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1.  Autologous bone marrow mononuclear cell transplantation in Duchenne muscular dystrophy – a case report 
Patient: Male, 9
Final Diagnosis: Duchenne muscular dystrophy
Symptoms: Hyporeflexia • hypotonia • weaknes of lower limbs
Medication: —
Clinical Procedure: —
Specialty: Neurology
Objective:
Congenital defects/diseases
Background:
Duchenne muscular dystrophy (DMD) is a fatal, genetic, progressive, degenerating muscle disorder. Current treatment options are palliative. Newer options of cellular therapy promise to alter the disease process. Preclinical studies have successfully tested myogenic, neurogenic potential and dystrophin expression of bone marrow mononuclear cells.
Case Report:
We treated a 9-year-old boy suffering from DMD with serial autologous bone marrow mononuclear cell transplantations followed by multidisciplinary rehabilitation. Brooke-Vignos score was 10 and he was wheelchair-bound. Over 36 months, gradual progressive improvement was noticed in muscle strength, ambulation with assistive devices, fine motor movements, Brooke-Vignos score, and functional independence measure score. Nine months after the transplantation, electromyography findings showed development of new normal motor unit potentials of the vastus medialis muscle.
Conclusions:
Magnetic resonance imaging scan of musculoskeletal systems showed no increase in fatty infiltration. This case report provides early investigative findings or the restorative effects of cellular therapy in DMD.
doi:10.12659/AJCR.890078
PMCID: PMC3976215
Stem Cell Transplantation; Autologous Bone Marrow Mono Nuclear Cells; Electromyography; Muscular Dystrophy; Duchenne – congenital
2.  Diagnosis and cell-based therapy for Duchenne muscular dystrophy in humans, mice, and zebrafish 
Journal of human genetics  2006;51(5):397-406.
The muscular dystrophies are a heterogeneous group of genetically caused muscle degenerative disorders. The Kunkel laboratory has had a longstanding research program into the pathogenesis and treatment of these diseases. Starting with our identification of dystrophin as the defective protein in Duchenne muscular dystrophy (DMD), we have continued our work on normal dystrophin function and how it is altered in muscular dystrophy. Our work has led to the identification of the defective genes in three forms of limb girdle muscular dystrophy (LGMD) and a better understanding of how muscle degenerates in many of the different dystrophies. The identification of mutations causing human forms of dystrophy has lead to improved diagnosis for patients with the disease. We are continuing to improve the molecular diagnosis of the dystrophies and have developed a high-throughput sequencing approach for the low-cost rapid diagnosis of all known forms of dystrophy. In addition, we are continuing to work on therapies using available animal models. Currently, there are a number of mouse models of the human dystrophies, the most notable being the mdx mouse with dystrophin deficiency. These mice are being used to test possible therapies, including stem-cell-based approaches. We have been able to systemically deliver human dystrophin to these mice via the arterial circulation and convert 8% of dystrophin-deficient fibers to fibers expressing human dystrophin. We are now expanding our research to identify new forms of LGMD by analyzing zebrafish models of muscular dystrophy. Currently, we have 14 different zebrafish mutants exhibiting various phenotypes of muscular dystrophy, including muscle weakness and inactivity. One of these mutants carries a stop codon mutation in dystrophin, and we have recently identified another carrying a mutation in titin. We are currently positionally cloning the disease-causative mutation in the remaining 12 mutant strains. We hope that one of these new mutant strains of fish will have a mutation in a gene not previously implicated in human muscular dystrophy. This gene would become a candidate gene to be analyzed in patients which do not carry a mutation in any of the known dystrophy-associated genes. By studying both disease pathology and investigating potential therapies, we hope to make a positive difference in the lives of people living with muscular dystrophy.
doi:10.1007/s10038-006-0374-9
PMCID: PMC3518425  PMID: 16583129
DNA sequencing; Muscle; Muscular dystrophy; Stem cells; Zebrafish
3.  Chronic Administration of a Leupeptin-Derived Calpain Inhibitor Fails to Ameliorate Severe Muscle Pathology in a Canine Model of Duchenne Muscular Dystrophy 
Calpains likely play a role in the pathogenesis of Duchenne muscular dystrophy (DMD). Accordingly, calpain inhibition may provide therapeutic benefit to DMD patients. In the present study, we sought to measure benefit from administration of a novel calpain inhibitor, C101, in a canine muscular dystrophy model. Specifically, we tested the hypothesis that treatment with C101 mitigates progressive weakness and severe muscle pathology observed in young dogs with golden retriever muscular dystrophy (GRMD). Young (6-week-old) GRMD dogs were treated daily with either C101 (17 mg/kg twice daily oral dose, n = 9) or placebo (vehicle only, n = 7) for 8 weeks. A battery of functional tests, including tibiotarsal joint angle, muscle/fat composition, and pelvic limb muscle strength were performed at baseline and every 2 weeks during the 8-week study. Results indicate that C101-treated GRMD dogs maintained strength in their cranial pelvic limb muscles (tibiotarsal flexors) while placebo-treated dogs progressively lost strength. However, concomitant improvement was not observed in posterior pelvic limb muscles (tibiotarsal extensors). C101 treatment did not mitigate force drop following repeated eccentric contractions and no improvement was seen in the development of joint contractures, lean muscle mass, or muscle histopathology. Taken together, these data do not support the hypothesis that treatment with C101 mitigates progressive weakness or ameliorates severe muscle pathology observed in young dogs with GRMD.
doi:10.3389/fphar.2011.00089
PMCID: PMC3253583  PMID: 22291646
muscular dystrophy; animal; calpain; skeletal muscle; protease; canine
4.  Low incidence of limb-girdle muscular dystrophy type 2C revealed by a mutation study in Japanese patients clinically diagnosed with DMD 
BMC Medical Genetics  2010;11:49.
