Maria Grazia Bruzzone

Gene therapy of experimental brain tumors using neural progenitor cells

Glioblastomas, the most frequent and malignant of primary brain tumors, have a very poor prognosis. Gene therapy of glioblastomas is limited by the short survival of viral vectors and by their difficulty in reaching glioblastoma cells infiltrating the brain parenchyma. Neural stem/progenitor cells can be engineered to produce therapeutic molecules and have the potential to overcome these limitations because they may travel along the white matter, like neoplastic cells, and engraft stably into the brain. Retrovirus-mediated transfer of the gene for interleukin-4 is an effective treatment for rat brain glioblastomas. Here, we transferred the gene for interleukin-4 into C57BL6J mouse primary neural progenitor cells and injected those cells into established syngeneic brain glioblastomas. This led to the survival of most tumor-bearing mice. We obtained similar results by implanting immortalized neural progenitor cells derived from Sprague-Dawley rats into C6 glioblastomas. We also documented by magnetic resonance imaging the progressive disappearance of large tumors, and detected 5-bromodeoxyuridine-labeled progenitor cells several weeks after the injection. These findings support a new approach for gene therapy of brain tumors, based on the grafting of neural stem cells producing therapeutic molecules.

Neurospheres Enriched in Cancer Stem–Like Cells Are Highly Effective in Eliciting a Dendritic Cell–Mediated Immune Response against Malignant Gliomas

Cancer stem-like cells (CSC) could be a novel target for cancer therapy, including dendritic cell (DC) immunotherapy. To address this, we developed experiments aimed at DC targeting of neurospheres (NS) from GL261 glioma cells because neurospheres can be enriched in CSC. We obtained murine neurospheres by growing GL261 cells in epidermal growth factor/basic fibroblast growth factor without serum. GL261-NS recapitulated important features of glioblastoma CSC and expressed higher levels of radial glia stem cell markers than GL261 cells growing under standard conditions (GL261 adherent cells, GL261-AC), as assessed by DNA microarray and real-time PCR. GL261-NS brain gliomas were highly infiltrating and more rapidly lethal than GL261-AC, as evidenced by survival analysis (P < 0.0001), magnetic resonance imaging and histology. DC from the bone marrow of syngeneic mice were then used for immunotherapy of GL261-NS and GL261-AC tumors. Strikingly, DC loaded with GL261-NS (DC-NS) cured 80% and 60% of GL261-AC and GL261-NS tumors, respectively (P < 0.0001), whereas DC-AC cured only 50% of GL261-AC tumors (P = 0.0022) and none of the GL261-NS tumors. GL261-NS expressed higher levels of MHC and costimulatory molecules (CD80 and CD86) than GL261-AC; the JAM assay indicated that DC-NS splenocytes had higher lytic activity than DC-AC splenocytes on both GL261-NS and GL261-AC, and immunohistochemistry showed that DC-NS vaccination was associated with robust tumor infiltration by CD8+ and CD4+ T lymphocytes. These findings suggest that DC targeting of CSC provides a higher level of protection against GL261 gliomas, a finding with potential implications for the design of clinical trials based on DC vaccination.

Reviews: Current Concepts in Alzheimer's Disease: A Multidisciplinary Review

This comprehensive, pedagogically-oriented review is aimed at a heterogeneous audience representative of the allied disciplines involved in research and patient care. After a foreword on epidemiology, genetics, and risk factors, the amyloid cascade model is introduced and the main neuropathological hallmarks are discussed. The progression of memory, language, visual processing, executive, attentional, and praxis deficits, and of behavioral symptoms is presented. After a summary on neuropsychological assessment, emerging biomarkers from cerebrospinal fluid assays, magnetic resonance imaging, nuclear medicine, and electrophysiology are discussed. Existing treatments are briefly reviewed, followed by an introduction to emerging disease-modifying therapies such as secretase modulators, inhibitors of Abeta aggregation, immunotherapy, inhibitors of tau protein phosphorylation, and delivery of nerve growth factor.

White Matter Involvement in Idiopathic Parkinson Disease: A Diffusion Tensor Imaging Study

