Neonatal Blood Cells Research Articles

Nucleated red blood cells in neonates with hypoxic ischaemic encephalopathy treated with hypothermia: A worthwhile prognostic biomarker for clinicians in LMIC?

Background. Neonatal hypoxic ischaemic encephalopathy (HIE) is a leading cause of term neonatal death worldwide, with a higherincidence in low- to middle-income settings.Objective. To investigate whether nucleated red blood cell (nRBC) counts could predict severity of HIE and outcomes in term neonatestreated with therapeutic hypothermia (TH).Methods. We conducted a retrospective sub-study at Tygerberg Hospital in Cape Town, South Africa. The review included all cooledneonates’ clinical records and blood samples from a National Health Laboratory Services database. One experienced neurodevelopmental expert assessed patients over a period of 12 months.Results. Twenty-five files out of a total of 100 were excluded owing to missing data. In accordance with the Thompson HIE score,the cohort was classified as mild (56%), moderate (27%), and severe (17%). All included patients (n=75) had full blood counts within6 hours of delivery. nRBC were detected in 52% of the samples. There was no correlation between nRBC category and HIE severity(p=0.265). Raised nRBCs (≥30 cells/100 white blood cells (WBCs)) were more frequent in infants who died than in those whosurvived (p=0.008). Infants with nRBC counts ≥30 cells/100 WBCs had an increased likelihood of having cerebral palsy or impairedneurodevelopment (p=0.013).Conclusion. The study demonstrated a significant association between an early increase in nRBC counts in HIE infants treated with TH,and both short- and long-term outcomes. A larger multicentre study is required to better understand the relationship between nRBCcounts and HIE in the era of cooling in our local setting.

WITHDRAWN: Comparing Nucleated Red Blood Cells in Neonates with and without Retinopathy

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Maternal Allergy Status Has No Impact on Neonatal Immune Responses to Allergen Stimuli

Due to the increasing incidence of allergic diseases, there is a strong need to identify a prognostic marker pointing to increased risk of allergy development allowing introduction of early preventive measures. Cord blood seems to be a good source for searching for such marker. The capacity of cord blood cells to respond to common allergens could point to increased predisposition to later allergy development. In our study, cytokines typical of Th1 (IFN-γ), Th2 (IL-5, IL-13) and Treg (IL-10) immune responses were followed at both the level of gene expression and cytokine secretion in cord blood cells of newborns of healthy mothers (children with relatively low risk of allergy development) and allergic mothers (children with relatively high risk of allergy development) stimulated by allergens (pollen from birch and timothy grass, house dust mite, ovalbumin). We have not observed any difference in the response of cord blood cells of neonates of healthy and allergic mothers to allergen in vitro. Both gene expression and secretion of cytokines in response to allergen stimulation were comparable with the unstimulated controls. It seems that early postnatal events will be more decisive for future allergy development than prenatal sensitization of the foetal immune system with allergen in utero in allergic mothers.

Effects of tobacco smoking during pregnancy on oxidative stress in the umbilical cord and mononuclear blood cells of neonates

BackgroundAlthough cigarette smoke is known to be a complex mixture of over 4000 substances that can lead to damage through active or passive smoking, its mechanisms and biochemical consequences in pregnancy and neonates are not yet fully understood. Therefore, in the present study, we propose to study the impact of smoking during gestation on the viability of blood mononuclear cells (MNC) from umbilical cords of newborns to assess the degree of oxidative stress and cell viability. After childbirth, the cord blood and the umbilical cord were immediately collected in public hospitals in Greater Vitoria, ES, Brazil. Flow cytometry was used to analyze the cord blood followed by biochemical and histological tests to analyze possible changes in the umbilical cord.ResultsPregnant smokers had a reduction of MNC viability from the umbilical cord (10%), an increase in the production of reactive oxygen species (ROS) and an increase in cell apoptosis (~2-fold) compared to pregnant non-smokers. In the umbilical cord, it was observed an increase of advanced oxidation protein products - AOPP (~2.5-fold) and a loss of the typical architecture and disposition of endothelial cells from the umbilical artery.ConclusionsThese data suggest that maternal cigarette smoking during pregnancy (even in small amounts) may compromise the viability of MNC cells and damage the umbilical cord structure, possibly by excessive ROS bioavailability.