Background
Limb-girdle muscular dystrophy type 2C (LGMD2C) is an autosomal recessive muscle dystrophy that resembles Duchenne muscular dystrophy (DMD). Although DMD is known to affect one in every 3500 males regardless of race, a widespread founder mutation causing LGMD2C has been described in North Africa. However, the incidence of LGMD2C in Japanese has been unknown because the genetic background remains uncharacterized in many patients clinically diagnosed with DMD.
Methods
We enrolled 324 patients referred to the Kobe University Hospital with suspected DMD. Mutations in the dystrophin or the SGCG genes were analyzed using not only genomic DNA but also cDNA.
Results
In 322 of the 324 patients, responsible mutations in the dystrophin were successfully revealed, confirming DMD diagnosis. The remaining two patients had normal dystrophin expression but absence of γ-sarcoglycan in skeletal muscle. Mutation analysis of the SGCG gene revealed homozygous deletion of exon 6 in one patient, while the other had a novel single nucleotide insertion in exon 7 in one allele and deletion of exon 6 in the other allele. These mutations created a stop codon that led to a γ-sarcoglycan deficiency, and we therefore diagnosed these two patients as having LGMD2C. Thus, the relative incidence of LGMD2C among Japanese DMD-like patients can be calculated as 1 in 161 patients suspected to have DMD (2 of 324 patients = 0.6%). Taking into consideration the DMD incidence for the overall population (1/3,500 males), the incidence of LGMD2C can be estimated as 1 per 560,000 or 1.8 per million.
Conclusions
To the best of our knowledge, this is the first study to demonstrate a low incidence of LGMD2C in the Japanese population.
doi:10.1186/1471-2350-11-49
PMCID: PMC2861025  PMID: 20350330
5.  The Natural History of Cardiac and Pulmonary Function Decline in Patients with Duchenne Muscular Dystrophy 
Spine  2011;36(15):E1009-E1017.
Study Design
Retrospective review of scoliosis progression, pulmonary and cardiac function in a series of patients with Duchenne Muscular Dystrophy (DMD).
Objective
To determine whether operative treatment of scoliosis decreases the rate of pulmonary function loss in patients with DMD.
Summary of Background Data
It is generally accepted that surgical intervention should be undertaken in DMD scoliosis once curve sizes reach 35 degrees to allow intervention before critical respiratory decline has occurred. There are conflicting reports, however, regarding the effect of scoliosis stabilization on the rate of pulmonary function decline when compared to non operative cohorts.
Methods
We reviewed spinal radiographs, echocardiograms, and spirometry, hospital, and operative records of all patients seen at our tertiary referral center from July 1, 1992 to June 1, 2007 Data was recorded to Microsoft Excel and analyzed with SAS and R statistical processing software.
Results
The percent predicted forced vital capacity (PPFVC) decreased 5% /year prior to operation. The mean PPFVC was 54% (sd=21%) prior to operation with a mean postoperative PPFVC of 43% (sd=14%). Surgical treatment was associated with a 12% decline in PPFVC independent of other treatment variables. PPFVC after operation declined at a rate of 1% per year and while this rate was lower, it was not significantly different than the rate of decline present prior to operation (p=0.18). Cardiac function as measured by left ventricular fractional shortening declined at a rate of 1%/year with most individuals exhibiting an LVFS rate of >30 prior to operation.
Conclusion
Operative treatment of scoliosis in DMD using the Luque Galveston method was associated with a reduction of FVC related to operation. The rate of pulmonary function decline after operation was not significantly reduced when compared to the rate of preoperative FVC decline.
doi:10.1097/BRS.0b013e3181fea1ed
PMCID: PMC3120906  PMID: 21289561
6.  Three Cases of Manifesting Female Carriers in Patients with Duchenne Muscular Dystrophy 
Yonsei Medical Journal  2010;52(1):192-195.
Duchenne muscular dystrophy usually affects males. However, females are also affected in rare instances. Approximately 8% of female Duchenne muscular dystrophy (DMD) carriers are manifesting carriers and have muscle weakness to some extent. We investigated the clinical features of 3 female patients with dystrophinopathy diagnosed by clinical, pathological, and genetic studies at our neuromuscular disease clinic. The onset age of manifesting symptoms varied (8-28 years). Muscle weakness grade varied as follows: patient 1 showed asymmetrical bilateral proximal upper and lower extremities weakness, patient 2 showed asymmetrical bilateral upper extremities weakness similar to scapulohumoral muscular dystrophy, and patient 3 had only bilateral asymmetric proximal lower extremities weakness. Two patients had familial histories of DMD (their sons were diagnosed with DMD), but the 1 remaining patient had no familial history of DMD. The serum creatine kinase level was elevated in all patients, but it was not correlated with muscular weakness. An electromyography study showed findings of myopathy in all patients. One patient was diagnosed with a DMD carrier by a muscle biopsy with an immunohistochemical stain (dystrophin). The remaining 2 patients with familial history of DMD were diagnosed by multiplex ligation-dependent probe amplification (MLPA). There were inconsistent clinical features in the female carriers. An immunohistochemical analysis of dystrophin could be useful for female carrier patients. Also, multiplex ligation-dependent probe amplification is essential for the diagnosis of a manifesting female carrier DMD in female myopathic patients because conventional multiplex PCR could not detect the duplication and is less accurate compared to MLPA.
