Summary Background Metachromatic leukodystrophy (a deficiency of arylsulfatase A ARSA) is a fatal demyelinating lysosomal disease with no approved treatment. We aimed to assess the long-term outcomes ...in a cohort of patients with early-onset metachromatic leukodystrophy who underwent haemopoietic stem-cell gene therapy (HSC-GT). Methods This is an ad-hoc analysis of data from an ongoing, non-randomised, open-label, single-arm phase 1/2 trial, in which we enrolled patients with a molecular and biochemical diagnosis of metachromatic leukodystrophy (presymptomatic late-infantile or early-juvenile disease or early-symptomatic early-juvenile disease) at the Paediatric Clinical Research Unit, Ospedale San Raffaele, in Milan. Trial participants received HSC-GT, which consisted of the infusion of autologous HSCs transduced with a lentiviral vector encoding ARSA cDNA, after exposure-targeted busulfan conditioning. The primary endpoints of the trial are safety (toxicity, absence of engraftment failure or delayed haematological reconstitution, and safety of lentiviral vector-tranduced cell infusion) and efficacy (improvement in Gross Motor Function Measure GMFM score relative to untreated historical controls, and ARSA activity, 24 months post-treatment) of HSC-GT. For this ad-hoc analysis, we assessed safety and efficacy outcomes in all patients who had received treatment and been followed up for at least 18 months post-treatment on June 1, 2015. This trial is registered with ClinicalTrials.gov , number NCT01560182. Findings Between April, 2010, and February, 2013, we had enrolled nine children with a diagnosis of early-onset disease (six had late-infantile disease, two had early-juvenile disease, and one had early-onset disease that could not be definitively classified). At the time of analysis all children had survived, with a median follow-up of 36 months (range 18–54). The most commonly reported adverse events were cytopenia (reported in all patients) and mucositis of different grades of severity (in five of nine patients grade 3 in four of five patients). No serious adverse events related to the medicinal product were reported. Stable, sustained engraftment of gene-corrected HSCs was observed (a median of 60·4% range 14·0–95·6 lentiviral vector-positive colony-forming cells across follow-up) and the engraftment level was stable during follow-up; engraftment determinants included the duration of absolute neutropenia and the vector copy number of the medicinal product. A progressive reconstitution of ARSA activity in circulating haemopoietic cells and in the cerebrospinal fluid was documented in all patients in association with a reduction of the storage material in peripheral nerve samples in six of seven patients. Eight patients, seven of whom received treatment when presymptomatic, had prevention of disease onset or halted disease progression as per clinical and instrumental assessment, compared with historical untreated control patients with early-onset disease. GMFM scores for six patients up to the last follow-up showed that gross motor performance was similar to that of normally developing children. The extent of benefit appeared to be influenced by the interval between HSC-GT and the expected time of disease onset. Treatment resulted in protection from CNS demyelination in eight patients and, in at least three patients, amelioration of peripheral nervous system abnormalities, with signs of remyelination at both sites. Interpretation Our ad-hoc findings provide preliminary evidence of safety and therapeutic benefit of HSC-GT in patients with early-onset metachromatic leukodystrophy who received treatment in the presymptomatic or very early-symptomatic stage. The results of this trial will be reported when all 20 patients have achieved 3 years of follow-up. Funding Italian Telethon Foundation and GlaxoSmithKline.
The present study aimed to perform a comprehensive data analysis of 47 consecutive patients treated in 8 years and to observe how clinical, radiologic, and surgical factors affect early and long-term ...outcomes, recurrence rate, and survival.
Clinical, radiologic, and surgical data were collected retrospectively from the review of a prospectively collected database. The neurologic disability was evaluated according to the modified Rankin Scale (mRS). Radiologic data were obtained by direct measurement performed on magnetic resonance imaging (MRI). Univariate and multivariate statistical analysis was performed.
