Search

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease affecting both upper and lower motoneurons. The transactive response DNA binding protein (TARDBP) gene, encoding for TDP-43, is one of the most commonly mutated gene associated with familial cases of ALS (10%). We generated a human induced pluripotent stem cell (hiPSC) line from the fibroblasts of an asymptomatic subject carrying the TARDBP p.G376D mutation. This mutation is very rare and was described in a large Apulian family, in which all ALS affected members are carriers of the mutation. The subject here described is the first identified asymptomatic carrier of the mutation.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of the upper and lower motor neurons (MNs). About 10% of patients have a family history (familial, fALS); however, most patients seem to develop the sporadic form of the disease (sALS). SOD1 (Cu/Zn superoxide dismutase-1) is the first studied gene among the ones related to ALS. Mutant SOD1 can adopt multiple misfolded conformation, lose the correct coordination of metal binding, decrease structural stability, and form aggregates. For all these reasons, it is complicated to characterize the conformational alterations of the ALS-associated mutant SOD1, and how they relate to toxicity. In this work, we performed a multilayered study on fibroblasts derived from two ALS patients, namely SOD1L145F and SOD1S135N, carrying the p.L145F and the p.S135N missense variants, respectively. The patients showed diverse symptoms and disease progression in accordance with our bioinformatic analysis, which predicted the different effects of the two mutations in terms of protein structure. Interestingly, both mutations had an effect on the fibroblast energy metabolisms. However, while the SOD1L145F fibroblasts still relied more on oxidative phosphorylation, the SOD1S135N fibroblasts showed a metabolic shift toward glycolysis. Our study suggests that SOD1 mutations might lead to alterations in the energy metabolism.

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

Herein, we explored the impact of the lysosome dysfunction during the progression of Amyotrophic Lateral Sclerosis type-1 (ALS1). We conducted the study in non-neural cells, primary fibroblasts (rFFFs), and bone marrow-mesenchymal stem cells (rBM-MSCs), isolated from the animal model ratG93A for ALS1 at two stages of the disease: Pre-symptomatic-stage (ALS1-PreS) and Terminal-stage (ALS1-EndS). We documented the storage of human mutant Superoxide Dismutase 1, SOD1G93A (SOD1*) in the lysosomes of ALS1-rFFFs and ALS1-rBM-MSCs and demonstrated the hallmarks of the disease in non-neural cells as in ratG93A-ALS1-tissues. We showed that the SOD1* storage is associated with the altered glycohydrolases and proteases levels in tissues and both cell types from ALS1-PreS to ALS1-EndS. Only in ALS1-rFFFs, the lysosomes lost homeostasis, enlarge drastically, and contribute to the cell metabolic damage. Contrariwise, in ALS1-rBM-MSCs, we found a negligible metabolic dysfunction, which makes these cells’ status similar to WT. We addressed this phenomenon to a safety mechanism perhaps associated with an enhanced lysosomal autophagic activity in ALS1-rBM-MSCs compared to ALS1-rFFFs, in which the lysosomal level of LC3-II/LC3I was comparable to that of WT-rFFFs. We suggested that the autophagic machinery could balance the storage of SOD1* aggregates and the lysosomal enzyme dysfunction even in ALS1-EndS-stem cells.

Introduction In central nervous system neurodegenerative disorders, stem cell-based therapies should be considered as a promising therapeutic approach. The safe use of human Neural Stem Cells (hNSCs) for the treatment of several neurological diseases is currently under evaluation of phase I/II clinical trials. Clinical application of hNSCs require the development of GMP standardized protocols capable of generating high quantities of reproducible and well characterized stem cells bearing stable functional and genetic properties. Aim The aim of this study was to evaluate possible instabilities or modifications of the microsatellite loci in different culture passages because high culture passages represent an in vitro replicative stress leading to senescence. Experimental method: The hNSCs were characterized at different culture time points, from passage 2 to passage 25, by genetic typing at ten microsatellite loci. Conclusion We showed that genetic stability at microsatellite loci is maintained by the cells even at high passages adding a further demonstration of the safety of our hNSCs GMP culture method.

Among the known causative genes of familial ALS, SOD1 mutation is one of the most common. It encodes for the ubiquitous detoxifying copper/zinc binding SOD1 enzyme, whose mutations selectively cause motor neuron death, although the mechanisms are not as yet clear. What is known is that mutant-mediated toxicity is not caused by loss of its detoxifying activity but by a gain-of-function. In order to better understand the pathogenic mechanisms of SOD1 mutation, a human induced pluripotent stem cell (hiPSC) line was generated from the somatic cells of a female patient carrying a missense variation in SOD1 (L145F).

