Mark G. Coulthard

Increased DNA Methylation at the AXIN1 Gene in a Monozygotic Twin from a Pair Discordant for a Caudal Duplication Anomaly

The AXIN1 gene has been implicated in caudal duplication anomalies. Its coding region was sequenced in both members of a monozygotic (MZ) twin pair discordant for a caudal duplication anomaly, but no mutation was found. Using bisulfite sequencing, we examined methylation at the promoter region of the AXIN1 gene in these twins and in twin and age-matched singleton controls. Methylation of the promoter region in peripheral blood mononucleated cells was variable among individuals, including MZ pairs. In the MZ pair discordant for the caudal duplication, this region of the affected twin was significantly more methylated than that of the unaffected twin (P < .0001), which was significantly more methylated than those of the controls (P = .02). We have confirmed that this CpG island does function as a promoter in vitro and that its activity is inversely proportional to the extent of methylation. This finding raises the possibility that hypermethylation of the AXIN1 promoter, by mechanisms as yet undetermined, is associated with the malformation. This case may be paradigmatic for some cases of MZ discordance.

Part 6: Pediatric basic life support and pediatric advanced life support. 2015 International Consensus on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science with Treatment Recommendations

The Pediatric Task Force reviewed all questions submitted by the International Liaison Committee on Resuscitation (ILCOR) member councils in 2010, reviewed all council training materials and resuscitation guidelines and algorithms, and conferred on recent areas of interest and controversy. We identified a few areas where there were key differences in council-specific guidelines based on historical recommendations, such as the A-B-C (Airway, Breathing, Circulation) versus C-A-B (Circulation, Airway, Breathing) sequence of provision of cardiopulmonary resuscitation (CPR), initial back blows versus abdominal thrusts for foreign-body airway obstruction, an upper limit for recommended chest compression rate, and initial defibrillation dose for shockable rhythms (2 versus 4 J/kg). We produced a working list of prioritized questions and topics, which was adjusted with the advent of new research evidence. This led to a prioritized palate of 21 PICO (population, intervention, comparator, outcome) questions for ILCOR task force focus. The 2015 process was supported by information specialists who performed in-depth systematic searches, liaising with pediatric content experts so that the most appropriate terms and outcomes and the most relevant publications were identified. Relevant adult literature was considered (extrapolated) in those PICO questions that overlapped with other task forces, or when there were insufficient pediatric data. In rare circumstances (in the absence of sufficient human data), appropriate animal studies were incorporated into reviews of the literature. However, these data were considered only when higher levels of evidence were not available and the topic was deemed critical. When formulating the PICO questions, the task force felt it important to evaluate patient outcomes that extend beyond return of spontaneous circulation (ROSC) or discharge from the pediatric intensive care unit (PICU). In recognition that the measures must have meaning, not only to clinicians but also to parents and caregivers, longer-term outcomes at 30 …

Eph/Ephrin signaling in injury and inflammation.

The Eph/ephrin receptor-ligand system plays an important role in embryogenesis and adult life, principally by influencing cell behavior through signaling pathways, resulting in modification of the cell cytoskeleton and cell adhesion. There are 10 EphA receptors, and six EphB receptors, distinguished on sequence difference and binding preferences, that interact with the six glycosylphosphatidylinositol-linked ephrin-A ligands and the three transmembrane ephrin-B ligands, respectively. The Eph/ephrin proteins, originally described as developmental regulators that are expressed at low levels postembryonically, are re-expressed after injury to the optic nerve, spinal cord, and brain in fish, amphibians, rodents, and humans. In rodent spinal cord injury, the up-regulation of EphA4 prevents recovery by inhibiting axons from crossing the injury site. Eph/ephrin proteins may be partly responsible for the phenotypic changes to the vascular endothelium in inflammation, which allows fluid and inflammatory cells to pass from the vascular space into the interstitial tissues. Specifically, EphA2/ephrin-A1 signaling in the lung may be responsible for pulmonary inflammation in acute lung injury. A role in T-cell maturation and chronic inflammation (heart failure, inflammatory bowel disease, and rheumatoid arthritis) is also reported. Although there remains much to learn about Eph/ephrin signaling in human disease, and specifically in injury and inflammation, this area of research raises the exciting prospect that novel therapies will be developed that precisely target these pathways.

