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Cancer stem–like cells (CSC) could be a novel target for cancer therapy, including dendritic cell (DC) immunotherapy. To address this, we developed experiments aimed at DC targeting of neurospheres (NS) from GL261 glioma cells because neurospheres can be enriched in CSC. We obtained murine neurospheres by growing GL261 cells in epidermal growth factor/basic fibroblast growth factor without serum. GL261-NS recapitulated important features of glioblastoma CSC and expressed higher levels of radial glia stem cell markers than GL261 cells growing under standard conditions (GL261 adherent cells, GL261-AC), as assessed by DNA microarray and real-time PCR. GL261-NS brain gliomas were highly infiltrating and more rapidly lethal than GL261-AC, as evidenced by survival analysis (P < 0.0001), magnetic resonance imaging and histology. DC from the bone marrow of syngeneic mice were then used for immunotherapy of GL261-NS and GL261-AC tumors. Strikingly, DC loaded with GL261-NS (DC-NS) cured 80% and 60% of GL261-AC and GL261-NS tumors, respectively (P < 0.0001), whereas DC-AC cured only 50% of GL261-AC tumors (P = 0.0022) and none of the GL261-NS tumors. GL261-NS expressed higher levels of MHC and costimulatory molecules (CD80 and CD86) than GL261-AC; the JAM assay indicated that DC-NS splenocytes had higher lytic activity than DC-AC splenocytes on both GL261-NS and GL261-AC, and immunohistochemistry showed that DC-NS vaccination was associated with robust tumor infiltration by CD8+ and CD4+ T lymphocytes. These findings suggest that DC targeting of CSC provides a higher level of protection against GL261 gliomas, a finding with potential implications for the design of clinical trials based on DC vaccination. (Cancer Res 2006; 66(21): 10247-52)

Supplementary Methods and Figures 1-8 from Neurospheres Enriched in Cancer Stem–Like Cells Are Highly Effective in Eliciting a Dendritic Cell–Mediated Immune Response against Malignant Gliomas

Supplementary Table S2 from Neurospheres Enriched in Cancer Stem–Like Cells Are Highly Effective in Eliciting a Dendritic Cell–Mediated Immune Response against Malignant Gliomas

We intend to investigate the indications, complications, and final results of amniotic membrane (AM) transplantation in ocular motility restrictions. Surgeons have tried to prevent restrictive adhesions between the extraocular muscles and surrounding tissues because they cause unpredictable results. AM transplantation wrapping extraocular musculature has been proposed as a technique with good results for this purpose. A search was carried out in Medline, Embase, Cochrane Library and Clinicaltrial, Lilac and Ibecs databases, using the indexed terms" amnion", "strabismus," "strabismus-subheading-surgery" and "ocular motility disorders." The only exclusion criteria were studies conducted in non-humans or studies with insufficient data on eye motility. No study was discarded for analysis because of language, age or methodology. This review includes 165 patients (223 eyes), with a mean follow-up of 11.49 months. The mean age was 21 years old, 47% were males and 57% were children. 14 studies conducted in humans were eligible: 4 single case, 8 case series, 1 cohort study and 1 randomized clinical trial. In the vast majority of these papers, use of AM transplantation to treat (12 out of 14 papers) or either to prevent (2 out of 14 papers) motility restrictions or restrictive strabismus secondary to muscular adhesions. All the studies except one presents very favorable results improving postsurgical eye motility. The cryopreservation method was more widely used, presenting a good safety profile with few adverse effects in the short and medium term. Significant improvement was reported in most patients after the use of amniotic membrane transplantation to treat or prevent ocular motility limitations. Very few complications or adverse effects were documented.

Puntos para una lectura rapida • La refraccion es el cambio de direccion que experimenta un rayo de luz al pasar de un medio a otro con diferente densidad. • Existen tres tipos basicos de ametropias: miopia, hipermetropia y astigmatismo. • La magnitud de la miopia se mide en dioptrias negativas y se corrige con lentes esfericas divergentes (concavas). • La magnitud de la hipermetropia se mide en dioptrias positivas y se corrige con lentes esfericas convergentes (convexas). • La receta de graduacion optica es un documento que describe la correccion que necesita un paciente para tener una vision lo mas nitida posible y alcanzar la mayor agudeza visual. • Es importante estar familiarizado con la receta de graduacion, pues su correcta interpretacion permite conocer el estado refractivo del paciente en cada momento y entender mejor la magnitud y caracteristicas de su deficit visual.

