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Background: Malignant Pleural Mesothelioma (MPM) is an aggressive tumor that has a significant incidence related to asbestos exposure with no effective therapy and poor prognosis. The role of mesenchymal stromal cells (MSCs) in cancer is controversial due to their opposite effects on tumor growth and in particular, only a few data are reported on MSCs and MPM. Methods: We investigated the in vitro efficacy of adipose tissue-derived MSCs, their lysates and secretome against different MPM cell lines. After large-scale production of MSCs in a bioreactor, their efficacy was also evaluated on a human MPM xenograft in mice. Results: MSCs, their lysate and secretome inhibited MPM cell proliferation in vitro with S or G0/G1 arrest of the cell cycle, respectively. MSC lysate induced cell death by apoptosis. The efficacy of MSC was confirmed in vivo by a significant inhibition of tumor growth, similar to that produced by systemic administration of paclitaxel. Interestingly, no tumor progression was observed after the last MSC treatment, while tumors started to grow again after stopping chemotherapeutic treatment. Conclusions: These data demonstrated for the first time that MSCs, both through paracrine and cell-to-cell interaction mechanisms, induced a significant inhibition of human mesothelioma growth. Since the prognosis for MPM patients is poor and the options of care are limited to chemotherapy, MSCs could provide a potential new therapeutic approach for this malignancy.

Moyamoya angiopathy (MMA) is a peculiar cerebrovascular condition characterized by progressive steno-occlusion of the terminal part of the internal carotid arteries (ICAs) and their proximal branches, associated with the development of a network of fragile collateral vessels at the base of the brain. The diagnosis is essentially made by radiological angiographic techniques. MMA is often idiopathic (moyamoya disease-MMD); conversely, it can be associated with acquired or hereditary conditions (moyamoya Syndrome-MMS); however, the pathophysiology underlying either MMD or MMS has not been fully elucidated to date, and this poor knowledge reflects uncertainties and heterogeneity in patient management. MMD and MMS also have similar clinical expressions, including, above all, ischemic and hemorrhagic strokes, then headaches, seizures, cognitive impairment, and movement disorders. The available treatment strategies are currently shared between idiopathic MMD and MMS, including pharmacological and surgical stroke prevention treatments and symptomatic drugs. No pharmacological treatment able to reverse the progressive disappearance of the ICAs has been found to date in both idiopathic and syndromic cases. Antithrombotic agents are usually prescribed in ischemic MMA, although the coexisting hemorrhagic risk should be considered. Surgical revascularization techniques, which are currently the best available treatment in symptomatic MMA, are associated with good long-term outcomes and reduced ischemic and hemorrhagic risks. Given the lack of dedicated randomized clinical trials, current treatment is mainly based on observational studies and physicians’ and surgeons’ expertise.

Background: Adipose tissue-derived MSCs engineered with the tumor necrosis factor- related apoptosis-inducing ligand protein (MSCs-TRAIL) have significant anticancer activity. MSCs, without any genetic modifications, exposed to high doses of chemotherapeutic agents are able to uptake the drug and release it in an amount affecting tumor proliferation. The purpose of this study was to verify the ability of MSCs-TRAIL to uptake and release paclitaxel (PTX) by providing an increased antitumor efficacy. Methods: MSCs and MSCs-TRAIL were tested for their sensitivity to Paclitaxel (PTX) by MTT assay, and the cells were loaded with PTX according to a standardized procedure. The secretome was analysed by HPLC for the presence of PTX, microarray assay for soluble TRAIL (s-TRAIL) and tested for in vitro anticancer activity. Results: MSCs-TRAIL were resistant to PTX and able to incorporate and then release the drug. The secretion of s-TRAIL by PTX loaded MSCs-TRAIL was not inhibited, and the PTX delivery together with s-TRAIL secretion resulted in increased antitumor efficacy of cell secretome as tested in vitro on human pancreatic carcinoma (CFPAC-1) and glioblastoma (U87-MG). Conclusions: Our result is the first demonstration of the possible merging of two new MSCs therapy approaches based on genetic manipulation and drug delivery. If confirmed in vivo, this could potentiate the efficacy of MSCs-TRAIL and strongly contribute to reducing the toxicity due to the systemic treatment of PTX.

Cerebral amyloid angiopathy (CAA), one of the main types of cerebral small vessel disease, is a major cause of spontaneous intracerebral haemorrhage and an important contributor to cognitive decline in elderly patients. Despite the number of experimental in vitro studies and animal models, the pathophysiology of CAA is still largely unknown. Although several pathogenic mechanisms including an unbalance between production and clearance of amyloid beta (Aβ) protein as well as ‘the prion hypothesis’ have been invoked as possible disease triggers, they do not explain completely the disease pathogenesis. This incomplete disease knowledge limits the implementation of treatments able to prevent or halt the clinical progression. The continuous increase of CAA patients makes imperative the development of suitable experimental in vitro or animal models to identify disease biomarkers and new pharmacological treatments that could be administered in the early disease stages to prevent irreversible changes and disease progression.

