Douglas F. Hacking

The transcription factor Erg is essential for definitive hematopoiesis and the function of adult hematopoietic stem cells

Ets-related gene (ERG), which encodes a member of the Ets family of transcription factors, is a potent oncogene. Chromosomal rearrangements involving ERG are found in acute myeloid leukemia, acute lymphoblastic leukemia, Ewings sarcoma and more than half of all prostate cancers; however, the normal physiological function of Erg is unknown. We did a sensitized genetic screen of the mouse for regulators of hematopoietic stem cell function and report here a germline mutation of Erg. We show that Erg is required for definitive hematopoiesis, adult hematopoietic stem cell function and the maintenance of normal peripheral blood platelet numbers.

Increased in vivo transcription of an IL-8 haplotype associated with respiratory syncytial virus disease-susceptibility.

Interleukin-8 (IL-8) has been implicated in the pathogenesis of RSV-induced bronchiolitis. Previously, we have described an association between bronchiolitis disease severity and a specific IL-8 haplotype comprising six single-nucleotide polymorphisms (SNPs) (−251A/+396G/+781T/+1238delA/+1633T/+2767T, haplotype 2). Here we investigated the functional basis for this association by measuring haplotype-specific transcription in vivo in human primary cells. We found a significant increase in transcript level derived from the IL-8 haplotype 2 relative to the mirror haplotype 1 (−251T/+396T/+781C/+1238insA/+1633C/+2767A) in respiratory epithelial cells but not in lymphocytes. A promoter polymorphism, −251A, present on the high producer haplotype, had no significant affect on the allele-specific level of transcription when analyzed in reporter gene experiments in human respiratory epithelial A549 cells. We proceeded to systematically screen for allele-specific protein-DNA binding in this functional haplotype, which revealed significant differential binding at the +781T/C polymorphism. C/EBP β was identified as being part of a transcription factor binding complex that preferentially bound in the presence of the +781 T allele. These results suggest that the mechanism for disease susceptibility to RSV-induced bronchiolitis may occur through a haplotype-specific increase in IL-8 transcription, which may be mediated by functional polymorphisms within that haplotype.

Agm1/Pgm3-Mediated Sugar Nucleotide Synthesis Is Essential for Hematopoiesis and Development

ABSTRACT Carbohydrate modification of proteins includes N-linked and O-linked glycosylation, proteoglycan formation, glycosylphosphatidylinositol anchor synthesis, and O-GlcNAc modification. Each of these modifications requires the sugar nucleotide UDP-GlcNAc, which is produced via the hexosamine biosynthesis pathway. A key step in this pathway is the interconversion of GlcNAc-6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, catalyzed by phosphoglucomutase 3 (Pgm3). In this paper, we describe two hypomorphic alleles of mouse Pgm3 and show there are specific physiological consequences of a graded reduction in Pgm3 activity and global UDP-GlcNAc levels. Whereas mice lacking Pgm3 die prior to implantation, animals with less severe reductions in enzyme activity are sterile, exhibit changes in pancreatic architecture, and are anemic, leukopenic, and thrombocytopenic. These phenotypes are accompanied by specific rather than wholesale changes in protein glycosylation, suggesting that while universally required, the functions of certain proteins and, as a consequence, certain cell types are especially sensitive to reductions in Pgm3 activity.

Elective decompression of the left ventricle in pediatric patients may reduce the duration of venoarterial extracorporeal membrane oxygenation.

