Outcome and management in neonatal thrombocytopenia due to maternal idiopathic thrombocytopenic purpura.

BACKGROUND AND OBJECTIVES: Pregnant women with Idiopathic thrombocytopenic purpura (ITP) can deliver neonates with severe thrombocytopenia. Clear evidence declaring the pathophysiological cause of this neonatal thrombocytopenia is lacking, as antiplatelet antibodies are not always detectable in maternal serum. Severe neonatal thrombocytopenia below 50 × 10(9) /l is reported in 8-13% of the neonates from mothers with ITP and intracranial haemorrhage (ICH) in 0-2·9%. Evidence about the optimal postnatal treatment is scarce. Our objective was to evaluate the outcome and management in neonates with passive ITP. MATERIALS AND METHODS: All neonates from mothers with ITP born between 1980 and 2011 were included. Platelet counts during the first 10 days, presence of ICH and postnatal treatment were recorded. Maternal characteristics were analysed as possible risk factors for severe neonatal thrombocytopenia. RESULTS: Sixty-seven neonates were included. Severe thrombocytopenia (<50 × 10(9) /l) occurred in 20/67 (29·9%) neonates. In three neonates, platelet count rose spontaneously, 18 neonates were treated (one with persistent moderate thrombocytopenia) with the following: platelet transfusions (3), prednisone (2), intravenous immunoglobulin (IVIG) (1), platelet transfusions and IVIG (11), platelet transfusion and prednisone (1). Recurrence of low platelet counts after transfusions was commonly seen. Risk factors for severe neonatal thrombocytopenia were a previous sibling with severe thrombocytopenia and low maternal platelet nadir during pregnancy. CONCLUSION: In this cohort, severe neonatal thrombocytopenia occurs more frequently than previously reported. To maintain a platelet count above 50 × 10(9) /l, often multiple transfusions and IVIG are required. Multiple transfusions may be avoided by starting IVIG, when platelet count falls below 50 × 10(9) /l after the first platelet transfusion.

Repeated courses of ibuprofen are effective in closure of a patent ductus arteriosus.

Patent ductus arteriosus (PDA) is a frequent complication in preterm infants. Ibuprofen and indomethacin (both COX inhibitors) are used for pharmacological closure of PDA. In most centers, a failed second course of COX inhibitors is followed by surgical closure. Our aim was to estimate the closure rate of clinically significant PDA after second and third courses of ibuprofen and record possible side effects. A study population, consisting of 164 preterm infants (<32 weeks' gestational age) with PDA admitted at our tertiary care center between November 2005 and September 2011, was retrospectively analyzed. Primary outcome was the closure rate after repeated courses of ibuprofen. The closure rate was similar after the first (109/164), second (24/43), and third (6/11) course of ibuprofen (X(2) = 2.1, p = 0.350). Late start of the first course of ibuprofen was a predictive factor for increased need of a second course (X(2) = 4.4, p = 0.036). No additional side effects of multiple courses of ibuprofen were detected. In conclusion, repeated courses of ibuprofen are an effective and safe alternative for surgical closure and should be considered after failure of the first course of ibuprofen.

Pim-1 levels determine the size of early B lymphoid compartments in bone marrow.

The mouse proto-oncogene Pim-1, which encodes two cytoplasmic serine-threonine-specific protein kinases, is frequently activated by proviral insertion in murine leukemia virus-induced hematopoietic tumors. Transgenic mice overexpressing Pim-1 show a low incidence of spontaneous T cell lymphomas, whereas null mutant mice lack an obvious phenotype. We have analyzed the early B lymphoid compartment from both null mutant and E mu-Pim-1 transgenic mice. The level of Pim-1 expression appears to be a determining factor in the ability of these cells to respond to the growth factors interleukin 7 (IL-7) and SF (steel factor). The impaired response in null mutant mice could be rescued by introduction of a functional Pim-1 transgene. Moreover, overexpression of Pim-1 facilitates the derivation of primitive lymphoid cell lines that are dependent on combined stimulation with IL-7 and SF or insulin-like growth factor 1. These results for the first time identify the involvement of Pim-1 in a normal cellular function, as an important regulator of early B lymphopoiesis in mice.