Mesenchymal stromal cells (MSCs) prepared as advanced therapies medicinal products (ATMPs) have been widely used for the treatment of different diseases. The latest developments concern the possibility to use MSCs as carrier of molecules, including chemotherapeutic drugs. Taking advantage of their intrinsic homing feature, MSCs may improve drugs localization in the disease area. However, for cell therapy applications, a significant number of MSCs loaded with the drug is required. We here investigate the possibility to produce a large amount of Good Manufacturing Practice (GMP)-compliant MSCs loaded with the chemotherapeutic drug Paclitaxel (MSCs-PTX), using a closed bioreactor system. Cells were obtained starting from 13 adipose tissue lipoaspirates. All samples were characterized in terms of number/viability, morphology, growth kinetics, and immunophenotype. The ability of MSCs to internalize PTX as well as the antiproliferative activity of the MSCs-PTX in vitro was also assessed. The results demonstrate that our approach allows a large scale expansion of cells within a week, the MSCs-PTX, despite a different morphology from MSCs, displayed the typical features of MSCs in terms of viability, adhesion capacity, and phenotype. In addition, MSCs showed the ability to internalize PTX and finally to kill cancer cells, inhibiting the proliferation of tumor lines in vitro. In summary our results demonstrate for the first time that it is possible to obtain, in a short time, large amounts of MSCs loaded with PTX to be used in clinical trials for the treatment of patients with oncological diseases.

Dendritic cells (DC) are the most potent antigen-presenting cells, strongly inducers of T cell-mediated immune responses and, as such, broadly used as vaccine adjuvant in experimental clinical settings. DC are widely generated from human monocytes following in vitro protocols which require 5&ndash, 7 days of differentiation with GM-CSF and IL-4 followed by 2&ndash, 3 days of activation/maturation. In attempts to shorten the vaccine&rsquo, s production, Fast-DC protocols have been developed. Here we reported a Fast-DC method in compliance with good manufacturing practices for the production of autologous mature dendritic cells loaded with antigens derived from whole tumor lysate, suitable for the immunotherapy in glioblastoma patients. The feasibility of generating Fast-DC pulsed with whole tumor lysate was assessed using a series of small-scale cultures performed in parallel with clinical grade large scale standard method preparations. Our results demonstrate that this Fast protocol is effective only in the presence of PGE2 in the maturation cocktail to guarantee that Fast-DC cells exhibit a mature phenotype and fulfill all requirements for in vivo use in immunotherapy approaches. Fast-DC generated following this protocol were equally potent to standard DC in inducing Ag-specific T cell proliferation in vitro. Generation of Fast-DC not only reduces labor, cost, and time required for in vitro clinical grade DC development, but can also minimizes inter-preparations variability and the risk of contamination.

The pathophysiological mechanisms of Moyamoya angiopathy (MA), which is a rare cerebrovascular condition characterized by recurrent ischemic/hemorrhagic strokes, are still largely unknown. An imbalance of vasculogenic/angiogenic mechanisms has been proposed as one possible disease aspect. Circulating endothelial progenitor cells (cEPCs) have been hypothesized to contribute to vascular remodeling of MA, but it remains unclear whether they might be considered a disease effect or have a role in disease pathogenesis. The aim of the present study was to provide a morphological, phenotypical, and functional characterization of the cEPCs from MA patients to uncover their role in the disease pathophysiology. cEPCs were identified from whole blood as CD45dimCD34+CD133+ mononuclear cells. Morphological, biochemical, and functional assays were performed to characterize cEPCs. A significant reduced level of cEPCs was found in blood samples collected from a homogeneous group of adult (mean age 46.86 &plusmn, 11.7, 86.36% females), Caucasian, non-operated MA patients with respect to healthy donors (HD, p = 0.032). Since no difference in cEPC characteristics and functionality was observed between MA patients and HD, a defective recruitment mechanism could be involved in the disease pathophysiology. Collectively, our results suggest that cEPC level more than endothelial progenitor cell (EPC) functionality seems to be a potential marker of MA. The validation of our results on a larger population and the correlation with clinical data as well as the use of more complex cellular model could help our understanding of EPC role in MA pathophysiology.

Cerebral amyloid angiopathy (CAA) is one of the major types of cerebral small vessel disease, and a leading cause of spontaneous intracerebral hemorrhage and cognitive decline in elderly patients. Although increasingly detected, a number of aspects including the pathophysiology, the clinical and neuroradiological phenotype, and the disease course are still under investigation. The incomplete knowledge of the disease limits the implementation of evidence-based guidelines on patient's clinical management and the development of treatments able to prevent or reduce disease progression. The SENECA (SEarchiNg biomarkErs of Cerebral Angiopathy) project is the first Italian multicenter cohort study aimed at better defining the disease natural history and identifying clinical and neuroradiological markers of disease progression. By a multidisciplinary approach and the collection of a large and well-phenotyped series and biorepository of CAA patients, the study is ultimately expected to improve the diagnosis and the knowledge of CAA pathophysiological mechanisms.

