Quantification of Hox and Surfactant Protein-B Transcription during Murine Lung Development.

Abstract BACKGROUND: Genetic processes underlying fetal lung development and maturation are incompletely understood. Better knowledge of these processes would provide insights into the causes of lung malformations and prevention of respiratory distress syndrome and the potential adverse effects of glucocorticoids. Hox genes are involved in the lung branching morphogenesis and maturation of respiratory epithelium, but their expression pattern remains to be defined. OBJECTIVES: We hypothesized that genes involved in lung branching would be downregulated during early development, whereas those involved in maturation would be unchanged or upregulated. METHODS: TaqMan real-time primers and probes were designed for all 39 murine Hox genes, and the murine SP-B gene and transcription profiles of these genes were obtained from whole lungs isolated at e14.5, e16.5, e18.5, e19.5 and postnatal days 1 and 20. RESULTS: Hox genes in clusters A and B, specifically those between paralog groups 3 and 7, were the most represented, with Hoxa4 and Hoxa5 being the most highly transcribed. A wave of reduced transcription in 16 Hox genes, coincident with increased SP-B transcription, was observed with advancing gestation. Consistently high transcription of Hoxa5 from e14.5 to postnatal day 20 may indicate that sustained transcription is required for normal lung maturation. When e15.5 lungs were cultured with dexamethasone, Hoxb6, Hoxb7 and Hoxb8 levels were significantly upregulated, creating the potential for modulation of diverse downstream target genes. CONCLUSIONS: Improved understanding of the genetic processes underlying lung development afforded by our Q-PCR platform may allow development of more specific methods for inducing fetal lung maturation.

High-dose adenosine overcomes the attenuation of myocardial perfusion reserve caused by caffeine.

Objectives:
We studied whether an increase in adenosine dose overcomes caffeine antagonism on adenosine-mediated coronary vasodilation.

Background:
Caffeine is a competitive antagonist at the adenosine receptors, but it is unclear whether caffeine in coffee alters the actions of exogenous adenosine, and whether the antagonism can be surmounted by increasing the adenosine dose.

Methods:
Myocardial perfusion scintigraphy (MPS) was used to assess adenosine-induced hyperemia in 30 patients before (baseline) and after coffee ingestion (caffeine). At baseline, patients received 140 µg/kg/min of adenosine combined with low-level exercise. For the caffeine study, 12 patients received 140 µg/kg/min of adenosine (standard) and 18 patients received 210 µg/kg/min (high dose) after caffeine intake (200 mg). Myocardial perfusion was assessed semiquantitatively and quantitatively, and perfusion defect was characterized according to the presence of reversibility.

Results:
Caffeine reduced the magnitude of perfusion abnormality induced by standard adenosine as measured by the summed difference score (SDS) (12.0 ± 4.4 at baseline vs. 4.1 ± 2.1 after caffeine, p < 0.001) as well as defect size (18% [3% to 38%] vs. 8% [0% to 22%], p < 0.01), whereas it had no effect on the abnormalities caused by high-dose adenosine (SDS, 7.7 ± 4.0 at baseline vs. 7.8 ± 4.2 after caffeine, p = 0.7). There was good agreement between baseline and caffeine studies for segmental defect category (kappa = 0.72, 95% confidence interval: 0.65 to 0.79) in the high-dose group. An increase in adenosine after caffeine intake was well tolerated.

Conclusions:
Caffeine in coffee attenuates adenosine-induced coronary hyperemia and, consequently, the detection of perfusion abnormality by adenosine MPS. This can be overcome by increasing the adenosine dose without compromising test tolerability.

From Rights to Compassion: The D Case and Contemporary Abortion Politics

This chapter is concerned with exploring the dynamics of contemporary debate on women’s reproductive choices and rights in the somewhat transformed social, political and economic context of the Republic of Ireland. News coverage of the events of April and May 2007 provide the focus of attention, as the case of ‘D’, a 17 year old in the temporary care of the state, seeking to terminate her pregnancy after a diagnosis of severe foetal abnormality, became yet again a focus of public debate on abortion access within the state. The analysis explores how the issues this case raised were framed in the public domain, in order to consider the shifting moral grammar shaping the debate. The paper explores the ways in which this case illustrates the ongoing tensions between changing characterisations of Irishness, and the social dynamics of access to reproductive rights, particularly for national minors in the care of the state.

