In the last few years some of the therapeutical approaches for Duchenne muscular dystrophy (DMD) are specifically targeting distinct groups of mutations, such as deletions eligible for skipping of individual exons. The aim of this observational study was to establish whether patients with distinct groups of mutations have different profiles of changes on the 6 minute walk test (6MWT) over a 12 month period.
The 6MWT was performed in 191 ambulant DMD boys at baseline and 12 months later. The results were analysed using a test for heterogeneity in order to establish possible differences among different types of mutations (deletions, duplications, point mutations) and among subgroups of deletions eligible to skip individual exons.
At baseline the 6MWD ranged between 180 and 560,80 metres (mean 378,06, SD 74,13). The 12 month changes ranged between −325 and 175 (mean −10.8 meters, SD 69.2). Although boys with duplications had better results than those with the other types of mutations, the difference was not significant.
Similarly, boys eligible for skipping of the exon 44 had better baseline results and less drastic changes than those eligible for skipping exon 45 or 53, but the difference was not significant.
even if there are some differences among subgroups, the mean 12 month changes in each subgroup were all within a narrow Range: from the mean of the whole DMD cohort. This information will be of help at the time of designing clinical trials with small numbers of eligible patients.
To test the effect of the single nucleotide polymorphism −66 T>G (rs28357094) in the osteopontin gene (SPP1) on functional measures over 12 months in Duchenne muscular dystrophy (DMD).
This study was conducted on a cohort of ambulatory patients with DMD from a network of Italian neuromuscular centers, evaluated longitudinally with the North Star Ambulatory Assessment (NSAA) and the 6-Minute Walk Test (6MWT) at study entry and after 12 months. Genotype at rs28357094 was determined after completion of the clinical evaluations. Patients were stratified in 2 groups according to a dominant model (TT homozygotes vs TG heterozygotes and GG homozygotes) and clinical data were retrospectively compared between groups.
Eighty patients were selected (age 4.1–19.3 years; mean 8.3 ± 2.7 SD). There were no differences in age or steroid treatment between the 2 subgroups. Paired t test showed a significant difference in both NSAA (p = 0.013) and 6MWT (p = 0.03) between baseline and follow-up after 12 months in patients with DMD carrying the G allele. The difference was not significant in the T subgroup. The analysis of covariance using age and baseline values as covariate and SPP1 genotype as fixed effect showed that these parameters are significantly correlated with the 12-month values.
These data provide evidence of the role of SPP1 genotype as a disease modifier in DMD and support its relevance in the selection of homogeneous groups of patients for future clinical trials.
The aim of the study was i) to assess the spectrum of changes over 24 months in ambulant boys affected by Duchenne muscular dystrophy, ii) to establish the difference between the first and the second year results and iii) to identify possible early markers of loss of ambulation.
One hundred and thirteen patients (age range 4.1–17, mean 8.2) fulfilled the inclusion criteria, 67 of the 113 were on daily and 40 on intermittent steroids, while 6 were not on steroids. All were assessed using the 6 Minute Walk Test (6MWT), the North Star Ambulatory Assessment (NSAA) and timed test.
On the 6MWT there was an average overall decline of −22.7 (SD 81.0) in the first year and of −64.7 (SD 123.1) in the second year. On the NSAA the average overall decline was of −1.86 (SD 4.21) in the first year and of −2.98 (SD 5.19) in the second year. Fourteen children lost ambulation, one in the first year and the other 13 in the second year of the study. A distance of at least 330 meters on the 6MWT, or a NSAA score of 18 at baseline reduced significantly the risk of losing ambulation within 2 years.
These results can be of help at the time of using inclusion criteria for a study in ambulant patients in order to minimize the risk of patients who may lose ambulation within the time of the trial.
Neurodegenerative diseases (NDDs) originate from a loss of neurons in the central nervous system and are severely debilitating. The incidence of NDDs increases with age, and they are expected to become more common due to extended life expectancy. Because no cure is available, these diseases have become a major challenge in neurobiology. The increasing relevance of microRNAs (miRNAs) in biology has prompted investigation into their possible involvement in neurodegeneration in order to identify new therapeutic targets. The idea of using miRNAs as therapeutic targets is not far from realization, but important issues need to be addressed before moving into the clinics. Here, we review what is known about the involvement of miRNAs in the pathogenesis of NDDs. We also report the miRNA expression levels in peripheral tissues of patients affected by NDDs in order to evaluate their application as biomarkers of disease. Finally, discrepancies, innovations, and the effectiveness of collected data will be elucidated and discussed.
microRNA; neurodegenerative diseases; biomarker; Parkinson's disease; Alzheimer's disease; amyotrophic lateral sclerosis; Huntington's disease
The aim of this study was to perform a longitudinal assessment using Quantitative Muscle Testing (QMT) in a cohort of ambulant boys affected by Duchenne muscular dystrophy (DMD) and to correlate the results of QMT with functional measures. This study is to date the most thorough long-term evaluation of QMT in a cohort of DMD patients correlated with other measures, such as the North Star Ambulatory Assessment (NSAA) or thee 6-min walk test (6MWT).