Objective We assessed sleep patterns in 85 patients with chronic disorders of consciousness (DOC) in order to reveal any relationship with the degree of the impairment. Methods Nocturnal polysomnography (PSG) was scored in patients classified as being in an unresponsive wakefulness syndrome/vegetative state (UWS/VS; n = 49) or a minimally conscious state (MCS; n = 36) in accordance with the rules of the American Academy of Sleep Medicine. The PSG data in the two diagnostic groups were compared, and the PSG parameters associated with the degree of impairment were analysed. Results In 19/49 UWS/VS patients, signal attenuation was the only EEG pattern detectable in sleep. Non-REM 2 (NREM2) and slow-wave sleep (SWS) (but not REM) stages were more frequent in the MCS patients. The presence of SWS was the most appropriate factor for classifying patients as UWS/VS or MCS, and the duration of SWS was the main factor that significantly correlated with revised Coma Recovery Scale scores. Conclusion The presence of NREM sleep (namely SWS) reflects better preservation of the circuitry and structures needed to sustain this stage of sleep in DOC patients. Significance PSG is a simple and effective technique, and sleep patterns may reflect the degree of impairment in chronic DOC patients.

Primary Angiitis of the Central Nervous System (PACNS) is a rare form of idiopathic CNS vasculitis. Neuroimaging is often abnormal and characterized by multifocal brain lesions, but brain biopsy definitely confirms the diagnosis. We report the rare case of a 45-year-old female presenting with symptoms of intracranial hypertension and leukodystrophy-like neuroimaging findings. A comprehensive diagnostic work-up led to the unexpected diagnosis of a definite PACNS which was successfully treated by immunosuppressive treatment. Although rarely, PACNS can present as diffuse leukoencephalopathy on neuroimaging, and mimic even an inherited leukodystrophy. Therefore, in adults with leukodystrophy-like neuroimaging findings, careful examination of clinical and non-clinical features is mandatory to avoid missing the diagnosis of a treatable acquired disease.

Background Several methodologies including neuroimaging and sleep evaluation are being developed to complement the clinical bedside examinations in patients with disorder of consciousness (DOC). Recently, we demonstrated a possible association between Period3 (Per3) variable number tandem repeat (VNTR) polymorphism and functional impairment of DOC patients, speculating a possible role of this gene in sleep regulation. Aim To assess whether the degree of structural and metabolic damage of the main brain areas involved in the sleep generation and homeostasis may influence the different outcome of DOC patients carrying the Per3 5/5 genotype in comparison to Per3 4/4 ones. Methods For the present study, we reviewed 44 DOC patients from the Coma Research Centre of the Fondazione IRCCS Istituto Neurologico “C. Besta” of Milan. All patients underwent to polysomnographic sleep evaluation, cerebral structural magnetic resonance imaging (MRI) and 18 F–fluoro-2-deoxyglucose positron emission tomography (FDG-PET) analysis. Results Our DOC patients presented a moderate anatomical (median score 2) and metabolic damage (median value 2.36 SUVmean) of the sleep areas at both MRI and FDG-PET evaluation. Total sleep time seemed to be higher in 5/5 genotype DOC patients (median value Per3 5/5 , 221 min, range 126–323 min; median value Per3 4/4 , 167 min, range 36–477 min; and median value Per3 4/5 , 187 min, range 29–422 min). However, the MRI scores and FDG-PET values of whole brain, overall sleep areas, hypothalamus, midbrain and thalamus did not differ by genotype distribution. Conclusion Although limited by the small sample size, our data might support the idea that Per3 genetic predisposition in DOC patients could affect impairment and residual cognitive functions through sleep homeostasis independently from structural and/or metabolic integrity of sleep areas.

Introduction Malignant pleural mesothelioma (MPM) is a rare fatal asbestos-related malignancy originating in the mesothelial cells of the pleura. A platinum-based doublet containing a third-generation antifolate is the front-line standard of care whilst there are no approved second-line treatments for MPM which remains a disease setting to test the efficacy of new therapeutic agents. Methods Bone marrow mesenchymal stromal cells (BM-MSCs) were loaded with pemetrexed (PMX) and paclitaxel (PTX) according to a standardized procedure. Drug release by both PMX- and PTX-primed BM-MSCs (BM-MSCs/PMX and BM-MSCs/PTX) was tested on the in vitro proliferation of a panel of tumor cell lines including NCI-H28 mesothelioma. Results The in vitro anticancer activity of pure PTX was significantly higher than that of PMX against all the cell lines tested (14.7 times higher than that of PMX against NCI-H28). Whereas BM-MSCs did not take up and release PMX in amounts effective on mesothelioma, PTX-loaded BM-MSCs dramatically inhibited mesothelioma proliferation. Conclusions PTX-primed mesenchymal stromal cells successfully inhibit the in vitro proliferation of human mesothelioma cells. Further studies and in vivo testing are required to confirm our preliminary in vitro results as a potential new mesothelioma therapy based on cell drug delivery.