Extracellular vesicles (EVs) are membrane particles involved in the exchange of a broad range of bioactive molecules between cells and the microenvironment. Although it has been shown that cells can traffic metabolic enzymes via EVs, much remains to be elucidated with regard to their intrinsic metabolic activity. Accordingly, herein we assessed the ability of neural stem/progenitor cell (NSC)-derived EVs to consume and produce metabolites. Our metabolomics and functional analyses both revealed that EVs harbor L-asparaginase activity, catalyzed by the enzyme asparaginase-like protein 1 (Asrgl1). Critically, we show that Asrgl1 activity is selective for asparagine and is devoid of glutaminase activity. We found that mouse and human NSC EVs traffic Asrgl1. Our results demonstrate, for the first time, that NSC EVs function as independent metabolic units that are able to modify the concentrations of critical nutrients, with the potential to affect the physiology of their microenvironment.

Stem cells are emerging as a therapeutic option for incurable diseases, such as Amyotrophic Lateral Sclerosis (ALS). However, critical issues are related to their origin as well as to the need to deepen our knowledge of the therapeutic actions exerted by these cells. Here, we investigate the therapeutic potential of clinical-grade human neural stem cells (hNSCs) that have been successfully used in a recently concluded phase I clinical trial for ALS patients (NCT01640067). The hNSCs were transplanted bilaterally into the anterior horns of the lumbar spinal cord (four grafts each, segments L3–L4) of superoxide dismutase 1 G93A transgenic rats (SOD1 rats) at the symptomatic stage. Controls included untreated SOD1 rats (CTRL) and those treated with HBSS (HBSS). Motor symptoms and histological hallmarks of the disease were evaluated at three progressive time points: 15 and 40 days after transplant (DAT), and end stage. Animals were treated by transient immunosuppression (for 15 days, starting at time of transplantation). Under these conditions, hNSCs integrated extensively within the cord, differentiated into neural phenotypes and migrated rostro-caudally, up to 3.77 ± 0.63 cm from the injection site. The transplanted cells delayed decreases in body weight and deterioration of motor performance in the SOD1 rats. At 40DAT, the anterior horns at L3–L4 revealed a higher density of motoneurons and fewer activated astroglial and microglial cells. Accordingly, the overall survival of transplanted rats was significantly enhanced with no rejection of hNSCs observed. We demonstrated that the beneficial effects observed after stem cell transplantation arises from multiple events that counteract several aspects of the disease, a crucial feature for multifactorial diseases, such as ALS. The combination of therapeutic approaches that target different pathogenic mechanisms of the disorder, including pharmacology, molecular therapy and cell transplantation, will increase the chances of a clinically successful therapy for ALS.

The main objective of this phase I trial was to assess the feasibility and safety of microtransplanting human neural stem cell (hNSC) lines into the spinal cord of patients with amyotrophic lateral sclerosis (ALS). Eighteen patients with a definite diagnosis of ALS received microinjections of hNSCs into the gray matter tracts of the lumbar or cervical spinal cord. Patients were monitored before and after transplantation by clinical, psychological, neuroradiological, and neurophysiological assessment. For up to 60 months after surgery, none of the patients manifested severe adverse effects or increased disease progression because of the treatment. Eleven patients died, and two underwent tracheotomy as a result of the natural history of the disease. We detected a transitory decrease in progression of ALS Functional Rating Scale Revised, starting within the first month after surgery and up to 4 months after transplantation. Our results show that transplantation of hNSC is a safe procedure that causes no major deleterious effects over the short or long term. This study is the first example of medical transplantation of a highly standardized cell drug product, which can be reproducibly and stably expanded ex vivo, comprising hNSC that are not immortalized, and are derived from the forebrain of the same two donors throughout this entire study as well as across future trials. Our experimental design provides benefits in terms of enhancing both intra- and interstudy reproducibility and homogeneity. Given the potential therapeutic effects of the hNSCs, our observations support undertaking future phase II clinical studies in which increased cell dosages are studied in larger cohorts of patients. Stem Cells Translational Medicine 2019;8:887–897

Introduction Amyotrophic Lateral Sclerosis (ALS) is a progressive, incurable neurodegenerative disease that targets motoneurons. Cell-based therapies have generated widespread interest as a potential therapeutic approach but no conclusive results have yet been reported either from pre-clinical or clinical studies. Areas covered This is an integrated review of pre-clinical and clinical studies focused on the development of cell-based therapies for ALS. We analyze the biology of stem cell treatments and results obtained from pre-clinical models of ALS and examine the methods and the results obtained to date from clinical trials. We discuss scientific, clinical, and ethical issues and propose some directions for future studies. Expert opinion While data from individual studies are encouraging, stem-cell-based therapies do not yet represent a satisfactory, reliable clinical option. The field will critically benefit from the introduction of well-designed, randomized and reproducible, powered clinical trials. Comparative studies addressing key issues such as the nature, properties, and number of donor cells, the delivery mode and the selection of proper patient populations that may benefit the most from cell-based therapies are now of the essence. Multidisciplinary networks of experts should be established to empower effective translation of research into the clinic.