This is a single centre, prospective, non-randomised, study assessing QMT using the Kin Com® 125 machine in a study cohort of 28 ambulant DMD boys, aged 5 to 12 years. This cohort was assessed longitudinally over a 12 months period of time with 3 monthly assessments for QMT and with assessment of functional abilities, using the NSAA and the 6MWT at baseline and at 12 months only. QMT was also used in a control group of 13 healthy age-matched boys examined at baseline and at 12 months.
There was an increase in QMT over 12 months in boys below the age of 7.5 years while in boys above the age of 7.5 years, QMT showed a significant decrease. All the average one-year changes were significantly different than those experienced by healthy controls. We also found a good correlation between quantitative tests and the other measures that was more obvious in the stronger children.
Our longitudinal data using QMT in a cohort of DMD patients suggest that this could be used as an additional tool to monitor changes, providing additional information on segmental strength.
MicroRNAs (miRNAs) have been recently involved in most of human diseases as targets for potential strategies to rescue the pathological phenotype. Since the skeletal muscle is a spread-wide highly differentiated and organized tissue, rescue of severely compromised muscle still remains distant from nowadays. For this reason, we aimed to identify a subset of miRNAs major involved in muscle remodelling and regeneration by analysing the miRNA-profile of single fibres isolated from dystrophic muscle, which was here considered as a model of chronic damage.
The miRNA-signature associated to regenerating (newly formed) and remodelling (resting) fibres was investigated in animal models of muscular dystrophies and acute damage, in order to distinguish which miRNAs are primary related to muscle regeneration. In this study we identify fourteen miRNAs associated to dystrophic fibres responsible for muscle regeneration and remodelling, and confirm over-expression of the previously identified regeneration-associated myomiR-206. In particular, a functional binding site for myomiR-206 was identified and validated in the 3′untranslated region (3′UTR) of an X-linked member of a family of sequence independent chromatin-binding proteins (Hmgb3) that is preferentially expressed in hematopoietic stem cells. During regeneration of single muscle fibres, Hmgb3 messenger RNA (mRNA) and protein expression was gradually reduced, concurrent with the up-regulation of miR-206.
Our results elucidate a negative feedback circuit in which myomiR-206 represses Hmgb3 expression to modulate the regeneration of single muscle fibres after acute and chronic muscle damage. These findings suggest that myomiR-206 may be a potential therapeutic target in muscle diseases.
Recently published reports have described possible cellular therapy approaches to regenerate muscle tissues using arterial route delivery. However, the kinetic of distribution of these migratory stem cells within injected animal muscular dystrophy models is unknown. Using living X-ray computed microtomography, we established that intra-arterially injected stem cells traffic to multiple muscle tissues for several hours until their migration within dystrophic muscles. Injected stem cells express multiple traffic molecules, including VLA-4, LFA-1, CD44, and the chemokine receptor CXCR4, which are likely to direct these cells into dystrophic muscles. In fact, the majority of intra-arterially injected stem cells access the muscle tissues not immediately after the injection, but after several rounds of recirculation. We set up a new, living, 3D-imaging approach, which appears to be an important way to investigate the kinetic of distribution of systemically injected stem cells within dystrophic muscle tissues, thereby providing supportive data for future clinical applications.
iron nanoparticles; micro-CT; CD133+ stem cells; dystrophic muscles
Treatment of dominantly inherited muscle disorders remains a difficult task considering the need to eliminate the pathogenic gene product in a body-wide fashion. We show here that it is possible to reverse dominant muscle disease in a mouse model of facioscapulohumeral muscular dystrophy (FSHD). FSHD is a common form of muscular dystrophy associated with a complex cascade of epigenetic events following reduction in copy number of D4Z4 macrosatellite repeats located on chromosome 4q35. Several 4q35 genes have been examined for their role in disease, including FRG1. Overexpression of FRG1 causes features related to FSHD in transgenic mice and the FRG1 mouse is currently the only available mouse model of FSHD. Here we show that systemic delivery of RNA interference expression cassettes in the FRG1 mouse, after the onset of disease, led to a dose-dependent long-term FRG1 knockdown without signs of toxicity. Histological features including centrally nucleated fibers, fiber size reduction, fibrosis, adipocyte accumulation, and inflammation were all significantly improved. FRG1 mRNA knockdown resulted in a dramatic restoration of muscle function. Through RNA interference (RNAi) expression cassette redesign, our method is amenable to targeting any pathogenic gene offering a viable option for long-term, body-wide treatment of dominant muscle disease in humans.