Pain assessment in patients with disorders of consciousness (DoC) is a controversial issue for clinicians, who require tools and standardised procedures for testing nociception in non-communicative patients. The aims of the present study were, first, to analyse the psychometric properties of the Italian version of the Nociception Coma Scale and, second, to evaluate pressure pain thresholds in a group of patients with DoC. The authors conducted a multi-centre study on 40 healthy participants and 60 DoC patients enrolled from six hospitals in Italy. For each group an electronic algometer was used to apply all nociceptive pressure stimuli. Our results show that the Italian version of the NCS retains the good psychometric properties of the original version and is therefore suitable for standardised pain assessment in clinical practice. In our study, pressure pain thresholds measured in a group of patients in vegetative and minimally conscious state were relatively lower than pain threshold values found in a group of healthy participants. Such findings motivate additional investigation on possible pain sensitisation in patients with severe brain injury and multiple co-morbidities, and on application of tailored therapeutic approaches useful for pain management in patients unable verbally to communicate their feelings.

Exploitation of the potential ability of human olfactory bulb (hOB) cells to carry, release, and deliver an effective, targeted anticancer therapy within the central nervous system (CNS) milieu remains elusive. Previous studies have demonstrated the marked ability of several types of stem cells (such as mesenchymal stem cells (MSCs) to carry and release different anti-cancer agents such as paclitaxel (PTX). Herein we investigate the ability of human olfactory bulb neural stem cells (Hu-OBNSCs) to carry and release paclitaxel, producing effective cytotoxic effects against cancer cells. We isolated Hu-OBNSCs from the hOB, uploaded them with PTX, and studied their potential cytotoxic effects against cancer cells in vitro. Interestingly, the Hu-OBNSCs displayed a five-fold increase in their resistance to the cytotoxicity of PTX, and the PTX-uploaded Hu-OBNSCs were able to inhibit proliferation and invasion, and to trigger marked cytotoxic effects on glioblastoma multiforme (GBM) cancer cells, and Human Caucasian fetal pancreatic adenocarcinoma 1 (CFPAC-1) in vitro. Despite their ability to resist the cytotoxic activity of PTX, the mechanism by which Hu-OBNSCs acquire resistance to PTX is not yet explained. Collectively our data indicate the ability of the Hu-OBNSCs to resist PTX, and to trigger effective cytotoxic effects against GBM cancer cells and CFPAC-1. This indicates their potential to be used as a carrier/vehicle for targeted anti-cancer therapy within the CNS.

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.

Stroke is the most common neurological cause of morbidity and mortality in industrialized countries, afflicting 15 million people every year. The numbers are expected to increase, mostly due to aging populations. One in five stroke patients dies, and one in three are left with permanent disabilities. Although some acute phase therapies such as intravenous recombinant tissue plasminogen activator (rt-PA) andendovascular treatment have been shown to improve ischemic stroke outcome, these therapies are available only for a small proportion of patients. The use of stem cells to replace brain cells lost during stroke is a long-term goal, and one which is difficult to achieve given that transplanted cells must integrate and restore neural pathways to regain function of damaged parts of the brain. Over the past decade the use of mesenchymal stromal cells (MSCs) as therapy has emerged as a particularly attractive option. MSCs are a class of multipotent, self-renewing cells that give rise to differentiated progeny when implanted into appropriate tissues. Herein, we present a review of the application of MSCs in ischemic stroke, including the source of MSCs, the route and timing of their delivery into the brain and the endpoints measured. Experimental data of transplantation of MSCs in animal stroke models suggest an improved functional recovery. The transplantation of MSCs influences a wide range of events by modulating the inflammatory environment, stimulating endogenous neurogenesis and angiogenesis and reducing the formation of glial scar, although the precise, underlying mechanism of this phenomenon remains unknown. The results from early clinical trials highlight the need to optimize variables such as cell selection and route of administration in order to translate these results into safe and successful clinical applications.

Background: The pathogenesis of moyamoya disease (MMD) is still unknown. The detection of inflammatory molecules such as cytokines, chemokines and growth factors in MMD patients' biological fluids supports the hypothesis that an abnormal angiogenesis is implicated in MMD pathogenesis. However, it is unclear whether these anomalies are the consequences of the disease or rather causal factors as well as these mechanisms remain insufficient to explain the pathophysiology of MMD. The presence of a family history in about 9-15% of Asian patients, the highly variable incidence rate between different ethnic and sex groups and the age of onset support the role of genetic factors in MMD pathogenesis. However, although some genetic loci have been associated with MMD, few of them have been replicated in independent series. Recently, RNF213 gene was shown to be strongly associated with MMD occurrence with a founder effect in East Asian patients. However, the mechanisms leading from RNF213 mutations to MMD clinical features are still unknown. Summary: The research on pathogenic mechanism of MMD is in its infancy. MMD is probably a complex and heterogeneous disorder, including different phenotypes and genotypes, in which more than a single factor is implicated. Key Message: Since the diagnosis of MMD is rapidly increasing worldwide, the development of more efficient stratifying risk systems, including both clinical but also biological drivers became imperative to improve our ability of predict prognosis and to develop mechanism-tailored interventions.