Adjustment of cerebrospinal fluid protein for red blood cells in neonates and young infants

To determine the relationship between cerebrospinal fluid (CSF) red blood cell (RBC) count and CSF protein in neonates and young infants undergoing lumbar puncture. Cross-sectional study. Urban tertiary care children's hospital. Infants 56 days of age and younger who had a lumbar puncture in the emergency department between January 1, 2005 and July 31, 2009 were eligible for inclusion. Infants with missing laboratory data, exceedingly high CSF red blood cell counts, or conditions known to elevate CSF protein were excluded. Linear regression was used to determine the association between CSF RBC and CSF protein. Of 1986 infants, 56 days of age or younger, who underwent lumbar puncture in the emergency department during the study period, 1241 (62.5%) met inclusion criteria. The median age was 34 days (interquartile range: 19-46 days); 45% of patients were male. The median CSF RBC count was 40 cells/mm(3) (interquartile range: 2-1080 cells/mm(3)); 11.8% of patients had a CSF RBC >10,000 cells/mm(3). CSF protein increased by 1.9 mg/dL (95% confidence interval: 1.7-2.1 mg/dL) per 1000 CSF RBCs for all included patients. Restricting analysis to patients without pleocytosis yielded comparable results, as did subanalyses by age and delivery type. We found that CSF protein concentrations increased by approximately 2 mg/dL for every 1000 CSF RBCs. These data may assist clinicians in interpreting CSF protein concentrations in infants 56 days of age and younger in the context of traumatic lumbar punctures.

CELL-DYN Sapphire and microscopic method comparison for nucleated red blood cells in neonatal blood

CELL-DYN Sapphire and microscopic method comparison for nucleated red blood cells in neonatal blood

Reference Ranges for Blood Concentrations of Nucleated Red Blood Cells in Neonates

Background: Previous studies reported a relationship between high nucleated red blood cells (NRBC) in neonates and the development of intraventricular hemorrhage (IVH) and/or retinopathy of prematurity (ROP). Objective: We sought to (1) establish reference ranges for NRBC in neonates based on a large data set, (2) compare NRBC from automated versus manual counts, (3) determine the effect of an elevated NRBC, on the day of birth, on the odds of developing grade ≧3 IVH or ROP. Methods: We analyzed all NRBC obtained during 8.5 years in a multihospital system, displaying the 5th and 95th percentile limits according to gestational age and postnatal age. Results: NRBC counts were retrieved from 61,932 neonates, 26,536 of which were excluded from the data set. Comparing 9,000 samples run simultaneously on manual versus automated methods, the manual counts yielded slightly higher counts, but the difference is likely insignificant clinically. Altitude of the birth hospital did not correlate with NRBC, and no correlations were observed with cord pH or 1- or 5-min Apgar. An NRBC count >95th percentile limit was associated with higher odds of developing a grade ≧3 IVH (OR 4.28; 95% CI 3.17–5.77) and grade ≧3 ROP (OR 4.18; 95% CI 2.74–6.38). Conclusion: The figures of this report display reference ranges for NRBC according to gestational age and postnatal age. An NRBC count above the 95% limit at birth is associated with a higher risk of subsequently developing severe IVH and severe ROP. We speculate that this association is because an elevated NRBC count is a marker for prenatal hypoxia.

