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Metastatic brain disease (MBD) has seen major advances in clinical management, focal radiation therapy approaches and knowledge of biological factors leading to improved prognosis. Extracellular vesicles (EVs) have been found to play a role in tumor cross-talk with the target organ, contributing to the formation of a premetastatic niche. Human lung and breast cancer cell lines were characterized for adhesion molecule expression and used to evaluate their migration ability in an in vitro model. Conditioned culture media and isolated EVs, characterized by super resolution and electron microscopy, were tested to evaluate their pro-apoptotic properties on human umbilical vein endothelial cells (HUVECs) and human cerebral microvascular endothelial cells (HCMEC/D3) by annexin V binding assay. Our data showed a direct correlation between expression of ICAM1, ICAM2, β3-integrin and α2-integrin and the ability to firmly adhere to the blood–brain barrier (BBB) model, whereas the same molecules were down-regulated at a later step. Extracellular vesicles released by tumor cell lines were shown to be able to induce apoptosis in HUVEC while brain endothelial cells showed to be more resistant.

The secreted products of the metastasis suppressor geneKiSS1-derived peptide levels in liquid biopsies from 60 NSCLC patients were assayed by ELISA. Preclinical experiments were carried out using quantitative real time polymerase chain reaction (qRT-PCR), ELISA, annexin V-binding and caspase activation assays.We compared KiSS1 release in 3 different matrices (serum, plasma and urine) and the highest levels were detectable in serum (range, 0-4.5 ng/mL). We observed increased levels of seric KiSS1 in NSCLC patients as compared to healthy donors. KiSS1 serum concentrations, after surgical procedure and/or adjuvant therapy. We observed differences among disease stages in urine samples. In preclinical models,Our results showing a peculiar modulation of KiSS1 levels in liquid biopsies of NSCLC patients and a regulation of cisplatin-induced apoptosis by KiSS1-derived peptides support an involvement of KiSS1 in cell response to treatment and highlight its promising features as a potential biomarker in NSCLC.

Background Altered microRNA profiles have been observed not only in tumour tissues but also in biofluids, where they circulate in a stable form thus representing interesting biomarker candidates. This study aimed to identify a microRNA signature as a non-invasive biomarker and to investigate its impact on glioma biology. Methods MicroRNAs were selected using a global expression profile in preoperative serum samples from 37 glioma patients. Comparison between serum samples from age and gender-matched controls was performed by using the droplet digital PCR. The ROC curve and Kaplan-Meier survival analyses were used to evaluate the diagnostic/prognostic values. The functional role of the identified signature was assessed by gain/loss of function strategies in glioma cells. Results A three-microRNA signature (miR-1-3p/−26a-1-3p/−487b-3p) was differentially expressed in the serum of patients according to the isocitrate dehydrogenase (IDH) genes mutation status and correlated with both patient Overall and Progression Free Survival. The identified signature was also downregulated in the serum of patients compared to controls. Consistent with these results, the signature expression and release in the conditioned medium of glioma cells was lower in IDH-wild type cells compared to the mutated counterpart. Furthermore, in silico analysis of glioma datasets showed a consistent deregulation of the signature according to the IDH mutation status in glioma tumour tissues. Ectopic expression of the signature negatively affects several glioma functions. Notably, it impacts the glioma invasive phenotype by directly targeting the invadopodia-related proteins TKS4, TKS5 and EFHD2. Conclusions We identified a three microRNA signature as a promising complementary or even an independent non-invasive diagnostic/prognostic biomarker. The signature displays oncosuppressive functions in glioma cells and impacts on proteins crucial for migration and invasion, providing potential targets for therapeutic intervention.

Background: The cerebrospinal fluid (CSF)/serum albumin quotient (Q-Alb) is a marker of the blood-CSF barrier (BCSFB) and possibly of the blood–brain barrier (BBB). The latter is known to be altered in Alzheimer’s disease (AD) based on neuropathological and neuroimaging studies. Following investigations performed on clinically diagnosed cohorts, we aimed at comparing Q-Alb in cognitively impaired patients with neurochemical demonstration of AD pathophysiology and neurological disease controls (NDCs). Methods: We evaluated N = 144 AD patients (MCI, N = 43; AD dementia — ADD, N = 101) and N = 132 NDCs. AD patients were all A + according to the A/T/N framework and were neurochemically classified based on T and N parameters. Results: Q-Alb did not significantly differ between AD patients and NDCs. Moreover, it was not associated with disease stage (MCI vs. ADD), MMSE score, or CSF AD biomarkers. Discussion: Our study indicates that BCSFB dysfunction is not a specific feature of AD. When interpreting Q-Alb as a marker of the BBB, the lack of difference from NDCs might be due to BBB dysfunction widely occurring in other neurological, non-degenerative, conditions or — more probably — to low sensitivity of this biochemical parameter towards subtle BBB alterations causing leakage of molecules smaller than albumin. Furthermore, Q-Alb is not associated with the degree of global cognitive deterioration in AD, nor with CSF AD neurochemical biomarkers.

Rheumatoid meningitis (RM) is a rare but often aggressive neurological complication of rheumatoid arthritis. The diagnosis of RM, besides the clinical, radiological, and laboratory criteria, usually requires a cerebral biopsy. Based on the two cases presented in this paper, we propose a new laboratory marker. Cerebrospinal fluid and serum anti-cyclic citrullinated peptide (CCP) IgG were measured, and the intrathecal synthesis of anti-CCP antibodies (anti-CCP antibody index) was calculated using the hyperbolic function. The anti-CCP antibody index was positive in both cases at first diagnosis and progressively decreased after treatments. Together with clinical and radiological criteria, the calculation of the anti-CCP intrathecal synthesis, more than the simple measurement of serum or cerebrospinal fluid anti-CCP antibody titers, may represent a useful tool for RM diagnosis and, possibly, for treatment response.