doi:10.3349/ymj.2011.52.1.192
PMCID: PMC3017697  PMID: 21155054
Dystrophinopathy; female carrier; multiplex ligation-dependent probe amplification
7.  Possible neurogenic factor in muscular dystrophy: its similarity to denervation atrophy1 
Muscle biopsy specimens from 179 cases of muscular dystrophies and from 140 cases of anterior horn cell disorders (from a total of 1,348 biopsied patients) were examined histologically. There were 72 cases of Duchenne type muscular dystrophy (DMD), five of Becker type MD, four girls with myopathy resembling DMD, 40 with limb-girdle, 10 with facioscapulohumeral, seven with late onset, 13 with congenital, and 28 with unclassifiable muscular dystrophies. Groups of small atrophied muscle fibres were encountered in 42 (23%) of the cases in this group, most frequently in patients with limb-girdle, facioscapulohumeral, and least frequently with DM dystrophy. In the second group there were 25 cases of infantile, 38 of juvenile, and 39 of adult spinal muscular atrophy (SMA); there were 21 patients with motor neurone disease (MND), six with poliomyelitis, and 11 with an unclassifiable type of anterior horn cell disorder. Pseudomyopathic changes were encountered in 43 (30%) of all cases in this group. They were most frequently present among patients with juvenile and adult SMA and in those with MND. The presence of group atrophy in muscular dystrophy is considered significant myopathological evidence of a denervation process. On the other hand, pseudomyopathic changes, variation in fibre size, rounding, central nuclei, and increase in connective tissue occurring in various anterior horn cell disorders are seen not to be specific `myopathic' changes. Thus there was an overlap of pathological reactions in muscles from the dystrophies and the neurogenic atrophies. Comparably atrophied fibres (much less than 2 SDs below the normal mean diameter) and hypertrophied fibres (much more than 2 SDs above the normal mean diameter) were encountered in both dystrophy and neurogenic atrophy, considering the large muscles of the limb. Likewise, the mean fibre diameters were comparable in DMD and in juvenile SMA. The fourth evidence of a neurogenic factor in muscular dystrophy was derived from an examination of SDH preparations of muscle. There was a preponderance of type I muscle fibres in dystrophic muscles compared with specimens from controls, suggesting depletion of type II fibres. It appears that the concept of muscular dystrophy as a primary muscle disease needs to be re-examined.
Images
PMCID: PMC494339  PMID: 4714102
8.  Antisense PMO Found in Dystrophic Dog Model Was Effective in Cells from Exon 7-Deleted DMD Patient 
PLoS ONE  2010;5(8):e12239.
Background
Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD). We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO) targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMDJ) lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells.
Methodology/Principal Findings
We converted fibroblasts of CXMDJ and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMDJ and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species.
Conclusion/Significance
Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.
doi:10.1371/journal.pone.0012239
PMCID: PMC2923599  PMID: 20805873
9.  The progress reports on the development of therapies of Duchenne muscular dystrophy 
Acta Myologica  2009;28(2):62-65.
Summary
The roots of the progress reports on the development of therapies for Duchenne muscular dystrophy (DMD) that since 2000 have been produced at Breitnau/Germany and distributed to the parents of DMD patients cover over 30 years of continual occupation with this disease. The beginning was marked by the development of an early detection programme for the genetic disposition for DMD in infant boys. The next step was the organisation of workshops on the management of DMD and the writing of progress reports on these and other relevant conferences. Getting acquainted with the ideas of the protagonists in the research field by holding interviews was a decisive prerequisite for this activity. This took place in tandem with the development of a new kind of multiplex “family letters” that attempted to answer frequently asked questions to many DMD families at the same time.
When – with the beginning of the new millennium – the endeavours towards gene therapies for DMD started to boom all over the scientific world, progress reports designed to keep the families informed about research on DMD treatment were added to the family letters. These reports that give an account of the latest state of the research are written in a plain language that can be understood by laypersons. In the meantime the reports have adopted the character of reviews that are updated annually. They are written in English and German and translated into Spanish and many other languages.
PMCID: PMC2858951  PMID: 20128138
Duchenne muscular dystrophy; CK screening; multiplex family letters; progress reports; gene therapy; exon skipping
10.  Long-term ventilation of patients with Duchenne muscular dystrophy: experiences at the Neuromuscular Centre Ulm 
Acta Myologica  2012;31(3):170-178.
The various measures used to treat the symptoms of Duchenne muscular dystrophy (DMD), i.e. medication with steroids, early operation on contractures and spine deformities as well as cardiac diagnostics and therapy, should always be accompanied by careful monitoring of the patient's respiratory status. Therapy for respiratory failure, in particular long-term ventilation, is now generally accepted as essential for DMD patients. The provision of assisted ventilation has made a decisive contribution to the quality of life for older patients and the stigma hitherto attached to it as being merely a means of keeping a patient comfortable towards the end of life has now been dispelled. Even outside the hospital, assisted ventilation has become routine. These days it is not uncommon for patients on assisted ventilation to have their life extended by 10 years or more.
