News in DMD

We need treatments to stop/slow the progression of DMD.....the parents need safe and complete information.....see the best informations of treatments in FFF -fight for a future....

26/11/03: Blood loss during posterior spinal fusion surgery in patients with neuromuscular disease: is there an increased risk?.

Abstract Background: Scoliosis surgery in paediatric patients can carry significant morbidity associated with intraoperative blood loss and the resultant transfusion therapy. Patients with neuromuscular disease may be at an increased risk for this intraoperative blood loss, but it is unclear if this is because of direct vascular pathophysiological changes or the fact that neuromuscular patients typically have more extensive orthopaedic disease and more vertebral segments involved. This study examined the risk of extensive blood loss (>50% of total blood volume) in patients with neuromuscular disease compared with patients who did not have neuromuscular disease when the extent of the surgery (number of segments fused), age and preoperative coagulation profile where taken into consideration.

Methods: Retrospective chart review of 163 paediatric patients was preformed. Patients who carried a diagnosis of preexisting neuromuscular disease were classified as such. Idiopathic, traumatic and iatrogenic scoliosis were classified as nonneuromuscular. Extensive blood loss was defined as >50% of estimated total blood volume. Logistic regression was used to predict the risk of extensive blood loss between the two groups when age, weight, extent of surgery was controlled for and anaesthetic and surgical techniques remained similar.

Results: Patients with neuromuscular disease did not vary significantly in age, weight, or preoperative haematocrit and platelet count from patients without neuromuscular disease. Neuromuscular patients did have significantly more vertebral segments fused. When this difference was controlled for statistically, neuromuscular patients had an almost seven times higher risk (adjusted odds ration 6.9, P < 0.05) of losing >50% of their estimated total blood volume during scoliosis surgery.

Conclusions: Patients with neuromuscular disease can present various anaesthetic challenges during scoliosis surgery, among these is the inherent risk of extensive blood loss. Recognizing this may help anaesthesiologists and surgeons more accurately prepare for and treat intraoperative blood loss during scoliosis surgery in patients with neuromuscular disease.

22/11/03 (IN PRESS: Neuromuscular Disorders): A-utrophin up-regulation in mdx skeletal muscle is independent of regeneration

Duchenne muscular dystrophy is a fatal childhood disease caused by mutations that abolish the expression of dystrophin in muscle. Utrophin is a paralogue of dystrophin and can functionally replace it in skeletal muscle. A method to induce utrophin up-regulation in muscle should therefore be therapeutically useful in Duchenne muscular dystrophy. The search for such a method needs to be informed by an understanding of the mechanisms controlling utrophin expression in muscle. Two full length utrophin isoforms are expressed: A and B. A-utrophin is up-regulated in dystrophin deficient skeletal muscle and we sought to test the hypothesis that this up-regulation occurs as a consequence of ongoing regeneration. We measured utrophin expression by immunohistochemistry and immunoblotting in the oesophageal outer muscular layer and in g-irradiated limb muscle from mdx mice. Skeletal muscle in these tissues is dystrophin deficient but not regenerating; we found that A-utrophin up-regulation still occurred. We conclude that utrophin up-regulation in skeletal muscle does not depend on regeneration. An alternative hypothesis involving competition for binding sites between utrophin and dystrophin is discussed. These results have important implications for future studies aiming to effect therapeutic utrophin up-regulation in Duchenne muscular dystrophy patients.

"Given that, it is possible, Rossi says, that they also could be used to artificially regenerate damaged muscle tissue in heart attack patients and people with diabetes or muscular dystrophy"

"Even if we were lucky and our first strategy to make them work well worked, by the time all the controls and clinical trials were done, it would take 10 years," Rossi, a member of UBC's Biomedical Research Centre

Perindopril Prevents the Onset and Progression of Left Ventricular Dysfunction in Children with Duchenne Muscular Dystrophy

Henri-Marc Becana, Pitie-Salpetriere Hospital, Paris, France; Christophe Meune, Cochin Hospital, Paris, France; Guy Lerebours, Pitie-Salpetriere Hospital, Paris, France; Jean-Yves Devaux, Denis Duboc, Cochin Hospital, Paris, France; The French Working Group on Heart Involvement in Myopathies

Background: Duchenne Muscular disease (DMD) is an inherited X-linked disease due to the absence of dystrophin and clinically characterized by progressive muscle weakness and constant myocardial involvement responsible for sudden death or end-stage heart failure in 40% of patients between the age of 15 and 30. The aim of this study was to evaluate the preventive effect of early ACEI treatment on left ventricular (LV) dysfunction in young patients with DMD and normal LVEF at inclusion. Methods: In phase I, children with genetically proven DMD and radionuclide ejection fraction (EF) superior than 55% were enrolled in a multicentric, controlled, randomized, double blind trial of perindopril 2-4 mg/day versus placebo for 3 years. In phase II, all patients then received open-label perindopril for 24 more months. Radionuclide LVEF was performed at 0, 36 and 60 months. Student's t-tests and chi-square analysis were used for comparisons. Results: 60 children asymptomatic for heart disease were included in the phase I study (10.6±1.2 years, EF 65.0±5.4%)(31 in the perindropil group and 29 in the placebo group) and 46 in the phase II study (n=23 in both groups). No adverse effect related to treatment was documented. At the end of phase I, LVEF remained stable in both groups (from 64.8±5.3% to 59.6±8.5% in the perindopril group and 65.5±5.4% to 64.5±9.9% in the placebo group, p=0.114). However, at 60 months, 6 patients in the control group (26%) had a LVEF of less than 45, versus one in the perindopril group (4%)(p=0.0319). Conclusions: Early treatment with ACEI perindopril is well tolerated in these young patients and delay onset of LVEF deterioration. This preventive efficacy of ACEI has to be evaluated in other groups of patients genetically exposed to develop LV dysfunction.

