In-vitro effects of the tyrosine kinase inhibitor imatinib on glioblastoma cell proliferation.

Glioblastoma (GBL) is the most malignant brain tumour in adults, causing the death of most patients within 9-12 months of diagnosis. Treatment is based on a combination of surgery, radiation therapy, and chemotherapy. With these treatment modalities, however, responses are extremely poor, so identification of novel treatment strategies is highly warranted. Platelet-derived growth factors (PDGF) and their receptors are commonly coexpressed in GBL, suggesting that stimulation of autocrine PDGF receptors may contribute to their growth. Interest in these receptors as drug target for glioblastoma treatment has increased with the clinical availability of the PDGFR kinase inhibitor antagonist imatinib mesylate (STI571). In this study, T98G and A172 human GBL cell lines were analysed for their sensitivity to treatment with imatinib. In particular, we focussed our attention on analysis of DNA distribution by flow cytometry at different times of incubation with different imatinib concentrations (1-30 microM: ). Our results show that imatinib induces growth arrest in T98G and A172 cells in the G(0)/G(1) phase of the cell cycle, at all the concentrations tested, as early as 24 h after treatment. However we have also seen, by means of annexin V staining, that at 20 and 30 microM: concentrations, in concomitance with a significant growth arrest in the G(0)/G(1) phase, there is an increase of apoptotic cells 48 h after treatment, suggesting that imatinib at low concentrations (1-10 microM: ) could act as a cytostatic agent whereas at high concentrations (20, 30 microM: ) it mainly behaves as a cytotoxic agent.

DNA fragmentation induced in human fibroblasts by 56Fe ions: experimental data and Monte Carlo simulations.

We studied the DNA fragmentation induced in human fibroblasts by iron-ion beams of two different energies: 115 MeV/nucleon and 414 MeV/nucleon. Experimental data were obtained in the fragment size range 1-5700 kbp; Monte Carlo simulations were performed with the PARTRAC code; data analysis was also performed through the Generalized Broken Stick (GBS) model. The comparison between experimental and simulated data for the number of fragments produced in two different size ranges, 1-23 kbp and 23-5700 kbp, gives a satisfactory agreement for both radiation qualities. The Monte Carlo simulations also allow the counting of fragments outside the experimental range: The number of fragments smaller than 1 kbp is large for both beams, although with a strong difference between the two cases. As a consequence, we can compute different RBEs depending on the size range considered for the fragment counting. The PARTRAC evaluation takes into account fragments of all sizes, while the evaluation from the experimental data considers only the fragments in the range of 1-5700 kbp. When the PARTRAC evaluation is restricted to this range, the agreement between experimental and computed RBE values is again good. When fragments smaller than 1 kbp are also considered, the RBE increases considerably, since gamma rays produce a small number of such fragments. The analysis performed with the GBS model proved to be quite sensitive to showing, with a phenomenological single parameter, variations in double-strand break (DSB) correlation.

Proliferation and programmed cell death: role of p53 protein in high and low grade astrocytoma.

p53 is a cell cycle regulator that has been well-recognized as the key molecule that triggers the induction and the control of cell proliferation and apoptosis in a wide variety of tumours, including astrocytoma. Apoptosis and proliferation are two processes intimately coupled, that occur simultaneously in tumour tissue. Previous studies of the correlations between proliferation and apoptotic index with p53 expression in astrocytic tumours have remained inconclusive. The aim of this study was to investigate the correlation of p53 expression with the apoptotic index (AI) and the cell proliferation index (PI) in pilocytic astrocytoma (PA) and glioblastoma multiforme (GBM). A correlation of p53 expression with AI and PI was found in pilocytic astrocytoma but not in glioblastoma, probably because of the mutated p53 phenotype in the latter.

Visual motion and rapid auditory processing are solid endophenotypes of developmental dyslexia.

