucose uptake, Glut 1 protein, and mRNA levels in females and males (P < 0.05). There was no difference in response between females and males. Short-term incubation (3 h) with sex hormones had no effect on glucose uptake. However, 3-h treatment with estradiol did increase Glut 1 mRNA levels (P < 0.05). Hexokinase activity was not affected by estradiol or DHT treatment. These findings indicate that estradiol and DHT differentially regulate glucose uptake in fetal rat lung tissue. This regulation of substrate supply (glucose) by estradiol and DHT may be another mechanism for the sexual dimorphism observed in lung development and surfactant synthesis.

Intrauterine growth retardation alters mitochondrial gene expression and function in fetal and juvenile rat skeletal muscle.

Uteroplacental insufficiency alters the anabolic metabolism of the fetus, resulting in intrauterine growth retardation (IUGR). The metabolic and physiologic factors that cause IUGR have long standing consequences after birth. Postnatal growth and glucose metabolism are altered in the IUGR infant. Skeletal muscle is an important component of growth and metabolizes up to 70% of i.v. glucose. The ability of skeletal muscle to metabolize glucose is affected by ATP availability. We hypothesized that gene expression and function of proteins involved in mitochondrial ATP production and distribution would be altered in juvenile IUGR muscle. To test this hypothesis, we used a model of IUGR, induced by bilateral uterine artery ligation in the pregnant rat, that mimics uteroplacental insufficiency in the human. RT-PCR was used to measure the mRNA levels of three important mitochondrial proteins; NADH-ubiquinone-oxireductase subunit 4L(ND-4L), subunit C of the F1F0-ATP synthase (SUC), and adenine nucleotide translocator 1 (ANT1) in IUGR and control rats in fetal and juvenile life. In the fetus, mRNA levels of all three proteins were significantly increased in IUGR skeletal muscle. In contrast, in juvenile animals, mRNA levels of all three proteins were significantly decreased. mRNA levels of other metabolically important proteins, glucose-6-phosphate dehydrogenase and carnitine-palmitoyl-transferase II, were not significantly altered in IUGR juvenile animals. To assess if decreased gene expression is associated with altered mitochondrial function, we measured the mitochondrial NAD+/NADH ratio in d 21 juvenile control and IUGR muscle. At d 21, decreased gene expression if ND-4L, SUC, and ANTI is associated with a decreased mitochondrial NAD+/NADH ratio. The results of our study suggest that the metabolic alterations associated with uteroplacental insufficiency in the rat result in altered fetal and postnatal muscle mitochondrial mRNA expression as well as altered postnatal mitochondrial function.

Cutaneous necrosis associated with interferon alpha-2b.

Cutaneous necrosis may occur as a complication of treatment with interferon. Here we report the first case of cutaneous necrosis developing in a patient receiving interferon alpha-2b for the treatment of chronic hepatitis C viral infection. The patient developed two necrotic lesions while receiving high doses of interferon. We suggest that discontinuation of treatment may be necessary to permit healing of such lesions. Although the exact mechanism involved in cutaneous necrosis remains unknown, our observations support earlier findings suggesting that intraarterial injection may be a factor.

Measurement of GLUT mRNA in liver of fetal and neonatal rats using a novel method of quantitative polymerase chain reaction.

Transfer of glucose into the hepatocyte is mediated by glucose transporters (GLUTs). GLUT mRNA levels are usually measured by Northern blot analysis. Reverse transcription-polymerase chain reaction (RT-PCR) is often used to measure RNA abundance. However, this method is only semiquantitative and has no internal control during first-strand synthesis. We designed a method of coreverse transcription and PCR amplification using bovine rhodopsin as an internal control for both cDNA synthesis and amplification. As part of the validation of this technique, we determined that there was no nonspecific amplification of bovine GLUTs by rhodopsin primers, that there were no differences in amplification due to different regions of the Glut gene amplified, and that there were no secondary structure effects on amplification. We applied our modified method of RT-PCR to measure the ontogeny of GLUT expression in liver of fetal and postnatal rats (d20 fetuses and d1, d4, d14, and d21 juvenile rat pups). GLUT 1 mRNA quantity decreased whereas GLUT 2 increased with age. We were able to detect small quantities of GLUT 3 in fetal liver and of GLUT 5 in postnatal liver. This method of RT-PCR provides an internal control and allows measurement of mRNA levels in small quantities of tissue, making it ideal for use in the fetus and any system in which mRNA levels are low.