Non-invasive ventilation is sufficient if used concomitantly with coughing aids. Before undergoing orthopaedic surgery the patient' s respiratory status has to be carefully assessed in order to minimize the risk of perioperative complications. Feeding and swallowing problems may develop if the patient has a scoliosis of the cervical spine region, even if he has had thoraco-lumbar spine surgery. There is still insufficient awareness of this potential problem in relation to respiratory care.
Interdisciplinary collaboration between hospitals, general practitioners, muscle and respiratory centres, as well as advocacies and self-help groups is vital. The administration of aids to support DMD patients is now facilitated by guidelines drawn up by several centres of excellence. Here we mainly describe the historic development of respiratory care at the Ulm Neuromuscular Centre.
PMCID: PMC3631799  PMID: 23620648
Duchenne muscular dystrophy; respiratory failure; non-invasive ventilation; increased survival
11.  Insights into bone health in Duchenne muscular dystrophy 
BoneKEy reports  2012;1:9.
Poor bone health is a significant problem for patients with Duchenne muscular dystrophy (DMD), a progressive, disabling disease. Although the primary focus of DMD disease pathogenesis is degeneration of striated muscle, impairment of bone health likely has a role in the disease that has only been superficially examined to date. Deficiency of bone mineral density and increased incidence of bone fractures are well-recognized clinical components of the DMD phenotype. Furthermore, therapy with corticosteroids, an approved treatment for DMD that prolongs ambulation, may have multiple effects on bone health in DMD patients. This review examines the evidence in preclinical models and in human DMD disease that provides insight into the role performed by bone in the disease pathogenesis and phenotype of DMD. The information reviewed here points toward the need for mechanistic and therapeutic studies to optimize bone health in DMD patients.
doi:10.1038/bonekey.2012.5
PMCID: PMC3727795  PMID: 23951421
12.  Progress in gene therapy of dystrophic heart disease 
Gene therapy  2012;19(6):678-685.
The heart is frequently afflicted in muscular dystrophy. In severe cases, cardiac lesion may directly result in death. Over the years, pharmacological and/or surgical interventions have been the mainstay to alleviate cardiac symptoms in muscular dystrophy patients. Although these traditional modalities remain useful, the emerging field of gene therapy has now provided an unprecedented opportunity to transform our thinking/approach in the treatment of dystrophic heart disease. In fact, the premise is already in place for genetic correction. Gene mutations have been identified and animal models are available for several types of muscular dystrophy. Most importantly, innovative strategies have been developed to effectively deliver therapeutic genes to the heart. Dystrophin-deficient Duchenne cardiomyopathy is associated with Duchenne muscular dystrophy (DMD), the most common lethal muscular dystrophy. Considering its high incidence, there has been a considerable interest and significant input in the development of Duchenne cardiomyopathy gene therapy. Using Duchenne cardiomyopathy as an example, here we illustrate the struggles and successes experienced in the burgeoning field of dystrophic heart disease gene therapy. In light of abundant and highly promising data with the adeno-associated virus (AAV) vector, we have specially emphasized on AAV-mediated gene therapy. Besides DMD, we have also discussed gene therapy for treating cardiac diseases in other muscular dystrophies such as limb-girdle muscular dystrophy.
doi:10.1038/gt.2012.10
PMCID: PMC3628728  PMID: 22318092
muscular dystrophy; heart; cardiomyopathy; Duchenne muscular dystrophy; dystrophin; sarcoglycan
13.  A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy 
Background
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation.
Methods
The capacity of AAV1, AAV6 or AAV8 to cross the vascular endothelial barrier carrying a micro-dystrophin cDNA was compared under identical conditions with delivery through a catheter placed in the femoral artery of the mdx mouse. Transduction efficiency was assessed by immuno-staining using an antibody (Manex1a) that recognizes the N-terminus of micro-dystrophin. The degree of physiologic correction was assessed by measuring tetanic force and protection from eccentric contraction in the extensor digitorum longus muscle (EDL). The vascular delivery paradigm found successful in the mouse was carried to the non-human primate to test its potential translation to boys with DMD.
Results
Regional vascular delivery resulted in transduction by rAAV8.micro-dystrophin reaching 94.5 ± 0.9 (1 month), 91.3 ± 3.1 (2 months), and 89.6 ± 1.6% (3 months). rAAV6.micro-dystrophin treated animals demonstrated 87.7 ± 6.8 (1 month), 78.9 ± 7.4 (2 months), and 81.2 ± 6.2% (3 months) transduction. In striking contrast, rAAV1 demonstrated very low transduction efficiency [0.9 ± 0.3 (1 month), 2.1 ± 0.8 (2 months), and 2.1 ± 0.7% (3 months)] by vascular delivery. Micro-dystrophin delivered by rAAV8 and rAAV6 through the femoral artery significantly improved tetanic force and protected against eccentric contraction. Mouse studies translated to the hindlimb of cynamologous macaques using a similar vascular delivery paradigm. rAAV8 carrying eGFP in doses proportional to the mouse (5 × 1012 vg/kg in mouse vs 2 × 1012 vg/kg in monkey) demonstrated widespread gene expression [medial gastrocnemius – 63.8 ± 4.9%, lateral gastrocnemius – 66.0 ± 4.5%, EDL – 80.2 ± 3.1%, soleus – 86.4 ± 1.9%, TA – 72.2 ± 4.0%.
