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International audience; Over the years, advances in immunohistochemistry techniques have been a critical step in detecting and mapping neuromodulatory substances in the central nervous system. The better quality and specificity of primary antibodies, new staining procedures and the spectacular development of imaging technologies have allowed such progress. Very recently, new methods permitting tissue transparency have been successfully used on brain tissues. In this work, we combined whole-mount immunostaining for tyrosine hydroxylase (TH), oxytocin (OXT) and arginine vasopressin (AVP), with iDISCO+ clearing method, light-sheet microscopy and semi automated counting of 3D-labelled neurons to obtain a 3D distribution of these neuronal populations in a 5-day postnatal (P5) mouse brain. Segmentation procedure and 3D reconstruction allowed us to map with high resolution TH staining the various catecholaminergic cell groups and their ascending and descending fiber pathways. We show that TH pathways are present in the whole P5 mouse brain, similar to what was observed in the adult rat brain.

This paper presents an analysis of the carbon–deuterium circulation and the resulting balance in Tore Supra over the period 2002–2007. Carbon balance combines the estimation of carbon gross erosion from spectroscopy, net erosion and deposition using confocal microscopy, lock-in thermography and SEM, and a measure of the amount of deposits collected in the vacuum chamber. Fuel retention is determined from post-mortem (PM) analyses and gas balance (GB) measurements. Special attention was paid to the deuterium outgassed during the nights and weekends of the experimental campaign (vessel under vacuum, Plasma Facing Components at 120 °C) and during vents (vessel at atmospheric pressure, PFCs at room temperature). It is shown that this outgassing is the main process reconciling the PM and GB estimations of fuel retention, closing the coupled carbon–deuterium balance. In particular, it explains why the deuterium concentration in deposits decreases with increasing depth.

Coexpression for c-Kit receptor and its ligand stem cell factor (SCF) has been described in neuroblastoma (NB) cell lines and tumors, suggesting the existence of an autocrine loop modulating tumor growth. We evaluated c-Kit and SCF expression by immunohistochemistry in a series of 75 primary newly diagnosed neuroblastic tumors. Immunostaining for c-Kit was found in 10/75 and for SCF in 17/75, with 5/10 c-Kit-positive tumors also expressing SCF. For both, c-Kit and SCF staining were predominantly found in the most aggressive subset of tumors, i.e., those amplified for MYCN: c-Kit was detected in 8/14 amplified vs. 2/61 single copy (p

Detectable levels of MAX messenger RNA were found in a set of human neuroblastoma tumors and established cell lines. MAX mRNA levels were independent of tumor stage and N-myc genomic amplification. By contrast, N-myc mRNA transcripts were detectable only in tumors with amplification of N-myc gene and in cell lines. Analysis by reverse transcriptase polymerase chain reaction and hybridization to specific oligodeoxynucleotide probes revealed approximately equal amounts of two MAX transcripts in all cases analyzed. Immunoprecipitations with a specific antibody to MAX detected two proteins of M(r) 21,000 and 22,000 in approximately equal amounts in all neuroblastoma lines regardless of N-myc amplification and/or expression. On the other hand, protein binding to the myc DNA consensus sequence correlated with N-myc expression in neuroblastoma cells. Thus, N-myc expression might be a limiting factor in the formation of the N-myc-MAX heterodimer in neuroblastomas.

Nerve growth factor (NGF), essential for differentiation and survival of sympathetic neurons is suggested to play a role in differentiation or regression of neuroblastoma. Expression of mRNA for the trk protooncogene, encoding a tyrosine kinase receptor essential for functional NGF signal transduction, and mRNA for the low affinity NGF receptor (LNGFR) was examined in 45 neuroblastomas and 3 benign ganglioneuromas using Northern blot analysis. Expression of trk mRNA and LNGFR mRNA correlated with young age, favorable clinical stages, and absence of N-myc amplification. All children (n = 19) with neuroblastomas coexpressing mRNA for trk and LNGFR are alive 8-84 months from diagnosis, regardless of age and stage. In contrast, no child (n = 15) with tumor lacking trk mRNA is alive without disease. Three subsets of patients were distinguished, one favorable (trk+, LNGFR+, n = 19, 100% survival probability), one intermediate (trk+, LNGFR-, n = 11, 62.3% survival probability), and one unfavorable (trk-, LNGFR +/-, n = 15, 0% survival probability, P0.001). In widespread neuroblastoma stage IVS prone to spontaneous regression, three tumors coexpressing trk and LNGFR mRNAs regressed after no or minimal therapy while the remaining tumor expressing trk but not LNGFR mRNA progressed to a fatal outcome. It is concluded that neuroblastomas coexpressing mRNA for both NGF receptor subtypes are favorable tumors likely to differentiate or regress spontaneously or respond to conventional therapy. It is further hypothesized that loss of functional NGF receptors is an important step in tumorigenesis of undifferentiated malignant childhood neuroblastoma. For these unfavorable tumors current therapy remains futile and first-line innovative therapy is justified.