We aimed to determine the effect of elective left heart decompression at the time of initiation of central venoarterial extracorporeal membrane oxygenation (VA ECMO) on VA ECMO duration and clinical outcomes in children in a single tertiary ECMO referral center with a large pediatric population from a national referral center for pediatric cardiac surgery. We studied 51 episodes of VA ECMO in a historical cohort of 49 pediatric patients treated between the years 1990 and 2013 in the Paediatric Intensive Care Unit (PICU) of the Royal Childrens Hospital, Melbourne. The cases had a variety of diagnoses including congenital cardiac abnormalities, sepsis, myocarditis, and cardiomyopathy. Left heart decompression as an elective treatment or an emergency intervention for left heart distension was effectively achieved by a number of methods, including left atrial venting, blade atrial septostomy, and left ventricular cannulation. Elective left heart decompression was associated with a reduction in time on ECMO (128 h) when compared with emergency decompression (236 h) (P = 0.013). Subgroup analysis showed that ECMO duration was greatest in noncardiac patients (elective 138 h, emergency 295 h; P = 0.02) and in patients who died despite both emergency decompression and ECMO (elective 133 h, emergency 354 h; P = 0.002). As the emergency cases had a lower pH, a higher PaCO2 , and a lower oxygenation index and were treated with a higher mean airway pressure, positive end-expiratory pressure, and respiratory rate prior to receiving VA ECMO, we undertook multivariate linear regression modeling to show that only PaCO2 and the timing of left heart decompression were associated with ECMO duration. However, elective left heart decompression was not associated with a reduction in length of PICU stay, duration of mechanical ventilation, or duration of oxygen therapy. Elective left heart decompression was not associated with improved ECMO survival or survival to PICU discharge. Elective left heart decompression may reduce ECMO duration and has therefore the potential to reduce ECMO-related complications. A prospective, randomized controlled trial is indicated to study this intervention further.

'Knock, and it shall be opened': knocking out and knocking in to reveal mechanisms of disease and novel therapies.

Recent significant advances in molecular biology have generated genetically modified bacteria, yeast, nematodes, fruit flies, and fish. However, it is the genetic modification of mammalian model organisms, particularly the mouse, that has the greatest potential to shed light on human development, physiology and pathology in ways that have significant implications for neonatal and paediatric clinical practice. Here, we review some of the techniques for knocking out (inactivating), mutating and knocking in (inserting) selected genes that are important to neonatology and show how this research will lead both to a better understanding of disease and to novel therapies for infants and children.

Frequency of respiratory deterioration after immunisation in preterm infants

Aim:  To determine the relationship between the initiation of respiratory support and the first routine immunisation of neonates at 2 months of age during primary hospitalisation.

Fetal inhibition of inflammation improves disease phenotypes in harlequin ichthyosis

Harlequin ichthyosis (HI) is a severe skin disease which leads to neonatal death in ∼50% of cases. It is the result of mutations in ABCA12, a protein that transports lipids required to establish the protective skin barrier needed after birth. To better understand the life-threatening newborn HI phenotype, we analysed the developing epidermis for consequences of lipid dysregulation in mouse models. We observed a pro-inflammatory signature which was characterized by chemokine upregulation in embryonic skin which is distinct from that seen in other types of ichthyosis. Inflammation also persisted in grafted HI skin. To examine the contribution of inflammation to disease development, we overexpressed interleukin-37b to globally suppress fetal inflammation, observing considerable improvements in keratinocyte differentiation. These studies highlight inflammation as an unexpected contributor to HI disease development in utero, and suggest that inhibiting inflammation may reduce disease severity.

06-P009 A role for Abca12 in regulating terminal differentiation and lipid balance in the developing epidermis

combinations, and identified those genes specifically involved in the disease and minimizing variations between samples and different cell cultures. The comparison between control and NSCL/P patients yielded 56 genes. Subsequent signaling pathway analyses involving the identified genes, suggested involvement of three different pathways. In particular, we concentrated on the up-regulation of 3 candidates genes (COL15A1, PPT2, ERAP2). All expression results were confirmed by real time PCR. Our results suggest that expression profile differences in stem cells isolated from normal and CLP patients can identify candidate genes which had not been previously detected in the multitude of association analyses performed to date. Our work is now moving towards functional studies involving these genes. CEPID/ FAPESP, CNPq.