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.

Malignant Pleural Mesothelioma (MPM) is a rare and aggressive neoplasm of the pleural mesothelium, mainly associated with asbestos exposure and still lacking effective therapies. Modern targeted biological strategies that have revolutionized the therapy of other solid tumors have not had success so far in the MPM. Combination immunotherapy might achieve better results over chemotherapy alone, but there is still a need for more effective therapeutic approaches. Based on the peculiar disease features of MPM, several strategies for local therapeutic delivery have been developed over the past years. The common rationale of these approaches is: (i) to reduce the risk of drug inactivation before reaching the target tumor cells; (ii) to increase the concentration of active drugs in the tumor micro-environment and their bioavailability; (iii) to reduce toxic effects on normal, non-transformed cells, because of much lower drug doses than those used for systemic chemotherapy. The complex interactions between drugs and the local immune-inflammatory micro-environment modulate the subsequent clinical response. In this perspective, the main interest is currently addressed to the development of local drug delivery platforms, both cell therapy and engineered nanotools. We here propose a review aimed at deep investigation of the biologic effects of the current local therapies for MPM, including cell therapies, and the mechanisms of interaction with the tumor micro-environment.

Background aims: Advanced therapy medicinal products (ATMPs) are novel drugs based on genes, cells or tissues developed to treat many different diseases. Stability studies of each new ATMP need to be performed to define its shelf life and guarantee efficacy and safety upon infusion, and these are presently based on guidelines originally drafted for standard pharmaceutical drugs, which have properties and are stored in conditions quite different from cell products. The aim of this report is to provide evidence-based information for stability studies on ATMPs that will facilitate the interlaboratory harmonization of practices in this area. Methods: We have collected and analyzed the results of stability studies on 19 different cell-based experimental ATMPs, produced by five authorized cell factories forming the Lombardy “Plagencell network” for use in 36 approved phase I/II clinical trials; most were cryopreserved and stored in liquid nitrogen vapors for 1 to 13 years. Results: The cell attributes collected in stability studies included cell viability, immunophenotype and potency assays, in particular immunosuppression, cytotoxicity, cytokine release and proliferation/differentiation capacity. Microbiological attributes including sterility, endotoxin levels and mycoplasma contamination were also analyzed. All drug products (DPs), cryopreserved in various excipients containing 10% DMSO and in different primary containers, were very stable long term at

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 The efficacy of dendritic cell (DC) immunotherapy as a single therapeutic modality for the treatment of glioblastoma (GBM) patients remains limited. In this study, we evaluated in patients with GBM recurrence the immune-mediated effects of DC loaded with autologous tumor lysate combined with temozolomide (TMZ) or tetanus toxoid (TT). Methods In the phase I-II clinical study DENDR2, 12 patients were treated with 5 DC vaccinations combined with dose-dense TMZ. Subsequently, in eight patients, here defined as Variant (V)-DENDR2, the vaccine site was preconditioned with TT 24 hours before DC vaccination and TMZ was avoided. As a survival endpoint for these studies, we considered overall survival 9 months (OS9) after second surgery. Patients were analyzed for the generation of effector, memory, and T helper immune response. Results Four of 12 DENDR2 patients reached OS9, but all failed to show an immunological response. Five of eight V-DENDR2 patients (62%) reached OS9, and one patient is still alive (OS >30 months). A robust CD8+ T-cell activation and memory T-cell formation were observed in V-DENDR2 OS>9. Only in these patients, the vaccine-specific CD4+ T-cell activation (CD38+/HLA-DR+) was paralleled by an increase in TT-induced CD4+/CD38low/CD127high memory T cells. Only V-DENDR2 patients showed the formation of a nodule at the DC injection site infiltrated by CCL3-expressing CD4+ T cells. Conclusions TT preconditioning of the vaccine site and lack of TMZ could contribute to the efficacy of DC immunotherapy by inducing an effector response, memory, and helper T-cell generation.