From 2008 to 2016, 47 consecutive patients underwent microsurgical resection of intramedullary lesions (28 males and 19 females; mean age, 41.2 years). Ependymoma (53.2%), astrocytoma (14.9%), hemangioblastoma (14.9%), and cavernous angioma (6.4%) were the most frequent tumor histology. The mean follow-up duration was 69.3 months. Gross total tumor resection was performed in 80.8% of cases. Forty-two patients (89.4%) were alive at last follow-up. Five-year overall survival and recurrence-free survival were 92% and 82%, respectively.
Among the examined variables, age seemed to strongly correlate with outcomes; better chances of recovery and a good postoperative outcome were observed in younger patients. Surfacing lesions had a better early functional outcome than did intramedullary located lesions. Patients' preoperative neurologic and functional status (mRS score ≤2) had a significant impact on late neurologic outcome. Progression-free survival correlated with the extent of tumor resection. Surgery should probably be performed before patients' neurologic decline, aiming to achieve maximal resection without compromising patients' quality of life.
In the peripheral nervous system (PNS) myelinating Schwann cells synthesize large amounts of myelin protein zero (P0) glycoprotein, an abundant component of peripheral nerve myelin. In humans, ...mutations in P0 cause the demyelinating Charcot-Marie-Tooth 1B (CMT1B) neuropathy, one of the most diffused genetic disorders of the PNS. We previously showed that several mutations, such as the deletion of serine 63 (P0-S63del), result in misfolding and accumulation of P0 in the endoplasmic reticulum (ER), with activation of the unfolded protein response (UPR). In addition, we observed that S63del mouse nerves display the upregulation of many ER-associated degradation (ERAD) genes, suggesting a possible involvement of this pathway in the clearance of the mutant P0. In ERAD in fact, misfolded proteins are dislocated from the ER and targeted for proteasomal degradation. Taking advantage of inducible cells that express the ER retained P0, here we show that the P0-S63del glycoprotein is degraded via ERAD. Moreover, we provide strong evidence that the Schwann cell-specific ablation of the ERAD factor Derlin-2 in S63del nerves exacerbates both the myelin defects and the UPR in vivo, unveiling a protective role for ERAD in CMT1B neuropathy. We also found that lack of Derlin-2 affects adult myelin maintenance in normal nerves, without compromising their development, pinpointing ERAD as a previously unrecognized player in preserving Schwann cells homeostasis in adulthood. Finally, we provide evidence that treatment of S63del peripheral nerve cultures with N-Acetyl-D-Glucosamine (GlcNAc), known to enhance protein quality control pathways in C.elegans, ameliorates S63del nerve myelination ex vivo. Overall, our study suggests that potentiating adaptive ER quality control pathways might represent an appealing strategy to treat both conformational and age-related PNS disorders.
Sensory trick may relieve dystonic symptoms in patients with idiopathic cervical dystonia (CD). We investigated the patterns of brain functional MRI (fMRI) during resting state, sensory trick ...simulation and sensory trick imagination in CD patients both with and without an effective sensory trick. We recruited 17 CD patients and 15 healthy controls. Nine patients (CD-trick) had an effective sensory trick, while 8 patients (CD-no-trick) did not. Cervical range of motion validated instrument assessed dystonic posture and sensory trick effect. Participants underwent resting state fMRI, which was repeated by patients while executing the sensory trick. Patients also performed an fMRI task in which they were asked to imagine a sensory trick execution. CD-trick and CD-no-trick patients were comparable in terms of CD severity. Applying the sensory trick, CD-trick patients significantly improved dystonic posture. CD-no-trick patients showed an increased functional connectivity of sensorimotor network relative to controls during classic resting state fMRI. During resting state fMRI with sensory trick, CD-trick patients showed a decrease of sensorimotor network connectivity. During the sensory trick imagination fMRI task, CD-trick relative to CD-no-trick patients increased the recruitment of cerebellum bilaterally. This study suggests a hyper-connectivity of sensorimotor areas during resting state in CD-no-trick subjects. In CD-trick patients, the sensory trick performance was associated with a decreased connectivity of the sensorimotor network. The increased activation of cerebellum in CD-trick patients during the sensory trick imagination suggests a possible role of this area in modulating cortical activity.