Clinical trials for Parkinson's disease, which used primary brain fetal tissue, have demonstrated that neural stem cell therapy could be suitable for neurodegenerative diseases. The use of fetal tissue presents several issues that have hampered the clinical development of this approach. In addition to the ethical concerns related to the required continuous supply of fetal specimen, the necessity to use cells from multiple fetuses in a single graft greatly compounded the problem. Cell viability and composition vary in different donors, and, further, the heterogeneity in the donor cells increased the probability of immunological rejection or contamination. An ideal cell source for cell therapy is one that is renewable, thus eliminating the need for transplantation of primary fetal tissue, and that also allows for viability, sterility, cell composition, and cell maturation to be controlled, while being inherently not tumorigenic. The availability of continuous and standardized clinical grade normal human neural cells, able to combine the plasticity of fetal tissue with an extensive proliferating capacity and functional stability, would be of paramount importance for the translation of cell therapy for central nervous system (CNS) disorders into the clinic. Here we describe a well-established protocol to produce human neural stem cells following GMP guidelines that allows us to obtain "clinical grade" cell lines.

Intraspinal stem cell (SC) transplantation represents a new therapeutic approach for amyotrophic lateral sclerosis (ALS) clinical trials. There are considerable difficulties in designing future efficacy trials, some related to the field of ALS and some that are specific to SCs or the mode of delivery. In October 2015, the most controversial points on SC transplantation were addressed during an international workshop intended to bring together international SC and ALS researchers in a public discussion on a topic for which expertise is limited. During the meeting, a discussion was started on the basic structure of the ideal clinical trial testing the efficacy and safety of SC transplantation. The current document includes a number of consensus points reflecting the design of phase II/III clinical trials.

Introduction: Despite knowledge on the molecular basis of amyotrophic lateral sclerosis (ALS) having quickly progressed over the last few years, such discoveries have not yet translated into new therapeutics. With the advancement of stem cell technologies there is hope for stem cell therapeutics as novel treatments for ALS.Areas covered: We discuss in detail the therapeutic potential of different types of stem cells in preclinical and clinical works. Moreover, we address many open questions in clinical translation.Expert opinion: SC therapy is a potentially promising new treatment for ALS and the need to better understand how to develop cell-based experimental treatments, and how to implement them in clinical trials, becomes more pressing. Mesenchymal stem cells and neural fetal stem cells have emerged as safe and potentially effective cell types, but there is a need to carry out appropriately designed experimental studies to verify their long-term safety and possibly efficacy. Moreover, the cost-benefit analysis of the results must take into account the quality of life of the patients as a major end point. It is our opinion that a multicenter international clinical program aime d at fine-tuning and coordinating transplantation procedures and protocols is mandatory.

In the present report, we have analysed the subgenus Vicia by karyological and molecular approaches with the aim to clarify the relationships among Vicia species included in this subgenus by previously evidenced morphological investigations. Multivariate analysis using several karyomorphological parameters in addition to symmetry indices has allowed the construction of a dendrogram of linkage distances very useful to compare and to include in a phylogenetic tree obtained by internal transcribed spacer (ITS) rDNA sequences. Moreover, a separate analysis was performed combining our molecular data on ITS sequences with those reported in the literature for the section Vicilla. Our analyses partly confirm the monophyletic status of the various sections in which the subgenus Vicia has been divided, however questioning, in some cases, the real need to maintain all the nine sections so far accepted and the placement of some individual species in the two subgenera Vicia and Vicilla.

NSCs have been demonstrated to be very useful in grafts into the mammalian central nervous system to investigate the exploitation of NSC for the therapy of neurodegenerative disorders in animal models of neurodegenerative diseases. To push cell therapy in CNS on stage of clinical application, it is necessary to establish a continuous and standardized, clinical grade (i.e., produced following the good manufacturing practice guidelines) human neural stem cell lines.In this chapter we will illustrate some of the protocols for the production and characterization routinely used into our GMP "cell factory" for the production of "clinical grade" human neural stem cell lines already in use in clinical trials on neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS- Clinicaltrials.gov number NCT01640067) and secondary progressive multiple sclerosis (SPMS- Clinicaltrials.gov number NCT03282760).

In questa introduzione viene presentata la struttura del libro, i temi trattati, gli autori.

Il contributo intende mostrare le radici (il passato), lo stato dell'arte (il presente) e le prospettive (il futuro) della pedagogia speciale. La disciplina viene presentata come epistemologicamente fondata e individuata in scenari futuri nazionali e internazionali, con mirati riferimenti alla più recente letteratura nazionale e internazionale.