Use of rifampin in persistent coagulase negative staphylococcal bacteremia in neonates.

BACKGROUND: Coagulase negative staphylococci (CoNS) are the most common cause of neonatal sepsis in the Neonatal Intensive Care Unit (NICU). A minority of neonates does not respond to vancomycin therapy and develops persistent bacteremia, which may be treated with rifampin. We evaluated the use of rifampin in persistent CoNS bacteremia. METHODS: Retrospective study of 137 neonates with CoNS bacteremia during admission to a tertiary NICU between July 2006 and July 2009. Main outcome measures were total duration of bacteremia and the adequacy of vancomycin and rifampin therapy. RESULTS: 137/1696 (8.0%) neonates developed a CoNS bacteremia. Eighteen were treated with rifampin because of persistent bacteremia (3 positive blood cultures at least 48 hours apart with clinical symptoms) or (a serious suspicion of) an intravascular thrombus. Duration of bacteremia prior to rifampin therapy (8.0 ± 3.6 days) was positively correlated (p < 0.001) to the total duration of bacteremia (10.3 ± 3.7 days). After starting rifampin therapy C-reactive protein (CRP) levels of all neonates declined and blood cultures became sterile after 2.3 ± 1.6 days. Vancomycin levels were not consistently measured in all neonates, resulting in late detection of subtherapeutic trough levels. CONCLUSION: Rifampin may be effective in the treatment of persistent CoNS infections in neonates. Outcome may be improved by adequate monitoring of vancomycin trough levels.

Short and long term outcome of neonatal hyperglycemia in very preterm infants: a retrospective follow-up study.

BACKGROUND: Hyperglycemia in premature infants is associated with increased morbidity and mortality, but data on long-term outcome are limited. We investigated the effects of neonatal hyperglycemia (blood glucose > or = 10 mmol/l, treated with insulin for > or = 12 hours) on growth and neurobehavioral outcome at 2 years of age. METHODS: Retrospective follow-up study at 2 years of age among 859 infants < or =32 weeks of gestation admitted to a tertiary neonatal center between January 2002 and December 2006. Thirty-three survivors treated with insulin for hyperglycemia and 63 matched controls without hyperglycemia were evaluated at a corrected age of 2 years. Outcome measures consisted of growth (weight, length, and head circumference) and neurological and behavioural development. RESULTS: 66/859 (8%) infants < or = 32 weeks of gestation developed hyperglycemia. Mortality during admission was 27/66 (41%) in the hyperglycemia group versus 62/793 (8%) in those without hyperglycemia (p < 0.001). Mortality was higher in infants with hyperglycemia with a birth weight < or =1,000 gram (p = 0.005) and/or gestational age of 24-28 weeks (p = 0.009) than in control infants without hyperglycemia. Sepsis was more prominent in infants with hyperglycemia and a birth weight of >1,000 gram (p = 0.002) and/or gestational age of 29-32 weeks (p = 0.009) than in control infants without hyperglycemia. Growth at 2 years of age was similar, but neurological and behavioural development was more frequently abnormal among those with neonatal hyperglycemia (p = 0.036 and 0.021 respectively). CONCLUSIONS: Mortality was higher in very preterm infants with hyperglycemia treated with insulin during the neonatal period. At 2 years of age survivors showed normal growth, but a higher incidence of neurological and behavioural problems. Better strategies to manage hyperglycemia may improve outcome of very preterm infants.

Dysglycaemia in small-for-gestational-age neonates: a matched case-control study in monochorionic twins.