Pericytes (PCs) are mesenchymal stromal cells (MSCs) that function as support cells and play a role in tissue regeneration and, in particular, vascular homeostasis. PCs promote endothelial cells (ECs) survival which is critical for vessel stabilization, maturation, and remodeling. In this study, PCs were isolated from human micro-fragmented adipose tissue (MFAT) obtained from fat lipoaspirate and were characterized as NG2

Glioblastoma multiforme (GBM) is considered the most malignant form of primary brain tumor. Despite multimodal treatment, prognosis remains poor. Ketogenic diet (KD) has been suggested for the treatment of GBM. In this study, the syngenic, orthotopic GL261 mouse glioma model was used to evaluate the effects of KD on the metabolic responses of the tumor using 7T magnetic resonance imaging/spectroscopy. GL261 cells were injected into the caudate nucleus of mice. Following implantation, animals were fed with standard chow or underwent a KD. 18 days after initiating the diet, mice fed with KD displayed significantly higher plasmatic levels of ketone bodies and survived longer than those fed with the standard diet. Decreased concentrations of gamma-aminobutyric acid, N-Acetyl-Aspartate and N-acetylaspartylglutamate were found in tumor tissue after 9 days into the KD, while a huge increase in beta-hydroxybutyrate (bHB) was detected in tumor tissue as compared to normal brain. The accumulation of bHB in the tumor tissue in mice undergoing the KD, may suggest either elevated uptake/release of bHB by tumor cells, or the inability of tumor cells in this context to use it for mitochondrial metabolism.

Translation of cell therapies into clinical practice requires the adoption of robust production protocols in order to optimize and standardize the manufacture and cryopreservation of cells, in compliance with good manufacturing practice regulations. Between 2012 and 2020, we conducted two phase I clinical trials (EudraCT 2009-014484-39, EudraCT 2015-004855-37) on amyotrophic lateral sclerosis secondary progressive multiple sclerosis patients, respectively, treating them with human neural stem cells. Our production process of a hNSC-based medicinal product is the first to use brain tissue samples extracted from fetuses that died in spontaneous abortion or miscarriage. It consists of selection, isolation and expansion of hNSCs and ends with the final pharmaceutical formulation tailored to a specific patient, in compliance with the approved clinical protocol. The cells used in these clinical trials were analyzed in order to confirm their microbiological safety; each batch was also tested to assess identity, potency and safety through morphological and functional assays. Preclinical, clinical and in vitro nonclinical data have proved that our cells are safe and stable, and that the production process can provide a high level of reproducibility of the cultures. Here, we describe the quality control strategy for the characterization of the hNSCs used in the above-mentioned clinical trials.

Moyamoya arteriopathy (MMA) is a rare cerebrovascular disorder that causes recurrent ischemic and hemorrhagic strokes, leading young patients to severe neurological deficits. The pathogenesis of MMA is still unknown. The disease onset in a wide number of pediatric cases raises the question of the role of genetic factors in the disease’s pathogenesis. In these patients, MMA’s clinical course, or progression, is largely unclear. By performing a comprehensive molecular and cellular profile in the plasma and CSF, respectively, of MMA pediatric patients, our study is aimed at assessing the levels of circulating endothelial progenitor cells (cEPC) and the release of selected proteins at an early disease stage to clarify MMA pathogenesis and progression. We employed cytofluorimetric methods and immunoassays in pediatric MMA patients and matched control subjects by age and sex. We detected increased levels of cEPC in peripheral blood and an upregulation of angiogenic markers in CSF (i.e., angiopoietin-2 and VEGF-A). This finding is probably associated with deregulated angiogenesis, as stated by the moderate severity of collateral vessel network development (Suzuki III-IV). The absence of significant modulation of neurofilament light in CSF led us to rule out the presence of substantial neuronal injury in MMA children. Despite the limited cohort of pediatric patients, we found some peculiar cellular and molecular characteristics in their blood and CSF samples. Our findings may be confirmed by wider and perspective studies to identify predictive or prognostic circulating biomarkers and potential therapeutic targets for personalized care of MMA pediatric patients.

Moyamoya arteriopathy (MA) is a rare cerebrovascular disorder characterized by ischemic/hemorrhagic strokes. The pathophysiology is unknown. A deregulation of vasculogenic/angiogenic/inflammatory pathways has been hypothesized as a possible pathophysiological mechanism. Since lipids are implicated in modulating neo-vascularization/angiogenesis and inflammation, their deregulation is potentially involved in MA. Our aim is to evaluate angiogenic/vasculogenic/inflammatory proteins and lipid profile in plasma of MA patients and control subjects (healthy donors HD or subjects with atherosclerotic cerebrovascular disease ACVD). Angiogenic and inflammatory protein levels were measured by ELISA and a complete lipidomic analysis was performed on plasma by mass spectrometry. ELISA showed a significant decrease for MMP-9 released in plasma of MA. The untargeted lipidomic analysis showed a cumulative depletion of lipid asset in plasma of MA as compared to HD. Specifically, a decrease in membrane complex glycosphingolipids peripherally circulating in MA plasma with respect to HD was observed, likely suggestive of cerebral cellular recruitment. The quantitative targeted approach demonstrated an increase in free sphingoid bases, likely associated with a deregulated angiogenesis. Our findings indicate that lipid signature could play a central role in MA and that a detailed biomarker profile may contribute to untangle the complex, and still obscure, pathogenesis of MA.

