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In 2002–2003, the Italian Association for Neuroimmunology (AINI) ran a program for procedure and method standardization in neuroimmunology. The main purposes of the program were: a) to improve the overall quality of analytical performance and, simultaneously, to reduce costs by resource optimization; b) to establish the bases for clinical guidelines in neurology; c) to promote the formation of laboratory networks and of joint research projects; d) to facilitate the procedures for certification required by governmental/non-governmental agencies. This report summarizes the consensus recommendations of a panel of AINI neuroimmunologists/biochemists involved in the field of cerebrospinal fluid examination. The collection process for said recommendations was guided by ‘‘impact-factored’’ literature and the knowledge of the experts involved. Communication was by email and face-to-face at two dedicated AINI workshops.

Background Meningioma are the second most common brain tumors in adults and can cause significant morbidity and mortality. The scarcity of in vitro and in vivo models represents the major obstacle to understand the molecular basis of meningioma tumorigenesis. The main aim of this study was to assess a method for radiobiology of meningioma cells colture by means of well-known meningioma lines. New method We carried out a protocol of cells culture for irradiation of meningioma cells. We used the immortalized cell lines IOMM-Lee and CH-157 to study their radiation-reponse by means of clonogenic assays and to evaluate their proliferation and apoptosis. We irradiated the cells with different total doses using two different linear accelerators. Results We observed a more radiation resistance of the IOMM-Lee than the CH-157. Indeed, the cellular death of CH-157 was obtained at a very low dose irradiation. Moreover, we showed a dose-response effect due to the early and late apoptosis, in fact the rate of apoptotic cells is greater than that of the necrotic cells at any dose of irradiation and at any time of analysis. Comparison with existing methods There is not a standardized method for radiobiology of meningioma experiments. Conclusions Our method of cells culture appears suitable for radiosensitivity studies on meningioma. We can confirm that the response to radiotherapy depends not only on irradiation features, but also on tumor radiosensitivity.

The XPO1/CRM1 inhibitor selinexor (KPT-330), is currently being evaluated in multiple clinical trials as an anticancer agent. XPO1 participates in the nuclear export of FoxO-1, which we previously found to be decreased in platinum-resistant ovarian carcinoma. The aim of this study was to determine whether enriching FoxO-1 nuclear localization using selinexor would increase ovarian cancer cell sensitivity to cisplatin. Selinexor, as a single agent, displayed a striking antiproliferative effect in different ovarian carcinoma cell lines. A schedule-dependent synergistic effect of selinexor in combination with cisplatin was found in cisplatin-sensitive IGROV-1, the combination efficacy being more evident in sensitive than in the resistant cells. In IGROV-1 cells, the combination was more effective when selinexor followed cisplatin exposure. A modulation of proteins involved in apoptosis (p53, Bax) and in cell cycle progression (p21WAF1) was found by Western blotting. Selinexor-treated cells exhibited enriched FoxO-1 nuclear staining. Knock-down experiments with RNA interference indicated that FOXO1-silenced cells displayed a reduced sensitivity to selinexor. FOXO1 silencing also tended to reduce the efficacy of the drug combination at selected cisplatin concentrations. Selinexor significantly inhibited tumor growth, induced FoxO-1 nuclear localization and improved the efficacy of cisplatin in IGROV-1 xenografts. Taken together, our results support FoxO-1 as one of the key factors promoting sensitivity towards selinexor and the synergistic interaction between cisplatin and selinexor in ovarian carcinoma cells with selected molecular backgrounds, highlighting the need for treatment regimens tailored to the molecular tumor features.

This document presents the guidelines for the cerebrospinal fluid (CSF) analysis and the determination of oligoclonal bands (OCBs) as pivotal tests in neuroin flammatory pathologies of the central nervous system. The guidelines have been developed following a consensus process built on questionnaire-based surveys, internet contacts, and discussions at workshops of the sponsoring Italian Association of Neuroimmunology (AINI) congresses. Essential clinical information on the pathologies in which the CSF analysis is indicated, and, particularly, on those characterized by the presence of OCBs in the intrathecal compartment, indications and limits of CSF analysis and OCB determination, instructions for result interpretation, and agreed laboratory protocols (Appendix) are reported for the communicative community of neurologists and clinical pathologists.

Progressive myoclonus epilepsy (PME) of Unverricht–Lundborg type (EPM1) is an autosomal recessive neurodegenerative disorder with the highest incidence of PME worldwide. Mutations in the gene encoding cystatin B (CSTB) are the primary genetic cause of EPM1. Here, we investigate the role of CSTB during neurogenesis in vivo in the developing mouse brain and in vitro in human cerebral organoids (hCOs) derived from EPM1 patients. We find that CSTB (but not one of its pathological variants) is secreted into the mouse cerebral spinal fluid and the conditioned media from hCOs. In embryonic mouse brain, we find that functional CSTB influences progenitors’ proliferation and modulates neuronal distribution by attracting interneurons to the site of secretion via cell‐non‐autonomous mechanisms. Similarly, in patient‐derived hCOs, low levels of functional CSTB result in an alteration of progenitor's proliferation, premature differentiation, and changes in interneurons migration. Secretion and extracellular matrix organization are the biological processes particularly affected as suggested by a proteomic analysis in patients’ hCOs. Overall, our study sheds new light on the cellular mechanisms underlying the development of EPM1., Mutations in the cystatin B (CSTB) gene cause EPM1 epilepsy in patients. CSTB secretion induces the recruitment of migrating interneurons and promotes progenitor cells expansion in the mouse cortex and human cerebral organoids (hCOs). Both functions are impaired in EPM1‐derived hCOs.