OBJECTIVE: Small-for-gestational-age (SGA) neonates (birth weight <10th centile) are at higher risk of altered glucose homeostasis compared to appropriate for gestational age (AGA) neonates. The aim of this matched case-control study was to estimate the incidence of hypoglycaemia and/or hyperglycaemia in monochorionic (MC) twins with selective intrauterine growth restriction (sIUGR). METHODS: We included all MC twins with sIUGR (2002-2013). Neonates in the SGA group were matched with their AGA co-twin. We recorded the occurrence of hypoglycaemia and hyperglycaemia in the first 48 h after birth and studied the association with SGA. RESULTS: In this retrospective study were 126 twin pairs included. The incidence of hypoglycaemia in the SGA group and AGA group was 29.6% and 17.4%, respectively, hyperglycaemia occurred in 8.7% of the SGA neonates and in 2.6% of the AGA co-twins. Multivariate analysis showed an independent association of SGA with hypoglycaemia (OR 1.97, CI 1.23-3.18, p ≤ 0.01), but not with hyperglycaemia (OR 2.57, CI 1.64-10.28, p = 0.182). Low gestational age (GA) at birth (OR 1.65, CI 1.09-2.48, p = 0.02) showed an independent association with hyperglycaemia. CONCLUSIONS: The risk of hypoglycaemia is almost twofold higher in SGA neonates compared to their MC AGA twins. Low GA appeared to be an independent risk factor for hyperglycaemia in SGA neonates.

Thymic lymphomas in interleukin 9 transgenic mice.

Transgenic mice overexpressing the interleukin 9 gene were generated to study the biological activity of this cytokine in vivo. Although no major histological or morphological modifications of the lymphoid system were observed in most animals, approximately 7% of transgenic mice developed thymic lymphomas at the age of 3-9 months. The tumor cells, which were clonal, with unique T cell rearrangements, were double positive for the expression of CD4 and CD8. The need for additional transforming events, suggested by the low incidence of spontaneous tumors, was further indicated by the high susceptibility of the transgenic animals to injections of low doses of N-methyl-N-nitrosourea, a chemical carcinogen with a thymic tropism. Expression of interleukin 9 was required for optimal tumor growth in vivo, as one of the tumors studied, which had lost the transgene, was much more efficiently transplanted into transgenic than in normal mice. Moreover, the in vitro proliferative activity of interleukin 9 on cell lines derived from such transgene-negative tumors suggests that an autocrine loop mediates the proliferation of these cells in vivo. Taken together, these results indicate that dysregulated IL-9 expression could be involved in the development of some T cell malignancies.

Enhancement of DNA replication by transcription factors NFI and NFIII/Oct-1 depends critically on the positions of their binding sites in the adenovirus origin of replication.

The origin of DNA replication of many human adenoviruses is composed of a highly conserved core origin and an auxiliary region, containing the binding sites for NFI and NFIII/Oct-1. We examined enhancement of DNA replication in vitro by the purified functional DNA-binding domains of NFI (NFI-BD) and NFIII/Oct-1 (the POU domain), using origins in which the positions of the binding sites for these proteins were transposed. Insertion or deletion of two or three base pairs between the core origin and the NFI binding site resulted in a 3-5-fold decrease of stimulation, whereas larger insertions gradually reduced the stimulation further. Mutants in which the NFI binding site was separated approximately one or two helical turns from the core origin by AT-rich sequences could still be stimulated by NFI. In contrast, insertion of two or more base pairs between the NFI and NFIII/Oct-1 binding sites abolished stimulation by NFIII/Oct-1 almost completely. Furthermore, stimulation by this protein was lost when the Ad2 NFIII/Oct-1 binding site was transposed to a position closer to the core origin, destroying the NFI binding site. This shows that the position of the NFIII/Oct-1 binding site is essential for stimulation. Models to explain these position-dependent effects on stimulation are discussed.

The Polycomb-group homolog Bmi-1 is a regulator of murine Hox gene expression.

Drosophila homeotic genes and vertebrate Hox genes are involved in the anteroposterior organization of the developing embryo. In Drosophila, the Polycomb- and trithorax-group genes are required to maintain the homeotic genes throughout development in the repressed or activated state, respectively. The murine Bmi-1 proto-oncogene was shown to exhibit homology to the Polycomb-group gene Posteior sex combs. Mice lacking the Bmi-1 gene revealed posterior transformations along the axial skeleton, whereas transgenic mice overexpressing Bmi-1 display anterior transformations. We have analysed the expression patterns of several Hox genes by RNA in situ hybridization on serial sections of 11.5- and 12.5-day Bmi-1 null mutant embryos. Furthermore, we have analysed the expression of a Hoxc-8/LacZ fusion gene in younger embryos. Our analyses show that Bmi-1 is involved in the repression of a subset of Hox genes from different clusters from at least day 9.5 onwards. We discuss the possibility that members of the murine Polycomb-group can form multimeric protein complexes of different compositions with varying affinity or specificity for different subsets of Hox genes.