Purpose: Clinicobiological characteristics of neuroblastic tumor (NT) expressing c-kit tyrosine kinase receptor and/or its ligand, stem cell factor (SCF), are debated. This study aimed at investigating the clinicobiological features of primary NTs expressing c-kit and/or SCF in order to define the clinical relevance of selective therapeutic targeting. Experimental Design: c-Kit and SCF expression was studied in 168 NTs using immunohistochemistry and in 106 of 168 using Northern blot. Quantitative determination of c-kit expression in 54 additional NTs was also done using real-time reverse transcription-PCR. Correlations between c-kit and SCF expression and clinicobiological features were analyzed using χ2 test, univariate, and multivariate regression analyses. Results: c-Kit protein was detected in 21 of 168 NTs (13%) and its mRNA in 23 of 106 NTs (22%). SCF protein was shown in 30 of 106 NTs (28%) and its mRNA in 33 of 106 NTs (31%). No mutations in exon 11 of c-kit gene were identified. By univariate analysis, c-kit and SCF expression correlated with advanced stage, MYCN amplification, and 1p36 allelic loss. Cox simple regression analysis showed that overall survival probability was 17% in the c-kit–positive subset versus 68% in the negative (P < 0.001), 43% in the SCF-positive subset versus 78% in the negative (P < 0.001). When using real-time reverse transcription-PCR, significant levels of c-kit mRNA were found in 35 of 54 NTs (65%), but the correlations with clinicobiological features were no longer documented. Conclusions: c-Kit expression can be detected in the majority of primary NTs. High levels of expression are preferentially found in tumors with unfavorable clinicobiological variables. c-Kit may represent a useful therapeutic target in a subset of otherwise untreatable NTs.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and accounts for about 5% of all malignant paediatric tumours. ��-Catenin, a multifunctional nuclear transcription factor in the canonical Wnt signaling pathway, is active in myogenesis and embryonal somite patterning. Dysregulation of Wnt signaling facilitates tumour invasion and metastasis. This study characterizes Wnt/��-catenin signaling and functional activity in paediatric embryonal and alveolar RMS. Immunohistochemical assessment of paraffin-embedded tissues from 44 RMS showed ��-catenin expression in 26 cases with cytoplasmic/membranous expression in 9/14 cases of alveolar RMS, and 15/30 cases of embryonal RMS, whereas nuclear expression was only seen in 2 cases of embryonal RMS. The potential functional significance of ��-catenin expression was tested in four RMS cell lines, two derived from embryonal (RD and RD18) RMS and two from alveolar (Rh4 and Rh30) RMS. Western blot analysis demonstrated the expression of Wnt-associated proteins including ��-catenin, glycogen synthase kinase-3��, disheveled, axin-1, naked, LRP-6 and cadherins in all cell lines. Cell fractionation and immunofluorescence studies of the cell lines (after stimulation by human recombinant Wnt3a) showed reduced phosphorylation of ��-catenin, stabilisation of the active cytosolic form and nuclear translocation of ��-catenin. Reporter gene assay demonstrated a T-cell factor/lymphoid-enhancing factor-mediated transactivation in these cells. In response to human recombinant Wnt3a, the alveolar RMS cells showed a significant decrease in proliferation rate and induction of myogenic differentiation (myogenin, MyoD1 and myf5). These data indicate that the central regulatory components of canonical Wnt/��-catenin signaling are expressed and that this pathway is functionally active in a significant subset of RMS tumours and might represent a novel therapeutic target.