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.

Localization of chemotherapy at the tumor site can improve therapeutic efficacy and reduce systemic toxicity. In previous studies we have shown that mesenchymal stromal cells (MSCs) isolated from bone marrow or adipose tissue can be loaded with the anti-cancer drug Paclitaxel (PTX) and kill cancer cells when localized nearby. We here investigated the capacity of human micro-fragmented adipose tissue (MFAT), used as a natural scaffold of MSCs, to deliver PTX with the idea to improve local drug concentration and to prolong the therapeutic activity. Surprisingly, we found that both fresh but also devitalized MFAT (DMFAT) (by freezing/thawing procedure) were able to deliver and release significant amount of PTX, killing several human cancer cell lines in vitro with a long lasting activity. In an orthotopic mice model of Neuroblastoma (NB) transplant, DMFAT loaded with PTX prevents or delays NB relapse when placed in the surgical area of tumor resection, without any collateral toxicity. We concluded that MFAT, but also DMFAT, may represent very innovative natural biomaterials able to localize and release anti-cancer molecules at the tumor site, helping to fight cancer in human.

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.

In a two-stage phase II study, 24 patients with first diagnosis of glioblastoma (GBM) were treated with dendritic cell (DC) immunotherapy associated to standard radiochemotherapy with temozolomide (TMZ) followed by adjuvant TMZ. Three intradermal injections of mature DC loaded with autologous GBM lysate were administered before adjuvant TMZ, while 4 injections were performed during adjuvant TMZ. According to a two-stage Simon design, to proceed to the second stage progression-free survival (PFS) 12 months after surgery was expected in at least 8 cases enrolled in the first stage. Evidence of immune response and interaction with chemotherapy were investigated. After a median follow up of 17.4 months, 9 patients reached PFS12. In these patients (responders, 37.5%), DC vaccination induced a significant, persistent activation of NK cells, whose increased response was significantly associated with prolonged survival. CD8+ T cells underwent rapid expansion and priming but, after the first administration of adjuvant TMZ, failed to generate a memory status. Resistance to TMZ was associated with robust expression of the multidrug resistance protein ABCC3 in NK but not CD8+ T cells. The negative effect of TMZ on the formation of T cell-associated antitumor memory deserves consideration in future clinical trials including immunotherapy.

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.

CADASIL is a life-threatening and disabling disease. Despite the progress achieved so far, no therapies able to limit the disease progression have been found and only empiric treatments can be employed to relieve the main disease symptoms. Further in vivo studies as well as data aggregation and multi-centre controlled clinical trials are needed to confirm the emerging findings in order to identify evidence-based therapies for CADASIL.

Background: Spontaneous intracerebral haemorrhage (ICH) is a relatively common fatal disease, with an overall global incidence estimated at 24.6 per 100,000 person- years. Given the high degree of morbidity and mortality associated with ICH, therapies that may have neuroprotective effects are of increasing interest to clinicians. In this last context, cell therapies offer the promise of improving the disease course which cannot be addressed adequately by existing treatments. Objective: The aim of this review is to evaluate the protective effects and molecular mechanisms of mesenchymal stem cells (MSCs) on haemorrhagic brain following ICH. We also discuss possible emerging therapeutic approaches worth of further research. Methods and Results: The available literature on the therapeutic potential of MSCs in ICH animal models clearly demonstrated that MSCs enhance the functional recovery and reduce the volume of the infarct size exerting anti-inflammatory and angiogenic properties. However, the quality of the original articles investigating the efficacy of stem cell therapies in ICH animal models is still poor and the lack of ICH clinical trial does not permit to reach any relevant conclusions. Conclusion: Further studies have to be implemented in order to achieve standardized methods of MSCs isolation, characterization and administration to improve ICH treatments with MSCs or MSC-derived products.

This is a protocol for a Cochrane Review (Intervention). The objectives are as follows: To assess efficacy and safety of invasive brain stimulation compared with conventional treatments for functional recovery and reducing complications in stroke patients.