BACKGROUND AND PURPOSE: Diffusion tensor imaging (DTI) offers a unique window on the connectivity changes, extending beyond the basal ganglia, which accompany the cognitive symptoms of Parkinson disease (PD). The primary purpose of this study was to assess the microstructural damage to cerebral white matter occurring in idiopathic PD. MATERIALS AND METHODS: Our sample included patients with PD without dementia (n = 10; Hoehn and Yahr stages I and II; Unified Parkinson Disease Rating Scale, 20.5 ± 8.3; and Mini-Mental State Examination, 28.3 ± 1.5) and age-matched healthy control subjects (n = 10). DTI was performed on a 1.5T scanner, and mean diffusivity (MD) and fractional anisotropy (FA) maps were obtained. Regions of interest (ROIs) were drawn on the major fiber bundles as well as on gray matter nuclei. RESULTS: In patients, the MD was increased at borderline significance in the substantia nigra but was unaltered in the thalamus, globus pallidus, putamen, and in the head of the caudate nucleus. The FA and MD were unaltered in the corticospinal tract in the midbrain and at the level of the internal capsule, and in the splenium of the corpus callosum. By contrast, the MD was increased and the FA was decreased in the genu of the corpus callosum and in the superior longitudinal fasciculus; in the cingulum, only the MD was altered. The observed changes were not significantly lateralized. CONCLUSIONS: Widespread microstructural damage to frontal and parietal white matter occurs already in the early stages of PD.

Age-related Iron Deposition in the Basal Ganglia: Quantitative Analysis in Healthy Subjects

PURPOSE To determine the values of iron accumulation in the basal ganglia of healthy volunteers of different ages with R2* and raw signal intensity measurements from T1-weighted magnetic resonance (MR) images, supported by voxel-based relaxometry (VBR), and to compare them with previously reported iron concentrations found in autopsy material. MATERIALS AND METHODS The ethics committee approved the study, and the participants or their parents gave written informed consent. Eighty subjects (41 female and 39 male subjects; age range, 1-80 years) were examined at 1.5 T. For each subject, R2* values were calculated. Curves for R2* versus age were obtained for globus pallidus (GP), putamen, caudate nucleus, substantia nigra (SN), and frontal white matter (FWM). To highlight possible differences in iron concentration among the age decades, VBR was applied. Signal intensity values were estimated on T1-weighted fast low-angle shot images, and regions of interest were drawn in each nucleus. R2* values were also compared with iron concentrations reported in a postmortem study. Statistical analysis was performed (t test), and a difference with P < .05 (FDR corrected) was significant. RESULTS The curves for R2* versus age showed an exponential increase with increasing age in all the basal ganglia. VBR demonstrated significant differences (P < .05, corrected) in the comparison between the 2nd and the following decades for lenticular nuclei. Good correlation coefficients were found for GP (R(2) = 0.64), putamen (R(2) = 0.51), and SN (R(2) = 0.53) when compared with findings in the postmortem study. Signal intensity curves were similar to the R2* curves. CONCLUSION R2* measurements can be used to quantify brain iron accumulation and thus may allow better evaluation of neurodegenerative diseases associated with iron deposition.

Tetracyclines affect prion infectivity

Prion diseases are transmissible neurodegenerative disorders of humans and animals for which no effective treatment is available. Conformationally altered, protease-resistant forms of the prion protein (PrP) termed PrPSc are critical for disease transmissibility and pathogenesis, thus representing a primary target for therapeutic strategies. Based on previous findings that tetracyclines revert abnormal physicochemical properties and abolish neurotoxicity of PrP peptides in vitro, we tested the ability of these compounds to interact with PrPSc from patients with the new variant of Creutzfeldt–Jakob disease (vCJD) and cattle with bovine spongiform encephalopathy (BSE). The incubation with tetracycline hydrochloride or doxycycline hyclate at concentrations ranging from 10 μM to 1 mM resulted in a dose-dependent decrease in protease resistance of PrPSc. This finding prompted us to investigate whether tetracyclines affect prion infectivity by using an animal model of disease. Syrian hamsters were injected intracerebrally with 263K scrapie-infected brain homogenate that was coincubated with 1 mM tetracycline hydrochloride, 1 mM doxycycline hyclate, or vehicle solution before inoculation. Hamsters injected with tetracycline-treated inoculum showed a significant delay in the onset of clinical signs of disease and prolonged survival time. These effects were paralleled by a delay in the appearance of magnetic-resonance abnormalities in the thalamus, neuropathological changes, and PrPSc accumulation. When tetracycline was preincubated with highly diluted scrapie-infected inoculum, one third of hamsters did not develop disease. Our data suggest that these well characterized antibiotics reduce prion infectivity through a direct interaction with PrPSc and are potentially useful for inactivation of BSE- or vCJD-contaminated products and prevention strategies.