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.

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 5–7 days of differentiation with GM-CSF and IL-4 followed by 2–3 days of activation/maturation. In attempts to shorten the vaccine’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.

Dendritic cells (DCs) are immune specialized cells playing a critical role in promoting immune response against antigens, and may represent important targets for therapeutic interventions in cancer. DCs can be stimulated ex vivo with pro-inflammatory molecules and loaded with tumor-specific antigen(s). Protocols describing the specific details of DCs vaccination manufacturing vary widely, but regardless of the employed protocol, the DCs vaccination safety and its ability to induce antitumor responses is clearly established. Many years of studies have focused on the ability of DCs to provide overall survival benefits at least for a selection of cancer patients. Lessons learned from early trials lead to the hypothesis that, to improve the efficacy of DCs-based immunotherapy, this should be combined with other treatments. Thus, the vaccine’s ultimate role may lie in the combinatorial approaches of DCs-based immunotherapy with chemotherapy and radiotherapy, more than in monotherapy. In this review, we address some key questions regarding the integration of DCs vaccination with multimodality therapy approaches for cancer treatment paradigms.

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: 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.

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.

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.

In the current study, we investigated whether naturally occurring CD4+CD25+ T cells, separated by immunomagnetic anti-CD4 and anti-CD25 Abs from naive animals, are able to protect from experimental autoimmune myasthenia gravis (EAMG) and modify the progression of ongoing disease when administered to Torpedo californica acetylcholine receptor (AChR)-immunized Lewis rats. Even though CD4+CD25+ and CD4+CD25high T cell frequencies were similar in the spleens and lymph nodes of EAMG and healthy rats, we observed that CD4+CD25+ T cells isolated from the spleens of naive animals inhibited in vitro the Ag-induced proliferation of T cell lines specific to the self-peptide 97–116 of the anti-AChR subunit (R97-116), an immunodominant and myasthenogenic T cell epitope, whereas CD4+CD25+ T cells purified from the spleens of EAMG rats were less effective. CD4+CD25+ T cells from EAMG rats expressed less forkhead box transcription factor P3 but more CTLA-4 mRNA than healthy rats. Naive CD4+CD25+ T cells, obtained from naive rats and administered to T. californica AChR-immunized animals according to a preventive schedule of treatment, reduced the severity of EAMG, whereas their administration 4 wk postinduction of the disease, corresponding to the onset of clinical symptoms (therapeutic treatment), was not effective. We think that the exogenous administration of CD4+CD25+ naive T cells prevents the early events underlying the induction of EAMG, events linked to the T cell compartment (Ag recognition, epitope spreading, and T cell expansion), but fails to ameliorate ongoing EAMG, when the IgG-mediated complement attack to the AChR at the neuromuscular junction has already taken place.

We investigated the effect of IgG immunoadsorption (IA) on cytokine network in patients with treatment-resistant Myasthenia Gravis (MG) and Lambert-Eaton Syndrome (LEMS). We observed upregulation of interleukin (IL)-10, an anti-inflammatory and B cells growth factor, and reduction of pro-inflammatory factors such as IL-18 and IL-17, in both MG and LEMS after IA. Our observation suggests that the massive removal of antibodies might induce modifications of the cytokine balance linked to T and B cells mediated autoimmunity.

In a mentally retarded and malformed boy who died at 6 months of age a ring chromosome 6 was identified by G banding. Clinical, cytogenetical and post-mortem findings are discussed.