Although a genetic component is known to have an important role in the etiology of developmental dyslexia (DD), we are far from understanding the molecular etiopathogenetic pathways. Reduced measures of neurobiological functioning related to reading (dis)ability, i.e. endophenotypes (EPs), are promising targets for gene finding and the elucidation of the underlying mechanisms. In a sample of 100 nuclear families with DD (229 offspring) and 83 unrelated typical readers, we tested whether a set of well-established, cognitive phenotypes related to DD [i.e. rapid auditory processing (RAP), rapid automatized naming (RAN), multisensory nonspatial attention and visual motion processing] fulfilled the criteria of the EP construct. Visual motion and RAP satisfied all testable criteria (i.e. they are heritable, associate with the disorder, co-segregate with the disorder within a family and represent reproducible measures) and are therefore solid EPs of DD. Multisensory nonspatial attention satisfied three of four criteria (i.e. it associates with the disorder, co-segregates with the disorder within a family and represents a reproducible measure) and is therefore a potential EP for DD. Rapid automatized naming is heritable but does not meet other criteria of the EP construct. We provide the first evidence of a methodologically and statistically sound approach for identifying EPs for DD to be exploited as a solid alternative basis to clinical phenotypes in neuroscience.

Sluggish dorsally-driven inhibition of return during orthographic processing in adults with dyslexia.

Dyslexia (D) is a neurodevelopmental reading disorder characterized by phonological and orthographic deficits. Before phonological decoding, reading requires a specialized orthographic system for parallel letter processing that assigns letter identities to different spatial locations. The magnocellular-dorsal (MD) stream rapidly process the spatial location of visual stimuli controlling visuo-spatial attention. To investigate the visuo-spatial attention efficiency during orthographic processing, inhibition of return (IOR) was measured in adults with and without D in a lexical decision task. IOR is the delay in responding to stimuli displayed in a cued location after a long cue-target interval. Only adults with D did not showed IOR effect during letter-string recognition, despite the typical left-hemisphere specialization for word identification. A specific deficit in coherent-dot-motion perception confirmed an MD-stream disorder in adults with D. Our results suggest that adults with D might develop an efficient visual word form area, but a dorsal-attentional dysfunction impairs their reading fluency.

Association of short-term memory with a variant within DYX1C1 in developmental dyslexia.

A substantial genetic contribution in the etiology of developmental dyslexia (DD) has been well documented with independent groups reporting a susceptibility locus on chromosome 15q. After the identification of the DYX1C1 gene as a potential candidate for DD, several independent association studies reported controversial results. We performed a family-based association study to determine whether the DYX1C1 single nucleotide polymorphisms (SNPs) that have been associated with DD before, that is SNPs '-3GA' and '1249GT', influence a broader phenotypic definition of DD. A significant linkage disequilibrium was observed with 'Single Letter Backward Span' (SLBS) in both single-marker and haplotype analyses. These results provide further support to the association between DD and DYX1C1 and it suggests that the linkage disequilibrium with DYX1C1 is more saliently explained in Italian dyslexics by short-term memory, as measured by 'SLBS', than by the categorical diagnosis of DD or other related phenotypes.

Exogenous platelet-derived growth factor (PDGF) induces human astrocytoma cell line proliferation.

Platelet-derived growth factor receptors (PDGFR) regulate several processes in normal cells including cellular proliferation, differentiation and migration, and are widely expressed in a variety of malignancies. In astrocytoma, PDGF ligand and receptor are often overexpressed and PDGFR activity deregulation has been linked to pathogenesis. The issue of the functional capacity of PDGFR has only occasionally been addressed in glioma cells by measuring the proliferative response induced by exogenous PDGF. In the present study, PDGFRalpha expression was evaluated in human grade 2 and 4 astrocytoma cell lines and tissue specimens by immunocytochemistry. The receptor responsiveness to exogenous PDGF was determined in astrocytoma cells with an MTT assay. It was found that astrocytoma cells express PDGFRalpha and respond to PDGF mitogenic action in a grade-dependent manner. The receptor was found to be functional since it induced cell proliferation at different ligand concentrations. We can thus conclude that the proliferative response of human astrocytoma cells is related to their malignancy and receptor status before PDGF stimulation, suggesting a role for PDGFRalpha inhibitors as blockers of malignant cell proliferation.