Analysis of Pim-1 function in mutant mice.

The Pim-1 gene has frequently been found activated by proviral insertion in haematopoietic tumors in mice. The fact that overexpression of Pim-1 can contribute to lymphomagenesis was formally proven by overexpressing a Pim-1 transgene in lymphoid cells. The transgene induces a low incidence of T cell lymphomas and an increased susceptibility to chemically (ENU) and virally (MoMuLV) induced lymphomas. The mouse Pim-1 gene encodes two cytoplasmic protein-serine/threonine kinases. Northern analysis shows the highest expression to be in haematopoietic tissues, especially early in development. High expression has also been noted in testis and ES cells. Expression can be induced by growth factors and mitogens. The gene is evolutionarily highly conserved. Inactivation of both Pim-1 alleles in ES cells or mice did not reveal any obvious abnormalities. In order to look more closely for possible haematopoietic abnormalities specific growth factor response were studied in vitro. The IL-3 response of bone marrow-derived mast-cell cultures (BMMC) was found to be severely impaired in mast cells derived from Pim-1 deficient mice.

Neonatal outcome in alloimmune thrombocytopenia after maternal treatment with intravenous immunoglobulin.

BACKGROUND: Weekly maternal intravenous immunoglobulin (IVIG) is the cornerstone of antenatal treatment of foetal and neonatal alloimmune thrombocytopenia (FNAIT). The aim of this study was to describe the neonatal outcome and management in neonates with FNAIT treated antenatally with IVIG. MATERIALS AND METHODS: All neonates treated antenatally and delivered at our centre between 2006 and 2012 were included in the study. We assessed the neonatal outcome and management, including the occurrence of intracranial haemorrhage, platelet count at birth and need for postnatal platelet transfusions or postnatal IVIG treatment. RESULTS: A total of 22 neonates were included of whom 12 (55%) had severe thrombocytopenia at birth (platelet count ≤50×10(9)/L). Most neonates (67%, 8/12) with severe thrombocytopenia received a platelet transfusion after birth. None of the neonates required postnatal treatment with IVIG. Three neonates had petechiae and haematomas, without clinical consequences. One foetus suffered from intracranial haemorrhage, which was detected just before the planned start of antenatal IVIG at 28 weeks' gestation. DISCUSSION: Our results suggest that antenatal maternal IVIG and, if necessary, postnatal matched platelet transfusions, are effective and safe for the treatment of FNAIT.

A pgk::hprt fusion as a selectable marker for targeting of genes in mouse embryonic stem cells: disruption of the T-cell receptor delta-chain-encoding gene.

We have constructed a hypoxanthine phosphoribosyl transferase-selectable marker (hprt) under the control of the phosphoglycerate kinase (pgk) promoter. This construct permits cell growth in hypoxanthine/aminopterin/thymidine media and confers 6-thioguanine sensitivity upon mouse Hprt- embryonic stem cells, allowing either positive or negative selection in gene-targeting experiments. We have successfully targeted the gene encoding the T-cell receptor delta-chain using the pgk::hprt fusion for counterselection.

Fetal, neonatal and developmental outcomes of lithium-exposed pregnancies.

INTRODUCTION: Many women with a bipolar disorder are of reproductive age and will need to continue lithium treatment during pregnancy. The teratogenic and perinatal effects of lithium are known, but not the long-term effects of lithium on neurodevelopment of the children. This study investigates growth, neurological, cognitive and behavioral development of children exposed to lithium in utero. METHOD: In an observational retrospective cohort study 15 children who were exposed to lithium in utero were investigated at 3-15 years of age. Neurological development was tested using the Hempel or Touwen examination. Cognitive development was assessed with the Bayley Scales of Infant Development III, Wechsler Preschool and Primary Scale of Intelligence or the Wechsler Intelligence Scale for Children. Parents completed the Child Behavior Checklist to assess behavioral development and a standard questionnaire about general development of the child since birth. RESULTS: One child had signs of a minor neurological dysfunction, but without further clinical implications. The results of the cognitive tests were within normal limits, although most children had lower scores on the performance IQ subtest. Growth, behavior and general development were within the normal range. CONCLUSIONS: Continuing lithium therapy during pregnancy did not cause adverse effects on growth, neurological, cognitive and behavioral development of exposed children.