Conclusion
These studies demonstrate regional vascular gene delivery with AAV serotype(s) in mouse and non-human primate at doses, pressures and volumes applicable for clinical trials in children with DMD.
doi:10.1186/1479-5876-5-45
PMCID: PMC2082019  PMID: 17892583
14.  Enalapril treatment discloses an early role of angiotensin II in inflammation- and oxidative stress-related muscle damage in dystrophic mdx mice☆ 
Pharmacological Research  2011;64(5):482-492.
Graphical abstract
Highlights
► An early treatment with enalapril was performed in exercised mdx mice. ► In vivo, enalapril increased mouse fore limb strength dose-dependently. ► Ex vivo, enalapril reduced muscular markers of oxidative stress and inflammation. ► Results corroborate an early role of angiotensin II in muscular dystrophy. ► Pre-clinical evidences of therapeutic interest of ACE inhibitors for therapy of DMD.
Inhibitors of angiotensin converting enzymes (ACE) are clinically used to control cardiomyopathy in patients of Duchenne muscular dystrophy. Various evidences suggest potential usefulness of long-term treatment with ACE inhibitors to reduce advanced fibrosis of dystrophic muscle in the mdx mouse model. However, angiotensin II is known to exert pro-inflammatory and pro-oxidative actions that might contribute to early events of dystrophic muscle degeneration. The present study has been aimed at evaluating the effects of an early treatment with enalapril on the pathology signs of exercised mdx mouse model. The effects of 1 and 5 mg/kg enalapril i.p. for 4–8 weeks have been compared with those of 1 mg/kg α-methyl-prednisolone (PDN), as positive control. Enalapril caused a dose-dependent increase in fore limb strength, the highest dose leading to a recovery score similar to that observed with PDN. A dose-dependent reduction of superoxide anion production was observed by dihydroethidium staining in tibialis anterior muscle of enalapril-treated mice, approaching the effect observed with PND. In parallel, a significant reduction of the activated form of the pro-inflammatory Nuclear Factor-kB has been observed in gastrocnemious muscle. Histologically, 5 mg/kg enalapril reduced the area of muscle necrosis in both gastrocnemious muscle and diaphragm, without significant effect on non-muscle area. In parallel no significant changes have been observed in both muscle TGF-β1 and myonuclei positive to phosphorylated Smad2/3. Myofiber functional indices were also monitored by microelectrodes recordings. A dose-dependent recovery of macroscopic chloride conductance has been observed upon enalapril treatment in EDL muscle, with minor effects being exerted in diaphragm. However a modest effect, if any, was found on mechanical threshold, a functional index of calcium homeostasis. No recovery was observed in creatine kinase and lactate dehydrogenase. Finally the results suggest the ability of enalapril to blunt angiotensin-II dependent activation of pro-inflammatory and pro-oxidant pathways which may be earlier events with respect to the pro-fibrotic ones, and may in part account for both functional impairment and muscle necrosis. The PDN-like profile may corroborate the combined use of the two classes of drugs in DMD patients so to potentiate the beneficial effects at skeletal muscle level, while reducing both spontaneous and PDN-aggravated cardiomyopathy.
doi:10.1016/j.phrs.2011.06.002
PMCID: PMC3184479  PMID: 21689754
DMD, Duchenne muscular dystrophy; ACE, angiotensin converting enzyme; RAS, renin-angiotensin system; Ang II, angiotensin II; PDN, α-methylprednisolone; NF-kB, nuclear factor-kB; TGF-β1, trasforming growth factor β1; EDL, extensor digitorum longus; CK, creatine kinase; LDH, lactate dehydrogenase; gCl, sarcolemmal chloride conductance; gK, sarcolemmal potassium conductance; MT, mechanical threshold; Muscular dystrophy; Pre-clinical pharmacological tests; Angiotensin-II; Inflammation; Oxidative stress
15.  Non-operative treatment for perforated gastro-duodenal peptic ulcer in Duchenne Muscular Dystrophy: a case report 
BMC Surgery  2004;4:1.
Background
Clinical characteristics and complications of Duchenne muscular dystrophy caused by skeletal and cardiac muscle degeneration are well known. Gastro-intestinal involvement has also been recognised in these patients. However an acute perforated gastro-duodenal peptic ulcer has not been documented up to now.
Case presentation
A 26-year-old male with Duchenne muscular dystrophy with a clinical and radiographic diagnosis of acute perforated gastro-duodenal peptic ulcer is treated non-operatively with naso-gastric suction and intravenous medication. Gastrointestinal involvement in Duchenne muscular dystrophy and therapeutic considerations in a high risk patient are discussed.
Conclusion
Non-surgical treatment for perforated gastro-duodenal peptic ulcer should be considered in high risk patients, as is the case in patients with Duchenne muscular dystrophy. Patients must be carefully observed and operated on if non-operative treatment is unsuccessful.
doi:10.1186/1471-2482-4-1
PMCID: PMC324410  PMID: 14713321
16.  Distinctive Serum miRNA Profile in Mouse Models of Striated Muscular Pathologies 
PLoS ONE  2013;8(2):e55281.