Purpose: Meningiomas account for one third of primary intracranial tumors; nevertheless information on meningioma cell lines and in vivo models is scant. Although radiotherapy is one of the most relevant therapeutic options for the treatment of patients with meningioma, radiobiological research to understand tumor response to this treatment is far from being thoroughly figured out. The aim of this report is to provide a comprehensive picture of the current literature on this field, so as to foster research in this regard. Methods: We carried out a review of meningioma radiobiology based on a peer-reviewed PubMed search. Results: Our findings confirm that the main limitation of radiobiological research into meningioma is the paucity of robust in vitro and in vivo models. Alternative approaches to overcome the already identified problems, and to allow better understanding of the entire histopathological spectrum of meningiomas have been explored. Conclusions: A radiobiological perspective of meningioma may help to improve clinical results both in terms of tumor control and healthy tissue sparing. Although we are far from drawing any conclusions, this review can lead researchers to identify some clues for future areas of study.

Purpose: Ménière disease (MD) is a multifactorial chronic disabling condition characterized by episodic vertigo, ear fullness, and hearing loss. MD patients often complain of aspecific gastrointestinal symptoms associated with autonomic dysregulation, frequently outweighed by the otological manifestations. Dietary modifications have been reported to improve the typical MD symptoms in some cases. Our purpose was to test the urinary levels of lactulose and mannitol (double sugar test) and the fecal calprotectin, both markers of altered intestinal permeability, in subjects with definite MD in an active and inactive stage. Materials and methods: Twenty-six with definite unilateral MD were studied: 14 patients were symptomatic for at least 3 months with moderate to severe vertigo spells and a functional level ≥ 4; 12 patients had been asymptomatic (no vertigo spells) for at least 3 months and had a functional level = 1 at the time of testing. Twenty healthy volunteers were recruited as “control group”. Results: Lactulose and mannitol absorption was significantly increased in the symptomatic M patients compared to the asymptomatic group (p < 0.02 and p < 0.004, respectively) and to the controls. FC were also higher than normal only in the symptomatic group. (p < 0.01). Conclusions: An altered intestinal permeability, according to the two assays, was found only in symptomatic MD patients. The rationale for a possible relationship between MD and intestinal permeability is forwarded. The double-sugar test and FC quantification might be implemented in the MD diagnostic workup

Background In vitro primary cultures of microvascular endothelial cells from endocrine pancreas are difficult to obtain, but can be a very helpful tool for studies of islet biology, transplantation and regenerative medicine. Methods We applied a protocol recently described for the isolation and culture of brain microvascular endothelial cells (EC) on human pancreatic islets. EC obtained were characterized in terms of morphological (light and transmission electron microscopy), phenotypical (by immunofluorescence and flow cytometry) and functional (cord formation assay and protein secretion by multiplex bead-based assay) characteristics. Results EC were obtained from 25% of islet preparations processed. Two primary endothelial cell lines showed high proliferative potential and were deeply characterized: they presented endothelial cell morphology and expressed CD31, CD49a, CD49e, CD34, von Willebrand Factor (vWF), Vascular Endothelial CAdherin (VE-CAD), Tyrosine Kinase with Ig and EGF Homology Domains-2 (TIE2), Vascular Endothelial Growth Factor Receptor 1 (VEGFR1), Ulex lectin and the endothelium endocrine-specific marker nephrin. Besides, they were able to form cordons in vitro and secreted factors involved in the process of angiogenesis such as Vascular Endothelial Growth Factor (VEGF), Monocyte Chemotactic Protein 1 (MCP-1), interleukin (IL)-8 and Melanoma Growth Stimulatory Activity Alpha (GROα). These cell lines were termed Human Islet Microvascular Endothelial Cells (HIMEC). Discussion This study establishes a simple and effective strategy for isolation and long-term culture of EC derived from human pancreatic islet. HIMEC in culture preserve phenotype and functional properties and are, therefore, a useful tool for future experiments of in vitro pancreas modelling, co-transplantation with pancreatic islets, re-vascularization of scaffold or matrix for regenerative medicine purposes. © 2017 International Society for Cellular Therapy