Systematic Review of the Diagnostic Accuracy of C-Reactive Protein to Detect Bacterial Infection in Nonhospitalized Infants and Children with Fever

OBJECTIVE To determine the accuracy of C-reactive protein (CRP) for diagnosing serious bacterial and bacterial infections in infants and children presenting with fever. STUDY DESIGN Systematic review of diagnostic accuracy studies. We included studies comparing the diagnostic accuracy of CRP with microbiologic confirmation of (a) serious bacterial and (b) bacterial infection. RESULTS For differentiating between serious bacterial infection and benign or nonbacterial infection (6 studies), the pooled estimate of sensitivity was 0.77 (95% CI, 0.68, 0.83); specificity, 0.79 (95% CI, 0.74, 0.83); positive likelihood ratio, 3.64 (95% CI, 2.99, 4.43); and negative likelihood ratio, 0.29 (95% CI, 0.22, 0.40). In multivariate analysis, CRP is an independent predictor of serious bacterial infection. 3 studies investigating the accuracy of CRP for diagnosing bacterial infection could not be pooled, but all showed a lower sensitivity compared with studies using serious bacterial infection as the reference diagnosis. CONCLUSIONS CRP provides moderate and independent information for both ruling in and ruling out serious bacterial infection in children with fever at first presentation. Poor sensitivity means that CRP cannot be used to exclude all bacterial infection.

Characterization of the Epha1 receptor tyrosine kinase: expression in epithelial tissues.

Abstract The Eph family of receptor tyrosine kinases plays a crucial role during development and is implicated in oncogenesis. Using a partial cDNA clone of an Eph-related kinase (Esk) we isolated the complete coding region of a gene which we show to be murine EphA1 by both structural and functional criteria. The chromosomal localization is shown to be syntenic to hEphA1 and the genomic organization also shows distinct features found in the hEphA1 gene. Functionally, in keeping with findings for the human homologue, both soluble recombinant and “native” mEphA1 show preferential binding to ephrin A1. However, we also observed significant binding to other A-type ligands as has been observed for other Eph receptors. We analysed the expression of mEphA1 mRNA by in situ hybridization on tissue sections. mEphA1 was expressed in epithelial elements of skin, adult thymus. kidney and adrenal cortex. Taken together with previous Northern blotting data these results suggest that mEphA1 is expressed widely in differentiated epithelial cells.

Part 6: Pediatric basic life support and pediatric advanced life support

The Pediatric Task Force reviewed all questions submitted by the International Liaison Committee on Resuscitation (ILCOR) member councils in 2010, reviewed all council training materials and resuscitation guidelines and algorithms, and conferred on recent areas of interest and controversy. We identified a few areas where there were key differences in council-specific guidelines based on historical recommendations, such as the A-B-C (Airway, Breathing, Circulation) versus C-A-B (Circulation, Airway, Breathing) sequence of provision of cardiopulmonary resuscitation (CPR), initial back blows versus abdominal thrusts for foreign-body airway obstruction, an upper limit for recommended chest compression rate, and initial defibrillation dose for shockable rhythms (2 versus 4 J/kg). We produced a working list of prioritized questions and topics, which was adjusted with the advent of new research evidence. This led to a prioritized palate of 21 PICO (population, intervention, comparator, outcome) questions for ILCOR task force focus. The 2015 process was supported by information specialists who performed in-depth systematic searches, liaising with pediatric content experts so that the most appropriate terms and outcomes and the most relevant publications were identified. Relevant adult literature was considered (extrapolated) in those PICO questions that overlapped with other task forces, or when there were insufficient pediatric data. In rare circumstances (in the absence of sufficient human data), appropriate animal studies were incorporated into reviews of the literature. However, these data were considered only when higher levels of evidence were not available and the topic was deemed critical. When formulating the PICO questions, the task force felt it important to evaluate patient outcomes that extend beyond return of spontaneous circulation (ROSC) or discharge from the pediatric intensive care unit (PICU). In recognition that the measures must have meaning, not only to clinicians but also to parents and caregivers, longer-term outcomes at 30 …

Generation and characterization of EphA1 receptor tyrosine kinase reporter knockout mice

Eph receptor tyrosine kinases (RTKs) are a highly conserved family of signaling proteins with functions in cellular migration, adhesion, apoptosis, and proliferation during both adult and embryonic life. Here, we describe a knock‐in mouse in which EphA1 expression is disrupted via the insertion of an internal ribosome entry site (IRES)‐human placental alkaline phosphatase (ALPP) reporter cassette into exon II of the EphA1 gene. This was shown to successfully knockout expression of endogenous EphA1 and enforce expression of the ALPP reporter by the EphA1 promoter. Staining for the ALPP reporter protein demonstrated an epithelially restricted expression pattern in mouse tissues. In EphA1 null mice, two separate phenotypes were identified: abnormal tail development manifesting as a kinky tail was found in ∼80% of homozygous adults. A second, distinct abnormality present in ∼18% of females was characterized by imperforate uterovaginal development with hydrometrocolpos and caused by a resistance of cells to apoptosis during reproductive tract canalization. These results indicate a possible role for EphA1 in tissue patterning and hormone‐induced apoptotic processes. genesis 46:553–561, 2008.