Differential gene expression in umbilical cord blood and maternal peripheral blood

Umbilical cord blood (UCB) has become a useful alternative source of hematopoietic stem cells for clinical and research applications. UCB represents neonatal blood and differs from adult blood in many aspects, displaying different cell composition and various features of cellular immaturity. To understand molecular basis of phenotypic differences between neonatal and adult blood, we studied variations in transcriptome of UCB and maternal peripheral blood (PB). Using Illumina microarrays, we determined gene expression profiles of UCB and PB samples obtained from 30 mothers giving birth to living baby. Out of 20,589 tested genes, 424 genes were down-regulated and 417 genes were up-regulated in UCB compared with PB. Reduced expression of many immunity-related pathways (e.g. TLR pathway, Jak-STAT pathway, cytokine-cytokine receptor interaction) in neonatal blood cells may contribute to the poor response to antigens, increasing susceptibility to infections at the time of disappearance of protective maternal antibodies. On the other hand, overexpression of erythropoiesis-related genes (glycophorins, fetal hemoglobins, enzymes catalysing heme synthesis and erythrocyte differentiation) in UCB probably enforces red cell production in newborns. Our study demonstrates that neonatal and maternal bloods show specific gene expression profiles, likely reflecting differences in phenotypes of immunologically immature and fully evolved hematopoietic cells.

Allogeneic neonatal blood transfusion ameliorates autoimmune diabetes in unconditioned non-obese diabetic mice by functionally deleting pathogenic T cells (99.19)

Abstract We show that transfusion of unconditioned, pre-diabetic non-obese diabetic mice with newborn blood containing multiple Sca-1+ stem/progenitor subsets derived from allogeneic diabetes-resistant mice can prevent diabetes. Diabetes protection was characterized by enhanced glucose-induced insulin release and diminished cellular infiltration of islets. Allogeneic neonatal blood transfusion induced only transient hematopoietic microchimerism, which did not correlate with the level of protection obtained. Splenocytes derived from cured mice failed to transfer diabetes into immunodeficient NODscid mice, indicating deletion/inactivation of diabetogenic T cells by transfused neonatal blood cells. Diabetes cure did not result in alloantigen-specific T cell unresponsiveness as assessed by T cell proliferation. Paradoxically, T cell anergy to allogeneic antigens was displayed by a majority of diabetic but not cured mice that received newborn blood cells. These results indicate that deletion/inactivation of diabetogenic T lymphocytes is instrumental in preventing the progression of insulitis to overt diabetes by allogeneic newborn blood cell transplantation.

Interferon regulatory factor 3-dependent responses to lipopolysaccharide are selectively blunted in cord blood cells

AbstractThe synthesis of interferon-β (IFNβ) and IFN-inducible factors elicited by lipopolysaccharide (LPS) depends on the transcriptional activity of interferon regulatory factor 3 (IRF-3) downstream of Toll-like receptor-4 (TLR4). To examine the ability of human newborns to mount TLR4-mediated IRF-3–dependent responses, we analyzed the pattern of genes expressed on the addition of LPS to cord blood or cord blood monocyte-derived dendritic cells (moDCs). Expression of IFNβ and IFN-inducible genes was selectively impaired in neonatal blood and moDCs as compared with their adult counterparts. This selective defect was confirmed by microarray experiments on moDCs. Altered expression of IFN-inducible genes was related to impaired IFNβ synthesis because IFNβ signaling was functional in neonatal moDCs. However, addition of exogenous IFNβ failed to restore LPS-induced IL-12p70 synthesis which was previously shown to be defective in neonatal moDCs. Although LPS-induced IRF-3 nuclear translocation was observed both in adult and neonatal moDCs, IRF-3 DNA-binding activity and association with the coactivator CREB-binding protein (CBP) were decreased in neonatal as compared with adult moDCs. We conclude that impaired IRF-3/CBP interaction in neonatal blood cells exposed to LPS is associated with impaired expression of IFNβ and IFN-inducible genes. Because IRF-3 activity is also required for IL-12p70 synthesis, our findings provide a molecular basis for the decreased ability of LPS-stimulated neonatal moDCs to elicit Th1-type responses.