Impaired Strain Rate Measurements in Patients With Duchenne Muscular Dystrophy

Nickolaos Giatrakos, Cardiology dept, Hammersmith Hospital, NHLI, ICSM, London, United Kingdom; Maria Kinali, Dubowitz Neuromuscular Centre, Department of Paediatrics, Hammersmith Hospital, ICSM, London, UK, London, United Kingdom; George Koutroulis, Cardiology dept, Hammersmith Hospital, NHLI, ICSM, London, United Kingdom; Francesco Muntoni, Dubowitz Neuromuscular Centre, Department of Paediatrics, Hammersmith Hospital, ICSM, London, UK, London, United Kingdom; Petros Nihoyannopoulos; Cardiology dept, Hammersmith Hospital, NHLI, ICSM, London, United Kingdom

Background: Patients with Duchenne muscular dystrophy (DMD) develop dilated cardiomyopathy at the later stages of the disease. Strain rate (SR) has been used to study myocardial function in ischeamia and cardiomyopathies. The aim of this study was to investigate the usefulness of SR for the early detection of cardiac involvement in young, asymptomatic patients with DMD.
Methods: We studied 53 patients with genetically confirmed diagnosis of DMD (mean age 8.7±2.8 years) without clinical symptoms from the cardiovascular system and normal conventional echocardiographic studies, and compared with 22 normal controls matched for age (mean age 8.5±2.5 years). We used the HDI 5000 (Philips Medical Systems) to acquire from the parasternal long axis the colour M-mode tissue Doppler (TDI) of the posterior wall of the LV. Images were digitally stored for offline analysis with dedicated software HDI-lab (Philips Medical Systems). We calculated the SR using the formula SR=Ua-Ub/d where U the velocities of the endocardium a and epicardium b, and d the distance of a and b at each time point.
Results: There was no significant difference for the parameters from conventional echocardiographic studies between the two groups. The velocities derived from the TDI, mean velocity at systole (26,99±7,12mm/sec vs. 33,4±7,3mm/sec, p<0,000), at early diastole (-45,79±13,93mm/sec vs.-60,46±7,58mm/sec, p<0,000) and late diastole (-10,93±3,41mm/sec vs.-13,32±6,4mm/sec, p<0,02) were significantly different in patients with DMD when compared with controls. SR was found to be significantly lower at systole (1,78±0,75s^-1 vs. 2,82±0,5s^-1, p<0,000) and early diastole (-5,17±1,98s^-1vs.-9,02±1,25s^-1, p<0,000) but not at late diastole (-1,52±0,84s^-1vs.-1.6±0,46s^-1, p<0,568) in patients with DMD.
Conclusions: Estimation of SR of the posterior wall of the LV showed systolic and diastolic dysfunction at early stages in asymptomatic patients with DMD and when the conventional echocardiography is still normal. Estimation of SR could be a sensitive method to investigate the pathophysiology of the disease and identify early impairment of the cardiac function.

Negamycin restores dystrophin expression in skeletal and cardiac muscles of mdx mice
M. SHIOZUKA,¹ M. ARAKAWA,¹ Y. NAKAYAMA,² T. HARA,² M. HAMADA,³ D. IKEDA,³ Y. TAKAHASHI,³ R. SAWA,³ Y. NONOMURA,³ K. SHEYKHOLESLAMI,⁴ K. KONDO,⁴ K. KAGA,⁴ S. TAKEDA,⁵ R. MATSUDA¹ ; ¹ Dept. of Life Sciences, The University of Tokyo, Tokyo, Japan, ² Dept. of Tumor Biochemistry, Tokyo Metropolitan Institute of Medical Sciences, Tokyo, Japan, ³ The Institute of Microbial Chemistry, Tokyo, Japan, ⁴ Dept. of Otolaryngology, The University of Tokyo, Tokyo, Japan, ⁵ Dept. of Molecular Therapy, National Institute of Neuroscience, Tokyo, Japan

The ability of aminoglycoside antibiotics to promote read-through of nonsense mutations has attracted interests in these drugs as potential therapeutic agents in genetic diseases. However, strong toxicity of aminoglycoside antibiotics may cause severe side effects during long-term treatment. In this study, we report that negamycin, a dipeptide antibiotic, also restores dystrophin expression in skeletal and cardiac muscles of mdx mouse; an animal model of Duchenne muscular dystrophy (DMD) with a nonsense mutation in dystrophin gene, and in cultured mdx myotubes. Dystrophin expression was confirmed by immunohistochemistry and immunoblotting. We also compared the toxicity of negamycin and gentamicin, and found negamycin to be less toxic. Furthermore, we demonstrated that negamycin bound to a partial sequence decording the eukaryotic rRNA A-site. We conclude that negamycin is a promising new candidate for chemotherapy for DMD and other genetic diseases caused by nonsense mutations.