ADAM12-cleaved ephrin-A1 contributes to lung metastasis

Eph receptor tyrosine kinases and their ephrin ligands have been implicated in neuronal development and neovascularization. Overexpression of ephrin-A1 has been implicated in tumor progression and poor prognosis. However, the mechanisms are not clear. Here, we report a role of the Eph/ephrin system in a cell adhesion mechanism. Clustered erythropoietin-producing hepatocellular receptor A1 (EphA1)/ephrin-A1 complexes on the plasma membrane did not undergo endocytosis, and the cell remained adherent to one another. The cell–cell contacts were maintained in an Eph tyrosine kinase activity-independent manner even in the absence of E-cadherin. EphA1 and ephrin-A1 co-localized in pulmonary endothelial cells, and regulated vascular permeability and metastasis in the lungs. We identified ADAM12 (A disintegrin and metalloproteinase 12) as an EphA1-binding partner by yeast two-hybrid screening and found that ADAM12 enhanced ephrin-A1 cleavage in response to transforming growth factor-β1 in primary tumors. Released soluble ephrin-A1 in the serum deteriorated the EphA1/ephrin-A1-mediated cell adhesion in the lungs in an endocrine manner, causing lung hyperpermeability that facilitated tumor cell entry into the lungs. Depletion of soluble ephrin-A1 by its neutralizing antibody significantly inhibited lung metastasis.

Evidence That the EphA2 Receptor Exacerbates Ischemic Brain Injury

Ephrin (Eph) signaling within the central nervous system is known to modulate axon guidance, synaptic plasticity, and to promote long-term potentiation. We investigated the potential involvement of EphA2 receptors in ischemic stroke-induced brain inflammation in a mouse model of focal stroke. Cerebral ischemia was induced in male C57Bl6/J wild-type (WT) and EphA2-deficient (EphA2−/−) mice by middle cerebral artery occlusion (MCAO; 60 min), followed by reperfusion (24 or 72 h). Brain infarction was measured using triphenyltetrazolium chloride staining. Neurological deficit scores and brain infarct volumes were significantly less in EphA2−/− mice compared with WT controls. This protection by EphA2 deletion was associated with a comparative decrease in brain edema, blood-brain barrier damage, MMP-9 expression and leukocyte infiltration, and higher expression levels of the tight junction protein, zona occludens-1. Moreover, EphA2−/− brains had significantly lower levels of the pro-apoptotic proteins, cleaved caspase-3 and BAX, and higher levels of the anti-apoptotic protein, Bcl-2 as compared to WT group. We confirmed that isolated WT cortical neurons express the EphA2 receptor and its ligands (ephrin-A1–A3). Furthermore, expression of all four proteins was increased in WT primary cortical neurons following 24 h of glucose deprivation, and in the brains of WT mice following stroke. Glucose deprivation induced less cell death in primary neurons from EphA2−/− compared with WT mice. In conclusion, our data provide the first evidence that the EphA2 receptor directly contributes to blood-brain barrier damage and neuronal death following ischemic stroke.

A randomised controlled trial of Hartmann's solution versus half normal saline in postoperative paediatric spinal instrumentation and craniotomy patients

Objective To compare the difference in plasma sodium at 16–18 h following major surgery in children who were prescribed either Hartmanns and 5% dextrose or 0.45% saline and 5% dextrose. Design A prospective, randomised, open label study. Setting The paediatric intensive care unit (650 admissions per annum) in a tertiary childrens hospital in Brisbane, Australia. Patients The study group comprised 82 children undergoing spinal instrumentation, craniotomy for brain tumour resection, or cranial vault remodelling. Interventions Patients received either Hartmanns and 5% dextrose at full maintenance rate or 0.45% saline and 5% dextrose at two-thirds maintenance rate. Main outcomes measures Primary outcome measure: plasma sodium at 16–18 h postoperatively; secondary outcome measure: number of fluid boluses administered. Results Mean postoperative plasma sodium levels of children receiving 0.45% saline and 5% dextrose were 1.4 mmol/l (95% CI 0.4 to 2.5) lower than those receiving Hartmanns and 5% dextrose (p=0.008). In the 0.45% saline group, seven patients (18%) became hyponatraemic (Na <135 mmol/l) at 16–18 h postoperatively; in the Hartmanns group no patient became hyponatraemic (p=0.01). No child in either fluid group became hypernatraemic. Conclusions The postoperative fall in plasma sodium was smaller in children who received Hartmanns and 5% dextrose compared to those who received 0.45% saline and 5% dextrose. It is suggested that Hartmanns and 5% dextrose should be administered at full maintenance rate postoperatively to children who have undergone major surgery in preference to hypotonic fluids.