Biomarkers are critically important for disease diagnosis and monitoring. In particular, close monitoring of disease evolution is eminently required for the evaluation of therapeutic treatments. Classical monitoring methods in muscular dystrophies are largely based on histological and molecular analyses of muscle biopsies. Such biopsies are invasive and therefore difficult to obtain. The serum protein creatine kinase is a useful biomarker, which is however not specific for a given pathology and correlates poorly with the severity or course of the muscular pathology. The aim of the present study was the systematic evaluation of serum microRNAs (miRNAs) as biomarkers in striated muscle pathologies. Mouse models for five striated muscle pathologies were investigated: Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophy type 2D (LGMD2D), limb-girdle muscular dystrophy type 2C (LGMD2C), Emery-Dreifuss muscular dystrophy (EDMD) and hypertrophic cardiomyopathy (HCM). Two-step RT-qPCR methodology was elaborated, using two different RT-qPCR miRNA quantification technologies. We identified miRNA modulation in the serum of all the five mouse models. The most highly dysregulated serum miRNAs were found to be commonly upregulated in DMD, LGMD2D and LGMD2C mouse models, which all exhibit massive destruction of striated muscle tissues. Some of these miRNAs were down rather than upregulated in the EDMD mice, a model without massive myofiber destruction. The dysregulated miRNAs identified in the HCM model were different, with the exception of one dysregulated miRNA common to all pathologies. Importantly, a specific and distinctive circulating miRNA profile was identified for each studied pathological mouse model. The differential expression of a few dysregulated miRNAs in the DMD mice was further evaluated in DMD patients, providing new candidates of circulating miRNA biomarkers for DMD.
doi:10.1371/journal.pone.0055281
PMCID: PMC3572119  PMID: 23418438
17.  Targeting Fibrosis in Duchenne Muscular Dystrophy 
Duchenne muscular dystrophy (DMD) is the most common genetic muscle disease affecting 1 in 3,500 live male births. It is an X-linked recessive disease caused by a defective dystrophin gene. The disease is characterized by progressive limb weakness, respiratory and cardiac failure and premature death. Fibrosis is a prominent pathological feature of muscle biopsies from patients with DMD. It directly causes muscle dysfunction and contributes to the lethal DMD phenotype. Although gene therapy and cell therapy may ultimately provide a cure for DMD, currently the disease is devastating, with no effective therapies. Recent studies have demonstrated that ameliorating muscle fibrosis may represent a viable therapeutic approach for DMD. By reducing scar formation, antifibrotic therapies may not only improve muscle function but also enhance muscle regeneration and promote gene and stem cell engraftment. Antifibrotic therapy may serve as a necessary addition to gene and cell therapies to treat DMD in the future. Therefore, understanding cellular and molecular mechanisms underlying muscle fibrogenesis associated with dystrophin deficiency is key to the development of effective antifibrotic therapies for DMD.
doi:10.1097/NEN.0b013e3181e9a34b
PMCID: PMC2916968  PMID: 20613637
Antifibrotic therapy; Duchenne muscular dystrophy; Muscle fibrosis
18.  Surgical treatment of spinal deformities in Duchenne muscular dystrophy: a long term follow-up study 
European Spine Journal  2004;13(5):441-448.
Background
Surgical treatment of spinal deformities in Duchenne muscular dystrophy (DMD) is influenced by a number of factors which have proven to be a difficult challenge. Each case should be carefully evaluated, considering not only the natural history of the spinal deformity, but also the patient’s general condition. These should be thoroughly assessed through clinical and radiographic investigations together with other medical specialists. Life expectancy should be determined according to the cardio-respiratory function, and both preoperative and postoperative quality of life should be taken into consideration, trying to imagine the functional status of each patient after surgery.
Methods
From February 1985 to February 2000, 58 patients with spinal deformity in DMD were surgically treated. Of 25 patients that were operated on between 1985 and 1995, only 20 were followed-up after 5 years because 5 of them had died during this time. Therefore, the present study focuses on the results obtained in 20 cases. The 20 cases reviewed presented with a mean angular value of scoliosis equal to 48° (range 10–92°). Spinal fusion with our modified Luque technique [6] was performed in 19 cases, whereas CD instrumentation was applied in only one case.
Results
At the 5 year follow-up (range 5.6–10 years), the age ranged from 18 to 24 years and averaged 20.4 years. The postoperative angular value of scoliosis averaged 22° (58%, range 0–43°), the mean correction at follow-up was 28° (range 0–60°), and the mean loss of correction was equal to 6° (range, 0–11°). Vital capacity showed a slow progression, slightly inferior to its natural evolution in untreated patients. The severest complication was the death that occurred in one of the patients.
Conclusions
According to the present study, an early surgery (angular value lower than 35–40°) dramatically reduces the rate of risk factors associated with spinal deformities in DMD, and its advantages far exceed the disadvantages, above all in terms of quality of life.
doi:10.1007/s00586-002-0515-6
PMCID: PMC3476590  PMID: 15108098
Duchenne muscular dystrophy; Scoliosis; Vital capacity; Arthrodesis
19.  Role of Telomere Dysfunction in Cardiac Failure in Duchenne Muscular Dystrophy 
Nature cell biology  2013;15(8):895-904.
Duchenne Muscular Dystrophy (DMD), the most common inherited muscular dystrophy of childhood, leads to death due to cardiorespiratory failure. Paradoxically, mdx mice with the same genetic deficiency of dystrophin, exhibit minimal cardiac dysfunction, impeding the development of therapies. We postulated that the difference between mdx and DMD might result from differences in telomere lengths in mice and humans. We show here that, like DMD patients, mice that lack dystrophin and have shortened telomeres (mdx/mTRKO) develop severe functional cardiac deficits including ventricular dilation, contractile and conductance dysfunction, and accelerated mortality. These cardiac defects are accompanied by telomere erosion, mitochondrial fragmentation, and increased oxidative stress. Treatment with anti-oxidants significantly retards the onset of cardiac dysfunction and death of mdx/mTRKO mice. In corroboration, of four DMD patients analyzed, all had 45% shorter telomeres in their cardiomyocytes relative to age- and sex-matched controls. We propose that the demands of contraction in the absence of dystrophin coupled with increased oxidative stress conspire to accelerate telomere erosion culminating in cardiac failure and death. These findings provide strong support for a link between telomere length and dystrophin deficiency in the etiology of dilated cardiomyopathy in DMD and suggest preventive interventions.
doi:10.1038/ncb2790
PMCID: PMC3774175  PMID: 23831727
20.  Markers of oxidative stress and aging in Duchene muscular dystrophy patients and the possible ameliorating effect of He:Ne laser 
Acta Myologica  2007;26(1):14-21.