Transformation of axial skeleton due to overexpression of bmi-1 in transgenic mice.

The oncogene bmi-1, which was originally found to be involved in B- and T-cell lymphoma formation encodes a protein with a domain of homology to the Drosophila protein Posterior sex combs (Psc) and its relative Suppressor 2 of Zeste (Su(z)2) (refs 4 and 5). Psc is a member of the Polycomb-group gene family, which is required to maintain the repression of homeotic genes that regulate the identities of Drosophila segments. The possibility that bmi-1 may play a similar role in vertebrates was suggested by our previous finding that mice lacking the bmi-1 gene show posterior transformations of the axial skeleton. Here we report that transgenic mice overexpressing Bmi-1 protein show the opposite phenotype, namely a dose-dependent anterior transformation of vertebral identity. The anterior expression boundary of the Hoxc-5 gene is shifted in the posterior direction, indicating that Bmi-1 is involved in the repression of Hox genes. We propose that Bmi-1 is a member of a vertebrate Polycomb complex that regulates segmental identity by repressing Hox genes throughout development.

Genetic interactions and dosage effects of Polycomb group genes in mice.

In Drosophila and mouse, Polycomb group genes are involved in the maintenance of homeotic gene expression patterns throughout development. Here we report the skeletal phenotypes of compound mutants for two Polycomb group genes bmi1 and M33. We show that mice deficient for both bmi1 and M33 present stronger homeotic transformations of the axial skeleton as compared to each single Polycomb group mutant, indicating strong dosage interactions between those two genes. These skeletal transformations are accompanied with an enhanced shift of the anterior limit of expression of several Hox genes in the somitic mesoderm. Our results demonstrate that in mice the Polycomb group genes act in synergy to control the nested expression pattern of some Hox genes in somitic mesodermal tissues during development.

In vivo analysis of Pim-1 deficiency.

The Pim-1 proto-oncogene encodes a highly conserved serine/threonine phosphokinase which is predominantly expressed in hematopoietic organs and gonads in mammals. Overexpression of Pim-1 predisposes to lymphomagenesis in mice. To develop a further understanding of Pim-1 in molecular terms, as well as in terms of its potential role in hematopoietic development, we have generated mice deficient in Pim-1 function. Pim-1-deficient mice are ostensibly normal, healthy and fertile. Detailed comparative analyses of the hematopoietic systems of the mutant mice and their wild-type littermates showed that they are indistinguishable for most of the parameters studied. Our analyses revealed one unexpected phenotype that correlated with the level of Pim-1 expression: Pim-1 deficiency correlated with a erythrocyte microcytosis, whereas overexpression of Pim-1 in E mu-Pim-1-transgenic mice resulted in erythrocyte macrocytosis. In order to confirm that the observed decrease in erythrocyte Mean Cell Volume (MCV) was attributable to the Pim-1 deficiency, we developed mice transgenic for a Pim-1 gene construct with its own promoter and showed that this transgene could restore the low erythrocyte Mean Cell Volume observed in the Pim-1-deficient mice to near wild-type levels. These results might be relevant to the observed involvement of the Pim-1 gene in mouse erythroleukemogenesis. The surprising lack of a readily observed phenotype in the lymphoid compartment of the Pim-1-deficient mice, suggests a heretofore unrecognized degree of in vivo functional redundancy of this highly conserved proto-oncogene.

Impaired interleukin-3 response in Pim-1-deficient bone marrow-derived mast cells.