Vicia barbazitae, a taxon belonging to section Vicia of subgenus Vicia, was recovered and analysed by cytological, karyological and molecular methods with the aim of both proposing a general characterisation of this species and studying the relationships among the species of section Vicia . Phylogenetic relationships among the species of the section Vicia and those of the sections Microcarinae, Wiggersia and Atossa were also analysed. Automated karyotype analysis has been determined after Feulgen's reaction; chromosome banding was performed by sequence-specific fluorochrome staining. Fluorescent chromosome banding showed CMA(+)/DAPI(-) NOR-associated heterochromatin in the satellite pair. Karyomorphological parameters, based on symmetry indices, the dendrogram of linkage distance constructed on 37 chromosome parameters, as well as the molecular data based on internal transcribed spacer sequences provided information about phylogenetic position of this species inside the section Vicia and among the species belonging to the sections Microcarinae, Wiggersia, Atossa and Vicia. From our karyological and molecular results, it emerges that V. barbazitae can be considered a natural member of section Vicia.

Il contributo riguarda il deficit visivo nel mondo, con una particolare attenzione ai processi di inclusione scolastica e sociale. Il contributo è corredato da fotografie inerenti le situazioni di deficit visivo in alcune parti del mondo e mostra le diverse forme di inclusione e di esclusione. Vi è una focalizzazione sull'intervento educativo sia nelle situazioni istituzionali che in quelle domiciliari.

Automated karyotype analyses and sequence of rDNA spacers have been analysed for the species belonging to sections Atossa, Microcarinae, Wiggersia and Vicia. Karyomorphological parameters, based on Rec, Syi and TF % indices, have been determined and evidenced that, in term of symmetry, the karyotype of Vicia lathyroides was the most asymmetric one. A multivariate analysis using 34 karyological parameters, in addition to the symmetry indices, has been carried out and the corresponding dendrogram of linkage distances showed six different groups. Molecular investigations on the inclusive group in study by employing ITS DNA sequences indicated a different pattern of relationships. The cladistic analysis combining the molecular data set with karyological parameters evidenced that the species of sections Vicia and Atossa join closely to each other in a paraphyletic group, which includes the monophyletic section Wiggersia. Therefore, our karyological and molecular data provide information about the phylogenetic position of the analysed species inside the subgenus Vicia and are discussed in relation to previous results obtained by morphology, isozymes and ribosomal genes analyses. L'articolo è disponibile sul sito dell'editore: http://www.springerlink.com

È dato ormai acquisito che la familiarità con i testi letterari, di narrativa e di poesia, sia foriera di apprendimenti fondamentali per lo sviluppo della personalità infantile, per la crescita in armonia delle sue diverse componenti. Ma certo, perché questo avvenga, perché la scuola non mortifichi il potenziale educativo dell’incontro e del confronto con le opere d’autore, è necessaria la mediazione di un adulto esperto, competente sul versante disciplinare non meno che su quello pedagogico e didattico. E dunque come si deve configurare l’insegnamento della letteratura italiana all’ ‘Università dei maestri’? Quali sono le priorità da osservare, i contenuti da proporre, le pratiche da incentivare, i pregiudizi e gli equivoci da scardinare? Ai quesiti l’articolo risponde sulla base di un’esperienza più che decennale, improntata a un difficile equilibrio fra le esigenze del sapere scientifico, specialistico, di cui occorre dotare i futuri docenti e le esigenze di un ordine di scuola in cui la letteratura non costituisce materia curricolare ma spazio formativo, che vive tanto dei contributi dei generi più diversi, dalla fiaba al romanzo, dalla filastrocca alla poesia lirica, quanto degli apporti complementari delle educazioni all’immagine, alla musica, al teatro.

Giustificazione teorica e valutazione critica dei criteri pedagogici ispiratori dell'esperienza formativa dei primi 10 anni di funzionamento del corso di laurera in scienze della formazione primaria dell'università di Palermo. Ipotesi progettuale per il nuovo corso di laurea magistrale quinquennale a ciclo unico.

Avvicinarsi al mondo della disabilità attraverso il materiale fornito dalle autobiografie di genitori di disabili obbliga a riflettere, a crescere, a progettare una società meno distratta nei confronti della sofferenza altrui e delle diversità. Molti sono i temi presenti nelle autobiografie prese in esame, ma in questo lavoro ci si limita a privilegiarne quattro: l’incontro dei genitori con la disabilità; i problemi connessi alla diagnosi riguardante le condizioni dei figli; l’amore dei genitori per questo figlio, tanto diverso da quello desiderato; il dolore provocato dagli «sguardi della gente», sguardi che non accolgono, ma condannano tutto ciò che viene considerato «fuori dalla norma». Obiettivo del contributo è di portare l’attenzione sui numerosi pregiudizi che colpiscono il mondo della disabilità., Rivista Italiana di Educazione Familiare, n°1 gennaio-giugno 2008