Robert D. Christensen

Erythropoietin and erythropoietin receptor in the developing human central nervous system

We have previously shown the presence of erythropoietin (Epo) within the spinal fluid of normal preterm and term infants, and the presence of Epo receptor (Epo-R) in the spinal cords of human fetuses. It is not known, however: 1) whether cells within the fetal central nervous system(CNS) express Epo; 2) if so, whether this expression changes with development; 3) which cells within the CNS express Epo-R;4) whether Epo-R expression within the CNS changes with development; and 5) whether Epo-R within the fetal CNS are functional. Expression of mRNA for Epo and Epo-R was sought by reverse transcription-PCR in mixed primary cultures of fetal spinal cords as well as NT2 and hNT cells, human cell lines of neuronal precursors and mature neurons, respectively. Epo was measured by ELISA in spent media from primary cell culture, and immunohistochemistry was used to identify Epo-R on neurons and glia in cell culture, and in brain sections. Developmental changes in Epo and Epo-R expression were sought in spinal cords and brains from fetuses of 7-24 wk postconception by semiquantitative PCR. To assess Epo-R function, NT2 cells were exposed to conditions which stimulate programmed cell death, and rescue from apoptosis by the addition of recombinant Epo was evaluated by nuclear matrix protein ELISA, cell counts, and by Klenow labeling of DNA fragments. Epo and Epo-R mRNA were expressed in mixed primary cultures of neural tissues and NT2 and hNT cells. Epo was detected by ELISA in media removed from mixed cell cultures, and immunohistochemical staining confirmed the presence of Epo-R on neurons and their supporting cells. Semiquantitative PCR revealed no significant change in expression of either Epo or Epo-R in spinal cords between 7 and 16 wk of gestation, with increased expression of Epo and Epo-R in brains from 8 to 24 wk of gestation. Epo mRNA expression from neurons doubled under conditions of hypoxia. Recombinant Epo decreased apoptotic cell death of neurons under conditions of hypoxia. Protein and mRNA for Epo and its receptor are expressed by human neurons and glial cells in spinal cord and brain during fetal development. These receptors appear to have a neuroprotective effect in conditions of hypoxia.

Tissue distribution of erythropoietin and erythropoietin receptor in the developing human fetus

OBJECTIVE Erythropoietin receptors (Epo-R) have been demonstrated on several nonhematopoietic cell types in animal models and in cell culture. Our objective was to determine the tissue distribution and cellular specificity of erythropoietin (Epo) and its receptor in the developing human fetus. STUDY DESIGN The expression of Epo and Epo-R mRNA was ascertained by RT-PCR for organs ranging in maturity from 5 to 24 weeks postconception. The cellular location of protein immunoreactivity was then determined using specific antiEpo and antiEpo-R antibodies. Antibody specificity was established by Western analysis. RESULTS mRNA for Epo and Epo-R was found in all organs in the first two trimesters. Immunolocalization of Epo was limited to the liver parenchymal cells, kidney interstitial cells and proximal tubules, neural retina of the eye, and adrenal cortex. As development progressed, immunoreactivity in the kidney became more prominent. In contrast, immunoreactivity for Epo-R was widespread throughout the body, in cell types including endothelial cells, myocardiocytes, macrophages, retinal cells, cells of the adrenal cortex and medulla, as well as in small bowel, spleen, liver, kidney, and lung. CONCLUSIONS The distribution of Epo and its receptor is more widespread in the developing human than was initially postulated. Epo-R is expressed on many cell types during early fetal development, leading us to speculate that Epo acts in concert with somatic growth and development factors during this period. Further investigation is required to understand the nonhematopoietic role of Epo during human development.