Neural stem cells (NSCs) transplantation has been proposed as a means of restoring damaged brain tissue, a possibility rendered more likely by reports of low NSCs immunogenicity in various experimental models because of low expression of MHC class I and II as well as co-stimulatory molecules. We investigated the immunogenicity of a human NSC line grown in normal culture conditions and in the presence of pro-inflammatory cytokines IFN-gamma and tumor necrosis factor alpha by one-way mixed lymphocyte reaction (MLR) experiments with peripheral blood lymphocytes from eight HLA-incompatible donors. NSCs stimulated lymphocyte proliferation in almost all donors tested, with stimulation indices in the range of the low-end distribution curve of MLR between donors. The healthy subject that gave negative MLR results was the best compatible donor with respect to NSC haplotype. Since we observed low MLR responses overall, we studied if NSCs might exert any immunomodulatory activity. We detected transcription and release of the immunomodulatory molecule transforming growth factor beta (TGF-beta)-1; moreover, the addition of TGF-beta1 in MLR experiments down-regulated proliferative responses. To further confirm the immunological potential of human NSCs, we studied xenogeneic recognition of NSCs by immunocompetent cells derived from C57BL/6 mice, showing that NSCs can elicit an allo(xeno) response ex vivo. Our data indicate that NSCs have low but not negligible immunogenic potential that is sufficient to activate peripheral lymphocytes. Secretion of TGF-beta1 might balance the immunogenicity of NSCs. Nevertheless, the possibility that allo-NSCs grafting might induce in the long term an immune activation, thus vanishing their therapeutical effect, should not be overlooked and deserves further investigation.

Background Moyamoya disease (MMD) is a slowly progressing steno-occlusive cerebrovascular disease. The typical moyamoya vessels, which originate from an initial stenosis of the internal carotid, highlight that increased and/or abnormal angiogenic, vasculogenic and arteriogenic processes are involved in the disease pathophysiology. Objective Herein, we summarize the current knowledge on the most important signaling pathways involved in MMD vessel formation, particularly focusing on the expression of growth factors and function of endothelial progenitor cells (EPCs). Methods and results Higher plasma concentrations of vascular endothelial growth factor, matrix metalloproteinase, hepatocyte growth factor, and interleukin-1β were reported in MMD. A specific higher level of basic fibroblast growth factor was also found in the cerebrospinal fluid of these patients. Finally, the number and the functionality of EPCs were found to be increased. In spite of the available data, the approaches and findings reported so far do not give an evident correlation between the expression levels of the aforementioned growth factors and MMD severity. Furthermore, the controversial results provided by studies on EPCs, do not permit to understand the true involvement of these cells in MMD pathophysiology. Conclusion Further studies should thus be implemented to extend our knowledge on processes regulating both the arterial stenosis and the excessive formation of collateral vessels. Moreover, we suggest advances of integrated approaches and functional assays to correlate biological and clinical data, arguing for the development of new therapeutic applications for MMD.

Cell therapy based on dendritic cells (DCs) pulsed with tumor lysate is a promising approach in addition to conventional therapy for the treatment of patients with glioblastoma (GB). The success of this approach strongly depends on the ability to generate high-quality, functionally mature DCs (mDCs), with a high level of standardization and in compliance with Good Manufacturing Practices. In the cell factory of the Carlo Besta Foundation, two phase I clinical trials on immunotherapy with tumor lysate-loaded DCs as treatment for GB are ongoing. From 2010 to 2014, 54 patients were enrolled in the studies and 54 batches of DCs were prepared. We retrospectively analyzed the results of the quality control tests carried out on each produced batch, evaluating yield of mDCs and their quality in terms of microbiological safety and immunological efficacy. The number of mDCs obtained allowed the treatment of all the enrolled patients. All 54 batches were sterile, conformed to acceptable endotoxin levels, and were free of Mycoplasma species and adventitious viruses. During culture, cells maintained a high percentage of viability (87%–98%), and all batches showed high viability after thawing (mean ± SD: 94.6% ± 2.9%). Phenotype evaluation of mDCs showed an evident upregulation of markers typical of DC maturation; mixed lymphocyte reaction tests for the functional evaluation of DCs demonstrated that all batches were able to induce lymphocyte responses. These results demonstrated that our protocol for DC preparation is highly reproducible and permits generation of large numbers of safe and functional DCs for in vivo use in immunotherapy approaches. Significance Cell therapy based on antigen-pulsed dendritic cells (DCs) is a promising approach for the treatment of glioblastoma patients. The success of this approach strongly depends on the ability to generate high-quality, functional DCs with a high level of standardization, ensuring reproducibility, efficacy, and safety of the final product. This article summarizes the results of the quality controls on 54 batches, to demonstrate the feasibility of producing a therapeutic cell-based vaccine via a well-controlled Good Manufacturing Practices (GMP)-compliant production process. The findings may be of scientific interest to those working in the field of preparation of GMP-compliant products for cell-therapy applications.