Runx2, p53, and pRB Status as Diagnostic Parameters for Deregulation of Osteoblast Growth and Differentiation in a New Pre-Chemotherapeutic Osteosarcoma Cell Line (OS1)

Osteosarcomas are the most prevalent primary bone tumors found in pediatric patients. To understand their molecular etiology, cell culture models are used to define disease mechanisms under controlled conditions. Many osteosarcoma cell lines (e.g., SAOS-2, U2OS, MG63) are derived from Caucasian patients. However, patients exhibit individual and ethnic differences in their responsiveness to irradiation and chemotherapy. This motivated the establishment of osteosarcoma cell lines (OS1, OS2, OS3) from three ethnically Chinese patients. OS1 cells, derived from a pre-chemotherapeutic tumor in the femur of a 6-year-old female, were examined for molecular markers characteristic for osteoblasts, stem cells, and cell cycle control by immunohistochemistry, reverse transcriptase-PCR, Western blotting and flow cytometry. OS I have aberrant G-banded karyotypes, possibly reflecting chromosomal abnormalities related to p53 deficiency. OS I had ossification profiles similar to human fetal osteoblasts rather than SAOS-2 which ossifies ab initio, (P

Elevated expression of Runx2 as a key parameter in the etiology of osteosarcoma

To understand the molecular etiology of osteosarcoma, we isolated and characterized a human osteosarcoma cell line (OS1). OS1 cells have high osteogenic potential in differentiation induction media. Molecular analysis reveals OS1 cells express the pocket protein pRB and the runt-related transcription factor Runx2. Strikingly, Runx2 is expressed at higher levels in OS1 cells than in human fetal osteoblasts. Both pRB and Runx2 have growth suppressive potential in osteoblasts and are key factors controlling competency for osteoblast differentiation. The high levels of Runx2 clearly suggest osteosarcomas may form from committed osteoblasts that have bypassed growth restrictions normally imposed by Runx2. Interestingly, OS1 cells do not exhibit p53 expression and thus lack a functional p53/p21 DNA damage response pathway as has been observed for other osteosarcoma cell types. Absence of this pathway predicts genomic instability and/or vulnerability to secondary mutations that may counteract the anti-proliferative activity of Runx2 that is normally observed in osteoblasts. We conclude OS1 cells provide a valuable cell culture model to examine molecular events that are responsible for the pathologic conversion of phenotypically normal osteoblast precursors into osteosarcoma cells.

Preconception care for women with diabetes: the midwife's role

Diabetes mellitus is a chronic illness which affects a significant number of childbearing women. Despite the potential for adverse consequences for both maternal and fetal wellbeing, few women with diabetes plan their pregnancies to ensure that they enter pregnancy in optimal health. Furthermore, whilst adverse pregnancy outcomes are well documented for women with type I diabetes, it is now apparent that an increasing number of women with type II diabetes are becoming pregnant with similar adverse associated risk. There is an increasing recognition that significant adverse pregnancy outcomes are determined prior to a woman initiating pregnancy care, many of which could be minimised with the introduction of preconception care. As formalised preconception care clinics remain scant across the United Kingdom, there is an urgent need to increase the opportunities for the provision of preconception care and advice to women with diabetes. Midwives are ideally placed to provide preconception advice to women and could provide the missing link in terms of preconception advice for women with diabetes.

Natural history of idiopathic erythrocytosis - A retrospective case series review