Altered hepatic gene expression of enzymes involved in energy metabolism in the growth-retarded fetal rat.

Intrauterine growth retardation (IUGR) resulting from placental insufficiency is a common complication of pregnancy. Bilateral uterine artery ligation of the pregnant rat is a model which mimics intrauterine growth retardation in the human. IUGR rat fetuses have altered hepatic energy and redox states, with reduced fetal hepatic ATP/ADP ratio, increased cytosolic NAD+/NADH ratio, and decreased mitochondrial NAD+/NADH ratio. These critical changes in energy metabolism contribute to IUGR. The effects of these changes at the molecular level are largely unknown. To address these effects we compared hepatic mRNA populations of IUGR and normal fetuses and neonates using mRNA differential display, a polymerase chain reaction-based method for assaying transcriptional differences under various conditions. We isolated and sequenced 18 cDNA products whose mRNA levels were elevated in IUGR compared with normal fetal and neonatal liver. These analyses demonstrated that NADH-ubiquinone oxireductase subunit 4L mRNA (ND-4L) was significantly increased in liver of IUGR fetuses and neonates. This suggested that IUGR may be associated with altered expression of genes involved in the generation of ATP and NADH. Therefore, we measured mRNA levels of adenine-nucleotide translocator-2 (ANT-2), glucose-6-phosphate dehydrogenase (G6PD), mitochondrial malate dehydrogenase (MMD), ornithine transcarbamylase (OTC), and phosphofructokinase-2 (PFK-2) using a semiquantitative reverse transcriptase-polymerase chain reaction-based technique. In the IUGR fetus, ND-4L, ANT-2, G6PD, and MMD mRNA levels were significantly elevated; PFK-2 mRNA levels were unchanged, and OTC levels were decreased. In the IUGR newborn rat, mRNA levels of all 6 enzymes were increased suggesting that the metabolic state of the growth retarded newborn remains abnormal after birth. Uteroplacental insufficiency affects the immediate and long-term metabolic milieu of the growth retarded animal, and forces specific adjustments, including the expression of mRNA encoding enzymes involved with hepatic energy production.

The effects of severe maternal diabetes on glucose transport in the fetal rat.

We mimicked the condition of severe maternal diabetes by administering high doses of streptozotocin (STZ) to the pregnant rat to determine the effects of increased glucose availability on fetal glucose transport and to assess whether a relationship might exist between glucose transport and altered fetal growth. Fetuses of STZ-treated pregnant rats were growth retarded (3.86 +/- 0.13 vs. 5.29 +/- 0.06 g), hyperglycemic (30.0 +/- 1.0 vs. 5.5 +/- 0.5 mM/liter), and hyperinsulinemic (1263.8 +/- 138.3 vs. 817.9 +/- 116.7 pM/liter). Glucose uptake, Glut 1 messenger RNA (mRNA), and Glut 1 protein were greater in STZ-treated fetal brain than in controls (50%, 83%, and 50%, respectively; P < 0.05). Glut 3 mRNA levels in STZ-treated and control fetal brain were equivalent and significantly less than levels of Glut 1. Glucose uptake in muscle of STZ fetuses was 70% greater than control values (P < 0.05). Glut 1 mRNA levels were increased by 93% in STZ fetal muscle (P < 0.05). Neither Glut 3 nor Glut 4 mRNA could be detected in STZ-treated and control fetal muscle. Glut 1 protein levels were increased by 70% in STZ-treated fetal muscle compared to control muscle (P < 0.05). These observations indicate that altered glucose transport per se does not directly contribute to fetal growth retardation with maternal STZ diabetes. Perturbations in other physiological and metabolic factors may contribute to the pathogenesis of fetal growth retardation in STZ-induced diabetes during pregnancy.