Edmund F. La Gamma

Preliminary Report: rhG-CSF May Reduce the Incidence of Neonatal Sepsis in Prolonged Preeclampsia-associated Neutropenia

Objectives. To determine whether adjunctive therapy with recombinant human granulocyte colony-stimulating factor (rhG-CSF) could reverse the neutropenia and reduce the incidence of sepsis (≤28 days postnatal age) in neonates with prolonged preeclampsia-associated neutropenia compared with conventional therapy. Study Design. An intravenous infusion of rhG-CSF (10 μg/kg/day × 3 days for 10 neonates or 5 μg/kg/day × 3 days for 5 neonates) was administered to ventilated patients with prolonged (≥3 consecutive days in the first postnatal week) preeclampsia-associated neutropenia (absolute neutrophil count [ANC] <1500/mm3). Neutrophilic responses and the incidence of neonatal sepsis in the next 28 postnatal days were compared with 13 case-matched control neonates who also had prolonged preeclampsia-associated neutropenia. Sepsis was defined as at least one positive blood culture in a newly symptomatic neonate treated with antibiotics for ≥7 days. Results. No significant differences existed among the three groups in the birth weight, gestational age, sex, growth retardation, method of delivery, magnitude of respiratory support, use of surfactant, usage of intravascular catheters, or in the initial (pretreatment) ANC. The average baseline ANC (pretreatment) in the 10-μg rhG-CSF group was 815 ± 169/mm3 (mean ± SEM), in the 5 μg group it was 786 ± 165/mm3, and in the conventional group it was 965 ± 283. Eighteen of 28 (64%) neonates with preeclampsia-associated neutropenia were neutropenic at birth, the other 10 (36%) had normal neutrophil counts at birth but subsequently developed ≥3 days of neutropenia between 24 and 120 hours after birth. The ANC increased by 2-fold at 24 hours, by 4-fold at 72 hours, and 14-fold by the 7th day in the 10-μg group. In the 5-μg group, a 2-fold and 5-fold increase occurred at 72 hours and 7 days, respectively. In the conventionally-treated group, only a 4-fold increase was seen as late as 7 days after achieving entry criteria. Sepsis was observed in 13% (2/15) of the rhG-CSF-treated neonates compared with an incidence of 54% (7/13) in the conventionally-treated neonates. Conclusions. rhG-CSF increases the ANC significantly (at 10 μg/kg/day × 3 days) and reduces the incidence of neonatal sepsis in critically ill ventilated neonates with prolonged preeclampsia-associated neutropenia when compared with conventional therapy. A future prospective, randomized, and blinded trial is needed to validate the beneficial effects of prophylactic rhG-CSF therapy in neonates with prolonged preeclampsia-associated neutropenia.

Effects of Prenatal Steroids on Water and Sodium Homeostasis in Extremely Low Birth Weight Neonates

Objective. We sought to determine if prenatal steroid (PNS) treatment affects water and sodium (Na) balance in extremely low birth weight infants (<1000 g). Methods. PNS treatment enhances lung maturation in preterm infants and induces maturation of renal tubular function and adenylate cyclase activity in animals. We compared water and Na homeostasis for the first week of life in those infants whose mothers received steroids before delivery (PNS: n = 16) to those who did not (nonsteroid group [NSG]: n = 14). The data were collected prospectively, but PNS treatment was not given in a randomized manner. Fluids were initiated at 100 to 125 mL/kg/d and adjusted every 8 to 12 hours to allow a daily weight loss of ≤4% of birth weight and to maintain normal serum electrolytes. Weight, serum and urine electrolytes, and urine output were frequently measured and fluid intake was adjusted by increasing the amount of free water to achieve these goals. Results. When using our fluid management protocol, the percent weight loss in both groups was equivalent during each of the 7 days (15% PNS vs 17% NSG maximum loss) as well as the cumulative urine output at 1 week of age (663 mL/kg/wk PNS vs 681 mL/kg/wk NSG). PNS infants had a higher urine output on the first 2 days of life and a lower daily fluid intake for the first week. PNS infants also had significantly less insensible water loss for each of the first 4 days of life. The PNS group had a significantly lower mean peak serum Na of 138 ± 1 mmol/L vs 144 ± 2 mmol/L and none had a peak serum Na >150 mmol/L compared with 36% of the NSG infants. PNS infants had a higher cumulative Na excretion at day 2 of life (10 ± 2 mmol/kg vs 6 ± 1 mmol/kg) but a less negative cumulative Na balance at 1 week (−10 mmol/kg vs −14 mmol/kg). Conclusion. PNS treatment was associated with lower estimated insensible water loss, a decreased incidence of hypernatremia, and an earlier diuresis and natriuresis in extremely low birth weight neonates. We speculate that PNS effects these changes through enhancement of epithelial cell maturation improving skin barrier function. PNS treatment may also enhance lung Na,K-ATPase activity leading to an earlier postnatal reabsorption of fetal lung fluid increasing extracellular volume expansion to help prevent hypernatremia.