A new cationic Pt(II) complex bearing 8-aminoquinoline as chelating ligand (called Pt-8AQ) was evaluated against two human carcinomas, one mesothelioma, and three glioblastoma cell lines. The in vitro comparison to the clinically approved CisPt showed a minor activity of Pt-8AQ against carcinoma and mesothelioma, whereas a significant activity of Pt-8AQ was observed on the proliferation of the three glioblastoma cell lines (U87-MG IC50 = 3.68 ± 0.69 µM; U373-MG IC50 = 11.53 ± 0.16 µM; U138-MG IC50 = 8.05 ± 0.23 µM) that was higher than that observed with the clinically approved CisPt (U87-MG IC50 = 7.27 + 1.80 µM; U373-MG IC50 = 22.69 ± 0.05 µM; U138-MG IC50 = 32.1 ± 4.44 µM). Cell cycle analysis proved that Pt-8AQ significantly affected the cell cycle pattern by increasing the apoptotic cells represented by the sub G0/G1 region related with a downregulation of p53 and Bcl-2. Moreover, an NMR investigation of Pt-8AQ interaction with 9-EtG, GSH, and Mets7 excluded DNA as the main target, suggesting a novel mechanism of action. Our study demonstrated the high stability of Pt-8AQ after incubation at 37 °C and a significant antineoplastic activity on glioblastomas. These features also make Pt-8AQ a good candidate for developing a new selective advanced cell chemotherapy approach in combination with MSCs.

Objective: Marycin is a porphyrin-type compound synthetically modified to spontaneously release fluorescence. This study is aimed at understanding possible mechanisms that could account for the antiproliferative effects observed in marycin. A proteomic approach was used to identify molecular effects. The proteome of proliferating MDA-MB-231 breast cancer cells was compared with that of marycin-treated cells. Methods: Label-free proteomic analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to reveal changes in protein expression and fluorescence microscopy and flow cytometry were used to detect subcellular organelle dysfunctions. Results: The bioinformatic analysis indicated an enhancement of the expression of proteins remodeling RNA splicing and more in general, of RNA metabolism. Marycin did not localize into the mitochondria and did not produce a dramatic increase of ROS levels in MDA-MB-231 cells. Marycin stained organelles probably peroxisomes. Conclusions: The results could support the possibility that the peroxisomes are involved in cell response to marycin.

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.

Simple Summary Strategies to overcome resistance to targeted therapy represent a clinical need for metastatic melanoma. Altered lipid metabolism has been identified in the metabolic reprogramming associated with melanoma progression. Lipid metabolism genes such as fatty acid synthase (FASN) and 24-dehydrocholesterol reductase (DHCR24) have been proposed to contribute to tumor aggressiveness, but the therapeutic value of lipid metabolism inhibitors, particularly in drug-resistant melanoma, is unknown. Here, we found that molecular targeting of FASN in melanoma cells resistant to the BRAF inhibitor PLX4032 increased the sensitivity to the drug. Up-regulation of DHCR24 upon FASN targeting revealed the activation of druggable compensatory pathways sustaining the growth of resistant cells. Abstract Metabolic changes promoting cell survival are involved in metastatic melanoma progression and in the development of drug resistance. In BRAF-inhibitor resistant melanoma cells, we explored the role of FASN, an enzyme involved in lipogenesis overexpressed in metastatic melanoma. Resistant melanoma cells displaying enhanced migratory and pro-invasive abilities increased sensitivity to the BRAF inhibitor PLX4032 upon the molecular targeting of FASN and upon treatment with the FASN inhibitor orlistat. This behavior was associated with a marked apoptosis and caspase 3/7 activation observed for the drug combination. The expression of FASN was found to be inversely associated with drug resistance in BRAF-mutant cell lines, both in a set of six resistant/sensitive matched lines and in the Cancer Cell Line Encyclopedia. A favorable drug interaction in resistant cells was also observed with U18666 A inhibiting DHCR24, which increased upon FASN targeting. The simultaneous combination of the two inhibitors showed a synergistic interaction with PLX4032 in resistant cells. In conclusion, FASN plays a role in BRAF-mutated melanoma progression, thereby creating novel therapeutic opportunities for the treatment of melanoma.

Glioblastoma is characterized by a high proliferative rate and drug resistance. The standard of care includes maximal safe surgery, followed by radiotherapy and temozolomide chemotherapy. The expression of glutamate receptors has been previously reported in human glioma cell lines. The aim of this study was to examine the cellular effects of perampanel, a broad-spectrum antiepileptic drug acting as an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) glutamate receptor antagonist, alone or in combination with temozolomide. Four human glioma cell lines were exposed to different concentrations of perampanel and temozolomide, alone or in combination. The type of drug interaction was assessed using the Chou-Talalay method. Apoptosis, cell cycle perturbation, and glutamate receptors (GluRs) subunit expression were assessed by flow cytometry. Perampanel significantly inhibited the growth, inducing high levels of apoptosis. A strong synergistic effect of the combination of perampanel with temozolomide was detected in U87 and A172, but not in U138. Treatment with perampanel resulted in an increased GluR2/3 subunit expression in U87 and U138. Perampanel displays a pro-apoptotic effect on human glioblastoma cell lines when used alone, possibly due to increased GluR2/3 expression. The observed synergistic effect of the combination of temozolomide with perampanel suggests further investigation on the impact of this combination on oncologic outcomes in glioblastoma.

The use of the ketogenic diet (KD) as an adjuvant therapy in high-grade gliomas (HGG) is supported by preclinical studies, but clinical data on its effects on metabolism are currently lacking. In this study, we describe the effects of a KD on glucose profile, ketonemia, energy metabolism, and nutritional status, in adults affected by HGG. This was a single-arm prospective study. An isocaloric 3:1 KD was administered for 1 mo. Glucose profile was assessed by using fasting glycemia, insulin, and glycated hemoglobin. To evaluate ketonemia changes, a hand-held ketone meter was used from home. Energy metabolism was assessed by indirect calorimetry. Nutritional status was evaluated through changes in body composition and in lipid and hepatic profile. No changes in fasting glycemia were observed; however, insulinemia dropped to half of baseline levels. The KD shifted the metabolism, rising ketonemia and decreasing glucose oxidation rate to a quarter of the initial values. Moreover, the KD was generally safe. One-month intervention with the KD was able to act upon key metabolic substrates potentially involved in HGG metabolism. The lack of a significant reduction in fasting glycemia should be investigated in future studies.