Glycated hemoglobin (HbA1c) is a suspected risk factor for sternal wound infection (SWI) after CABG.Methods and Results:Data on preoperative HbA1c and SWI were available in 2,130 patients undergoing isolated CABG from the prospective E-CABG registry. SWI occurred in 114 (5.4%). Baseline HbA1c was significantly higher in patients with SWI (mean, 54±17 vs. 45±13 mmol/mol, P0.0001). This difference was also observed in patients without a diagnosis of diabetes (P=0.027), in insulin-dependent diabetic (P=0.023) and non-insulin-dependent diabetic patients (P=0.034). In the overall series, HbA1c70 mmol/mol (NGSP units, 8.6%) was associated with the highest risk of SWI (20.6% vs. 4.6%; adjusted OR, 5.01; 95% CI: 2.47-10.15). When dichotomized according to the cut-off 53 mmol/mol (NGSP units, 7.0%) as suggested both for diagnosis and optimal glycemic control of diabetes, HbA1c was associated with increased risk of SWI in the overall series (10.6% vs. 3.9%; adjusted OR, 2.09; 95% CI: 1.24-3.52), in diabetic patients (11.7% vs. 5.1%; adjusted OR, 2.69; 95% CI: 1.38-5.25), in patients undergoing elective surgery (9.9% vs. 2.7%; adjusted OR, 2.09; 95% CI: 1.24-3.52) and in patients with bilateral mammary artery grafts (13.7% vs. 4.8%; adjusted OR, 2.35; 95% CI: 1.17-4.69).Screening for HbA1c before CABG may identify untreated diabetic patients, as well as diabetic patients with suboptimal glycemic control, at high risk of SWI.

Childhood neuroblastic tumors are characterized by heterogeneous clinical courses, ranging from benign ganglioneuroma (GN) to highly lethal neuroblastoma (NB). Although a refined prognostic evaluation and risk stratification of each tumor patient is becoming increasingly essential to personalize treatment options, currently only few biomolecular markers (essentially MYCN amplification, chromosome 11q status and DNA ploidy) are validated for this purpose in neuroblastic tumors. Here we report that Galectin-3 (Gal-3), a β-galactoside-binding lectin involved in multiple biological functions that has already acquired diagnostic relevance in specific clinical settings, is variably expressed in most differentiated and less aggressive neuroblastic tumors, such as GN and ganglioneuroblastoma, as well as in a subset of NB cases. Gal-3 expression is associated with the INPC histopathological categorization (P

MYCN amplification occurs in approximately 20% of human neuroblastomas and is associated with early tumor progression and poor outcome, despite intensive multimodal treatment. However, MYCN overexpression also sensitizes neuroblastoma cells to apoptosis. Thus, uncovering the molecular mechanisms linking MYCN to apoptosis might contribute to designing more efficient therapies for MYCN-amplified tumors. Here we show that MYCN-dependent sensitization to apoptosis requires activation of p53 and its phosphorylation at serine 46. The p53S46 kinase HIPK2 accumulates on MYCN expression, and its depletion by RNA interference impairs p53S46 phosphorylation and apoptosis. Remarkably, MYCN induces a DNA damage response that accounts for the inhibition of HIPK2 degradation through an ATM- and NBS1-dependent pathway. Prompted by the rare occurrence of p53 mutations and by the broad expression of HIPK2 in our human neuroblastoma series, we evaluated the effects of the p53-reactivating compound Nutlin-3 on this pathway. At variance from other tumor histotypes, in MYCN-amplified neuroblastoma, Nutlin-3 further induced HIPK2 accumulation, p53S46 phosphorylation, and apoptosis, and in combination with clastogenic agents purged virtually the entire cell population. Altogether, our data uncover a novel mechanism linking MYCN to apoptosis that can be triggered by the p53-reactivating compound Nutlin-3, supporting its use in the most difficult-to-treat subset of neuroblastoma. Mol Cancer Res; 9(1); 67–77 ©2010 AACR.

Dalla revisione della casistica del gruppo di Studio Italiano sui Tumori epatici del Bambino (GSTEB), risulta un miglioramento dell'Outcome di questi pazienti. Gli Autori presentano i risultati dello Studio.