Effect of granulocyte colony-stimulating factor on preeclampsia-associated neonatal neutropenia ☆ ☆☆ ★

Absolute neutropenia lasting longer than 72 hours after birth occurred in four very low birth weight neonates with a maternal history of severe pregnancy-induced hypertension, and was treated with recombinant granulocyte colony-stimulating factor for 3 days. Absolute neutrophil counts increased nearly four-fold within 48 hours; maximal values were recorded on the ninth day after the infusion was started. Total leukocyte counts subsequently decreased but remained in the normal range. It appears that recombinant human granulocyte colony-stimulating factor promotes a rapid increase in circulating neutrophils in these patients despite the possible presence of a circulating preeclampsia-associated inhibitor of neutrophil production.

Genetically Modified Primary Astrocytes as Cellular Vehicles for Gene Therapy in the Brain

Combining genetic engineering and cell transplantation has been proposed as one way to overcome the limited availability of donor tissue that may restrict the application of graft therapy to neurological diseases. Important issues in this approach concern the choice of a suitable cellular vehicle, and the method of gene insertion. In this regard, we have investigated the use of brain-derived primary astrocytes as cellular vehicles for gene therapy, because they can be transfected, divide in culture, are brain-region specific, possess a secretory mechanism, and may migrate several mm from the transplant site. To address the issue of gene insertion, we have generated stably transfected primary rat astrocytes using the nonviral calcium phosphate method to co-transfect a reporter construct (RSV-chloramphenicol acetyltransferase (CAT), or human enkephalin promoter CAT, plus a neomycin resistance plasmid (pRSVNeo). Modified astrocytes were then propagated by transfer to selective media containing G418 (300 μg/mL) for 3 wk. The presence of the reporter gene product (CAT) was demonstrated by immunocytochemistry, and by biochemical assay of CAT enzyme catalytic activity. These genetically modified astrocytes were followed for up to 3 wk after transplantation into the rat striatum. Criteria used to distinguish transplanted astrocytes included histological evidence of abundant nuclei interrupting the normal cytoarchitecture of the striatum, astrocyte morphology, and the presence of CAT enzyme activity. Our data indicates that genetically modified astrocytes are an important candidate vehicle for use in transplantation therapy in neurological diseases. We suggest that genetically modified astrocytes can also be used for studying the human enkephalin promoter, other promoters, and expressed proteins using this paradigm.

Development of transsynaptic regulation of adrenal enkephalin

Transsynaptic activity differentially regulates biosynthesis of sympathoadrenal catecholamines and co-localized opiate peptides in the rat. We determined whether similar mechanisms were operative during development. Adrenal Leu-enkephalin (LEU), was first detected at E16.5, then increased 5-fold during maturation from birth to adulthood while adrenal weight increased 10-fold. Since medullary cells do not divide after the first postnatal week, this represents a specific maturational increase in LEU content per chromaffin cell. In adult medullae, decreasing transsynaptic activity through adrenal denervation or explantation results in a 30-50-fold increase in LEU. In contrast, LEU levels in denervated or explanted medullae from neonatal rats (less than or equal to 10 days) do not. Prolonged denervation (day 5-21) prevented even the normal maturational increase in LEU. However, depolarizing medullae with KCl lowered LEU levels at all ages tested with an increased magnitude of effect after 10 days postnatal age. Specific deficits in signal-transduction mechanisms or immaturity of opiate biosynthetic pathways may account for these observations. Thus, during development, adrenal opiate peptides are not under transsynaptic control yet require presynaptic terminals to mature normally. Therefore, like catecholamines, co-localized adrenal opiate peptides require presynaptic regulatory signals to achieve normal development and function.

Dopaminergic regulation of a transfected preproenkephalin promoter in primary rat astrocytes in vitro and in vivo.

The clinical benefit of transplantation therapies utilizing genetically modified cells could be enhanced if expression of engineered genes was regulated by clinically useful pharmacological agents. Toward this end, we examined pharmacologic effects on the expression of hybrid gene constructs transfected into primary rat striatal astrocytes. These astrocytes are known to express receptors for the neurotransmitter dopamine (DA). In vitro, we found that expression of a transiently transfected human ppEnk promoter-driven chloramphenicol acetyltransferase (CAT) reporter construct was induced by DAergic agonists, as much as 20-fold. This induction was blocked by a DA receptor antagonist. The same concentration of DA also increased the endogenous rat ppEnk mRNA, by > 2-fold. In vivo, regulation of CAT expression by DA was tested by implanting the genetically modified astrocytes into the normal striatum and the contralateral striatum which had > 95% DA depletion induced by a previous 6-hydroxy-DA lesion of the substantia nigra. As hypothesized on the basis of the in vitro data, CAT activity on the lesioned side, where the stimulating effect of endogenous DA was lacking, was 30% lower than on the control side where the normal DA content was present. The data suggest that control of the enkephalin gene in astrocytes may involve second messenger pathways activated by DA receptors. Moreover, the evidence that clinically applicable drugs can regulate inducible genes introduced into the brain by astrocyte implantation is of potential importance in development of therapeutic strategies.