Background Patients with neuroimmunological conditions such as multiple sclerosis (MS) often receive disease-modifying therapies (DMTs) or immunosuppressants which may reduce the response to vaccines. BNT162b2 (Pfizer-BioNTech) is the first COVID-19 vaccine authorized in Italy. Its clinical efficacy and serological response were not evaluated in MS patients receiving DMTs or immunosuppressants. This early multicenter study evaluated serological response to BNT162b2 and safety in these patients. Methods From February 2021 we enrolled consecutive MS patients, treated with at least one DMT and all healthcare workers (HCWs), having received or being scheduled to receive the first dose of BNT162b2. Blood samples were collected after the second vaccine dose and analyzed to quantitatively detect the presence of anti-Spike antibodies. Serological response was compared to the one from a control population of HCWs, with neither neuroimmunological conditions nor receiving immunosuppressants. Patients receiving treatments associated with a possible reduced response (Under-scrutiny treatment group) were also compared to those undergoing other treatments. Anti-Spike levels were described as median and interquartile range (IQR). Comparisons were performed with Wilcoxon-Mann-Whitney test. Solicited and unsolicited adverse events (AEs) were collected. Results 39 MS patients and a control population of 273 HCWs were included. One patient, under treatment with ocrelizumab, did not respond to BNT162b2, while all the remaining patients and all controls developed a serological response to the vaccine. Median anti-Spike levels were similar between patients (1471.0 BAU/ml; IQR 779.7 to 2357.0) and controls (1479.0 BAU/ml; IQR 813.1 to 2528.0) (p = 0.53). Patients included in the Under-scrutiny treatments group showed reduced anti-Spike levels (156.4 BAU/ml; IQR 33.4 to 559.1) compared to those receiving other treatments (1582.4 BAU/ml; IQR 1296.5 to 2219.0) (p = 0.001). Solicited AEs were all mild to moderate in severity, generally reported in the first days after vaccination, and resolved in the following days. Two MS patients reported a clinical relapse after the second vaccine dose. Conclusion BNT162b2 induced a serological response in MS patients treated with DMTs similar to controls not receiving DMTs or immunosuppressants. Some treatments were associated with reduced levels of anti-Spike antibodies in patients. These observations have relevant implications for treated patients receiving BNT162b2 and the community.

In spite of new knowledge on prostate cancer molecular landscape, this has been only partially translated to the therapeutic setting. The activation of Ras/Mitogen-activated protein kinase (MAPK) signaling plays an important role in progression of prostate cancer in which deregulation of histone deacetylases (HDAC) is frequent. Based on the notion that HDAC inhibitors may reactivate the expression of genes favoring cell response to drugs, the aim of this study was to investigate the interaction between the HDAC6-specific inhibitor ricolinostat (ACY1215) and the MEK-inhibitor selumetinib (AZD6244) to identify effective combinations in prostate cancer models. Using cell lines exhibiting differential activation of survival pathways (PC3, DU145, 22Rv1) and following different treatment schedules, a synergistic interaction was observed in all cell models, the drug combination being particularly effective in 22Rv1 cells. Marginal levels of apoptosis were observed in PC3 cells after combined treatment, whereas higher levels were achieved in DU145 and 22Rv1 cells. RNAi-mediated knockdown of HDAC6 in selumetinib-treated 22Rv1 cells resulted in increased apoptosis. Combined treatment suppressed the constitutively deregulated survival pathways in all cell lines. A decrease of androgen receptor (AR)-dependent gene (KLK2, DUSP1) mRNA levels was observed in 22Rv1 treated cells, associated with increased AR cytoplasmatic expression, suggesting AR signaling down-regulation, not involving Hsp90 acetylation. When a taxane was used in combination with AZD6244 and ACY1215 by a simultaneous schedule, a synergistic cytotoxic effect together with increased apoptosis was evidenced in all cell models. These results support a rational use of targeted agents to improve prostate cancer cell apoptotic response.

An innovative spectroscopic technique to determine the drug concentration in biological fluids is discussed. We introduce the context of drugs with narrow therapeutic index in relation to epilepsy and Parkinson’s disease. We then recapitulate the essentials of Raman and enhanced Raman spectroscopy that makes use of a corrugated metallic surface. Optimizing the intensification of the spectroscopic signatures of a given analyte critically depends on the metal choice and on the fine details of the induced surface nanostructuring. We review the topic with emphasis on noble metal surfaces synthesized by pulsed laser ablation in inert gas at high pressure. The performance of optimized surfaces to determine the drug concentration in different fluids, including human blood, is discussed with reference to carbamazepine, an anti-epileptic drug widely adopted in Developing Countries and to apomorphine, a drug used to treat via subcutaneous injection patients with important manifestations of Parkinson’s disease.

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.

OBJECTIVES We evaluated a prospective cohort of 150 patients under observation in our centre for lacunar strokes. The purpose of this study was to evaluate the outcome at time of discharge and 6 months after lacunar stroke, as well as the correlation with cardiovascular risk factors and selected biochemical parameters already evaluated on admission. Focus was to identify possible prognostic factors, which might be targeted through appropriate intervention concentrating on reduction in the incidence and impact of early clinical deterioration. METHODS 150 patients with a lacunar stroke were included in the present study. A clinical 6-month follow-up was available for 98.7% of the patients. Infarcts were classified by size, shape and location. RESULTS The most important predictors of high NIHSS score at time of discharge resulted NIHSS on admission (P

Surface-enhanced Raman scattering is a well-established technique for molecular detection at low concentration, which is becoming increasingly popular in the field of biotechnology and health sciences. Since the process is understood in depth, the technique is becoming reliable. In this contribution, we consider another aspect of SERS besides molecular detection, focusing on the binding mechanisms of a complex system such as a protein to the noble metal substrates required by the technique itself. We also show that using a solid nanostructured substrate produced by controlled pulsed laser deposition SERS enables label-free detection of a protein. This is checked on lysozyme as a well-known prototype. Use of solid substrates with controlled morphology proves advantageous over colloidal systems for SERS applications. Moreover, such substrates are superior in terms of shelf life, packaging and ease of shipment.