Immunohistochemical Localization of Erythropoietin and Its Receptor in the Developing Human Brain

ABSTRACT We have previously shown erythropoietin (Epo) and its receptor (Epo-R) to be present in the fetal human central nervous system (CNS), and Epo to be present in the spinal fluid of normal preterm and term infants. To investigate the cellular specificities and developmental patterns of expression of these polypeptides in the human brain—areas that have not been well researched—we designed the following study. Human brains ranging in maturity from 5 weeks post-conception to adult were preserved at the time of elective abortion, surgical removal (tubal pregnancy, or removal for temporal lobe epilepsy), or autopsy. Immunohistochemistry was used to localize Epo and Epo-R reactivity in brains of different stages of development. Astrocytes, neurons, and microglia were identified in sequential tissue sections by specific antibodies. At 5 to 6 weeks post-conception, both Epo and Epo-R localized to cells in the periventricular germinal zone. At 10 weeks post-conception, Epo immunoreactivity was present throughout the cortical wall, with the most intense immunoreactivity present in the ventricular and subventricular zones. Epo-R, in contrast, was localized primarily to the subventricular zone, with little staining evident in the ventricular zone. In late fetal brains, Epo-R reactivity was most prominent in astrocytic cells, although modest reactivity was observed in certain neuron populations. In contrast, Epo staining localized primarily to neurons in fetal brains, although a subpopulation of astrocytes was also immunoreactive. In postnatal brains, both astrocyte and neuron populations were immunoreactive with antibodies to Epo-R and Epo. From these results it is clear that Epo and its receptor are present in the developing human brain as early as 5 weeks post-conception, and each protein shows a specific distribution that changes with development. We speculate that Epo is important in neurodevelopment, and that it also plays a role in brain homeostasis later in life, functioning in an autocrine or paracrine manner.

Identifying patients, on the first day of life, at high-risk of developing parenteral nutrition-associated liver disease.

Background:Prolonged use of parenteral nutrition (PN) in neonates can lead to parenteral nutrition-associated liver disease (PNALD), manifested by elevated direct bilirubin concentrations, and in some cases progressing to hepatic failure. When new potential means of preventing PNALD in the neonatal intensive care unit (NICU), such as Omegaven usage, are tested in clinical trials, the studies should enroll neonates at a very high risk of developing PNALD. However, it is not always clear, in the first days of life, which neonates are most likely to develop PNALD. Therefore, preparatory to devising studies of prophylaxis against PNALD, we conducted an evaluation of all NICU patients who received PN for ⩾14 day, assessing their likelihood of developing PNALD.Methods:We performed an historic cohort analysis of all neonates in the Intermountain Healthcare system, receiving PN for 14 days or more during their stay, with dates of birth between 1 January, 2002 and 30 June, 2006.Results:During the 4½-year period, 9861 neonates were cared for in the Intermountain Healthcare NICUs. Of these, 9547 (96.8%) survived for at least 28 days, and of these 6543 (68.5%) received PN. Twenty-one percent (1366 patients) of those receiving PN, received it for ⩾14 days. PNALD was ascertained in this group by a direct bilirubin ⩾2.0 mg/dl. Neonates receiving PN for 14–28 days had a 14% incidence of PNALD, those receiving PN for 29–56 days had a 43% incidence, those receiving PN for 57–100 days had a 72% incidence and those receiving PN for >100 days had a 85% incidence. Groups of patients identifiable on the first day of life as having the highest risk of developing PNALD were birth weight <500 g (odds ratio (OR), 30.7), birth weight 500–749 g (OR, 13.1), gastrochisis (OR, 20.3) and jejunal atresia (OR, 24.0). Among 357 patients who developed PNALD, the highest direct bilirubin concentrations correlated with the highest serum alkaline phosphatase and transaminase concentrations. Deaths after 28 days were much more common in those with the highest direct bilirubin and transaminase concentrations (P<0.0001).Conclusions:In the first days of life, certain NICU patients can be identified as being at very high risk for developing PNALD. These are patients <750 g birth weight, those with gastrochisis and those with jejunal atresia. We speculate that these groups would be reasonable subjects for including in a PNALD prophylaxis trial, testing new preventative strategies such as Omegaven usage.

Is “transfusion-associated necrotizing enterocolitis” an authentic pathogenic entity?

BACKGROUND: Necrotizing enterocolitis (NEC) sometimes occurs after a transfusion, but it is unclear whether this is a chance association or cause and effect.

