Evidence for a role of TGF-beta1 in the expression and regulation of alpha-SMA in fetal growth restricted placentae.

There is evidence that alpha-smooth muscle actin (alpha-SMA) is a protein that plays a pivotal role in the production of contractile forces and it is induced by transforming growth factor-beta1 (TGF-beta1). We have analysed the expression of alpha-SMA, TGF-beta1, its receptor RI and the activator phospho-Smad2 in (a) fetal growth restriction pre-eclamptic placentae characterised by early onset and absence of end diastolic velocities in the umbilical arteries (FGR-AED) and (b) control placentae accurately matched for gestational age. The study was performed by immunohistochemical, quantitative Western blotting, ELISA, RT-PCR and in vitro analyses. We found that TGF-beta1 stimulates alpha-SMA production in chorionic villi cultured in vitro. In addition, we observed that in vivo TGF-beta1 concentration is significantly higher in FGR-AED placental samples than in control placentae and that this growth factor could have a paracrine action on villous stroma myofibroblasts expressing TGF-beta1 receptors and phospho-Smad2. Indeed, we report that alpha-SMA undergoes a redistribution in FGR-AED placental villous tree, i.e. we show that alpha-SMA is enhanced in medium and small stem villi and significantly decreased in the peripheral villi. Our data allow us to consider TGF-beta1 and alpha-SMA as key molecules related to FGR-AED placental villous tree phenotypic changes responsible for increased impedance to blood flow observable in this pathology.

JunB/cyclin-D1 imbalance in placental mesenchymal stromal cells derived from preeclamptic pregnancies with fetal-placental compromise.

INTRODUCTION: In the present study, we characterized the expression of Activating Protein 1 (AP-1) factors, key cell cycle regulators, in primary placental mesenchymal stromal cells (PDMSCs) derived from normal and preeclamptic (PE) pregnancies with fetal-placental compromise. METHODS: PDMSCs were isolated from control (n = 20) and preeclamptic (n = 24) placentae. AP-1 expression was determined by semi-quantitative RT-PCR (sqRT-PCR), Real Time PCR and Western Blot assay. PDMSCs were plated and JunB siRNA was performed. JunB and Cyclin-D1 expression were assessed by Real Time and Western Blot analyses. RESULTS: JunB expression was significantly increased while Cyclin-D1 expression was significantly down-regulated in PE relative to control PDMSCs. JunB siRNA was accompanied by JunB down-regulation and increased Cyclin-D1 in normal PDMSCs. CONCLUSIONS: We described, for the first time, AP-1 expression in PDMSCs derived from physiological and PE placentae. Importantly, we demonstrated that JunB over-expression in PE-PDMSCs affects Cyclin-D1 regulation. Our data suggest a possible contribution of these pathological placental cells to the altered cell cycle regulation typical of preeclamptic placentae.

IFPA Meeting 2013 Workshop Report I: diabetes in pregnancy, maternal dyslipidemia in pregnancy, oxygen in placental development, stem cells and pregnancy pathology.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2013 there were twelve themed workshops, four of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of pregnancy pathologies and placental metabolism: 1) diabetes in pregnancy; 2) lipids, fatty acids and the placenta; 3) oxygen in placental development and pathologies; 4) stem cells and pathologies.

The double life of MULE in preeclamptic and IUGR placentae.

The E3 ubiquitin ligase MULE (Mcl-1 Ubiquitin Ligases E3) targets myeloid cell leukemia factor 1 (Mcl-1) and tumor suppressor p53 for proteasomal degradation. Although Mcl-1 and p53 have been implicated in trophoblast cell death in preeclampsia (PE) and intrauterine growth restriction (IUGR), the mechanisms regulating their expression in the human placenta remains elusive. Herein, we investigated MULE's involvement in regulating Mcl-1 and p53 degradation during normal and abnormal (PE, IUGR) placental development. MULE expression peaked at 5-7 weeks of gestation, when oxygen tension is low and inversely correlated with that of Mcl-1 and p53. MULE efficiently bound to Mcl-1 and p53 and regulated their ubiquitination during placental development. Exposure of first trimester villous explants to 3% O(2) resulted in elevated MULE expression compared with 20% O(2). Low-oxygen-induced MULE expression in JEG3 choriocarcinoma cells was abolished by hypoxia-inducible factor (HIF)-1α siRNA. MULE was overexpressed in both PE and IUGR placentae. In PE, MULE preferentially targeted p53 for degradation, allowing accumulation of pro-apoptotic Mcl-1 isoforms. In IUGR, however, MULE targeted pro-survival Mcl-1, allowing p53 to accumulate and exert its apoptotic function. These data demonstrate that oxygen regulates Mcl-1 and p53 stability during placentation via HIF-1-controlled MULE expression. The different preferential targets of MULE in PE and IUGR placentae classify early-onset PE and IUGR as distinct molecular pathologies.