Regulation of Trafficking Receptor Expression in Human Forkhead Box P3+ Regulatory T Cells

Forkhead Box P3(+) (FOXP3(+)) T cells are regulatory cells important for maintaining immune tolerance. While chemokine- and other homing-receptors are important for T cell migration, it has been unclear how they are regulated in FOXP3(+) T cells. We thoroughly investigated, ex vivo and in vitro, the regulation of chemokine receptor expression on human FOXP3(+) T cells in neonatal cord blood, adult peripheral blood, and tonsils. We found that human FOXP3(+) T cells undergo changes in trafficking receptors according to their stages of activation and differentiation. FOXP3(+) T cells are divided into CD45RA(+) (naive type) and CD45RO(+) (memory type) FOXP3(+) T cells in neonatal blood, adult blood, and tonsils. CD45RA(+)FOXP3(+) T cells mainly express lymphoid tissue homing receptors (CD62L, CCR7, and CXCR4), while CD45RO(+)FOXP3(+) T cells highly express both Th1 and Th2-associated trafficking receptors along with the lymphoid tissue homing receptors at reduced frequencies. Up-regulation of Th1/Th2-associated trafficking receptors begins with activation of CD45RA(+)FOXP3(+) T cells and is completed after their differentiation to CD45RO(+) cells. Some chemokine receptors such as CXCR5 and CXCR6 are preferentially expressed by many FOXP3(+) cells at a specific stage (CD69(+)CD45RO(+)) in tonsils. Our in vitro differentiation study demonstrated that CD45RA(+)FOXP3(+) T cells indeed undergo chemokine receptor switch from CD45RA(+) (secondary lymphoid tissue homing) to CD45RO(+) type (lymphoid and nonlymphoid tissue homing). The orderly regulation of trafficking receptors in FOXP3(+) T cells according to stages of differentiation and activation is potentially important for their tissue-specific migration and regulation of immune responses in humans.

Shorter PFA‐100® closure times in neonates than in adults: role of red cells, white cells, platelets and von Willebrand factor

In neonates, despite poor platelet function in various in vitro tests, closure times (CTs) in PFA‐100 measurements are shorter than in adults. Neonates have a higher polymeric von Willebrand factor (vWF). They also have a higher haematocrit and higher white blood cell count than adults, which may interfere with the evaluation of platelet and vWF function by means of the PFA‐100 in neonates. To assess the role of different blood constituents on neonatal CTs, red blood cell, platelet and white blood cell counts in cord blood were modified. These modifications did not provide any evidence that the difference in number between adult and neonatal blood cells was responsible for shorter neonatal CTs. In further experiments, platelets and/or vWF were inhibited by means of abciximab and anti‐vWF antibody, and mixing experiments with neonatal platelet‐rich and platelet‐poor plasma were performed. The results showed that short cord blood PFA‐100 CTs were caused by a constituent of neonatal platelet‐poor plasma, probably the neonatal high multimeric vWF. Conclusion: This study demonstrates that CTs in neonates are dependent on the same components, platelets and vWF, as in adults, making it likely that the PFA‐100 can be used in neonates in the same way as in adults to investigate platelet and vWF function.

Autologous transplantation of retrovirally transduced bone marrow or neonatal blood cells into cats can lead to long-term engraftment in the absence of myeloablation.

Autologous transplantation of retrovirally transduced bone marrow (BM) or neonatal blood cells was carried out on eight cats (ranging in age from 2 weeks to 12 months) with mucopolysaccharidosis type VI (MPS VI). The transducing vector contained the full-length cDNA encoding human arylsulfatase B (hASB), the enzymatic activity deficient in this lysosomal storage disorder. Following transplantation, the persistence of transduced cells and enzymatic expression were monitored for more than 2 years. Five of the cats received no myeloablative preconditioning, two cats received 370-390 cGy of total body irradiation (TBI), and one cat received 190 cGy TBI. Evidence of transduced cells, as judged by enzymatic activity and PCR detection of the provirus, was demonstrated in granulocytes, lymphocytes, or BM cells of the treated animals up to 31 months after transplantation. Radiation preconditioning was not required to achieve these results, nor were they dependent on the recipient's age. However, despite the long-term persistence of transduced cells, the levels of ASB activity in the transplanted animals was low, and no clinical improvements were detected. These data provide evidence for the long-term persistence of retrovirally transduced feline hematopoietic cells, and further documentation that engraftment of transduced cells can be achieved in the absence of myeloablation. Consistent with previous bone marrow transplantation studies, these results also suggest that to achieve clinical improvement of MPS VI, particularly in the skeletal system, high-level expression of ASB must be achieved in the treated animals and improved techniques for targeting the expressed enzyme to specific sites of pathology (e.g. chondrocytes) must be developed.