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

Brain microvascular endothelial cells (BMVECs) have an important role in the constitution of the blood-brain barrier (BBB). The BBB is involved in the disease processes of a number of neurological disorders in which its permeability increases. Isolation of BMVECs could elucidate the mechanism involved in these processes. This protocol describes how to isolate and expand human and mouse BMVECs. The procedure covers brain-tissue dissociation, digestion and cell selection. Cells are selected on the basis of time-responsive differential adhesiveness to a collagen type I-precoated surface. The protocol also describes immunophenotypic characterization, cord formation and functional assays to confirm that these cells in endothelial proliferation medium (EndoPM) have an endothelial origin. The entire technique requires ∼7 h of active time. Endothelial cell clusters are readily visible after 48 h, and expansion of BMVECs occurs over the course of ∼60 d.

Pixantrone (BBR2778) (PIX) and mitoxantrone share the same mechanism of action because both drugs act as DNA intercalants and inhibitors of topoisomerase II. PIX is an interesting candidate immunosuppressant for the treatment of autoimmune diseases because of its reduced cardiotoxicity compared with mitoxantrone. The clinical response to conventional immunosuppressive treatments is poor in some patients affected by myasthenia gravis (MG), and new but well-tolerated drugs are needed for treatment-resistant MG. PIX was tested in vitro on rat T cell lines specific for the immunodominant peptide 97–116 derived from rat acetylcholine receptor (AChR), and showed strong antiproliferative activity in the nanomolar range. We demonstrate in this study that PIX administration reduced the severity of experimental autoimmune MG in Lewis rats. Biological and immunological analysis confirmed the effect of PIX, compared with vehicle-treated as well as mitoxantrone-treated experimental autoimmune MG rats. Anti-rat AChR Abs were significantly reduced in PIX-treated rats, and AChR content in muscles were found increased. Torpedo AChR-induced T cell proliferation tests were found reduced in both in vitro and ex vivo experiments. The effectiveness and the reduced cardiotoxicity make PIX a promising immunosuppressant agent suitable for clinical investigation in MG, although additional experiments are needed to confirm its safety profile in prolonged treatments.

Cancer stem–like cells (CSC) could be a novel target for cancer therapy, including dendritic cell (DC) immunotherapy. To address this, we developed experiments aimed at DC targeting of neurospheres (NS) from GL261 glioma cells because neurospheres can be enriched in CSC. We obtained murine neurospheres by growing GL261 cells in epidermal growth factor/basic fibroblast growth factor without serum. GL261-NS recapitulated important features of glioblastoma CSC and expressed higher levels of radial glia stem cell markers than GL261 cells growing under standard conditions (GL261 adherent cells, GL261-AC), as assessed by DNA microarray and real-time PCR. GL261-NS brain gliomas were highly infiltrating and more rapidly lethal than GL261-AC, as evidenced by survival analysis (P < 0.0001), magnetic resonance imaging and histology. DC from the bone marrow of syngeneic mice were then used for immunotherapy of GL261-NS and GL261-AC tumors. Strikingly, DC loaded with GL261-NS (DC-NS) cured 80% and 60% of GL261-AC and GL261-NS tumors, respectively (P < 0.0001), whereas DC-AC cured only 50% of GL261-AC tumors (P = 0.0022) and none of the GL261-NS tumors. GL261-NS expressed higher levels of MHC and costimulatory molecules (CD80 and CD86) than GL261-AC; the JAM assay indicated that DC-NS splenocytes had higher lytic activity than DC-AC splenocytes on both GL261-NS and GL261-AC, and immunohistochemistry showed that DC-NS vaccination was associated with robust tumor infiltration by CD8+ and CD4+ T lymphocytes. These findings suggest that DC targeting of CSC provides a higher level of protection against GL261 gliomas, a finding with potential implications for the design of clinical trials based on DC vaccination. (Cancer Res 2006; 66(21): 10247-52)