Bladder cancer radiotherapy margins: a comparison of daily alignment using skin, bone or soft tissue.

AIMS: To determine the clinical target volume (CTV) to planning target volume (PTV) margins required for bladder coverage when using skin or bony or soft tissue matching on a daily basis. MATERIALS AND METHODS: Twenty-seven patients with T2-T4 transitional cell carcinoma of the bladder were treated with daily online adaptive image-guided radiotherapy using cone beam computed tomography. All daily CTVs were contoured by a single observer. A retrospective comparison of coverage of the bladder CTV using skin, bone and soft tissue matching was conducted. RESULTS: With the skin set-up, bladder CTV coverage with a margin of 0.5, 1.0, 1.5, 2.0 and 2.5 cm was 0, 19, 56, 93 and 96%, respectively. For the daily set-up based on bone, the respective coverage was 0, 41, 63, 89 and 96%. For soft tissue set-up based on the geometric centre of the bladder, coverage was 52, 89, 96, 100 and 100%, respectively. CONCLUSIONS: Based on coverage of the CTV, the required CTV to PTV margins are smaller for the daily online soft tissue set-up compared with skin or bone.

Cost minimisation analysis: kilovoltage imaging with automated repositioning versus electronic portal imaging in image-guided radiotherapy for prostate cancer.

AIMS: To compare the treatment time and cost of prostate cancer fiducial marker image-guided radiotherapy (IGRT) using orthogonal kilovoltage imaging (KVI) and automated couch shifts and orthogonal electronic portal imaging (EPI) and manual couch shifts. MATERIALS AND METHODS: IGRT treatment delivery times were recorded automatically on either unit. Costing was calculated from real costs derived from the implementation of a new radiotherapy centre. To derive cost per minute for EPI and KVI units the total annual setting up and running costs were divided by the total annual working time. The cost per IGRT fraction was calculated by multiplying the cost per minute by the duration of treatment. A sensitivity analysis was conducted to test the robustness of our analysis. Treatment times without couch shift were compared. RESULTS: Time data were analysed for 8648 fractions, 6057 from KVI treatment and 2591 from EPI treatment from a total of 294 patients. The median time for KVI treatment was 6.0 min (interquartile range 5.1-7.4 min) and for EPI treatment it was 10.0 min (interquartile range 8.3-11.8 min) (P value < 0.0001). The cost per fraction for KVI was A$258.79 and for EPI was A$345.50. The cost saving per fraction for KVI varied between A$66.09 and A$101.64 by sensitivity analysis. In patients where no couch shift was made, the median treatment delivery time for EPI was 8.8 min and for KVI was 5.1 min. CONCLUSIONS: Treatment time is less on KVI units compared with EPI units. This is probably due to automation of couch shift and faster evaluation of imaging on KVI units. Annual running costs greatly outweigh initial setting up costs and therefore the cost per fraction was less with KVI, despite higher initial costs. The selection of appropriate IGRT equipment can make IGRT practical within radiotherapy departments.

IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.

Review: Feto-placental vascularization: a multifaceted approach.

Doppler Ultrasound allows the in vivo study of feto-placental hemodynamics. Doppler flow velocity waveforms (FVW's) obtained from the umbilical arteries reflect downstream blood flow impedance, thus giving indirect evidence of vascular villous tree characteristics. Pulsatility Index, which quantifies FVW's, decreases throughout normal pregnancy, indicating decreasing impedance and is often higher in cases of fetal growth restriction (FGR). Different approaches (morphometrical, morphological, mathematical, immunohistochemical and molecular) have contributed to elucidation of which anomalies of the vascular villous tree underlie Doppler findings. 3D ultrasound may be useful in the study of feto-placental perfusion. However, the unsolved question is why developmental villous tree anomalies occur. Crucial to the success of future research is definition of the population studied based on the uniform and correct definition of FGR.

Activating protein-1 family of transcription factors in the human placenta complicated by preeclampsia with and without fetal growth restriction.