The mouse Pim-1 gene encodes two cytoplasmic serine-threonine-specific protein kinases. The gene has been found to be activated (overexpressed) by retroviral insertion in hematopoietic tumors in mice. Transgenic mice that overexpress Pim-1 (E mu-Pim-1) have a low incidence of spontaneous T-cell lymphomas and an increased susceptibility to Moloney murine leukemia virus and N-ethyl-N-nitrosourea-induced lymphomas. Apart from a slight enlargement of the spleen, no abnormalities were found in prelymphomatous transgenic mice. Inactivation of the Pim-1 gene in the germline of mice resulted in mice with a surprisingly subtle phenotype. Therefore, we investigated whether subtle effects of the absence of Pim-1 could be made visible during in vitro culturing of hematopoietic cells. We found that bone marrow-derived mast cells (BMMC) lacking Pim-1 had a distinct growth disadvantage when grown on interleukin (IL)-3, but not when stimulated by the factors IL-4, IL-9, or Steel factor (SF). This indicates a role for Pim-1 as a modulator of the IL-3 signal transduction pathway.

Proviral tagging in E mu-myc transgenic mice lacking the Pim-1 proto-oncogene leads to compensatory activation of Pim-2.

The Pim-1 proto-oncogene is one of the most potent collaborators of the myc proto-oncogenes in inducing lymphomagenesis in mice. Contrary to the profound effects when overexpressed in vivo, Pim-1-deficient mice showed only subtle phenotypic alterations, which could indicate the presence of redundantly acting genes. In line with this, a PCR-based screen has led to the identification of a closely homologous gene, Pim-2. The X-linked Pim-2 gene is 53% identical to Pim-1 at the amino acid level and shares substrate preference and the usage of non-AUG initiation codons with Pim-1. We have used these data to test whether the strong synergistic interaction between Pim-1 and c-myc can be utilized to gain access to Pim-1 compensatory pathways. We reasoned that, upon proviral tagging in compound mutant mice (E mu-myc/Pim-1-/- mice), the selective advantage of cells carrying provirally activated genes, that act downstream from or parallel to Pim-1, would increase. We show here that this is the case. A dramatic increase (from 15 to 80%) was found in the frequency of proviral activation of the Pim-2 gene. These data show that the described strategy of 'complementation tagging' represents a powerful new tool to identify components of pathways involved in processes as complex as multistep tumorigenesis.

Mice doubly deficient for the Polycomb Group genes Mel18 and Bmi1 reveal synergy and requirement for maintenance but not initiation of Hox gene expression.

Polycomb group genes were identified as a conserved group of genes whose products are required in multimeric complexes to maintain spatially restricted expression of Hox cluster genes. Unlike in Drosophila, in mammals Polycomb group (PcG) genes are represented as highly related gene pairs, indicative of duplication during metazoan evolution. Mel18 and Bmi1 are mammalian homologs of Drosophila Posterior sex combs. Mice deficient for Mel18 or Bmi1 exhibit similar posterior transformations of the axial skeleton and display severe immune deficiency, suggesting that their gene products act on overlapping pathways/target genes. However unique phenotypes upon loss of either Mel18 or Bmi1 are also observed. We show using embryos doubly deficient for Mel18 and Bmi1 that Mel18 and Bmi1 act in synergy and in a dose-dependent and cell type-specific manner to repress Hox cluster genes and mediate cell survival of embryos during development. In addition, we demonstrate that Mel18 and Bmi1, although essential for maintenance of the appropriate expression domains of Hox cluster genes, are not required for the initial establishment of Hox gene expression. Furthermore, we show an unexpected requirement for Mel18 and Bmi1 gene products to maintain stable expression of Hox cluster genes in regions caudal to the prospective anterior expression boundaries during subsequent development.

Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease.

Two types of P-glycoprotein have been found in mammals: the drug-transporting P-glycoproteins and a second type, unable to transport hydrophobic anticancer drugs. The latter is encoded by the human MDR3 (also called MDR2) and the mouse mdr2 genes, and its tissue distribution (bile canalicular membrane of hepatocytes, B cells, heart, and muscle) suggests a specialized metabolic function. We have generated mice homozygous for a disruption of the mdr2 gene. These mice develop a liver disease that appears to be caused by the complete inability of the liver to secrete phospholipid into the bile. Mice heterozygous for the disrupted allele had no detectable liver pathology, but half the level of phospholipid in bile. We conclude that the mdr2 P-glycoprotein has an essential role in the secretion of phosphatidylcholine into bile and hypothesize that it may be a phospholipid transport protein or phospholipid flippase.