Erythropoietin (EPO) mediates a wide range of neuroprotective activities, including amelioration of disease and neuroinflammation in rat models of EAE. However, optimum dosing parameters are currently unknown. In the present study, we used a chronic EAE model induced in mice by immunization with the myelin oligodendrocyte glycoprotein peptide (MOG35-55) to compare the effect of EPO given with different treatment schedules. EPO was administered intraperitoneally at 0.5, 5.0 or 50 microg/kg three times weekly starting from day 3 after immunization (preventive schedule), at the onset of clinical disease (therapeutic schedule) or 15 days after the onset of symptoms (late therapeutic schedule). The results show that EPO is effective even when given after the appearance of clinical signs of EAE, but with a reduced efficacy compared to the preventative schedule. To determine whether this effect requires the homodimeric EPO receptor (EPOR2)-mediated hematopoietic effect of EPO, we studied the effect of carbamylated EPO (CEPO) that does not bind EPOR2. CEPO, ameliorated EAE without changing the hemoglobin concentration. Another non-erythropoietic derivative, asialoEPO was also effective. Both EPO and CEPO equivalently decreased the EAE-associated production of TNF-alpha, IL-1beta and IL-1Ra in the spinal cord, and IFN-gamma by peripheral lymphocytes, indicating that their action involves targeting neuroinflammation. The lowest dosage tested appeared fully effective. The possibility to dissociate the anti-neuroinflammatory action of EPO from its hematopoietic action, which may cause undesired side effects in non-anemic patients, present new avenues to the therapy of multiple sclerosis.

Recurrent glioblastoma (GB) are highly aggressive tumors allowing 6-8 month survival. Here we evaluated the possible benefits of immunotherapy with mature dendritic cells (DC) loaded with autologous tumor lysate in 15 patients with recurrent GB. After a median follow-up of 8 months median progression free survival (PFS) was 4.4 months and median overall survival (OS) 8.0 months. Patients with small tumors at first vaccination (< 20 cc; n = 8) had significantly longer PFS and OS than others: 6.0 vs 3.0 months (p = 0.01) and 16.5 vs 7.0 months (p = 0.003), respectively. Patients were analysed for CD8+ T cells, CD56+ NK cells and other relevant immunological parameters in peripheral blood before and after immunization, defining a vaccination/baseline ratio (V/B ratio). Increased V/B ratio of NK but not CD8+ T cells was significantly associated to longer PFS and OS. Patients showing NK cell responses had higher levels of IFN-γ and E4BP4, the NK cell transcription factor. Furthermore, NK V/B ratio was inversely correlated with TGF-β2 and VEGF V/B ratio. The results suggest that tumor-loaded DC may increase survival of recurrent GB after effective tumor debulking and emphasize the role of NK cell responses in this therapeutic setting. Citation Format: Serena Pellegatta, Marica Eoli, Simona Frigerio, Carlo Antozzi, Maria Grazia Bruzzone, Gabriele Cantini, Sara Nava, Elena Anghileri, Lucia Cuppini, Valeria Cuccarini, Emilio Ciusani, Marta Dossena, Bianca Pollo, Renato Mantegazza, Eugenio A. Parati, Gaetano Finocchiaro. NK cell response and tumor debulking are associated to prolonged survival in recurrent glioblastoma treated by dendritic cells loaded with autologous tumor lysate. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2839. doi:10.1158/1538-7445.AM2013-2839 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Recurrent glioblastomas (GBs) are highly aggressive tumors associated with a 6–8 mo survival rate. In this study, we evaluated the possible benefits of an immunotherapeutic strategy based on mature dendritic cells (D cs) loaded with autologous tumor-cell lysates in 15 patients affected by recurrent GB. The median progression-free survival ( pFs) of this patient cohort was 4.4 mo, and the median overall survival (O s) was 8.0 mo. p atients with small tumors at the time of the first vaccination (< 20 cm 3 ; n = 8) had significantly longer p Fs and Os than the other patients (6.0 vs. 3.0 mo, p = 0.01; and 16.5 vs. 7.0 mo, p = 0.003, respectively). c D8 + T cells, cD56 + natural killer (NK) cells and other immune parameters, such as the levels of transforming growth factor β , vascular endothelial growth factor, interleukin-12 and interferon γ (IFN γ), were measured in the peripheral blood and serum of patients before and after immunization, which enabled us to obtain a vaccination/baseline ratio (V/B ratio). an increased V/B ratio for NK cells, but not cD8 + T cells, was significantly associated with prolonged pFs and Os. patients exhibiting NK-cell responses were characterized by high levels of circulating IFN γ and e4Bp4, an NK-cell transcription factor. Furthermore, the NK cell V/B ratio was inversely correlated with the TGF β2 and VeGF V/B ratios. These results suggest that tumor-loaded D cs may increase the survival rate of patients with recurrent GB after effective tumor debulking, and emphasize the role of the NK-cell response in this therapeutic setting.