Incidence, Neutrophil Kinetics, and Natural History of Neonatal Neutropenia Associated with Maternal Hypertension

Abstract Neutropenia occurs often among the newborns of women with hypertension, but its cause, mechanism, and clinical consequences have not been adequately studied. Of 72 infants whose mothers had hypertension during pregnancy, 35 (49 percent) had neutropenia, which persisted from 1 hour to 30 days. The disorder was more prevalent among newborns whose growth had been retarded in utero (P<0.01), those who had been delivered prematurely (P<0.001), and those whose mothers had had severe hypertension (P<0.002) or hypertension and the HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets) (P<0.01). Kinetic investigations of circulating, marginated, storage, and proliferative neutrophils and their progenitors suggested that the neutropenia was the result of diminished neutrophil production. Nosocomial infections occurred during the first 2 1/2 weeks of life in eight (23 percent) of the newborns with neutropenia, but in only one (3 percent) of those without this disorder (P<0.01). We conclude th...

Comparing two methods of delivering high-flow gas therapy by nasal cannula following endotracheal extubation: a prospective, randomized, masked, crossover trial

Objective:We compared two methods of delivering high-flow gas therapy by nasal cannula, applied immediately after planned endotracheal extubations of NICU patients.Study design and methods:Thirty NICU patients who were about to be extubated from mechanical ventilation were randomized into two groups; Group 1 received Vapotherm® for the first 24 h after extubation, then standard high-flow nasal cannula for the next 24 h, and Group 2 received standard high-flow therapy for the first 24 h, then Vapotherm® for the next 24 h. At 24 h after extubation and again 48 h after extubation, a neonatologist who was not aware which modality the patient had been receiving examined the nasal mucosa and applied a scoring system. A research nurse who was unaware of the modality abstracted respiratory rates and respiratory effort scores from a specific study-bedside record. The experimental design was such that a patient could ‘fail’ extubation either by reintubation for mechanical ventilation, or by rescue to the opposite modality before completing the 24-h test period.Results:Fifteen patients were randomized to Group 1 and 15 to Group 2. No differences were apparent between the groups in birth weight, gestational age, age at study entry, gender or underlying pulmonary disorder. Respiratory rates were similar while on Vapotherm® (52±13 breaths/min, mean±s.d.) and high-flow (54±14/min). At 24 h after starting the modality, those on Vapotherm® had more normal examinations of the nasal mucosa (2.7±1.2 vs 7.8±1.7, P<0.0005) and lower respiratory effort scores (1.2±0.6 vs 2.0±0.9, P<0.05) than did those on high-flow. No patients failed while on Vapotherm®, but seven failed while on high-flow (two reintubations and five rescue switches to Vapotherm®, P<0.005).Conclusions:Among NICU patients immediately following extubation, Vapotherm® performed better than a standard high-flow nasal cannula in maintaining a normal appearing nasal mucosa, a lower respiratory effort score, and averting reintubation.

Platelet reference ranges for neonates, defined using data from over 47 000 patients in a multihospital healthcare system

Objective:Identifying a platelet count as abnormal (thrombocytopenia or thrombocytosis) can facilitate recognizing various disease states. However, the published reference ranges for platelet counts in neonates may be imprecise, as they were generated from relatively small sample sizes and compiled before modern platelet enumeration methods.Study design:We derived new neonatal reference ranges for platelet counts and mean platelet volume (MPV) measurements using electronic data accumulated during a recent 6-year period from a multihospital healthcare system.Result:Platelet counts were obtained between the first and the 90th day after birth, from 47 291 neonates delivered at 22 to 42 weeks gestation. The first platelet counts obtained in the first 3 days of life, increased over the range of 22 to 42 weeks gestation. In those born ⩽32 weeks gestation, the lower reference range (5th percentile) was 104 200 μl−1, but it was 123 100 μl−1 in late-preterm and -term neonates. Advancing postnatal age had a significant effect on platelet counts; during the first 9 weeks, the counts fit a sinusoidal pattern with two peaks; one at 2 to 3 weeks and a second at 6 to 7 weeks. The upper limit of expected counts (95th percentile) during these peaks were as high as 750 000 μl−1.Conclusion:The figures herein describe reference ranges for platelet counts and MPV determinations of neonates at various gestational ages during their first 90 days. Expected values differ substantially from the 150 000 μl−1 to 450 000 μl−1 range previously used to define neonatal thrombocytopenia and thrombocytosis. The new definitions will render the diagnoses of neonatal thrombocytopenia and thrombocytosis less commonly than when the old definitions were used, because the new ranges are wider than 150 000 μl−1 to 450 000 μl−1.

