Genetic alterations and in vivo tumorigenicity of neurospheres derived from an adult glioblastoma

Pediatric brain tumors may originate from cells endowed with neural stem/precursor cell properties, growing in vitro as neurospheres. We have found that these cells can also be present in adult brain tumors and form highly infiltrating gliomas in the brain of immunodeficient mice. Neurospheres were grown from three adult brain tumors and two pediatric gliomas. Differentiation of the neurospheres from one adult glioblastoma decreased nestin expression and increased that of glial and neuronal markers. Loss of heterozygosity of 10q and 9p was present in the original glioblastoma, in the neurospheres and in tumors grown into mice, suggesting that PTEN and CDKN2A alterations are key genetic events in tumor initiating cells with neural precursor properties

Nitric Oxide Synthetic Pathway in Patients with Microvascular Angina and Its Relations with Oxidative Stress

A decreased nitric oxide (NO) bioavailability and an increased oxidative stress play a pivotal role in different cardiovascular pathologies. As red blood cells (RBCs) participate in NO formation in the bloodstream, the aim of this study was to outline the metabolic profile of L-arginine (Arg)/NO pathway and of oxidative stress status in RBCs and in plasma of patients with microvascular angina (MVA), investigating similarities and differences with respect to coronary artery disease (CAD) patients or healthy controls (Ctrl). Analytes involved in Arg/NO pathway and the ratio of oxidized and reduced forms of glutathione were measured by LC-MS/MS. The arginase and the NO synthase (NOS) expression were evaluated by immunofluorescence staining. RBCs from MVA patients show increased levels of NO synthesis inhibitors, parallel to that found in plasma, and a reduction of NO synthase expression. When summary scores were computed, both patient groups were associated with a positive oxidative score and a negative NO score, with the CAD group located in a more extreme position with respect to Ctrl. This finding points out to an impairment of the capacity of RBCs to produce NO in a pathological condition characterized mostly by alterations at the microvascular bed with no significant coronary stenosis

Pleiotropic modes of action in tumor cells of RNASET2, an evolutionary highly conserved extracellular RNase

As widely recognized, tumor growth entails a close and complex cross-talk among cancer cells and the surrounding tumor microenvironment. We recently described the human RNASET2 gene as one key player of such microenvironmental cross-talk. Indeed, the protein encoded by this gene is an extracellular RNase which is able to control cancer growth in a non-cell autonomous mode by inducing a sustained recruitment of immune-competent cells belonging to the monocyte/macrophage lineage within a growing tumor mass. Here, we asked whether this oncosuppressor gene is sensitive to stress challenges and whether it can trigger cell-intrinsic processes as well. Indeed, RNASET2 expression levels were consistently found to increase following stress induction. Moreover, changes in RNASET2 expression levels turned out to affect several cancer-related parameters in vitro in an ovarian cancer cell line model. Of note, a remarkable rearrangement of the actin cytoskeleton organization, together with changes in cell adhesion and motility, emerged as putative mechanisms by which such cell-autonomous role could occur. Altogether, these biological features allow to put forward the hypothesis that the RNASET2 protein can act as a molecular barrier for limiting the damages and tissue remodeling events occurring during the earlier step of cell transformation

Unadjusted simple Pearson correlation pattern between plasma and RBC analytes.

*<p>P<0.05,</p>†<p>P<0.01,</p>‡<p>P<0.001.</p

RBC levels of analytes involved in Arginine/NO pathway.

<p>(A) L-arginine (Arg), L-citrulline (Cit), L-ornithine (Orn) were simultaneously determined by LC-MS/MS. (B) Arg bioavailability and the relative activity of arginase and RBC-NOS enzymes are expressed as Arg/(Orn+Cit) and Orn/Cit ratios, respectively. (C) The endogenous inhibitors asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) were determined by LC-MS/MS. (D) Tetrahydrobiopterin (BH₄) and oxidized biopterins (Box) were detected by HPLC after selective oxidation with iodine. Data are presented as age and sex adjusted geometric means and 95% C.I. Comparisons between groups (CAD, n = 22; Ctrl, n = 20) were performed by covariance analysis, adjusting for age and sex.</p

Partial Pearson correlation pattern between plasma and RBC analytes, adjusted for the effects of all other analytes.

*<p>P<0.05,</p>†<p>P<0.01,</p>‡<p>P<0.001.</p

Demographic and clinical characteristics of CAD patients and Ctrl subjects.

<p>Quantitative variables were expressed as mean ± SD and categorical variables as n (%).</p><p>P value: Wilcoxon test for continous variables and Chi Square test for categorical variables.</p><p>P value adjusted for sex and age after log-transformation of the data.</p

Immunostaining of NO synthase (NOS) protein in human red blood cells (RBCs).

<p>NOS was detected in RBCs from CAD patients (A) and from Ctrl (B). RBCs were incubated with a monoclonal anti eNOS antibody (2.5 µg/ml) and with an anti-mouse conjugated secondary antibody (40 µg/ml; Alexa Fluor488). The samples were mounted with fluorescent mounting medium and examined by laser scanning confocal microscope (LSM710, Carl Zeiss) using a 63×/1.3 oil immersion objective lens. Fluorescent images were captured with a digital camera using the image processor Zen (Carl Zeiss). (C) Fluorescence intensity (densitometric sum of grey) was quantified. Data are expressed as the log median of total fluorescence intensity per field ± interquartile range subtracted of the negative control value. Means derive from n = 10 CAD and n = 10 Ctrl.</p