Effectiveness of stereotactic body radiotherapy in the treatment of inoperable early-stage lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Stereotactic body irradiation offers a non-invasive treatment modality for patients with early stage NSCLC who are not amenable to surgery or other invasive approaches because of their poor medical condition. PATIENTS AND METHODS: Forty-three inoperable patients with NSCLC were treated with SBRT at our institution. A mean total dose of 30.5 Gy in 1-4 fractions was applied. The median follow-up duration was 14 months (range 6-36 months). RESULTS: The actuarial survival at two years was 53%: two patients died from cancer progression whereas a further 8 patients died from comorbidities. Acute toxicity was practically absent, with 7 (16.3%) patients suffering from grade 1 symptoms and two from (4.6%) grade II effects. At the time of this report, only 1 patient had grade II and 6 patients (13.9%) grade I chronic symptoms. CONCLUSION: Our results compare favourably with recently published studies and confirm that stereotactic radiotherapy has the potential to produce high local control rates with a low risk of lung toxicity in patients not amenable to curative resection. The low grade of side-effects is encouraging for shortening the treatment using a greater dose per fraction.

Surface properties and implantation site affect the capsular fibrotic overgrowth.

Transplantation of encapsulated pancreatic islets is a promising approach for the treatment of type 1 diabetes. Large-scale application of this technique, however, is hampered by insufficient biocompatibility of the capsules. In this study, we have evaluated the biocompatibility of a new synthetic material with six different chemical groups on their surface (amino, carboxy-sulfate, carboxylate, hydroxylate, sulfate, and PMMA) used for the fabrication of the microcapsules. Eight Lewis rats were inoculated with a suspension of empty capsules made for each candidate material in the retroperitoneal ileopsoas muscle and renal subcapsular space. Four weeks later kidney and muscle containing the capsules were explanted, paraffin embedded, sectioned and stained with Sirius Red and Masson's Trichrome for histological analysis. The amount of fibrosis was also ultrastructurally evaluated with scanning electron microscopy. The samples were then subjected to digitalized quantitative analysis using specific software to determine the degree of fibrotic overgrowth. The quantification of collagen deposition, calculated in proximity of the microcapsules, was expressed as a percentage of the total area and can be considered a good index for the biocompatibility, an essential prerequisite for functional pancreatic islet transplantation. The results show that subcapsular renal space is the best implantation site and the positive surface charge induces a more intense collagen synthesis.

Minichromosome maintenance protein 7: a reliable tool for glioblastoma proliferation index.

At present there is increasing evidence concerning the value of minichromosome maintenance (MCM) protein expression as a novel indicator of proliferation. In the present study, 15 glioblastoma samples, classified according to WHO, were analysed to evaluate the expression of the principal proliferation markers. The samples examined were subdivided into 2 cytological subsets, small cell (SC) or multiforme cell (MC) glioblastoma, according to the predominant cell type defined in individual specimens. MCM7 detected more cells in the cycle than Ki67 and PCNA and all cases of SC glioblastoma, the most aggressive subset, displayed a significant increase of MCM7-stained nuclei versus those stained with Ki67. These results suggest that the cell cycle-associated proteins MCM are not only useful markers of proliferation, but also valid aids for diagnosis in cerebral glioblastoma.

Immunohistochemical evaluation of minichromosome maintenance protein 7 in astrocytoma grading.

The minichromosome maintenance (MCM) proteins, which play an important role in eukaryotic DNA replication, represent a group of proteins that are currently under investigation as novel diagnostic tumor markers. Several studies have proved a greater reliability of MCM proteins to stain proliferating cells compared to Ki67 protein, a routinely used proliferation marker in histopathology. In the present study, the expressions of MCM7 and Ki67 were estimated in 66 primary human astrocytomas in relation to tumor grade (Grade I-IV, WHO). MCM7 significantly stained more nuclei compared to Ki67 in all the histopathological grades investigated. In addition, a stronger increase of the MCM7 labelling index, in relation to the tumor aggressiveness, was observed.

Histochemical study of cardiac mast cells degranulation and collagen deposition: interaction with the cathecolaminergic system in the rat.