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.

By nanosecond pulsed laser ablation in an ambient gas gold nanoparticles (NPs) were produced that self assemble on a substrate resulting in increasingly elaborated architectures of growing thickness, from isolated NP arrays up to percolated films. NPs nucleate and grow in the plasma plume propagating through the gas. Process parameters including laser wavelength, laser energy density, target to substrate distance, nature and pressure of the gas affect plasma expansion, thus asymptotic NP size and kinetic energy. NP size, energy and mobility at landing determine film growth and morphology that affect the physicochemical properties of the film. Keeping fixed the other process parameters, we discuss the sensitive dependence of film surface nanostructure on Ar pressure and on laser pulse number. The initial plume velocity and average ablated mass per pulse allow predicting the asymptotic NP size. The control of growth parameters favors fine-tuning of NP aggregation, relevant to plasmonics to get optimized substrates for surface enhanced Raman spectroscopy (SERS). Their behavior is discussed for testing conditions of interest for clinical application. Both in aqueous and in biological solutions we obtained good sensitivity and reproducibility of the SERS signals for the anti-Parkinson drug apomorphine, and for the anti-epilepsy drug carbamazepine. (C) 2016 Elsevier B.V. All rights reserved.

Metastatic spread is mainly sustained by cancer stem cells (CSC), a subpopulation of cancer cells that displays stemness features. CSC are thought to be derived from cancer cells that undergo epithelial to mesenchymal transition (EMT), thus acquiring resistance to anoikis and anti-cancer drugs. After detachment from the primary tumor mass, CSC reach the blood and lymphatic flow, and disseminate to the target tissue. This process is by nature dynamic and in vitro models are quite far from the in vivo situation. In this study, we have tried to reproduce the adhesion process of CSC to a target tissue by using a 3D dynamic cell culture system. We isolated two populations of 3D tumor spheroids displaying CSC-like features from breast carcinoma (MCF-7) and lung carcinoma (A549) cell lines. Human fibroblasts were layered on a polystyrene scaffold placed in a dynamically perfused millifluidic system and then the adhesion of tumor cell derived from spheroids to fibroblasts was investigated under continuous perfusion. After 24 h of perfusion, we found that spheroid cells tightly adhered to fibroblasts layered on the scaffold, as assessed by a scanning electron microscope (SEM). To further investigate mechanisms involved in spheroid cell adhesion to fibroblasts, we tested the effect of three RGD integrin antagonists with different molecular structures on cell adhesion, when injected into the circuit, only cilengitide was able to inhibit cell adhesion to fibroblasts. Although our model needs further refinements and improvements, we do believe this study could represent a promising approach in improving current models to study metastatic infiltration in vitro and a new tool to screen new potential anti-metastatic molecules.

Glioblastoma multiforme is the most malignant subtype of brain tumor. Despite multimodal treatment (surgical resection and chemo/radiotherapy) the prognosis remains unsatisfactory. Based on the Warburg hypothesis, ketogenic diet (KD) has been suggested in the treatment of GBM. The syngenic, orthotopic GL261 mouse glioma model was used to evaluate the effects of KD on 7T magnetic resonance imaging/spectroscopy and metabolic response of the tumor to diet. Mice were injected with 10^5 GL261 cells into the caudate nucleus. Following implantation, animals were fed standard chow for five days then were randomly assigned to standard diet or ketogenic diet. 18 days after diet start, mice fed at KD displayed significantly higher plasmatic levels of ketone bodies. Mice fed with KD survived longer than those fed with standard diet (p< 0.05). Decreased concentrations of gamma-aminobutyric acid, N Acetyl Aspartate and N-acetylaspartylglutamate were found in tumor tissue after 9 days from the beginning of the KD diet while a huge increase in beta-hydroxybutyrate (bHB) was detected in tumor tissue as compared to normal brain. The addition of bHB at various concentrations to low-glucose culture medium did not significantly improve GL261 in vitro growth suggesting that this cell line has a limited ability to use bHB as a carbon source. The accumulation of bHB in the tumor tissue in KD fed mice, may suggest either elevated uptake of/release of bHB by tumor cells or inability of tumor cells in this context to use it in mitochondrial metabolism; the latter hypothesis is supported by the observation that GL261 cells did not display an increase in in vitro proliferation when exposed to bHB. The far less evident peak of bHB at MRI spectroscopy in the healthy brain tissue of KD fed mice, on the other hand suggests that normal brain is able to use bHB as energy source.

International audience; Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the corticospinal tract and leading to motor neuron death. According to a recent study, magnetic resonance imaging-visible changes suggestive of neurodegeneration seem absent in the motor cortex of G93A-SOD1 ALS mice. However, it has not yet been ascertained whether the cortical neural activity is intact, or alterations are present, perhaps even from an early stage. Here, cortical neurons from this model were isolated at post-natal day 1 and cultured on multielectrode arrays. Their activity was studied with a comprehensive pool of neurophysiological analyses probing excitability, criticality and network architecture, alongside immunocytochemistry and molecular investigations. Significant hyperexcitability was visible through increased network firing rate and bursting, whereas topological changes in the synchronization patterns were apparently absent. The number of dendritic spines was increased, accompanied by elevated transcriptional levels of the DLG4 gene, NMDA receptor 1 and the early pro-apoptotic APAF1 gene. The extracellular Na+, Ca2+, K+ and Cl- concentrations were elevated, pointing to perturbations in the culture micro-environment. Our findings highlight remarkable early changes in ALS cortical neuron activity and physiology. These changes suggest that the causative factors of hyperexcitability and associated toxicity could become established much earlier than the appearance of disease symptoms, with implications for the discovery of new hypothetical therapeutic targets.