Summary
Replicative aging and oxidative stress are two plausible theories explaining the etiology of muscular dystrophy. The first theory indicates that replicative aging of myogenic cells (satellite cells), owing to enhanced myofiber turnover, is a plausible explanation of the progression of Duchenne muscular dystrophy (DMD). The oxidative stress theory indicates that failure of muscle regeneration to keep up with the ongoing apoptosis and necrosis following oxidative stress, that normally associates muscular exercise, leads to muscle atrophy in DMD.
To test for these two theories, markers of replicative aging and oxidative stress were assessed in the blood of 30 DMD patients vs. 20 normal healthy age matching controls. Markers of replicative aging showed significantly lower telomerase activity, significantly increased expression of receptors for advanced glycation end products (RAGEs) mRNA and Bax mRNA (an apoptotic gene) in DMD compared to controls. There was a significant increase in markers of oxidative stress among DMD patients compared to controls, measured in terms of increased apoptotic percentage in circulating mononuclear cells, increased lipid peroxidation measured in terms of plasma malondialdehyde (MDA) and increased protein carbonyls. Levels of plasma nitric oxide (NO), which neutralizes oxygen radicals, and expression of inducible nitric oxide synthase (iNOS) mRNA in neutrophils was significantly lower among DMD compared to controls.
Biostimulation of WBC by helium neon (He:Ne) laser irradiation induced a significant increase in the expression of iNOS mRNA and plasma NO levels, but still at a lower level compared to controls. He:Ne laser irradiation induced a marked decrease in markers of oxidative stress among DMD patients compared to their level before irradiation, measured in terms of decreased plasma protein carbonyls, decreased plasma MDA, and decreased apoptosis percentage.
Conclusion: This study points to that oxidative stress is the prime cause for muscle degeneration in DMD and points out to the possible ameliorative effect of He:Ne laser on this stress.
PMCID: PMC2949317  PMID: 17915565
Apoptosis; Bax mRNA; He:Ne laser; lipid peroxidation; nitric oxide; nitric oxide synthase; receptors for advanced glycation end products (RAGEs); telomerase reverse transcriptase
21.  Muscle histology vs MRI in Duchenne muscular dystrophy 
Neurology  2011;76(4):346-353.
Objective:
There are currently no effective treatments to halt the muscle breakdown in Duchenne muscular dystrophy (DMD), although genetic-based clinical trials are being piloted. Most of these trials have as an endpoint the restoration of dystrophin in muscle fibers, hence requiring sufficiently well-preserved muscle of recruited patients. The choice of the muscles to be studied and the role of noninvasive methods to assess muscle preservation therefore require further evaluation.
Methods:
We studied the degree of muscle involvement in the lower leg muscles of 34 patients with DMD >8 years, using muscle MRI. In a subgroup of 15 patients we correlated the muscle MRI findings with the histology of open extensor digitorum brevis (EDB) muscle biopsies. Muscle MRI involvement was assigned using a scale 0–4 (normal–severe).
Results:
In all patients we documented a gradient of involvement of the lower leg muscles: the posterior compartment (gastrocnemius > soleus) was most severely affected; the anterior compartment (tibialis anterior/posterior, popliteus, extensor digitorum longus) least affected. Muscle MRI showed EDB involvement that correlated with the patient's age (p = 0.055). We show a correlation between the MRI and EDB histopathologic changes, with MRI 3–4 grades associated with a more severe fibro-adipose tissue replacement. The EDB was sufficiently preserved for bulk and signal intensity in 18/22 wheelchair users aged 10–16.6 years.
Conclusion:
This study provides a detailed correlation between muscle histology and MRI changes in DMD and demonstrates the value of this imaging technique as a reliable tool for the selection of muscles in patients recruited into clinical trials.
doi:10.1212/WNL.0b013e318208811f
PMCID: PMC3034418  PMID: 21263136
22.  Physical training in boys with Duchenne Muscular Dystrophy: the protocol of the No Use is Disuse study 
BMC Pediatrics  2010;10:55.
Background
"Use it or lose it" is a well known saying which is applicable to boys with Duchenne Muscular Dystrophy (DMD). Besides the direct effects of the muscular dystrophy, the increasing effort to perform activities, the fear of falling and the use of personal aids indirectly impair leg and arm functions as a result of disuse. Physical training could oppose this secondary physical deterioration. The No Use is Disuse (NUD) study is the first study in human subjects with DMD that will examine whether a low-intensity physical training is beneficial in terms of preservation of muscle endurance and functional abilities. The study consists of two training intervention studies: study 1 "Dynamic leg and arm training for ambulant and recently wheelchair-dependent boys with DMD and, study 2 "Functional training with arm support for boys with DMD who have been confined to a wheelchair for several years". This paper describes the hypotheses and methods of the NUD study.