Idiopathic Erythrocytosis (IE) is a diagnosis given to patients who have an absolute erythrocytosis (red cell mass more than 25% above their mean normal predicted value) but who do not have a known form of primary or secondary erythrocytosis (BCSH guideline, 2005). We report here the results of a follow-up study of 80 patients (44 male and 36 female) diagnosed with IE from the United Kingdom and the Republic of Ireland over a 10 year period. Baseline information was initially collected when investigating for molecular causes of erythrocytosis in this group. The diagnosis of IE was made on the basis of a raised red cell mass >25% above mean normal predicted value, absence of Polycythaemia Vera (PV) based on the criteria of Pearson and Messinezy (1996), and the exclusion of secondary erythrocytosis (oxygen saturation >92% on pulse oximetry, no history of sleep apnoea, no renal or hepatic pathology, and a normal oxygen dissociation curve (if indicated). The average age at diagnosis of erythrocytosis was 34.5 (2–74 years). Erythropoietin levels were available for 77/80 of the patients and were low in 18 (23%) and normal or high in 59 (74%). Ultrasound imaging was carried out in 67 patients (84%) at time of diagnosis and no significant abnormalities found. Fourteen patients had a family history of erythrocytosis. These patients have now been followed up for an average of 9.4 years (range 1–39). Out of 80 patients 56 patients can still be classified as having IE, of whom 52 are living (cause of death in the other 4 - lung cancer, RTA, sepsis, unknown). Thirty-five of these patients are regularly venesected, 3 take hydroxyurea (one also venesected), 11 receive no treatment while treatment is unknown in 2. Twenty take aspirin, 1 warfarin and 31 no thromboprophylaxis. Four of these patients had suffered thromboembolic complications (3 with CVA/TIAs and 1 with recurrent DVT) at or before their original diagnosis. Since diagnosis 8 patients have had 9 thrombotic events of which 7 were arterial (1 CVA, 3 TIAs, 1 MI, 2 PVD) and 2 venous (DVT/PE). Twenty take aspirin, 1 dipyridamole, 1 warfarin and 30 take no thromboprophylaxis. Out of the 24 patients who now have a diagnosis other than IE, 8 have been diagnosed with myelo-proliferative disease. Thirteen patients have a molecular abnormality which is likely to account for their erythrocytosis (11 VHL, 1 PHD-2, 1 EPO-receptor mutations). Three patients have secondary erythrocytosis. Older case studies identified a heterogenous group of patients, some of whom probably had apparent erythrocytosis and some who had either primary polycythaemia or secondary causes later identified (Modan and Modan, Najean et al). More recent reviews have identified a more homogenous group with low rates of transformation to myelofibrosis/acute leukaemia and low rates of thrombosis of around 1% patient-year. Follow up of our initial patient group does indeed reveal a heterogeneous group of patients with 10% now diagnosed with an MPD, although when analysis is confined to those patients who continue to fulfil the criteria for IE, the clinical course has been more stable. There has been no progression to MDS or leukaemia in this group (one patient with PV progressed to AML). The rate of thrombosis is 1.6% patient-years which is lower than the rate seen in PV and is consistent with the rate identified in other series. Molecular defects continue to be identified in this group and future investigation is likely to reveal further abnormalities.

The incidences of trisomy 8, trisomy 9 and D20S108 deletion in polycythaemia vera: an analysis of blood granulocytes using interphase fluorescence in situ hybridization:an analysis of blood granulocytes using interphase fluorescence in situ hybridization

We have used interphase fluorescence in situ hybridization (IFISH) to detect trisomy 8, trisomy 9 and 20q deletion in circulating granulocytes from patients with polycythaemia vera (PV). Out of 64 PV patients, 15 (23%) exhibited an abnormality. Two patients had trisomy 9, three had trisomy 8 and 10 patients had hemizygous deletion of D20S108 (a locus in the 20q common deleted region). Aberrant nuclei ranged from 10% to 80% in these 15 cases. There was no correlation between the presence of a marker and sex, age, interval between presentation and IFISH analysis, neutrophil or platelet count or therapy. Conventional marrow cytogenetic karyotype results were available in 23 cases and there was concurrence between these and blood IFISH in 16 cases (13 normal and three with 20q/D20S108 deletion by both methods). Three patients with D20S108 deletion by IFISH were normal by previous marrow cytogenetic testing and four cases with 20q deletion by previous marrow cytogenetics had normal blood granulocytes according to IFISH. Thus, we confirm that trisomies 8 and 9 and deletion of 20q are diagnostically useful markers of PV. IFISH analysis of blood granulocytes is a practical method for detecting these markers, but as an adjunct to, not as a substitute for, conventional marrow cytogenetics.

Erythropoietin receptor and hematological disease

This review will discuss evidence for the role of the erythropoietin (Epo) receptor in the development of erythrocytosis and other hematological disorders, The possible causative role of mutations of other genes in the pathogenesis of idiopathic erythrocytosis will be considered, Polycythemia vera (PV) is a myeloproliferative disorder that is caused by an undefined stem cell abnormality, characterized by a significant erythrocytosis, leukocytosis, and thrombocytosis. However, erythrocytosis may arise from apparent (or relative) polycythemia in which the hematocrit is raised due to a low plasma volume. In such cases the red cell mass is normal. A group of disorders with increased red cell mass caused by stimulation of erythrocyte production is known as secondary polycythemia, Investigation of such patients may reveal a congenital abnormality such as high affinity hemoglobin or an acquired abnormality caused, for example, by smoking, renal Vascular impairment, or an Epo-producing tumor. Even after thorough examination there remains a cohort of patients for whom no definite cause for the erythrocytosis can be established, A careful clinical history may reveal whether this idiopathic erythrocytosis is likely to be congenital and/or familial, in which case the term