Although their role in the cardiovascular system is still largely unknown, mast cells are present in the myocardium of both experimental animals and humans. Interestingly, cathecolaminergic nerve fibres and mast cells are often described in close morphological and functional interactions in various organs. In the present study we investigated the effects of chronic interference with beta-adrenergic receptors (via either sympathectomy or beta-blockade) on cardiac mast cell morphology/activation and on interstitial collagen deposition. In rats subjected to chemical sympathectomizy with the neurotoxin 6-hydroxydopamine (6-OHDA) we observed a significant increase of mast cell density, and in particular of degranulating mast cells, suggesting a close relationship between the cardiac catecholaminergic system and mast cell activation. In parallel, chronic 6-OHDA treatment was associated with increased collagen deposition. The influence of the beta-adrenergic receptor component was investigated in rats subjected to chronic propranolol administration, that caused a further significant increase in mast cell activation associated with a lower extent of collagen deposition when compared to chemical sympathectomy. These data are the first demonstration of a close relationship between rat cardiac mast cell activation and the catecholaminergic system, with a complex interplay with cardiac collagen deposition. Specifically, abrogation of the cardiac sympathetic efferent drive by chemical sympathectomy causes mast cell activation and interstitial fibrosis, possibly due to the local effects of the neurotoxin 6-hydroxydopamine. In contrast, beta-adrenergic blockade is associated with enhanced mast cell degranulation and a lower extent of collagen deposition in the normal myocardium. In conclusion, cardiac mast cell activation is influenced by beta-adrenergic influences.

Modelling radiation-induced bystander effect and cellular communication.

In the last 10 years evidence has accumulated on the so-called radiation-induced 'non-targeted effects' and in particular on bystander effects, consisting of damage induction in non-irradiated cells most likely following the release of soluble factors by the irradiated ones. These phenomena were observed for different biological endpoints, both lethal and non-lethal for the cell. Although the underlying mechanisms are largely unknown, it is now widely recognised that two types of cellular communication (i.e. via gap junctions and/or release of molecular messengers into the extracellular environment) play a pivotal role. Furthermore, the effects can be significantly modulated by parameters such as cell type and cell-cycle stage, cell density, time after irradiation etc. Theoretical models and simulation codes can be of help to improve our knowledge of the mechanisms, as well as to investigate the possible role of these effects in determining deviations from the linear relationship between dose and risk which is generally applied in radiation protection. In this paper three models, including an approach under development at the University of Pavia, will be presented in detail. The focus will be on the various adopted assumptions, together with their implications in terms of non-targeted radiobiological damage and, more generally, low-dose radiation risk. Comparisons with experimental data will also be discussed.

Gamma ray-induced bystander effect in tumour glioblastoma cells: a specific study on cell survival, cytokine release and cytokine receptors.

Recent experimental evidence has challenged the paradigm according to which radiation traversal through the nucleus of a cell is a prerequisite for producing genetic changes or biological responses. Thus, unexposed cells in the vicinity of directly irradiated cells or recipient cells of medium from irradiated cultures can also be affected. The aim of the present study was to evaluate, by means of the medium transfer technique, whether interleukin-8 and its receptor (CXCR1) may play a role in the bystander effect after gamma irradiation of T98G cells in vitro. In fact the cell specificity in inducing the bystander effect and in receiving the secreted signals that has been described suggests that not only the ability to release the cytokines but also the receptor profiles are likely to modulate the cell responses and the final outcome. The dose and time dependence of the cytokine release into the medium, quantified using an enzyme linked immunosorbent assay, showed that radiation causes alteration in the release of interleukin-8 from exposed cells in a dose-independent but time-dependent manner. The relative receptor expression was also affected in exposed and bystander cells.

In vivo radiobiological assessment of the new clinical carbon ion beams at CNAO.

In this article, the in vivo study performed to evaluate the uniformity of biological doses within an hypothetical target volume and calculate the values of relative biological effectiveness (RBE) at different depths in the spread-out Bragg peak (SOBP) of the new CNAO (National Centre for Oncological Hadrontherapy) carbon beams is presented, in the framework of a typical radiobiological beam calibration procedure. The RBE values (relative to (60)Co γ rays) of the CNAO active scanning carbon ion beams were determined using jejunal crypt regeneration in mice as biological system at the entrance, centre and distal end of a 6-cm SOBP. The RBE values calculated from the iso-effective doses to reduce crypt survival per circumference to 10, ranged from 1.52 at the middle of the SOBP to 1.75 at the distal position and are in agreement with those previously reported from other carbon ion facilities. In conclusion, this first set of in vivo experiments shows that the CNAO carbon beam is radiobiologically comparable with the NIRS (National Institute of Radiological Sciences, Chiba, Japan) and GSI (Helmholtzzentrum für Schwerionenforschung, Darmstadt, Germany) ones.