Human neonatal blood: stem cell content, kinetics of CD34+ cell decline and ex vivo expansion capacity.

Haemopoietic stem cells are present in fetal blood but their levels decline rapidly in the peripheral circulation of the infant after birth. We previously reported a case of stem cell transplant in a beta-thalassaemia boy using a combination of the cord blood (CB) and neonatal blood (NB) of his sister. This transplant resulted in a successful engraftment. To investigate the possibility of using NB to supplement CB for related transplants, we further characterized stem and progenitor cells and lymphocyte subsets in 20 NB samples, comparing the findings with those in 20 CB samples. Our data showed that NB contained substantial levels of CD34+ cells, CD34+CD38- cells, colony-forming units-granulocyte macrophage (CFU-GM), colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E) and long-term culture initiating cells (LTCIC). NB was similar to CB in the levels of T lymphocytes, but the amounts of B lymphocytes and natural killer cells were higher in CB (P = 0.033, P= 0.001, respectively). The kinetics of CD34+ cells in NB was investigated in serial blood samples obtained from 10 full-term infants at 2, 4, 6, 8, 24 and 48h after birth. CD34+ cells decreased rapidly after birth, declining to only 30% of the 2h level at 48h (P<0012). The rate of decline was greatest in the first 4 h of life. NB from four infants was expanded by culturing the blood samples in the presence of thrombopoietin (Tpo), interleukin 1beta (IL1beta), IL-3, IL-6, flt-3 ligand and stem cell factor (SCF) for 7 d. This resulted in the increase of CD34+ cells, CFU-GM and CFU-MK by 271+/-179, 556+/-385 and 113+/-75 fold respectively. Three of the five samples expanded for 7 d contained LTCIC. These findings suggest that NB might be a supplementary or alternative source of stem cells to CB for transplant. The ethics and practicality of this approach deserve further exploration.

Preliminary experience with a prospective protocol for planned vaginal delivery of triplet gestations