To investigate the prognostic role and the major determinants of serum phosphorylated neurofilament heavy -chain (pNfH) concentration across a large cohort of motor neuron disease (MND) phenotypes. ...Enzyme-linked immunosorbent assay (ELISA) was used to measure serum pNfH concentration in 219 MND patients consecutively enrolled in our tertiary MND clinic. A multifactorial analysis was carried out to investigate the major clinical determinants of serum pNfH. Kaplan–Meier survival curves and Cox regression analysis were performed to explore the prognostic value of serum pNfH. Serum pNfH levels were not homogenous among MND phenotypes; higher concentrations in pyramidal, bulbar, and classic phenotypes were observed.
C9orf72-
MND exhibited higher pNfH concentrations compared to non-
C9orf72
MND. Multiple linear regression analysis revealed mean MEP/cMAP and disease progression rate as the two major predictors of serum pNfH levels (
R
2
= 0.188;
p
≤ 0.001). Kaplan–Meier curves showed a significant difference of survival among MND subgroups when divided into quartiles based on pNfH concentrations, log-rank
X
2
= 53.0,
p
≤ 0.0001. Our study evidenced that higher serum pNfH concentration is a negative independent prognostic factor for survival. In Cox multivariate model, pNfH concentration showed the highest hazard ratio compared to the other factors influencing survival included in the analysis. pNfH differs among the MND phenotypes and is an independent prognostic factor for survival. This study provides supporting evidence of the role of pNfH as useful prognostic biomarker for MND patients. Neurofilament measurements should be considered in the future prognostic models and in clinical trials for biomarker-based stratification, and to evaluate treatment response.
Although many nerve prostheses have been proposed in recent years, in the case of consistent loss of nervous tissue peripheral nerve injury is still a traumatic pathology that may impair patient's ...movements by interrupting his motor-sensory pathways. In the last few decades tissue engineering has opened the door to new approaches;: however most of them make use of rigid channel guides that may cause cell loss due to the lack of physiological local stresses exerted over the nervous tissue during patient's movement. Electrospinning technique makes it possible to spin microfiber and nanofiber flexible tubular scaffolds composed of a number of natural and synthetic components, showing high porosity and remarkable surface/volume ratio.
In this study we used electrospun tubes made of biodegradable polymers (a blend of PLGA/PCL) to regenerate a 10-mm nerve gap in a rat sciatic nerve in vivo. Experimental groups comprise lesioned animals (control group) and lesioned animals subjected to guide conduits implantated at the severed nerve stumps, where the tubular scaffolds are filled with saline solution. Four months after surgery, sciatic nerves failed to reconnect the two stumps of transected nerves in the control animal group. In most of the treated animals the electrospun tubes induced nervous regeneration and functional reconnection of the two severed sciatic nerve tracts. Myelination and collagen IV deposition have been detected in concurrence with regenerated fibers. No significant inflammatory response has been found. Neural tracers revealed the re-establishment of functional neuronal connections and evoked potential results showed the reinnervation of the target muscles in the majority of the treated animals.
Corroborating previous works, this study indicates that electrospun tubes, with no additional biological coating or drug loading treatment, are promising scaffolds for functional nervous regeneration. They can be knitted in meshes and various frames depending on the cytoarchitecture of the tissue to be regenerated. The versatility of this technique gives room for further scaffold improvements, like tuning the mechanical properties of the tubular structure or providing biomimetic functionalization. Moreover, these guidance conduits can be loaded with various fillers like collagen, fibrin, or self-assembling peptide gels or loaded with neurotrophic factors and seeded with cells. Electrospun scaffolds can also be synthesized in different micro-architectures to regenerate lesions in other tissues like skin and bone.