Defective Production of Interleukin-6 by Monocytes: A Possible Mechanism Underlying Several Host Defense Deficiencies of Neonates

ABSTRACT: Several deficiencies in antibacterial defense have been described in neonates. Among those best characterized are delayed maturation of B cells into antibody producing cells, deficient T-cell maturation, and delayed cycling of hematopoietic progenitor cells after an infectious challenge. No unifying theory has been forwarded, however, to explain the concomitance of these three developmental deficiencies. IL-6, a cytokine produced primarily by monocytes and macrophages in response to stimulation by IL-1, is involved in the regulation of these three processes. Thus, we postulated that defective production of IL-6 could be a mechanism underlying these immune deficiencies of neonates. Indeed, we observed that at peak production, cells of five term neonates produced only one half as much IL-6 (14 120 ± 2590 pg IL-6/108 monocytes) as those of five adults (28 940 ± 1680 pg, p < 0.001). Peak production was lower still by monocytes of six preterm neonates (7190 ± 1400 pg, p < 0.001 versus term). Production of IL-6 protein was inhibited by actinomycin D and the IL-6 mRNA content of monocytes from neonates, as assessed by competitive polymerase chain reaction, was less than that of adult monocytes. We speculate that defective IL-6 transcription might underlie some of the defects in immune regulation observed in neonates.

Blood and marrow neutrophils during experimental group B streptococcal infection: quantification of the stem cell, proliferative, storage and circulating pools.

Summary: In an attempt to explain the profound neutropenia and depletion of marrow neutrophil reserves observed in human and experimental group B streptococcal infection, we have studied the stem cell, proliferative, storage, and circulating neutrophil pools in groups of adult and neonatal rats inoculated with 3 × 106 type II group B streptococci/g body weight. At intervals after inoculation, animals were sacrificed and the unipotent granulocytic stem cells, (Colony Forming Units in culture, CFUC) from the femoral marrow were quantified. In addition, the total body neutrophil proliferative pool, neutrophil storage pool, circulating blood neutrophil concentration and blood immature to total neutrophil ratio were measured. Adult animals developed neutrophilia with a peak blood neutrophil concentration of 3.4 times control by 24 h after inoculation (P < 0.001). A left shift was seen in these mature animals as early as 2 h but by 24 h was no longer present. The neutrophil storage pool diminished to 65% of control by 15 h (P < 0.001) but then increased to exceed the control in all animals by 48 h (P < 0.01). The neutrophil proliferative pool did not change during infection in the adult animals, but CFUC increased to three times control 48 h after inoculation (P < 0.001) and returned to baseline by 72 h.The myeloid response of the neonatal rats was markedly different from that of the adults. Neonates developed profound neutropenia with an extreme left shift, exhaustion of the neutrophil storage pool (less than 10% of control by 15 h, P < 0.001), and diminution of the proliferative pool to 58% of control by 10 h (P < 0.05). In contrast to the adult animals, neonatal rats infected with group B streptococci failed to increase CFUC, and in fact a diminution of CFUC to 48% of control was seen by 15 h (P < 0.05).Speculation: When adults develop bacterial infection, they often demonstrate neutrophilia and a leukocyte left shift. We observed these findings in infected adult rats accompanied by a moderate, temporary diminution of storage neutrophils and by an elevation in granulocytic stem cells. The adult response enables the animal to deliver large numbers of neutrophils to the infected tissues. In contrast, infected neonates developed profound neutropenia, exhaustion of neutrophil reserves and decreased neutrophii production. The neonatal response results in diminished delivery of neutrophils to infected tissues and a marked increase in mortality. The decreased production of neutrophils accompanying neutrophil storage pool depletion and neutropenia in neonates may constitute a condition for which neutrophil transfusion may represent a reasonable therapeutic approach.