The administration of rituximab (RTX) in vivo results in B-cell depletion, but evidence for multiple mechanisms of action have been reported. Surprisingly, B cell depletion produced a response in patients with polymyositis, which is characterized as a T cell-mediated autoimmune disorder with biopsy findings similar to Miyoshi myopathy (MM). Indeed, in dysferlinopathies, there is evidence of immune system involvement including the presence of muscle inflammation and a down regulation of the complement inhibitory factor, CD55.
Two patients were treated with four weekly infusions of RTX 375 mg/m2. To measure the improvement in muscle strength after treatment, the isometric hand grip maximal voluntary contraction (MVC) was measured by load cell four times during treatment, and again after one year. In order to assess the reproducibility of our grip assessment, we determined the hand MVC analysis in 16 healthy subjects. Moreover, we measured the number of B cells present in patients by flow cytometric analysis during the course of treatment.
The analysis of B cell number during the course of treatment showed that CD20- and CD19-positive cells were depleted to 0-0.01%. The decrease in B cells was followed by an improvement in the mobility of the pelvic and shoulder girdles as shown by the MRC%. The MVC values of both patients began at values lower than normal whereas during treatment patients had improved percentage of muscle strength. The strength peak in both patients coincided with the minimum B cell values. There were no severe adverse events associated with an infusion of RTX.
We consider the increase in muscle strength observed in both treated patients to be a consequence of their treatment with RTX. To our knowledge, these are the first cases of increased muscle strength in patients with MM. Furthermore, the results of this study indicate that B cell depletion with RTX may be useful in the treatment of patients affected by MM, suggesting a possible role for B cells in the pathophysiology of this muscle disorder.
Advances in stem cell research have provided important understanding of the cell biology and offered great promise for developing new strategies for tissue regeneration. The beneficial effects of stem cell therapy depend also by the development of new approachs for the track of stem cells in living subjects over time after transplantation. Recent developments in the use of nanotechnologies have contributed to advance of the high-resolution in vivo imaging methods, including positron emission tomography (PET), single-photon emission tomography (SPECT), magnetic resonance (MR) imaging, and X-Ray computed microtomography (microCT). This review examines the use of nanotechnologies for stem cell tracking.
stem cells; nanotechnologies; SPIO nanoparticles; X-ray microCT; in vivo imaging
Hepatocyte Growth Factor (HGF) is a pleiotropic cytokine of mesenchymal origin that mediates a characteristic array of biological activities including cell proliferation, survival, motility and morphogenesis. Its high affinity receptor, the tyrosine kinase Met, is expressed by a wide range of tissues and can be activated by either paracrine or autocrine stimulation. Adult myogenic precursor cells, the so called satellite cells, express both HGF and Met. Following muscle injury, autocrine HGF-Met stimulation plays a key role in promoting activation and early division of satellite cells, but is shut off in a second phase to allow myogenic differentiation. In culture, HGF stimulation promotes proliferation of muscle precursors thereby inhibiting their differentiation.
Magic-Factor 1 (Met-Activating Genetically Improved Chimeric Factor-1 or Magic-F1) is an HGF-derived, engineered protein that contains two Met-binding domains repeated in tandem. It has a reduced affinity for Met and, in contrast to HGF it elicits activation of the AKT but not the ERK signaling pathway. As a result, Magic-F1 is not mitogenic but conserves the ability to promote cell survival. Here we show that Magic-F1 protects myogenic precursors against apoptosis, thus increasing their fusion ability and enhancing muscular differentiation. Electrotransfer of Magic-F1 gene into adult mice promoted muscular hypertrophy and decreased myocyte apoptosis. Magic-F1 transgenic mice displayed constitutive muscular hypertrophy, improved running performance and accelerated muscle regeneration following injury. Crossing of Magic-F1 transgenic mice with α-sarcoglycan knock-out mice –a mouse model of muscular dystrophy– or adenovirus-mediated Magic-F1 gene delivery resulted in amelioration of the dystrophic phenotype as measured by both anatomical/histological analysis and functional tests.