Metachromatic leukodystrophy (MLD) is a demyelinating lysosomal storage disorder for which new treatments are urgently needed. We previously showed that transplantation of gene-corrected ...hematopoietic stem progenitor cells (HSPCs) in presymptomatic myeloablated MLD mice prevented disease manifestations. Here we show that HSC gene therapy can reverse neurological deficits and neuropathological damage in affected mice, thus correcting an overt neurological disease. The efficacy of gene therapy was dependent on and proportional to arylsulfatase A (ARSA) overexpression in the microglia progeny of transplanted HSPCs. We demonstrate a widespread enzyme distribution from these cells through the CNS and a robust cross-correction of neurons and glia in vivo. Conversely, a peripheral source of enzyme, established by transplanting ARSA-overexpressing hepatocytes from transgenic donors, failed to effectively deliver the enzyme to the CNS. These results indicate that the recruitment of gene-modified, enzyme-overexpressing microglia makes the enzyme bioavailable to the brain and makes therapeutic efficacy and disease correction attainable. Overall, our data provide a strong rationale for implementing HSPC gene therapy in MLD patients.
Abstract
Charcot–Marie–Tooth (CMT) neuropathies are a group of genetic disorders that affect the peripheral nervous system with heterogeneous pathogenesis and no available treatment. Axonal ...neuregulin 1 type III (Nrg1TIII) drives peripheral nerve myelination by activating downstream signaling pathways such as PI3K/Akt and MAPK/Erk that converge on master transcriptional regulators of myelin genes, such as Krox20. We reasoned that modulating Nrg1TIII activity may constitute a general therapeutic strategy to treat CMTs that are characterized by reduced levels of myelination. Here we show that genetic overexpression of Nrg1TIII ameliorates neurophysiological and morphological parameters in a mouse model of demyelinating CMT1B, without exacerbating the toxic gain-of-function that underlies the neuropathy. Intriguingly, the mechanism appears not to be related to Krox20 or myelin gene upregulation, but rather to a beneficial rebalancing in the stoichiometry of myelin lipids and proteins. Finally, we provide proof of principle that stimulating Nrg1TIII signaling, by pharmacological suppression of the Nrg1TIII inhibitor tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17), also ameliorates the neuropathy. Thus, modulation of Nrg1TIII by TACE/ADAM17 inhibition may represent a general treatment for hypomyelinating neuropathies.
The aim of this study was to assess the diagnostic accuracy (sensitivity and specificity) of clinical, imaging and motor evoked potentials (MEP) for predicting the short-term prognosis of multiple ...sclerosis (MS).
We obtained clinical data, MRI and MEP from a prospective cohort of 51 patients and 20 matched controls followed for two years. Clinical end-points recorded were: 1) expanded disability status scale (EDSS), 2) disability progression, and 3) new relapses. We constructed computational classifiers (Bayesian, random decision-trees, simple logistic-linear regression-and neural networks) and calculated their accuracy by means of a 10-fold cross-validation method. We also validated our findings with a second cohort of 96 MS patients from a second center.
We found that disability at baseline, grey matter volume and MEP were the variables that better correlated with clinical end-points, although their diagnostic accuracy was low. However, classifiers combining the most informative variables, namely baseline disability (EDSS), MRI lesion load and central motor conduction time (CMCT), were much more accurate in predicting future disability. Using the most informative variables (especially EDSS and CMCT) we developed a neural network (NNet) that attained a good performance for predicting the EDSS change. The predictive ability of the neural network was validated in an independent cohort obtaining similar accuracy (80%) for predicting the change in the EDSS two years later.
The usefulness of clinical variables for predicting the course of MS on an individual basis is limited, despite being associated with the disease course. By training a NNet with the most informative variables we achieved a good accuracy for predicting short-term disability.
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