The complex architecture of adult brain derives from tightly regulated migration and differentiation of precursor cells generated during embryonic neurogenesis. Changes at transcriptional level of genes that regulate migration and differentiation may lead to neurodevelopmental disorders. Androgen receptor (AR) is a transcription factor that is already expressed during early embryonic days. However, AR role in the regulation of gene expression at early embryonic stage is yet to be determinate. Long non-coding RNA (lncRNA) Sox2 overlapping transcript (Sox2OT) plays a crucial role in gene expression control during development but its transcriptional regulation is still to be clearly defined. Here, using Bicalutamide in order to pharmacologically inactivated AR, we investigated whether AR participates in the regulation of the transcription of the lncRNASox2OTat early embryonic stage. We identified a new DNA binding region upstream of Sox2 locus containing three androgen response elements (ARE), and found that AR binds such a sequence in embryonic neural stem cells and in mouse embryonic brain. Our data suggest that through this binding, AR can promote the RNA polymerase II dependent transcription of Sox2OT. Our findings also suggest that AR participates in embryonic neurogenesis through transcriptional control of the long non-coding RNA Sox2OT.

This study investigated whether canine mesenchymal stromal cells (cMSCs) are able to take up and release paclitaxel (PTX) in active form, and therefore whether they have potential as a tool for therapeutic delivery of this drug. cMSCs from bone marrow and adipose tissue were isolated, expanded and characterised phenotypically. cMSCs were loaded with PTX (cMSCs-PTX) and their capacity for release of PTX was determined by their effect on proliferation of cancer cells. cMSCs-PTX derived from bone marrow and adipose tissue were able to take up and then release active PTX. cMSCs-PTC inhibited proliferation of the canine glioma cell line J3T, and the human glioblastoma cell lines T98G and U87MG. The potential of canine cMSCs-PTX for treatment of canine gliomas should be investigated further.

Differential diagnosis between Vegetative State and Minimally Conscious State is a challenging task that requires specific assessment scales, involvement of expert neuropsychologists or physicians and use of tailored stimuli for eliciting behavioural responses. Although misdiagnosis rate as high as 40 % has been reported, no clear guidelines are available in literature on the optimal setting for assessment. The present study aims to analyse score differences in behavioural assessments of persons with disorders of consciousness (DOC) with or without family members and to determine whether the presence of caregivers could improve clinical accuracy in diagnostic evaluation. The research was conducted on 92 adults with DOC among 153 consecutive patients enrolled in the Coma Research Centre of the Neurological Institute C. Besta of Milan between January 2011 and May 2013. The results indicate that in almost half of the sample the scoring, thus the performance, observed with caregivers was better than without them. Furthermore, in 16 % of the sample, when assessment was performed with caregivers there was a change in diagnosis, from Vegetative to Minimally Conscious State or from that to Severe Disability. Finally, statistical differences were found in relation to diagnosis between mean scores in the “visual function” Coma Recovery Scale revised’s subscale obtained by raters plus caregiver and rates only assessment. This study demonstrates how the presence of caregivers can positively affect behavioural assessments of persons with DOC, thus contributing to the definition of the optimal setting for behavioural evaluation of patients, to decrease misdiagnosis rates.

Assessment of cerebrovascular reactivity (CVR) is essential in cerebrovascular diseases, as exhausted CVR may enhance the risk of cerebral ischemic events. Transcranial Doppler (TCD) with a vasodilatory stimulus is currently used for CVR evaluation. Scanty data are available for Quantitative Magnetic Resonance Angiography (QMRA), which supplies higher spatial resolution and quantitative cerebral blood flow values. Aims of our pilot study were: (a) to assess safety and feasibility of CO 2 administration during QMRA, (b) evaluation of CVR under QMRA compared to TCD, and (c) quantitative evaluation of blood flow from the major intracranial arterial vessels both at rest and after CO 2 . CVR during 5% CO 2 air breathing was measured with TCD as a reference method and compared with QMRA. Fifteen healthy subjects (age 60.47 ± 2.24; male 11/15) were evaluated at rest and during CO 2 challenge. Feasibility and safety of QMRA under CO 2 were ensured in all subjects. CVR from middle cerebral artery territory was not statistically different between TCD and MRI ( p > 0.05). Mean arterial pressure (MAP) and heart rate (HR) increased during QMRA and TCD (MAP p = 0.007 and p = 0.001; HR p = 0.043 and p = 0.068, respectively). Blood flow values from all intracranial vessels increased after CO 2 inhalation ( p CO 2 administration during QMRA sessions is safe and feasible. Good correlation in terms of CVR was obtained comparing TCD and QMRA. Blood flow values significantly increased from all intracranial arterial vessels after CO 2 . Studies regarding CVR in physiopathological conditions might consider the utilization of QMRA both in routine clinical settings and in research projects.

