Murine adipose-derived mesenchymal stromal cell vesicles: in vitro clues for neuroprotective and neuroregenerative approaches.

BACKGROUND AIMS: Adipose-derived mesenchymal stromal cells (ASC) are known to promote neuroprotection and neuroregeneration in vitro and in vivo. These biological effects are probably mediated by paracrine mechanisms. In recent years, nanovesicles (NV) and microvesicles (MV) have been shown to play a major role in cell-to-cell communication. We tested the efficacy of NV and MV obtained from ASC in mediating neuroprotection and neuroregeneration in vitro. METHODS: We exposed neuronal cells (both cell line and primary cultures) to oxidative stress in the presence or not of NV or MV. RESULTS: In this experimental setting, we found that low doses of NV or MV protected neurons from apoptotic cell death. We then assessed the neuroregenerative effect of NV/MV in cerebellar slice cultures demyelinated with lysophosphatidylcholine. We observed that low but not higher doses of NV and MV increased the process of remyelination and activated nestin-positive oligodendroglial precursors. CONCLUSIONS: Taken together, our data in vitro support the relevance of ASC vesicles as a source of protecting and regenerating factors that might modulate the microenvironment in neuro-inflammatory as well as in neurodegenerative disorders. The present findings may suggest that stromal cell-derived vesicles might represent a potential therapeutic tool, enabling the safe administration of stromal cell effector factors, avoiding the cellular counterpart.

Predictors of survival and effect of short (40 Gy) or standard-course (60 Gy) irradiation plus concomitant temozolomide in elderly patients with glioblastoma: a multicenter retrospective study of AINO (Italian Association of Neuro-Oncology).

The efficacy of temozolomide (TMZ) plus radiation therapy (RT) in elderly patients with glioblastoma is unclear. We performed a large multicenter retrospective study to analyze prognostic factors and clinical outcome in these patients. Inclusion criteria were age ≥65 years, newly histologically confirmed glioblastoma, ECOG PS 0-2, adjuvant treatment with RT plus TMZ. We enrolled 237 patients; the average age was 71 and ECOG PS was 0-1 in 196 patients; gross total resection was performed in 174 cases. MGMT was analyzed in 151 persons and was methylated in 56 %. IDH1 was assessed in 100 patients and was mutated in 6 %. Seventy-one patients were treated with RT 40 Gy and 166 with RT 60 Gy. Progression-free survival and overall survival (OS) were 11.3 and 17.3 months, respectively. Overall survival was 19.4 vs 13.8 months for patients treated with RT 60 Gy and 40 Gy (p = 0.02); OS was 17.7 versus 16.1 months for patients treated with gross total resection vs partial surgery (p = 0.02); OS was 21.2 versus 13.6 months for methylated and unmethylated MGMT (p < 0.001). On multivariate analysis, gross total resection, RT 60 Gy, methylated MGMT and ECOG PS 0-1 were independent predictors of longer survival. Twenty-five patients (10 %) had grade 3-4 haematological toxicity during the concomitant treatment. We showed that, in elderly patients in good clinical condition treated with concomitant treatment, standard-course irradiation might be more effective than short-course irradiation. Methylated MGMT remains the most important prognostic factor.

Uptake by human glioma cell lines and biological effects of a peptide-nucleic acids targeting miR-221.

MicroRNAs are a family of small noncoding RNAs regulating gene expression by sequence-selective mRNA targeting, leading to a translational repression or mRNA degradation. The oncomiR miR-221 is highly expressed in human gliomas, as confirmed in this study in samples of low and high grade gliomas, as well in the cell lines U251, U373 and T98G. In order to alter the biological functions of miR-221, a peptide nucleic acid targeting miR-221 (R8-PNA-a221) was produced, bearing a oligoarginine peptide (R8) to facilitate uptake by glioma cells. The effects of R8-PNA-a221 were analyzed in U251, U373 and T98G glioma cells and found to strongly inhibit miR-221. In addition, the effects of R8-PNA-a221 on p27(Kip1) (a target of miR-221) were analyzed in U251 and T98G cells by RT-qPCR and by Western blotting. No change of p27(Kip1) mRNA content occurs in U251 cells in the presence of PNA-a221 (lacking the R8 peptide), whereas significant increase of p27(Kip1) mRNA was observed with the R8-PNA-a221. These data were confirmed by Western blot assay. A clear increment of p27(Kip1) protein expression in the samples treated with R8-PNA-a221 was detected. In addition, R8-PNA-a221 was found able to increase TIMP3 expression (another target of miR-221) in T98G cells. These results suggest that PNAs against oncomiRNA miR-221 might be proposed for experimental treatment of human gliomas.

Single-agent Bevacizumab in Recurrent Glioblastoma After Second-line Chemotherapy With Fotemustine: The Experience of the Italian Association of Neuro-Oncology.