Immune-based treatments represent a promising new class of therapy designed to boost the immune system to specifically eradicate malignant cells. Immunotherapy may generate specific anti-tumor immune responses, and dendritic cells (DC), professional antigen-presenting cells, are widely used in experimental cancer immunotherapy. Several reports describe methods for the generation of mature, antigen-pulsed DC for clinical use. Improved quality and standardization are desirable to obtain GMP-compliant protocols. In this study we describe the generation of DC from 31 Glioblastoma (GB) patients starting from their monocytes isolated by immunomagnetic CD14 selection using the CliniMACS® device. Upon differentiation of CD14+ with IL-4 and GM-CSF, DC were induced to maturation with TNF-α, PGE(2), IL-1β, and IL-6. Whole tumor lysate was obtained, for the first time, in a closed system using the semi-automated dissociator GentleMACS®. The yield of proteins improved by 130% compared to the manual dissociation method. Interestingly the Mean Fluorescence Intensity for CD83 increased significantly in DC pulsed with "new method" lysate compared to DC pulsed with "classical method" lysate. Our results indicate that immunomagnetic isolation of CD14(+) monocytes using the CliniMACS® device and their pulsing with whole tumor lysate proteins is a suitable method for clinical-scale generation of high quality, functional DC under GMP-grade conditions.

Mesenchymal stem cell (MSC) therapy is considered one of the most promising approaches for treating different neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). We previously characterized a subpopulation of human skeletal muscle-derived stem cells (SkmSCs) with MSC-like characteristics that differentiate into the neurogenic lineage in vitro. In the present study, we evaluated the SkmSC therapeutic effects in the most characterized model of spontaneous motor neuron degeneration, the Wobbler (Wr) mouse. Before evaluating the therapeutic efficacy in the Wr mouse, we followed the route of Skm-SCs at different times after intracerebroventricular injection. Two exogenous tracers, superparamagnetic iron oxide (SPIO) nanoparticles and Hoechst 33258, were used for the in vivo and ex vivo tracking of SkmSCs. We found that the loading of both Hoechst and SPIO was not toxic and efficiently labeled SkmSCs. The magnetic resonance imaging (MRI) system 7 Tesla allowed us to localize transplanted SkmSCs along the whole ventricular system up to 18 wks after injection. The ex vivo Hoechst 33258 visualization confirmed the in vivo results obtained by MRI analyses. Behavioral observations revealed a fast and sustained improvement of motor efficacy in SkmSC-treated Wr mice associated with a relevant protection of functional neuromuscular junctions. Moreover, we found that in SkmSC-treated Wr mice, a significant increase of important human antiinflammatory cytokines occurred. This evidence is in accordance with previous findings showing the bystander effect of stem cell transplantation in neurodegenerative disorders and further strengthens the hypothesis of the possible link between inflammation, cytotoxicity and ALS.