Background Glioblastoma (GBM) represents the most aggressive malignant brain tumor in adults, with a risible median life expectancy despite gold standard treatment. Novel drug-delivery methods have been explored. Here we evaluated the possibility to use mononuclear cells (MCs) belonging to the monocytic-dendritic lineage as drug-carrier. Methods MCs were obtained from 10 patients harboring a GBM, and from healthy volunteers, considered as controls. GBM tissue was also obtained from patients. MCs were cultured and the adherent population on fibronectin (FN-MCs), after immunocytochemistry and flow cytometry characterization, was loaded with Paclitaxel (FN-MCs-PTX). Antiproliferative and migration activity of FN-MCs-PTX was evaluated in two-dimensional (2D) and three-dimensional (3D) co-culture assays with red fluorescent U87 Malignant Glioma cells and primary GBM cells. Antiangiogenic properties of FN-MCs-PTX were tested on cultures with endothelial cells. Results Phenotypical characterization showed a high expression of monocytic-dendritic markers in GBM cells and FN-MCs. FN-MCs demonstrated to effectively uptake PTX and to strongly inhibit GBM growth in vitro ( P 0.01). Moreover, tumor-induced migration of MCs, although partially affected by the PTX cargo, remained statistically significant when compared with unprimed cells and this was confirmed in a 3D Matrigel model ( P 0.01) and in a Trans-well assay ( P 0.01). FN-MCs-PTX also disclosed considerable antiangiogenic properties. Discussion Our results suggest that the fibronectin-adherent population of MCs isolated from peripheral blood can be an effective tool to inhibit GBM growth. Given the relative facility to obtain such cells and the short time needed for their culture and drug loading this approach may have potential as an adjuvant therapy for GBM.

Localization of chemotherapy at the tumor site can improve therapeutic efficacy and reduce systemic toxicity. In previous studies we have shown that mesenchymal stromal cells (MSCs) isolated from bone marrow or fat tissue, can be loaded with the anti-cancer drug Paclitaxel (PTX) and kill cancer cells when localized nearby. We here investigated the capacity of human micro-fragmented adipose tissue (MFAT), used as a natural container of MSCs, to delivery PTX with the idea to improve local drug concentration and to prolong the therapeutic activity. Surprisingly, we found that both fresh but also devitalized MFAT (DMFAT) (by freezing/thawing procedure) were very effective biomaterials able to deliver and release significant amount of PTX, killing several human cancer cell lines in vitro with an impressive long lasting activity. In an orthotopic mice model of Neuroblastoma (NB) transplant, we found that DMFAT loaded with PTX prevents or delay NB relapse when placed in the surgical area of tumor resection, without any collateral toxicity. We concluded that MFAT, but also DMFAT, may work as natural biomaterials that can be used to localize and release anti-cancer molecules at the tumor site. In addition, the procedure to easily prepare DMFAT did not alter its natural scaffold structure. This suggest that such biomaterial may have a prominent role in the near future in many oncological applications to delivery anti-cancer drugs, helping to fight cancer in human. Citation Format: Giulio Alessandri, Valentina Coccè, Fabio Pastorino, Rita Paroni, Michele Deicas, Francesco Restelli, Carlo Tremolada, Angiola Berenzi, Eugenio Parati, Anna Teresa Brini, Giampietro Bondiolotti, Augusto Pessina, Mirco Ponzoni. Microfragmented human fat tissue is a natural scaffold for drug delivery: potential application in cancer chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2160.

Epidemiological and family studies have provided evidence on the role of genetic factors in stroke, particularly in stroke occurring at young age. However, despite its impact, young stroke continues to be understudied. This article reviews the existing literature on the most investigated monogenic disorders (CADASIL, Fabry disease, MELAS, RVCL, COL4A1, Marfan and Ehlers-Danlos syndromes) causing stroke in young and a number of candidate genes associated with stroke occurring in patients younger than 50 years. Although our study failed in identifying strong and reliable associations between specific genes and young stroke, our detailed literature revision on the field allowed us to compile a panel of genes possibly generating a susceptibility to stroke, which could be a starting point for future research. Since stroke is a potentially preventable disease, the identification of genes associated with young stroke may promote novel prevention strategies and allow the identification of therapeutic disease targets.

Background and Purpose— Recently, a novel locus at 17q25 was associated with white matter hyperintensities (WMH) on MRI in stroke-free individuals. We aimed to replicate the association with WMH volume (WMHV) in patients with ischemic stroke. If the association acts by promoting a small vessel arteriopathy, it might be expected to also associate with lacunar stroke. Methods— We quantified WMH on MRI in the stroke-free hemisphere of 2588 ischemic stroke cases. Association between WMHV and 6 single-nucleotide polymorphisms at chromosome 17q25 was assessed by linear regression. These single-nucleotide polymorphisms were also investigated for association with lacunar stroke in 1854 cases and 51 939 stroke-free controls from METASTROKE. Meta-analyses with previous reports and a genetic risk score approach were applied to identify other novel WMHV risk variants and uncover shared genetic contributions to WMHV in community participants without stroke and ischemic stroke. Results— Single-nucleotide polymorphisms at 17q25 were associated with WMHV in ischemic stroke, the most significant being rs9894383 ( P =0.0006). In contrast, there was no association between any single-nucleotide polymorphism and lacunar stroke. A genetic risk score analysis revealed further genetic components to WMHV shared between community participants without stroke and ischemic stroke. Conclusions— This study provides support for an association between the 17q25 locus and WMH. In contrast, it is not associated with lacunar stroke, suggesting that the association does not act by promoting small-vessel arteriopathy or the same arteriopathy responsible for lacunar infarction.