Because of these features Magic-F1 represents a novel molecular tool to counteract muscle wasting in major muscular diseases such as cachexia or muscular dystrophy.
Various prognostic serum and cellular markers have been identified for many diseases, such as cardiovascular diseases and tumor pathologies. Here we assessed whether the levels of certain stem cells may predict the progression of Duchenne muscular dystrophy (DMD).
Methods and Findings
The levels of several subpopulations of circulating stem cells expressing the CD133 antigen were determined by flow cytometry in 70 DMD patients. The correlation between the levels and clinical status was assessed by statistical analysis. The median (±SD) age of the population was 10.66±3.81 (range 3 to 20 years). The levels of CD133+CXCR4+CD34- stem cells were significantly higher in DMD patients compared to healthy controls (mean±standard deviation: 17.38±1.38 vs. 11.0±1.70; P = 0.03) with a tendency towards decreased levels in older patients. Moreover, the levels of this subpopulation of cells correlated with the clinical condition. In a subgroup of 19 DMD patients after 24 months of follow-up, increased levels of CD133+CXCR4+CD34- cells was shown to be associated with a phenotype characterised by slower disease progression. The circulating CD133+CXCR4+CD34- cells in patients from different ages did not exhibit significant differences in their myogenic and endothelial in vitro differentiation capacity.
Our results suggest that levels of CD133+CXCR4+CD34- could function as a new prognostic clinical marker for the progression of DMD.
Efficient delivery of cells to target tissues is a major problem in cell therapy. We report that enhancing delivery of mesoangioblasts leads to a complete reconstitution of downstream skeletal muscles in a mouse model of severe muscular dystrophy (α-sarcoglycan ko). Mesoangioblasts, vessel-associated stem cells, were exposed to several cytokines, among which stromal- derived factor (SDF) 1 or tumor necrosis factor (TNF) α were the most potent in enhancing transmigration in vitro and migration into dystrophic muscle in vivo. Transient expression of α4 integrins or L-selectin also increased several fold migration both in vitro and in vivo. Therefore, combined pretreatment with SDF-1 or TNF-α and expression of α4 integrin leads to massive colonization (>50%) followed by reconstitution of >80% of α-sarcoglycan–expressing fibers, with a fivefold increase in efficiency in comparison with control cells. This study defines the requirements for efficient engraftment of mesoangioblasts and offers a new potent tool to optimize future cell therapy protocols for muscular dystrophies.
Duchenne muscular dystrophy (DMD) is a common X-linked disease characterized by widespread muscle damage that invariably leads to paralysis and death. There is currently no therapy for this disease. Here we report that a subpopulation of circulating cells expressing AC133, a well-characterized marker of hematopoietic stem cells, also expresses early myogenic markers. Freshly isolated, circulating AC133+ cells were induced to undergo myogenesis when cocultured with myogenic cells or exposed to Wnt-producing cells in vitro and when delivered in vivo through the arterial circulation or directly into the muscles of transgenic scid/mdx mice (which allow survival of human cells). Injected cells also localized under the basal lamina of host muscle fibers and expressed satellite cell markers such as M-cadherin and MYF5. Furthermore, functional tests of injected muscles revealed a substantial recovery of force after treatment. As these cells can be isolated from the blood, manipulated in vitro, and delivered through the circulation, they represent a possible tool for future cell therapy applications in DMD disease or other muscular dystrophies.
Attempts to repair muscle damage in Duchenne muscular dystrophy (DMD) by transplanting skeletal myoblasts directly into muscles are faced with the problem of the limited migration of these cells in the muscles. The delivery of myogenic stem cells to the sites of muscle lesions via the systemic circulation is a potential alternative approach to treat this disease. Muscle-derived stem cells (MDSCs) were obtained by a MACS® multisort method. Clones of MDSCs, which were Sca-1+/CD34−/L-selectin+, were found to adhere firmly to the endothelium of mdx dystrophic muscles after i.v. or i.m. injections. The subpopulation of Sca-1+/CD34− MDSCs expressing L-selectin was called homing MDSCs (HMDSCs). Treatment of HMDSCs with antibodies against L-selectin prevented adhesion to the muscle endothelium. Importantly, we found that vascular endothelium from striate muscle of young mdx mice expresses mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a ligand for L-selectin. Our results showed for the first time that the expression of the adhesion molecule L-selectin is important for muscle homing of MDSCs. This discovery will aid in the improvement of a potential therapy for muscular dystrophy based on the systemic delivery of MDSCs.
gene therapy; muscle derived stem cell; transplantation; muscle homing; dystrophin