Background Platinum-based therapy represents the main pharmacological treatment for ovarian carcinoma. Since molecular targeting of receptor tyrosine kinases (RTK) affects factors that may modulate drug response, the aim of this study was to examine whether downstream targets of AXL RTK could be exploited to improve cell response to cisplatin. Materials and methods Inhibitors of p38 (SB203580) and of signal transducer and activator of transcription 3 (stattic) were employed in combination with cisplatin in ovarian carcinoma cell lines. Apoptosis assay and western blot analysis were performed to evaluate cell response after treatment. Results SB203580 produced a synergistic effect in combination with cisplatin in cisplatin-resistant IGROV-1/Pt1 cells. In addition, a favorable drug interaction was observed in A2780 cells when pre-incubated with cisplatin prior to stattic. The analysis of cell response after combined treatment showed down-regulation of the pro-apoptotic protein BCL2-associated agonist of cell death (BAD). Conclusion Our results support the notion that downstream targets of AXL in ovarian carcinoma cells can be exploited to increase cisplatin activity in ovarian carcinoma models.

A delicate balance of reactive oxygen species (ROS) exists inside the cell: when the mechanisms that control the level of ROS fail, the cell is in an oxidative stress state, a condition that can accelerate aging processes. To contrast the pro-aging effect of ROS, the supplementation of antioxidants has been recently proposed. Sulforaphane (SFN) is an isothiocyanate isolated from Brassica plants that has been shown to modulate many critical factors inside the cells helping to counteract aging processes. In the present work, we exposed human dermal fibroblast to short, sublethal and repeated treatments with hydrogen peroxide for eight days, without or in combination with low concentration of SFN. Hydrogen peroxide treatments did not affect the oxidative status of the cells, without any significant change of the intracellular ROS levels or the number of mitochondria or thiols in total proteins. However, our regime promoted cell cycle progression and cell viability, increased the anti-apoptotic factor survivin and increased DNA damage, measured as number of foci positive for &gamma, H2AX. On the other hand, the treatment with SFN alone seemed to exert a protective effect, increasing the level of p53, which can block the expansion of possible DNA damaged cells. However, continued exposure to SFN at this concentration could not protect the cells from stress induced by hydrogen peroxide.

Preoperative prognostic nutritional index (PNI) is linked to the clinical outcome of patients with malignant tumours, however few studies have investigated its utility in predicting outcome in glioblastoma multiforme (GBM). We performed a retrospective study on adult patients with GBM in order to evaluate the impact of PNI on overall survival (OS), after adjusting for known prognostic factor (age, extent of surgery, Karnofsky performance status, radiochemotherapy). This is an Italian, multicentre, retrospective, cohort study. The patient's cohort includes 282 individuals with a newly diagnosed GBM followed in 3 Lombardia Hospitals In all cases the diagnosis was supported by histological data. Patient's information including sex, age at onset, Karnofsky performance status (KPS), extension of surgical resection (EOR), adjuvant treatment, antiepileptic treatment, serum variables and survival data were collected. Univariate and multivariate analysis did not reveal an association between PNI and overall survival in our series of GBM patients. PNI is a controversial marker for prognosis in GBM patients and further prospective studies are necessary to elucidate its role.

The synthesis by pulsed laser ablation and the characterization of both the surface nanostructure and the optical properties of noble metal nanoparticle-based substrates used in Surface Enhanced Raman Spectroscopy are discussed with reference to application in the detection of anti-epileptic drugs. Results on two representative drugs, namely Carbamazepine and Perampanel, are critically addressed.

GENetics of mOyaMoyA (GEN-O-MA) project is a multicenter observational study implemented in Italy aimed at creating a network of centers involved in moyamoya angiopathy (MA) care and research and at collecting a large series and bio-repository of MA patients, finally aimed at describing the disease phenotype and clinical course as well as at identifying biological or cellular markers for disease progression. The present paper resumes the most important study methodological issues and preliminary results. Nineteen centers are participating to the study. Patients with both bilateral and unilateral radiologically defined MA are included in the study. For each patient, detailed demographic and clinical as well as neuroimaging data are being collected. When available, biological samples (blood, DNA, CSF, middle cerebral artery samples) are being also collected for biological and cellular studies. Ninety-eight patients (age of onset mean ± SD 35.5 ± 19.6 years; 68.4% females) have been collected so far. 65.3% of patients presented ischemic (50%) and haemorrhagic (15.3%) stroke. A higher female predominance concomitantly with a similar age of onset and clinical features to what was reported in previous studies on Western patients has been confirmed. An accurate and detailed clinical and neuroimaging classification represents the best strategy to provide the characterization of the disease phenotype and clinical course. The collection of a large number of biological samples will permit the identification of biological markers and genetic factors associated with the disease susceptibility in Italy.

Background aims Pancreatic cancer (pCa) is a tumor characterized by a fibrotic state and associated with a poor prognosis. The observation that mesenchymal stromal cells (MSCs) migrate toward inflammatory micro-environments and engraft into tumor stroma after systemic administration suggested new therapeutic approaches with the use of engineered MSCs to deliver and produce anti-cancer molecules directly within the tumor. Previously, we demonstrated that without any genetic modifications, MSCs are able to deliver anti-cancer drugs. MSCs loaded with paclitaxel by exposure to high concentrations release the drug both in vitro and in vivo , inhibiting tumor proliferation. On the basis of these observations, we evaluated the ability of MSCs (from bone marrow and pancreas) to uptake and release gemcitabine (GCB), a drug widely used in pCa treatment. Methods MSCs were primed by 24-h exposure to 2000 ng/mL of GCB. The anti-tumor potential of primed MSCs was then investigated by in vitro anti-proliferation assays with the use of CFPAC-1, a pancreatic tumor cell line sensitive to GCB. The uptake/release ability was confirmed by means of high-performance liquid chromatography analysis. A cell-cycle study and secretome evaluation were also conducted to better understand the characteristics of primed MSCs. Results GCB-releasing MSCs inhibit the growth of a human pCa cell line in vitro . Conclusions The use of MSCs as a "trojan horse" can open the way to a new pCa therapeutic approach; GCB-loaded MSCs that integrate into the tumor mass could deliver much higher concentrations of the drug in situ than can be achieved by intravenous injection.