The main goal in cancer chemotherapy is to drive the drug into the tumor microenvironment to kill as many cancer cells as possible while producing the lowest collateral toxicity. Previously, we have shown that human bone marrow derived mesenchymal stromal cells (hBM-MSCs) exposed to Paclitaxel (PTX) were able to uptake and subsequently release the drug in the culture medium. PTX primed hBM-MSCs (hBM-MSCsPTX) located in the vicinity of cancer cells produced a strong inhibition of tumor cell growth both in vitro and in vivo. To expand these observations, in the present study we exposed human skin derived fibroblasts (hSDFs) to 2,000 ng/ml of PTX and then tested both cells and their conditioned medium (CM) in vitro for their capacity to inhibit the proliferation of human tumor cell lines (MOLT-4, DU-145, U87-MG, SH-SY5Y(+) and LAN-5). We found that hSDFs primed with PTX (hSDFsPTX) were able to uptake and subsequently release PTX in a time dependent manner. hSDFsPTX-derived CM(hSDFsPTX-CM) from 1:4 to 1:10 dilutions produced a significant (p

Background A new tool for the drug delivery is based on the use of Mesenchymal Stromal Cells (MSCs) loaded in vitro with anti-cancer drugs. Unfortunately, the restricted lifespan of MSCs represents a significant limitation to produce them in high amounts and for long time studies. Immortalized MSCs from adipose tissue (hASCs) have been generated as good source of cells with stable features. These cells could improve the development of standardized procedures for both in vitro and preclinical studies. Furthermore they facilitate procedures for preparing large amounts of secretome containing microvesicles (MVs). Method We used human adipose tissue derived MSCs immortalized with hTERT+SV40 (TS) genes and transfected with GFP (hASCs-TS/GFP+). This line was investigated for its ability to uptake and release anticancer drugs. Microvesicles associated to paclitaxel (MVs/PTX) were isolated, quantified, and tested on pancreatic cancer cells. Results The line hASCs-TS/GFP+ maintained the main mesenchymal characters and was able to uptake and release, in active form, both paclitaxel and gemcitabine. From paclitaxel loaded hASCs-TS/GFP+ cells were isolated microvesicles in sufficient amount to inhibit "in vitro" the proliferation of pancreatic tumor cells. Conclusion Our study suggests that human immortalized MSCs could be used for a large scale production of cells for mediated drug delivery. Moreover, the secretion of drug-associated MVs could represent a new way for producing new drug formulation by "biogenesis". In the context of the "advanced cell therapy procedure", the MVs/PTX production would use less resource and time and it could possibly contribute to simplification of GMP procedures.

Fabry disease is an X-linked, lysosomal storage disorder caused by a mutation in the GLA gene leading to a deficiency in alpha-galactosidase A enzyme (α-Gal A) activity, which in turn results in accumulation of globotriaosylceramide in the vascular endothelium and smooth muscle cells of different organs, including kidney and heart, finally leading to impairment or failure of organ function. The central and peripheral nervous systems are also affected leading to neurological manifestations such as cerebrovascular diseases, small fiber neuropathy (SFN), and dysautonomic disorders that may be the presenting clinical features in a proportion of patients. This review offers a complete update of all neurological aspects of Fabry disease and therapeutic options. The rarity of disease, as well as the incomplete knowledge regarding natural history, pathogenic mechanisms, and the uncertain efficacy of available therapies, make imperative the acquisition of standardized data on natural disease course. These data are fundamental for the development of new treatments better able to access the central nervous system, to bypass the neutralizing antibodies and to improve the heart and kidney function.

Background and Purpose— Although a genetic contribution to ischemic stroke is well recognized, only a handful of stroke loci have been identified by large-scale genetic association studies to date. Hypothesizing that genetic effects might be stronger for early- versus late-onset stroke, we conducted a 2-stage meta-analysis of genome-wide association studies, focusing on stroke cases with an age of onset Methods— The discovery stage of our genome-wide association studies included 4505 cases and 21 968 controls of European, South-Asian, and African ancestry, drawn from 6 studies. In Stage 2, we selected the lead genetic variants at loci with association P −6 and performed in silico association analyses in an independent sample of ≤1003 cases and 7745 controls. Results— One stroke susceptibility locus at 10q25 reached genome-wide significance in the combined analysis of all samples from the discovery and follow-up stages (rs11196288; odds ratio =1.41; P =9.5×10 −9 ). The associated locus is in an intergenic region between TCF7L2 and HABP2 . In a further analysis in an independent sample, we found that 2 single nucleotide polymorphisms in high linkage disequilibrium with rs11196288 were significantly associated with total plasma factor VII–activating protease levels, a product of HABP2 . Conclusions— HABP2 , which encodes an extracellular serine protease involved in coagulation, fibrinolysis, and inflammatory pathways, may be a genetic susceptibility locus for early-onset stroke.