Role of Ca2+ in induction of neurotransmitter-related gene expression by butyrate

We examined the effect of butyrate on neurotransmitter-related gene expression and calcium homeostasis in PC12 cells. Pretreatment with Ca2+ chelators (EGTA or BAPTA-AM) attenuated the butyrate-triggered accumulation of TH and ppEnk mRNA indicating that Ca2+ plays a role in butyrate-induced regulation of neuronal genes. Butyrate alone did not alter intracellular Ca2+ levels as determined by Fura-PE3 fluorescence; however, pretreatment with butyrate (18–24 h) reduced the first Ca2+ peak and prevented the second sustained rise in [Ca2+]i as induced by nicotine or ryanodine. In contrast, butyrate had no effect on Ca2+ transients when added shortly before or during nicotine or ryanodine stimulation. These results suggest that chronic butyrate exposure can modulate cell responses by affecting intracellular Ca2+ signaling.

Does Hyponatremia Result in Pontine Myelinolysis and Neurological injury in Extremely Low Birth Weight (ELBW) Micropremies? † 825

Does Hyponatremia Result in Pontine Myelinolysis and Neurological injury in Extremely Low Birth Weight (ELBW) Micropremies? † 825

Umbilical Arterial Blood Sampling Alters Cerebral Tissue Oxygenation in Very Low Birth Weight Neonates

OBJECTIVE To evaluate the magnitude, consistency, and natural history of reductions in cerebral regional tissue oxygenation (CrSO2) during umbilical arterial (UA) blood sampling in very low birth weight neonates. STUDY DESIGN Data were collected during a prospective observational near-infrared spectroscopy survey conducted on a convenience sample of 500-1250 g neonates during the first 10 postnatal days. A before-after analysis of UA blood sampling effects on CrSO2 absolute values and variability was performed. The present analysis was not designed a priori and was conducted following the bedside observation of CrSO2 decrements contiguous with UA blood draws. RESULTS Fifteen very low birth weight neonates had 201 UA blood draws. Baseline CrSO2 (mean ± SEM) decreased following UA blood sampling, from 70 ± 1% to a nadir of 63 ± 1% (P < .001) occurring 4 ± 3 (range 2-24) minutes following blood draws. CrSO2 subsequently increased to 70 ± 1% (P < .001 compared with nadir) at 10 ± 4 (range 4-28) minutes following UA blood sampling. Coefficients of variation (mean ± SEM) increased from 0.02 ± 0.001 at baseline to 0.05 ± 0.004 (P < .001), followed by a decrease to 0.03 ± 0.003 (P < .001 for all comparisons), thus denoting increased CrSO2 variability following UA blood sampling. CONCLUSIONS UA blood sampling is associated with significant CrSO2 decrements with increased variability over clinically significant intervals. Whether these changes impact complications of prematurity, including intraventricular hemorrhage and periventricular leukomalacia, remain unknown.

Correction: Whole genome expression profiling associates activation of unfolded protein response with impaired production and release of epinephrine after recurrent hypoglycemia

Recurrent hypoglycemia can occur as a major complication of insulin replacement therapy, limiting the long-term health benefits of intense glycemic control in type 1 and advanced type 2 diabetic patients. It impairs the normal counter-regulatory hormonal and behavioral responses to glucose deprivation, a phenomenon known as hypoglycemia associated autonomic failure (HAAF). The molecular mechanisms leading to defective counter-regulation are not completely understood. We hypothesized that both neuronal (excessive cholinergic signaling between the splanchnic nerve fibers and the adrenal medulla) and humoral factors contribute to the impaired epinephrine production and release in HAAF. To gain further insight into the molecular mechanism(s) mediating the blunted epinephrine responses following recurrent hypoglycemia, we utilized a global gene expression profiling approach. We characterized the transcriptomes during recurrent (defective counter-regulation model) and acute hypoglycemia (normal counter-regulation group) in the adrenal medulla of normal Sprague-Dawley rats. Based on comparison analysis of differentially expressed genes, a set of unique genes that are activated only at specific time points after recurrent hypoglycemia were revealed. A complementary bioinformatics analysis of the functional category, pathway, and integrated network indicated activation of the unfolded protein response. Furthermore, at least three additional pathways/interaction networks altered in the adrenal medulla following recurrent hypoglycemia were identified, which may contribute to the impaired epinephrine secretion in HAAF: greatly increased neuropeptide signaling (proenkephalin, neuropeptide Y, galanin); altered ion homeostasis (Na+, K+, Ca2+) and downregulation of genes involved in Ca2+-dependent exocytosis of secretory vesicles. Given the pleiotropic effects of the unfolded protein response in different organs, involved in maintaining glucose homeostasis, these findings uncover broader general mechanisms that arise following recurrent hypoglycemia which may afford clinicians an opportunity to modulate the magnitude of HAAF syndrome.