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The malignancy of glioblastoma (GB) is primarily due to the ability of glioma cancer stem cells (GSC) to disseminate into surrounding brain tissues, despite surgery and chemotherapy, and to form new tumoral masses. Members of the RGD-binding integrin family, which recognize the arginine-glycine-aspartic acid (RGD) sequence present in components of the extracellular matrix, and which serve a crucial function in the dissemination of GCS, are overexpressed in GB. Small-molecule integrin antagonists (SMIAs) designed to recognize RGD-integrins may therefore be an effective tool for decreasing GB infiltration and recurrence. In the present study, in vitro pro-apoptotic and infiltrative effects elicited by the SMIA 1a‑RGD in human GSC were investigated. Reverse transcription-quantitative polymerase chain reaction analysis revealed that, compared with normal human astrocytes, GSC grown on laminin-coated dishes overexpressed stemness markers as well as αvβ3 and αvβ5 integrins. In addition, dissociated GSC were identified to exhibit tumorigenic capacity when injected into immunodeficient mice. Using annexin/fluorescence-activated cell sorting analysis and ELISA nucleosome assays, it was identified that treatment of GSC with 25 µM 1a‑RGD for 48 h elicited detachment‑dependent anoikis not accompanied by necrosis-dependent cell death. A colorimetric proliferation assay indicated that 1a‑RGD did not affect cell viability, but that, instead, it markedly inhibited GSC migration as assessed using a Transwell assay. Western blot experiments revealed a decrease in focal adhesion kinase and protein kinase B phosphorylation with a concomitant increase in caspase-9 and -3/7 activity following 1a‑RGD treatment, suggesting that the pro-anoikis effects of 1a‑RGD may be mediated by these molecular mechanisms. Western blot analysis revealed no changes in specific markers of autophagy, suggesting further that 1a‑RGD-induced cell death is primarily sustained by anoikis-associated mechanisms. In conclusion, the results of the present study indicate that SMIA have potential as a therapeutic tool for decreasing GSC dissemination.

Over the last few years, human microfragmented adipose tissue (MFAT), containing significant levels of mesenchymal stromal cells (MSCs) and obtained from fat lipoaspirate (LP) through a minimal manipulation in a closed system device, has been successfully used in aesthetic medicine as well as in orthopedic and general surgery. Interestingly, in orthopedic diseases, this ready-to-use adipose tissue cell derivative seems to have a prolonged time efficacy even upon a single shot injection into osteoarthritic tissues. Here, we investigated the long-term survival and content of MSCs as well the anti-inflammatory activity of LP and its derived MFAT in vitro, with the aim to better understand a possible in vivo mechanism of action. MFAT and LP specimens from 17 human donors were investigated side by side. During a long-term culture in serum-free medium, we found that the total cell number as well the MSC content in MFAT decreased more slowly if compared to those from LP specimens. The analysis of cytokines and growth factors secreted into the conditioned medium (CM) was similar in MFAT and LP during the first week of culture, but the total amount of cytokines secreted by LP decreased much more rapidly than those produced by MFAT during prolonged culture (up to 28 days). Similarly, the addition of MFAT-CM recovered at early (3-7 days) and late stage (14-28 days) of culture strongly inhibited inflammatory function of U937 monocyte cell line, whereas the anti-inflammatory activity of LP-CM was drastically reduced after only 7 days of culture. We conclude that MFAT is an effective preparation with a long-lasting anti-inflammatory activity probably mediated by a long-term survival of their MSC content that releases a combination of cytokines that affect several mechanisms involved in inflammation processes.

In this study, the in vitro stability of cisplatin (CisPt) and cationic platinum(II)-complex (caPt(II)-complex) and their in vitro activity (antiproliferative and anti-angiogenic properties) were investigated against three aggressive human tumor cell lines. caPt(II)-complex shown a high stability until 9 days of treatment and displayed a significant and higher activity than CisPt against both NCI-H28 mesothelioma (19.37 ± 9.57 μM versus 34.66 ± 7.65 μM for CisPt) and U87 MG glioblastoma (19.85 ± 0.97 μM versus 54.14 ± 3.19 for CisPt). Mesenchymal Stromal Cells (AT-MSCs) showed a significant different sensitivity (IC 50 = 71.9 ± 15.1 μM for caPt(II)-complex and 8.7 ± 4.5 μM for CisPt) to the antiproliferative activity of caPt(II)-complex and CisPt. The ability of MSCs to uptake both the drugs in a similar amount of 2.49 pM /cell, suggested a possible development of new therapies based on cell mediated drug delivery.

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.

One of the crucial and unsolved problems of the airborne carbon nanoparticles is the role played by the adsorbed environmental pollutants on their toxicological effect. Indeed, in the urban areas, the carbon nanoparticles usually adsorb some atmospheric contaminants, whose one of the leading representatives is the benzo(α)pyrene. Herein, we used the proteomics to investigate the alteration of toxicological pathways due to the carbon nanopowder-benzo(α)pyrene complex in comparison with the two contaminants administered alone on human skin-derived fibroblasts (hSDFs) exposed for 8 days in semi-static conditions. The preliminary confocal microscopy observations highlighted that carbon-nanopowder was able to pass through the cell membranes and accumulate into the cytoplasm both when administered alone and with the adsorbed benzo(α)pyrene. Proteomics revealed that the effect of carbon nanopowder-benzo(α)pyrene complex seems to be related to a new toxicological behavior instead of simple additive or synergistic effects. In detail, the cellular pathways modulated by the complex were mainly related to energy shift (glycolysis and pentose phosphate pathway), apoptosis, stress response and cellular trafficking.