Early effects comparison of X-rays delivered at high-dose-rate pulses by a plasma focus device and at low dose rate on human tumour cells.

A comparative study has been performed on the effects of high-dose-rate (DR) X-ray beams produced by a plasma focus device (PFMA-3), to exploit its potential medical applications (e.g. radiotherapy), and low-DR X-ray beams produced by a conventional source (XRT). Experiments have been performed at 0.5 and 2 Gy doses on a human glioblastoma cell line (T98G). Cell proliferation rate and potassium outward currents (IK) have been investigated by time lapse imaging and patch clamp recordings. The results showed that PFMA-3 irradiation has a greater capability to reduce the proliferation rate activity with respect to XRT, while it does not affect IK of T98G cells at any of the dose levels tested. XRT irradiation significantly reduces the mean IK amplitude of T98G cells only at 0.5 Gy. This work confirms that the DR, and therefore the source of radiation, is crucial for the planning and optimisation of radiotherapy applications.

The gradient of visual attention in developmental dyslexia.

This study investigated the gradient of visual attention in 21 children, 11 children with specific reading disorder (SRD) or dyslexia and 10 children with normal reading skills. We recorded reaction times (RTs) at the onset of a small point along the horizontal axis in the two visual fields. In 70% of the cases the target appeared inside a circle acting as focusing cue and in 30% of the cases it appeared outside, allowing us to study the distribution of attentional resources outside the selected area. Normally reading children showed a normal symmetric distribution of attention. Indeed, RTs were directly proportional to the eccentricity of the target, and no visual field effect was observable. In contrast, children with SRD showed an anomalous and asymmetric distribution. The effect of the target eccentricity influenced RTs only when the stimulus was projected in the left visual field, whereas no effect was observable when the stimulus was projected in the right visual field. Findings allowed us to discuss the relation between this anomalous spatial distribution of visual attentional resources and dyslexia. To interpret the visual perceptual difficulties of children with SRD the hypothesis was made of a selective disorder of spatial attention (left inattention and right over-distractibility) related to a right parietal cortex dysfunction.

Is attentional focusing an inhibitory process at distractor location?

This study investigated whether attentional focusing is an inhibitory mechanism of lateral distractor stimuli. Attentional focusing is defined as the visual system capacity to vary the spatial extent of the attended area without shifting attention. A two-choice-reaction time task with compatible and incompatible flankers was used. Results were interpreted on the basis of the time course of attentional focusing.

Visuospatial attention in myopia.

Four experiments were conducted to ascertain whether myopia is associated with deficits of visuospatial attention. In myopic and emmetropic control subjects, we studied: (1) automatic and voluntary orienting of attention, (2) focusing of attention and (3) performance on a visual search task. The results indicated that automatic orienting was defective in myopics and their performance in visual search was less efficient than that of controls. By contrast, myopics showed no deficits in voluntary orienting and in focusing.

Facilitation and inhibition mechanisms of human visuospatial attention in a non-search task.

This study investigated both facilitation and inhibition mechanisms of attentional orienting. Orienting consists in shifting the attentional focus in the visual field. To study the distribution of attentional resources, a two-choice-reaction time task with compatible and incompatible flankers was used. When an irrelevant flanker is presented adjacent to a target stimulus, interference is observed when the two stimuli are associated with conflicting responses (flanker effect). Orienting of attention, at cued and uncued flanker locations, was manipulated by an exogenous cueing paradigm (automatic capture following a peripheral visual transient). Results showed that orienting of attention at cued distractor location produces an increased flanker effect as compared to the no-cue condition. In contrast, the flanker effect was absent when the attentional focus shifted to the uncued distractor location. These effects were interpreted in terms of facilitation and inhibition processes of visuospatial selection by early attentional competition models.