OBJECTIVES: Bevacizumab is an anti-vascular endothelial growth factor antibody used in the treatment of recurrent glioblastoma (GBM). Despite the large number of studies carried out in patients with recurrent GBM, little is known about the administration of this angiogenesis inhibitor after the failure of the second-line chemotherapy. MATERIALS AND METHODS: In this retrospective multicenter study, on behalf of the Italian Association of Neuro-Oncology, we reported the results obtained in 51 patients with recurrent GBM treated with single-agent bevacizumab after the failure of second-line chemotherapy with fotemustine. RESULTS: In March 2016, at the time of data analysis, 3 patients (14.4%) were still alive with stable disease, whereas 48 died due to disease progression. Kaplan-Meier estimated median survival from the diagnosis of GBM was 28 months (95% confidence interval [CI], 22.1-33.9 mo). Median survival measured from the beginning of fotemustine and bevacizumab therapy were 11.3 (95% CI, 8.4-13.6 mo) and 6 months (95% CI, 3.8-8.1 mo), respectively. The 6- and 12-month progression free survival rates from the beginning of bevacizumab treatment were 18% and 13%, respectively. CONCLUSIONS: On the basis of our data, in patients with recurrent GBM, the failure of a second-line chemotherapy with cytotoxic agents might not exclude the administration of bevacizumab as third-line chemotherapy.

ABCG2 regulates self-renewal and stem cell marker expression but not tumorigenicity or radiation resistance of glioma cells.

Glioma cells with stem cell traits are thought to be responsible for tumor maintenance and therapeutic failure. Such cells can be enriched based on their inherent drug efflux capability mediated by the ABC transporter ABCG2 using the side population assay, and their characteristics include increased self-renewal, high stem cell marker expression and high tumorigenic capacity in vivo. Here, we show that ABCG2 can actively drive expression of stem cell markers and self-renewal in glioma cells. Stem cell markers and self-renewal was enriched in cells with high ABCG2 activity, and could be specifically inhibited by pharmacological and genetic ABCG2 inhibition. Importantly, despite regulating these key characteristics of stem-like tumor cells, ABCG2 activity did not affect radiation resistance or tumorigenicity in vivo. ABCG2 effects were Notch-independent and mediated by diverse mechanisms including the transcription factor Mef. Our data demonstrate that characteristics of tumor stem cells are separable, and highlight ABCG2 as a potential driver of glioma stemness.

Synergistic effect of apolipoprotein E polymorphisms and cigarette smoking on risk of ischemic stroke in young adults.

BACKGROUND AND PURPOSE: The effect of apolipoprotein E (APOE) polymorphisms on stroke risk may be influenced by the coexistence of modifiable predisposing conditions. We explored the interactions of APOE genotypes and conventional risk factors in a case-control study of young adults with cerebral infarct. METHODS: We analyzed 124 consecutive patients (age, 34.7+/-7.3 years) and 147 age- and sex-matched controls. APOE genotypes were determined by restriction fragment-length polymorphism analysis. RESULTS: The prevalence of the epsilon4 allele and epsilon34 genotype was slightly higher in cases than in controls (0.125 versus 0.071 and 0.242 versus 0.136, respectively). Carriers of the epsilon34 genotype and epsilon4 allele were associated with an increased risk of stroke on multivariate analysis compared with the epsilon33 genotype and non-epsilon4 carriers, respectively (odds ratio [OR], 2.29; 95% confidence interval [CI], 1.10 to 4.76; and OR, 2.27; 95% CI, 1.13 to 4.56). ORs for stroke were 2.99 (95% CI, 1.64 to 5.45), 2.69 (95% CI, 1.25 to 5.77), and 5.39 (95% CI, 1.59 to 18.30) for smokers with the epsilon33 genotype, nonsmokers with the epsilon34 genotype, and smokers with the epsilon34 genotype, respectively, compared with nonsmokers with the epsilon33 genotype. Similar results were obtained when epsilon4 carriers and non-epsilon4 carriers were compared in the same interaction model. No significant interaction between APOE and hypertension was found. CONCLUSIONS: In young adults, the APOE epsilon4 allele and cigarette smoking act synergistically, increasing an individual's propensity to have a cerebral ischemic event. This finding may help in determining an individual's predisposition to stroke and more targeted preventive interventions.

Candidate pathways for promoting differentiation or quiescence of oligodendrocyte progenitor-like cells in glioma.

Platelet-derived growth factor receptor alpha-positive oligodendrocyte progenitor cells (OPC) located within the mature central nervous system may remain quiescent, proliferate, or differentiate into oligodendrocytes. Human glioblastoma multiforme tumors often contain rapidly proliferating oligodendrocyte lineage transcription factor 2 (Olig2)-positive cells that resemble OPCs. In this study, we sought to identify candidate pathways that promote OPC differentiation or quiescence rather than proliferation. Gene expression profiling conducted in both normal murine OPCs and highly proliferative Olig2-positive glioma cells identified all the transcripts associated with the highly proliferative state of these cells and showed that among the various cell types found within the brain, Olig2-positive tumor cells are most similar to OPCs. We then subtracted OPC transcripts found in tumor samples from those found in normal brain samples and identified 28 OPC transcripts as candidates for promoting differentiation or quiescence. Systematic analysis of human glioma data revealed that these genes have similar expression profiles in human tumors and were significantly enriched in genomic deletions, suggesting an antiproliferative role. Treatment of primary murine glioblastoma cells with agonists of one candidate gene, Gpr17, resulted in a decreased number of neurospheres. Together, our findings show that comparison of the molecular phenotype of progenitor cells in tumors to the equivalent cells in the normal brain represents a novel approach for the identification of targeted therapies.