SummaryBackground Drug toxicity currently represents the main challenge of tumour chemotherapy. Our group recently developed a new method for drug delivery inspired by the ‘Trojan Horse’ concept. Human mesenchymal stem cells (hMSCs) have been shown to play the role of new ‘horses’ in delivering anti-tumour agents, without involving any genetic manipulation. As human stromal dermal fibroblasts (hSDFs) represent an interesting alternative to hMSCs, being easy to isolate, they could be an ideal candidate for this kind of procedure. Aim To investigate whether hSDFs can take up and deliver paclitaxel (PTX) in sufficient concentrations to inhibit a very aggressive melanoma tumour (IgR39) in vitro. Methods hSDFs were primed with high doses of PTX, and then the effect of drug delivery on IgR39 melanoma proliferation in vitro was evaluated using several assays (antiproliferation, transwell cocultures, rosette assays and colony growth assays). Furthermore, the cell cycle and PTX uptake/release mechanism of hSDFs were studied both under both normal and hypoxic conditions. Results hSDFs incorporated PTX and then released it with unaffected pharmacological activity, inhibiting human IgR39 melanoma growth in vitro. The hypoxic conditions did not induce changes in cell cycle pattern and the uptake–release mechanism with PTX was not affected. Conclusions hSDFs can be used as a Trojan horse, as the released drug was functionally active. These results indicated that these cells could be used for clinical treatment as the drug was released into the cellular environment and the primed cells underwent apoptosis.

BACKGROUND: Brain microvascular endothelial cells (BMVECs) constitute the primary limitation for passage of ions and molecules from the blood into the brain through the blood brain barrier. Numerous multi-step procedures for isolating and culturing BMVECs have been described. However, each one demonstrates major limitations in purity of culture and/or low proliferation rate. Our goal was to study the efficiency of our pending patent medium, Endothelial Proliferation Medium (EndoPM), on the isolation and purification of human and murine BMVECs. METHODS: BMVECs, cultured in EndoPM were compared to those cultured in a commercial medium EBM. Cultures were characterized by flow cytometric analysis, lineage differentiation, the ability to form tube-like structure, immunofluorescence, molecular analyses and also in an in vivo model assay. Moreover permeability was assayed by monitoring the passage of Dextran-FITC through a tight monolayer of BMVECs grown to confluence in Boyden chambers. One way Anova two-tailed test was utilized for all statistical analyses. RESULTS: The properties of ECs in human and murine BMVECs is confirmed by the expression of endothelial markers (CD31, CD105, CD146, Tie-2 and vWF), of representative proangiogenic genes (ICAM1, VCAM1 and integrin ITGAV), of considerable tube-forming ability, with low-density lipoprotein uptake, eNOS and GLUT-1 expression. Furthermore cells are able to express markers of the junctional architecture as VE-cadherin, β-catenin and Claudin-5 and greatly reduce dextran permeability as barrier functional test. Moreover BMVECs spontaneously organize in vascular-like structures and maintain the expression of endothelial markers in an in vivo xenograft model assay. The significant effect of EndoPM is confirmed by the study of proliferation index, survival index and the behaviour of BMVECs and fibroblasts in co-culture conditions. CONCLUSION: Herein we describe a simple and reproducible method for the isolation and expansion of human and mouse BMVECs, based on a newly formulated medium (EndoPM) with optimized concentration of growth factors (EGF, FGF-2 and Bovine Brain Extract-BBE). This procedure should facilitate the isolation and expansion of human and mouse BMVECs with extended lifetime, good viability and purity. This approach may provide an effective strategy to aid phenotypical and functional studies of brain vessels under physiological and pathological conditions.

Pantothenate kinase-associated neurodegeneration (PKAN) is a rare, inborn error of metabolism characterized by iron accumulation in the basal ganglia and by the presence of dystonia, dysarthria, and retinal degeneration. Mutations in pantothenate kinase 2 (PANK2), the rate-limiting enzyme in mitochondrial coenzyme A biosynthesis, represent the most common genetic cause of this disorder. How mutations in this core metabolic enzyme give rise to such a broad clinical spectrum of pathology remains a mystery. To systematically explore its pathogenesis, we performed global metabolic profiling on plasma from a cohort of 14 genetically defined patients and 18 controls. Notably, lactate is elevated in PKAN patients, suggesting dysfunctional mitochondrial metabolism. As predicted, but never previously reported, pantothenate levels are higher in patients with premature stop mutations in PANK2. Global metabolic profiling and follow-up studies in patient-derived fibroblasts also reveal defects in bile acid conjugation and lipid metabolism, pathways that require coenzyme A. These findings raise a novel therapeutic hypothesis, namely, that dietary fats and bile acid supplements may hold potential as disease-modifying interventions. Our study illustrates the value of metabolic profiling as a tool for systematically exploring the biochemical basis of inherited metabolic diseases. © 2011 Elsevier Inc.

MicroRNAs (miRNAs) are posttranscriptional regulatory noncoding RNAs used to profile human hematopoietic tumors. In this study, some mature miRNAs was quantitated in peripheral blood from dogs with chronic lymphocytic leukemia (CLL). Relative expression data were normalised against four endogenous controls (let-7a, miR-17-5p, miR-26b, and miR-223) selected by geNorm analysis. The results revealed distinct miRNA patterns in CLL depending on the immunophenotype. Also in dogs, the different miRNAs expression could reflect developmental lineage and tumor differentiation. The similar genetics, physiology and exposure to environment in dogs and humans make the miRNA expression study in canine CLL attractive for comparative oncology. (C) 2011 Elsevier B.V. All rights reserved.