Preeclampsia (PE) is a serious disorder of human pregnancy, it is often associated with fetal growth restriction (FGR) which is a failure of the fetus to reach its own growth potential. Activator protein-1 (AP-1) is a family of transcription factors inducible in response to a variety of extracellular stimuli and functions. AP-1 plays a complex role in the regulation of different fundamental cellular processes, including cell proliferation, survival, death and transformation. We investigate the expression pattern of AP-1 transcription factors in normal placentas during gestation and in placentas from PE without and with FGR using semiquantitative RT-PCR and immunohistochemistry techniques. The most interesting data concern the alterations of protein expression patterns of c-fos, Jun D and c-jun in normal gestation as well as in PE and PE-FGR pathologies. In addition, alterations but not significant changes are detected in mRNA expressions for these transcription factors. We strongly suggest that c-fos is implicated in regulating invasiveness mechanism of extravillous trophoblast in normal gestation as well as in PE placentas. In addition, we suggest that the opposite modulation of Jun D and c-jun in PE and PE-FGR supports the recent hypothesis that PE and PE-FGR could be considered two pathologies with different origin (maternal and placental) each of which has a different molecular pattern of expression.

The effect of an intensive nutritional program on daily set-up variations and radiotherapy planning margins of head and neck cancer patients.

This is a prospective case-control study to assess nutritional supplementation in limiting weight loss and its impact on daily set-up variations and planning target volume (PTV) margins in head and neck (H&N) radiotherapy (RT). Twenty sequential H&N patients were recruited for this study. Ten patients had a percutaneous endoscopic gastrostomy (PEG) tube inserted prior to RT and 10 did not. PEG use was determined by departmental guidelines for patients considered at high risk for weight loss. Daily 2D electronic portal images were taken for orthogonal verification. Set-up variations were determined for both PEG and non-PEG patients by calculating systematic (Sigma) and random (sigma) errors, and PTV margins were derived. PEG patients lost less weight (P = 0.04) over the course of RT and had a reduction in set-up variation in the superior-inferior (SI) and anterior-posterior (AP) planes compared to those without. Mean correctional shifts in mm (range) for PEG patients were: Right-Left (RL) 0.1 (-1.9-2.1), SI -1.7 (-2.9-0.0), AP -0.4 (-2.0-0.8), and for non-PEG patients were: RL -0.2 (-2.7-1.3), SI -1.3 (-3.1-1.0), AP 0.4 (-1.5-2.8). The adapted PTV margins (mm) in the RL, SI and AP planes, respectively, for PEG patients were 4.1, 3.3 and 3.6, and for non-PEG were 3.9, 4.9 and 4.8. Intensive enteral support maintained weight stability in H&N patients considered at risk of weight loss during RT and this was associated with reduced set-up variation.

Offline adaptive radiotherapy for bladder cancer using cone beam computed tomography.

We investigated if an adaptive radiotherapy approach based on cone beam CT (CBCT) acquired during radical treatment was feasible and resulted in improved dosimetric outcomes for bladder cancer patients compared to conventional planning and treatment protocol. A secondary aim was to compare a conventional plan with a theoretical online process where positioning is based on soft tissue position on a daily basis and treatment plan choice is based on bladder size. A conventional treatment plan was derived from a planning CT scan in the radical radiotherapy of five patients with muscle invasive bladder cancer. In this offline adaptive protocol using CBCT, the patients had 10 CBCT: daily CBCT for the first five fractions and then CBCT scan on a weekly basis. The first five daily CBCT in each patient were used to create a single adaptive plan for treatment from fraction eight onwards. A different process using the planning CT and the first five daily CBCT was used to create small, average and large bladder volumes, giving rise to small, average and large adaptive bladder treatment plans, respectively. In a retrospective analysis using the CBCT scans, we compared the clinical target volume (CTV) coverage using three protocols: (i) conventional; (ii) offline adaptive; and (iii) online adaptive with choice of 'plan of the day'. Daily CBCT prolonged treatment time by an average of 7 min. Two of the five patients demonstrated such variation in CTV that an offline adaptive plan was used for treatment after the first five CBCT. Comparing the offline adaptive plan with the conventional plan, the CTV coverage improved from a minimum of 60.1 to 94.7% in subsequent weekly CBCT. Using the CBCT data, modelling an online adaptive protocol showed that coverage of the CTV by the 95% prescribed dose line by small, medium and large adaptive plans were 34.9, 67.4 and 90.7% of occasions, respectively. More normal tissue was irradiated using a conventional CTV to planning target volume margin (1.5 cm) compared to an online adaptive process (0.5 cm). An offline adaptive strategy improves dose coverage in certain patients to the CTV and results in a higher conformity index compared to conventional planning. Further research in online adaptive radiation therapy for bladder cancer is indicated.

Hematologic values in healthy and small for gestational age newborns.