Methods
Study 1 is an explorative randomized controlled trial with multiple baseline measurements. Thirty boys with a DNA-established diagnosis of DMD will be included. The intervention consists of a six-months physical training during which boys train their legs and arms with active and/or assisted cycling training equipment. The primary study outcomes are muscle endurance and functional abilities, assessed with a Six-Minute Bicycle Test and the Motor Function Measure. Study 2 has a within-group repeated measurements design and will include ten boys with DMD who have already been confined to a wheelchair for several years. The six-months physical training program consists of 1) a computer-assisted training and 2) a functional training with an arm support. The primary study outcome is functional abilities of the upper extremity, assessed with the Action Research Arm Test.
Discussion
The NUD study will fill part of the gap in the current knowledge about the possible effects of training in boys with DMD and will increase insight into what type of exercise should be recommended to boys with DMD. The study will finish at the end of 2010 and results are expected in 2011.
Trial registration
The Netherlands National Trial Register1631
doi:10.1186/1471-2431-10-55
PMCID: PMC2929216  PMID: 20691042
23.  Requiem 
Acta Myologica  2012;31(1):48-60.
"In never considering neuromuscular disease to be untreatable, Yves Rideau has found ways to ameliorate every aspect of these conditions. His work has resulted in immeasurably enhancing the quality of life of his patients". This dedication included into the Guide to Evaluation in Management of Neuromuscular Diseases, 1999, made by a coworker who studied in Poitiers from 1981 to 1983, summarizes the content of this paper.
PMCID: PMC3440801  PMID: 22655517
Duchenne muscular dystrophy; rehabilitation; surgery; ventilation
24.  Injection of Vessel-Derived Stem Cells Prevents Dilated Cardiomyopathy and Promotes Angiogenesis and Endogenous Cardiac Stem Cell Proliferation in mdx/utrn−/− but Not Aged mdx Mouse Models for Duchenne Muscular Dystrophy 
It was hypothesized that mesoangioblast stem cells (aorta-derived mesoangioblasts [ADMs]) would restore dystrophin and alleviate or prevent dilated cardiomyopathy (DCM) in animal models of Duchenne muscular dystrophy (DMD). It was found that ADMs delay or prevent development of DCM in dystrophin-deficient heart, resulting in dystrophin expression, angiogenesis, stimulation of endogenous cardiac stem cell division, and the appearance of nestin+ cardiomyocytes of host origin. It was also found that timing of stem cell transplantation may be critical for achieving benefit with cell therapy in DMD cardiac muscle.
Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy. DMD patients lack dystrophin protein and develop skeletal muscle pathology and dilated cardiomyopathy (DCM). Approximately 20% succumb to cardiac involvement. We hypothesized that mesoangioblast stem cells (aorta-derived mesoangioblasts [ADMs]) would restore dystrophin and alleviate or prevent DCM in animal models of DMD. ADMs can be induced to express cardiac markers, including Nkx2.5, cardiac tropomyosin, cardiac troponin I, and α-actinin, and adopt cardiomyocyte morphology. Transplantation of ADMs into the heart of mdx/utrn−/− mice prior to development of DCM prevented onset of cardiomyopathy, as measured by echocardiography, and resulted in significantly higher CD31 expression, consistent with new vessel formation. Dystrophin-positive cardiomyocytes and increased proliferation of endogenous Nestin+ cardiac stem cells were detected in ADM-injected heart. Nestin+ striated cells were also detected in four of five mdx/utrn−/− hearts injected with ADMs. In contrast, when ADMs were injected into the heart of aged mdx mice with advanced fibrosis, no functional improvement was detected by echocardiography. Instead, ADMs exacerbated some features of DCM. No dystrophin protein, increase in CD31 expression, or increase in Nestin+ cell proliferation was detected following ADM injection in aged mdx heart. Dystrophin was observed following transplantation of ADMs into the hearts of young mdx mice, however, suggesting that pathology in aged mdx heart may alter the fate of donor stem cells. In summary, ADMs delay or prevent development of DCM in dystrophin-deficient heart, but timing of stem cell transplantation may be critical for achieving benefit with cell therapy in DMD cardiac muscle.
doi:10.5966/sctm.2012-0107
PMCID: PMC3659745  PMID: 23283493
Cellular therapy; Muscular dystrophy; Angiogenesis; Cardiac; Cellular proliferation; Neural stem cell; Cell transplantation
25.  Duchenne and Becker Muscular Dystrophy: Contribution of a Molecular and Immunohistochemical Analysis in Diagnosis in Morocco 
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders caused by mutations of the DMD gene located at Xp21. In DMD patients, dystrophin is virtually absent; whereas BMD patients have 10% to 40% of the normal amount. Deletions in the dystrophin gene represent 65% of mutations in DMD/BMD patients. To explain the contribution of immunohistochemical and genetic analysis in the diagnosis of these dystrophies, we present 10 cases of DMD/BMD with particular features. We have analyzed the patients with immunohistochemical staining and PCR multiplex to screen for exons deletions. Determination of the quantity and distribution of dystrophin by immunohistochemical staining can confirm the presence of dystrophinopathy and allows differentiation between DMD and BMD, but dystrophin staining is not always conclusive in BMD. Therefore, only identification involved mutation by genetic analysis can establish a correct diagnosis.
doi:10.1155/2009/325210
PMCID: PMC2683945  PMID: 19461958

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