Objective: The objective of the study was to evaluate a protocol for vaginal delivery of triplet gestations. Study Design: All women with triplet gestations managed between January 1, 1995, and December 31, 1997, by University Medical Center’s perinatal practice were offered enrollment in our vaginal delivery protocol. Our protocol offered attempt of vaginal delivery if triplet A was in vertex presentation, fetal monitoring was possible, and there were no other obstetric contraindications. Twenty-three triplet gestations were identified; 8 achieved vaginal delivery. Outcome parameters investigated included neonatal mortality, Apgar scores, neonatal intracranial hemorrhage, arterial cord pH, neonatal weight, and length of postpartum hospital stays of mother and neonates. All parameters were analyzed with analysis of variance and the Student t test as appropriate with the JMP 3.1 statistics program (Cary, NC). Results: Twenty-three sets of triplets were enrolled. Eight sets were delivered vaginally. Eight of 9 patients (88.9%) who attempted trial of labor were delivered vaginally, 1 of which was a vaginal birth after cesarean section. The remaining triplet gestation failed to progress at 4-cm dilation. Twelve sets of triplets had a nonvertex-presenting triplet and were delivered by the cesarean route. The remaining 2 triplet gestations were delivered by the cesarean route because of inadequate fetal monitoring. Neonatal survivals were 100% for both groups. No significant differences in neonatal mortality, Apgar scores, intracranial hemorrhage, arterial cord blood pH, hospital or neonatal intensive care unit stay of neonate, neonatal weight, and change in maternal or neonatal blood cell count were noted. There were no cases of grade III or IV intraventricular hemorrhage in either group. A significant reduction in postpartum hospital stay of mother was noted in the vaginal delivery group (2.8 vs 4.5 days, P < .001). The mean gestational age at delivery was significantly lower for the vaginal delivery group (31.3 vs 34.0 weeks, P < .02). The mean neonatal weight for the vaginal delivery group was significantly lower (1758 ± 473 vs 2022 ± 407 g, P < .02). There were no significant differences in outcome parameters for the first, second, and third triplets within each group when compared with each other or with the other study group. One patient who underwent vaginal delivery had retained products of conception and required curettage. A single fetal death occurred at 22 weeks’ gestation from twin-twin transfusion, with the remaining triplets being delivered vaginally at 35 weeks’ gestation. Cesarean hysterectomy was required in 1 case for uncontrollable bleeding at the time of cesarean delivery. Perinatal complications occurred in a large number of patients, with the incidence of premature labor 47.8% (n = 11), that of preterm premature rupture of membranes 26.1% (n = 6), and that of preeclampsia 34.8% (n = 8). Conclusion: In selected cases vaginal delivery of triplet gestations can be accomplished without increased maternal or neonatal morbidity and mortality and may significantly decrease maternal hospital stay and postoperative morbidity. (Am J Obstet Gynecol 1998;179:1133-5.)

Regulation of Interleukin-10 Gene Expression: Possible Mechanisms Accounting for Its Upregulation and for Maturational Differences in Its Expression by Blood Mononuclear Cells

AbstractInterleukin-10 (IL-10) downmodulates phagocytic immune responses and accentuates humoral responses. Human neonates exhibit broad immune deficits that parallel actions of IL-10. We postulated that IL-10 production would be diminished in neonatal blood cells. We found that IL-10 production by lipopolysaccharide-stimulated peripheral blood mononuclear cells (PBMNCs) in vitro was greater by adult cells than by term cells and preterm cells. Additional studies were undertaken to identify mechanisms responsible for the developmental differences in IL-10 gene expression. IL-10 transcription was present in freshly isolated adult and neonatal cells in the absence of detectable levels of transcript. Transcription rates were not different between adult and neonatal cells. IL-10 transcripts were approximately 40% more abundant in adult cells than in term cells and were consistent with differences in secreted protein; however, no differences were noted in mRNA stability. IL-10 half-life was 60 minutes for both adult and term PBMNCs. We conclude that up-regulation of IL-10 gene expression in PBMNCs is modulated at the post-transcriptional level, that IL-10 protein production and mRNA content are greater in activated cells from adults compared with those from neonates, and that maturational differences in IL-10 expression are not due to differences in transcription rate or mRNA stability. Maturational differences in IL-10 expression might be due to differences in subpopulations of cytokine-producing cells or differences in nucleo-cytoplasmic transport.

Should frusemide be prescribed after packed red cell transfusions in the newborn?

We have observed that it is a common practice in the United Kingdom to follow transfusions of packed red blood cells in neonates with an injection of frusemide. The purpose of this paper is not to comment on whether or when transfusions should be given, but on the use of frusemide with blood transfusions. On reviewing manuals of neonatal intensive care we were only able to find one that recommended giving frusemide with blood transfusions [10]. When we asked clinicians for their rationale for this practice, a consistent answer was that it would diminish the vascular overload provoked by the transfusion. Is this reasonable? The origins of this practice appear to be reports describing the haemodynamic eects of blood transfusion in chronic anaemia. Duke et al. [4] pointed out that in anaemic but euvolaemic patients transfusion may produce hypervolaemia. Gupta et al. [6] measured pulmonary capillary wedge pressure as an index of left ventricular filling pressure before and after the transfusion of one unit of blood, at a rate of 300 ml/h, in adults with chronic severe anaemia. Patients who recevied frusemide at the start of the transfusion showed a reduction in wedge pressure, in contrast to those who did not, in whom there was a rise in wedge pressure. DePalma and Luban [3] have recommended that patients in heart failure should be given no more than 2 ml/kg per hour of red blood cells. However, most preterm newborn babies receiving ‘‘top up’’ red cell transfusions are not in heart failure. In addition, Duke et al. [4] published their report at a time when only whole blood was used for transfusion. Present day practice, based on specific component therapy, is for ‘‘top up’’ transfusions to consist of red cell concentrates with an haematocrit of around 50%‐70%.