In this chapter are illustrated the context and activities that have been pursued to develop a surface enhanced Raman spectroscopy technique for clinical applications in therapeutic drug monitoring with focus on anti-epileptic and anti-Parkinson drugs. We discuss how the surface nanostructure of sensors made of arrays of noble metal nanoparticle obtained by pulsed laser ablation in inert gas and in liquid affects their optical properties, in particular the wavelength, intensity and width of the surface plasmon. These in turn are shown to strongly influence the intensity and the spatial reproducibility of the enhanced Raman signals as functions of drug concentration.

Artificially roughened gold surfaces with controlled nanostructure produced by pulsed laser deposition have been investigated as sensors for apomorphine detection aiming at clinical application. The use of such gold surfaces has been optimized using aqueous solutions of apomorphine in the concentration range between 3.3 × 10−4 M and 3.3 × 10−7 M. The experimental parameters have been investigated and the dynamic concentration range of the sensor has been assessed by the selection of two apomorphine surface enhanced Raman scattering (SERS) peaks. The sensor behavior used to detect apomorphine in unfiltered human blood plasma is presented and discussed.

Background aims In attempting to develop new strategies to circumvent the immunosuppression associated with glioblastoma (GB), novel approaches have been designed using dendritic cell (DC)-based vaccination, which is considered a promising strategy to attack high-grade glioma. In previous studies, we demonstrated that human mesenchymal stromal cells without genetic manipulation but primed with Paclitaxel (PTX) acquire a potent anti-tumor activity, providing an interesting new biological approach for drug delivery. On the basis of these results, we here investigated whether both CD14+ and their derived DCs may behave like mesenchymal stromal cells acquiring anti-tumor activity on priming with PTX. Methods Human CD14+ cells were isolated from peripheral blood. Fluorescence-activated cell sorter analysis was performed to determine the purity of CD14+ and their differentiation into mature DCs. Cells were primed by incubation with 1 μg/mL of PTX for 24 h, and the PTX released by cells was assessed by mass spectrometry analysis. Anti-tumor activity was checked by testing the conditioned medium (CM) on the proliferation of U87 MG, a GB cell line. Results Both CD14+ and DCs were able to incorporate PTX and release the drug in the CM in a time-dependent manner (maximal release over 24 h). The addition of CM from CD14+ and DCs loaded with PTX strongly inhibits proliferation of U87 MG cells. Conclusions Our results are the first demonstration that peripheral blood–derived CD14+ and DCs, in addition to their application for immunotherapy for GB, could also be used to delivery anti-cancer drugs, such as PTX, to kill GB cells.

We developed anin vitrocontact through-feet blood brain barrier (BBB) model built using type IV collagen, rat astrocytes, and human umbilical vein endothelial cells (HUVECs) cocultured through Transwell porous polycarbonate membrane. The contact between astrocytes and HUVECs was demonstrated by electron microscopy: astrocytes endfeet pass through the 8.0 μm pores inducing HUVECs to assume a cerebral phenotype. Using this model we evaluated transmigration of melanoma cells from two different patients (M1 and M2) selected among seven melanoma primary cultures. M2 cells showed a statistically significant higher capability to pass across thein vitroBBB model, compared to M1. Expression of adhesion molecules was evaluated by flow cytometry: a statistically significant increased expression of MCAM,αvβ3, and CD49b was detected in M1. PCR array data showed that M2 had a higher expression of several matrix metalloproteinase proteins (MMPs) compared to M1. Specifically, data suggest that MMP2 and MMP9 could be directly involved in BBB permeability and that brain invasion by melanoma cells could be related to the overexpression of many MMPs. Future studies will be necessary to deepen the mechanisms of central nervous system invasion.

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

Botulin toxin (BTX) is widely used for treating skeletal muscle spasticity. Experimental reports on BTX treatment were mainly focused on the neuromuscular junction, while relatively little is known about toxin effects on the muscle cell itself. We investigated possible impact of BTX type A on skeletal muscle cell transcriptome by microarray analysis in muscle-derived cell cultures (fibroblasts, myoblasts and myotubes) from controls and spastic patients, and results were then validated at transcript and protein level. BTX-A treatment of control cells induced major changes in the myogenic component of the transcriptome, whereas the same treatment had a negligible effect in the fibrogenic component. BTX-A treatment of cell cultures from spastic patients induced an increased number of genes differentially expressed both in the fibrogenic and myogenic components. Specifically, BTX-A had a major effect on cell cycle-related genes in myoblasts, on muscle contraction-related genes in myotubes, and on extracellular matrix-related genes in fibroblasts from spastic patients. Our findings show that in vitro BTX-A treatment differentially affects transcript expression in muscle cells from spastic patients compared to those from controls suggesting a direct effect of BTX-A on muscle-specific functional pathways.

Chronic migraine (CM) is a disabling condition arising from a complex mixture of interconnected biological, psychological and social factors, and is often associated with medication overuse (MO). Mindfulness is emerging as a helpful treatment for pain, and one study showed that the longitudinal 12 months’ course of CM-MO patients that attended mindfulness-based treatment alone was similar to that of patients receiving medical prophylaxis alone; in this study, we describe the course of biomarkers of inflammation. Our results provide initial evidence of sustained similar effects on reduced concentration of biomarkers of inflammation, although not sizeable enough to reach statistical significance. Whether more intensive treatment and/or larger samples would lead to greater changes is unknown, but these encouraging preliminary findings suggest further research is warranted.