MEF promotes stemness in the pathogenesis of gliomas.

High-grade gliomas are aggressive and uniformly fatal tumors, composed of a heterogeneous population of cells that include many with stem-cell-like properties. The acquisition of stem-like traits might contribute to glioma initiation, growth, and recurrence. Here we investigated the role of the transcription factor myeloid Elf-1 like factor (MEF, also known as ELF4) in gliomas. We found that MEF is highly expressed in both human and mouse glioblastomas and its absence impairs gliomagenesis in a PDGF-driven glioma mouse model. We show that modulation of MEF levels in both mouse neural stem cells and human glioblastoma cells has a significant impact on neurosphere formation. Moreover, we identify Sox2 as a direct downstream target of MEF. Taken together, our studies implicate MEF as a previously unrecognized gatekeeper gene in gliomagenesis that promotes stem cell characteristics through Sox2 activation.

The mef/elf4 transcription factor fine tunes the DNA damage response.

The ATM kinase plays a critical role in initiating the DNA damage response that is triggered by genotoxic stresses capable of inducing DNA double-strand breaks. Here, we show that ELF4/MEF, a member of the ETS family of transcription factors, contributes to the persistence of γH2AX DNA damage foci and promotes the DNA damage response leading to the induction of apoptosis. Conversely, the absence of ELF4 promotes the faster repair of damaged DNA and more rapid disappearance of γH2AX foci in response to γ-irradiation, leading to a radio-resistant phenotype despite normal ATM phosphorylation. Following γ-irradiation, ATM phosphorylates ELF4, leading to its degradation; a mutant form of ELF4 that cannot be phosphorylated by ATM persists following γ-irradiation, delaying the resolution of γH2AX foci and triggering an excessive DNA damage response. Thus, although ELF4 promotes the phosphorylation of H2AX by ATM, its activity must be dampened by ATM-dependent phosphorylation and degradation to avoid an excessive DNA damage response.

The oncogenic role of the ETS transcription factors MEF and ERG.

Several ETS transcription factors, including MEF/ELF4 and ERG, can function as oncogenes and are overexpressed in human cancer. MEF cooperates in tumorigenesis in retroviral insertional mutagenesis-based mouse models of cancer and MEF is overexpressed in human lymphoma and ovarian cancer tissues via unknown mechanisms. ERG (Ets related gene) overexpression or increased activity has been found in various human cancers, including sarcomas, acute myeloid leukemia and prostate cancer, where the ERG gene is rearranged due to chromosomal translocations. We have been examining how MEF functions as an oncogene and recently showed that MEF can cooperate with H-Ras(G12V) and can inhibit both p53 and p16 expression thereby promoting transformation. In fact, in cells lacking p53, the absence of Mef abrogates H-Ras(G12V)-induced transformation of mouse embryonic fibroblasts, at least in part due to increased p16 expression. We discuss the known mechanisms by which the ETS transcription factors MEF and ERG contribute to the malignant transformation of cells.

Progressive multifocal leukoencephalopathy in a patient with Good's syndrome.

Good's syndrome (GS) is an immunodeficiency characterized by thymoma, hypogammaglobulinemia, and impaired T-cell function. The clinical manifestations of GS include recurrent or chronic infections from common or opportunistic pathogens. Encephalitis is a rare event, with only anecdotal reports of cytomegalovirus infection. Herein we report the case of a 79-year-old woman with GS who developed subacute motor deficits and cognitive changes. Magnetic resonance imaging (MRI) of the brain disclosed white- and gray-matter lesions, mostly in the right frontal and parietal areas. Polyoma virus JC, the agent of progressive multifocal encephalopathy (PML), was identified in cerebrospinal fluid samples and brain biopsy specimens. After diagnosis, the disease had a rapid fatal course. The present case represents the first reported association between GS and PML.

Paradoxical brain embolism in a young man with isolated pulmonary arteriovenous fistula.

We herein report a case of ischemic stroke due to paradoxical brain embolism in a young man, a trumpet player. Extensive diagnostic investigations revealed the presence of an isolated pulmonary arteriovenous fistula as the only risk factor for stroke. The peculiarity of this case is the early onset of neurological symptoms in the absence of Hereditary Hemorrhagic Teleangiectasia. The Authors suppose the repeated Valsalva maneuvers as a possible factor promoting fistula enlargement and symptoms development.