Prognostic factors for survival in 676 consecutive patients with newly diagnosed primary glioblastoma

Reliable data on large cohorts of patients with glioblastoma are needed because such studies differ importantly from trials that have a strong bias toward the recruitment of younger patients with a higher performance status. We analyzed the outcome of 676 patients with histologically confirmed newly diagnosed glioblastoma who were treated consecutively at a single institution over a 7-year period (1997-2003) with follow-up to April 30, 2006. Survival probabilities were 57% at 1 year, 16% at 2 years, and 7% at 3 years. Progression-free survival was 15% at 1 year. Prolongation of survival was significantly associated with surgery in patients with a good performance status, whatever the patients age, with an adjusted hazard ratio of 0.55 (p < 0.001) or a 45% relative decrease in the risk of death. Radiotherapy and chemotherapy improved survival, with adjusted hazard ratios of 0.61 (p = 0.001) and 0.89 (p = 0.04), respectively, regardless of age, performance status, or residual tumor volume. Recurrence occurred in 99% of patients throughout the follow-up. Reoperation was performed in one-fourth of these patients but was not effective, whether performed within 9 months (hazard ratio, 0.86; p = 0.256) or after 9 months (hazard ratio, 0.98; p = 0.860) of initial surgery, whereas second-line chemotherapy with procarbazine, lomustine, and vincristine (PCV) or with temozolomide improved survival (hazard ratio, 0.77; p = 0.008). Surgery followed by radiotherapy and chemotherapy should be considered in all patients with glioblastoma, and these treatments should not be withheld because of increasing age alone. The benefit of second surgery at recurrence is uncertain, and new trials are needed to assess its effectiveness. Chemotherapy with PCV or temozolomide seems to be a reasonable option at tumor recurrence.

Conversion of the BASE prion strain into the BSE strain: the origin of BSE?

Atypical neuropathological and molecular phenotypes of bovine spongiform encephalopathy (BSE) have recently been identified in different countries. One of these phenotypes, named bovine “amyloidotic” spongiform encephalopathy (BASE), differs from classical BSE for the occurrence of a distinct type of the disease-associated prion protein (PrP), termed PrPSc, and the presence of PrP amyloid plaques. Here, we show that the agents responsible for BSE and BASE possess different biological properties upon transmission to transgenic mice expressing bovine PrP and inbred lines of nontransgenic mice. Strikingly, serial passages of the BASE strain to nontransgenic mice induced a neuropathological and molecular disease phenotype indistinguishable from that of BSE-infected mice. The existence of more than one agent associated with prion disease in cattle and the ability of the BASE strain to convert into the BSE strain may have important implications with respect to the origin of BSE and spongiform encephalopathies in other species, including humans.

Methylation of O6-Methylguanine DNA Methyltransferase and Loss of Heterozygosity on 19q and/or 17p Are Overlapping Features of Secondary Glioblastomas with Prolonged Survival

Purpose: Recent data suggest that methylation of the DNA repair gene O6-methylguanine DNA methyltransferase (MGMT), by increasing the chemosensitivity of glioblastoma multiforme, is significantly associated with improved prognosis. Results in contradiction with these findings, however, are present in the literature and the clinical and genetic context framing MGMT methylation is poorly characterized. Experimental Design: To address these issues, we have investigated the MGMT methylation status, clinical and magnetic resonance imaging characteristics, and relevant genetic features (loss of heterozygosity on 17p and 19q, EGFR amplification, and p53 mutations) in a retrospective study on 86 patients affected by glioblastoma multiforme: 72 patients had a clinical history indicating de novo insurgence of the tumor and the remaining 14 were secondary glioblastoma multiforme. Results: MGMT methylation was detected by methylation-specific PCR in 41 of 86 cases (47.7%; Meth+). Progression-free survival and overall survival were significantly longer in Meth+ than in Meth− patients [10 versus 7 months (P = 0.003, log-rank test) and 18 versus 14 months (P = 0.0003, log-rank test), respectively]. Mixed-nodular enhancement at magnetic resonance imaging was significantly more frequent in Meth+ and secondary glioblastoma multiforme and ring enhancement in Meth− and primary glioblastoma multiforme (P < 0.005). MGMT methylation was more present in secondary glioblastoma multiforme (P = 0.006) and associated with loss of heterozygosity on 17p and/or 19q (P = 0.005). Conclusions: These observations suggest that MGMT methylation is part of a genetic signature of glioblastomas that developed from lower-grade gliomas.

MR spectroscopy, functional MRI, and diffusion-tensor imaging in the aging brain: a conceptual review.

In vivo magnetic resonance spectroscopy (MRS), functional magnetic resonance imaging (fMRI), and diffusion-tensor imaging (DTI) have recently opened new possibilities for noninvasively assessing the metabolic, functional, and connectivity correlates of aging in research and clinical settings. The purpose of this article is to provide a conceptual review intended for a multidisciplinary audience, covering physical principles and main findings related to normal aging and senile cognitive impairment. This article is divided into 3 sections, dedicated to MRS, to fMRI, and to DTI. The spectroscopy section surveys physiological function of the observable metabolites, concentration changes in normal aging and their interpretation, and correlation with cognitive performance. The functional MRI section surveys the hemispheric asymmetry reduction model from compensation and de-differentiation viewpoints, memory encoding, retrieval and consolidation, inhibitory control, perception and action, resting-state networks, and functional deactivations. The DTI section surveys age-related changes, correlation with behavioral scores, and transition to cognitive impairment.