Many of the published reports of reference values in neonates are found in older medical literature. Recognition of abnormalities in blood cell morphology and hematologic parameters depend on well-established normative data; it is essential that each neonatal medical unit have its own reference ranges. We give the umbilical cord blood complete blood count reference values from 142 healthy, appropriate for gestational age (AGA) newborns and 58 small for gestational age (SGA) newborns (term and preterm). Our data, obtained by automated blood cell counter analysis of umbilical cord blood samples taken at birth, are comparable to other previously published data. The correlation between previous data and our reference data confirms that in term AGA newborns, values for red blood cells, hemoglobin, and hematocrit are higher and mean corpuscular volume values lower than in preterm AGA newborns. Also, we found that platelet levels are reduced in SGA newborns, in accordance with the literature. These findings further support the fact that preterm SGA infants are truly growth restricted, whereas term SGA infants are most likely small but otherwise healthy babies.

[Effect of estradiol on carbohydrate metabolism in the uterine musculature]. / Effetto dell'estradiolo sul metabolismo glicidico del muscolo uterino.

The Effect of 7.5 micrograms i.v. 17-beta-oestradiol on the sugar metabolism of the uterine musculature was studied in 180-200 g Wistar rats. The myometrial metabolic reaction consisted of increased glucose phosphorylation, enhanced utilisation of creatine phosphate, and increased production of adenosine triphosphate and creatine phosphate initially, followed by their consumption. The gradual fall in the lactate: pyruvate ratio showed that this stimulation of glycolysis was not occurring in the absence of oxygen.

Effects of papaveroline-monosulphate on the systemic and regional circulation in the dog.

The effects of papaveroline 6'-sulphonic of N-methylglucamine (UTEN), administered by intravenous injection in doses ranging from 10 to 20 mg/kg, on cardiac dynamics and peripheral blood flow distribution were studied in conscious and anesthetized dogs, using electromagnetic flowmeters implanted around the ascending aorta, pulmonary artery, left coronary artery, superior mesenteric artery, and renal and external iliac arteries. In the conscious dogs, blood pressure after the injection of the drug showed an initial fall and returned to normal control values after a transitory increase. The changes in aortic pressure were accompanied by an increase in heart rate, cardiac output, stroke volume and cardiac work. The administration of UTEN was also associated with an increase in the peripheral blood flow and a decrease in their calculated resistances and in total peripheral resistances. Similar variations were observed in the anesthetized dogs. The effects of UTEN were compared with those of other vasodilator drugs (papaverine, isoxsuprine and D.E.D.). From the results obtained it is possible to conclude that UTEN produces a vasodilation in all investigated vessels, whereas other vasodilator agents only act in some particular vascular beds; moreover, the effect of UTEN is longer-lasting. There is also a probable constriction in the capacitance vessels, as shown by haemodynamic changes in the pulmonary circulation.

[Hormonal regulation of uterine contraction]. / Regolazione ormonale della contrattilità uterina.

A personal method has been used to study spontaneous kinetic activity of the uterus in 50 women during puerperium following miscarriage between the VIIIth and XIIth weeks. Modifications induced were evaluated in several sessions (510 recordings) and at various periods of time, following administration of: 50 mg of 17B oestradiol, 200 mg natural progesterone, 500 mg of natural progesterone, 250 mg of 17 hydroxyprogesterone caproate and 500 mg of 17 hydroxyprogesterone caproate. In the case of some of the patients, hysterotonometry was evaluated following intravenous oxytocin loading. The results pointed to an activation of the uterine pacemaker after oestrogenic loading, and a clear-cut progestinic block of contractile activity.

Australian and New Zealand three-dimensional conformal radiation therapy consensus guidelines for prostate cancer.

Three-dimensional conformal radiation therapy (3DCRT) has been shown to reduce normal tissue toxicity and allow dose escalation in the curative treatment of prostate cancer. The Faculty of Radiation Oncology Genito-Urinary Group initiated a consensus process to generate evidence-based guidelines for the safe and effective implementation of 3DCRT. All radiation oncology departments in Australia and New Zealand were invited to complete a survey of their prostate practice and to send representatives to a consensus workshop. After a review of the evidence, key issues were identified and debated. If agreement was not reached, working parties were formed to make recommendations. Draft guidelines were circulated to workshop participants for approval prior to publication. Where possible, evidence-based recommendations have been made with regard to patient selection, risk stratification, simulation, planning, treatment delivery and toxicity reporting. This is the first time a group of radiation therapists, physicists and oncologists representing professional radiotherapy practice across Australia and New Zealand have worked together to develop best-practice guidelines. These guidelines should serve as a baseline for prospective clinical trials, outcome research and quality assurance.