Umbilical vein endothelial cells are an important source of c-kit and stem cell factor which regulate the proliferation of haemopoietic progenitor cells.

The expression and production of c-kit and its ligand, stem cell factor (SCF), in cord blood and neonates were studied. Serum SCF levels were significantly higher in cord blood, neonates aged 1-30 d, and in 4-month-old infants than in the maternal serum (P < 0.01). SCF levels decreased in children from 7 months to 15 years of age (P < 0.01). The serum soluble c-kit levels were significantly higher in cord (P < 0.01) and neonatal blood (P < 0.05) than in the maternal blood. SCF and c-kit levels in placental tissue homogenates and the culture media of decidual cells and trophoblasts were low. To determine the sites of high SCF and c-kit production in cord blood and in early neonates. SCF and c-kit mRNA expression was analysed in various tissues by polymerase chain reaction. High SCF mRNA expression was observed in human umbilical vein endothelial cells (HUVEC). Moderate c-kit mRNA expression was detected in HUVEC, the bone marrow, and cord blood. These findings suggest that endothelial cells mainly produce the SCF in cord blood and in early neonates. To confirm the role of endothelial cells in haemopoiesis, colony-forming assays were performed in the presence of HUVEC culture media, which induced the formation of high numbers of granulocyte and erythroid colonies in cord blood. IL-3, IL-6 and SCF levels were elevated in the media. Our findings suggest that endothelial cells have an important role in the maintenance and proliferation of progenitor cells in neonatal blood via the interaction of c-kit and SCF with other factors. The ex vivo expansion of cord progenitor cells in the presence of endothelial cells needs to be investigated further.

Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency.

Haematopoietic stem cells in umbilical cord blood are an attractive target for gene therapy of inborn errors of metabolism. Three neonates with severe combined immunodeficiency were treated by retroviral-mediated transduction of the CD34+ cells from their umbilical cord blood with a normal human adenosine deaminase complementary DNA followed by autologous transplantation. The continued presence and expression of the introduced gene in leukocytes from bone marrow and peripheral blood for 18 months demonstrates that umbilical cord blood cells may be genetically modified with retroviral vectors and engrafted in neonates for gene therapy.

The production of large ‘signalling competent’ myeloid cells from circulating CD34 + cells in neonatal blood

A method is described for the production of large myeloid cells, expressing functional formylated peptide receptors. CD34 + cells were isolated from neonatal cord blood by a two stage cell sorting method. Inclusion of SCF, together with IL-3, GM-CSF or both cytokines stimulated growth of these cells over 14 day period. The resultant cells, which ranged from 30 μm to 100 μm in size, were maintained in culture for up to 5 weeks, during which time the cell population increasingly displayed myeloid characteristics, including expression of formylated peptide receptors, phagocytosis and oxidase activation. These large cells had a functioning Ca 2+ signalling system in response to the chemotactic peptide, f-Met-Leu-Phe. The large size of the cells enabled the rise in cytosolic free Ca 2+ which resulted from either transmembrane influx of extracellular Ca 2+ and release of Ca 2+ from intracellular stores to be visualised. These large myeloid cells thus provide a model system for investigating the spatial characteristics of Ca 2+ signalling by formylated peptide receptors on human myeloid cells.