Calcineurin-Induced Upregulation of Utrophin Attenuates the Dystrophic Pathology in mdx Mouse Muscle
J. V. Chakkalakal,¹ M. Harrison,² E. R. Chin,³ R. N. Michel,² B. J. Jasmin¹ ; ¹ Cellular & Molecular Medicine, University of Ottawa, Ottawa, ON, Canada, ² Neuromuscular Lab, Laurentian University, Sudbury, ON, Canada, ³ Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton, CT

We recently showed that mice expressing a constitutively active form of calcineurin (CnA*) have elevated levels of utrophin A in their muscles (Proc. Natl. Acad. Sci. USA, 100: 7791-7796, 2003). In the present study, we crossed these transgenic animals with mdx mice to determine whether mdx/CnA* mice would be less affected by the dystrophic process. Since expression of CnA* has been shown previously to stimulate the slow/oxidative myofiber program, we examined the expression of myosin heavy chain isoforms in mdx/CnA* and mdx mice. By immunofluorecence and RT-PCR assays, we observed in mdx/CnA* a shift in myosin heavy chain profile towards a slower, more oxidative phenotype compared to mdx muscles. In addition, we determined that expression of utrophin A and its transcript were increased by ~ 2 fold in mdx/CnA* mouse muscles. Immunodetection of IgM inside myofibers, used in this case as an index of sarcolemmal disruption, showed that the number of IgM-positive fibers was significantly reduced in muscles from mdx/CnA* mice. Consistent with these findings, we also observed that muscles from mdx/CnA* mice showed less variability in fiber size and contained fewer central nuclei. Together, these results demonstrate that enhanced calcineurin activity can have important beneficial effects on the dystrophic phenotype by stimulating the expression of utrophin A. Furthermore, these findings provide specific targets for which pharmacological strategies may be designed to enhance utrophin levels in muscles from patients with Duchenne muscular dystrophy. Funded by the MDA and CIHR.

Young children with Duchenne muscular dystrophy (DMD) show improved muscle strength and functional status and evidence of a slowing of disease progression with creatine supplements, researchers reported at the 128th annual meeting of the American Neurological Association.

A study of 60 children with DMD between the ages of five and nine years was conducted, based on studies of animal models of DMD that showed that glutamine and creatine supplementation prevented loss of muscle strength compared with prednisone.

Lead investigator Diana M. Escolar, MD, from the Children's National Medical Center at George Washington University in Washington, D.C., and the Cooperative International Neuromuscular Research Group, randomized the children to one of three arms: creatine 5 g daily plus placebo twice daily, glutamine 0.3 mg/kg daily plus placebo twice daily, or placebo twice daily alone. Dr. Escolar noted that the investigators were comparing creatine versus placebo and glutamine versus placebo, but not creatine versus glutamine.

The primary outcome measure was manual muscle strength (MMT, 34 muscle groups) and the secondary outcome measure was quantitative muscle strength (QMT, seven muscle groups), which measured functional status using various physical activities.

"We found very different outcomes in the 30 children in the younger age group [between the ages of five and seven years] and the 30 children in the older group [between the ages of seven and nine years] on the primary outcome measure," Dr. Escolar told Medscape.

In the six-month study, MMT scores did not significantly differ between the three groups of the study, Dr. Escolar reported. But on QMT scores, children in the placebo group showed more deterioration in muscle strength compared with children in the two treatment groups. Children randomized to creatine showed a lesser degree of deterioration than children receiving glutamine. Children in the younger age group scored higher on measures of functionality such as standing, climbing, and running than the older children receiving creatine.

"I have a theory to explain this," Dr. Escolar said. "These compounds improve muscle energy. Younger kids have muscles that respond more to an increase in energy than older kids.... They don't need more strength, they just need more energy. For the older kids, an increase in strength translates to an increase in functionality." Dr. Escolar said it is possible that the energy supplements may even slow the progression of DMD.

While she stressed that there was only a trend toward increased functionality in the younger children rather than a statistically strong outcome, she pointed out that creatine and glutamine have very minimal adverse effects. "If parents want to go out and buy these supplements, I wouldn't care if they do," Dr. Escolar asserted.

18/10/03: Abstracts from Neuroscience 2003, the Society for Neuroscience 33rd Annual Meeting, New Orleans, November 8 - 12, 2003

1) C. Zhang, S. Chen, Y. Xie, W. Huang, S. Li, W. Zhang, Q. Li, X. Liu. THERAPEUTIC EFFECT OF DUCHENNE MUSCULAR DYSTROPHY MICE WITH BONE MARROW STEM CELLS TRANSPLANTATION. Program No. 413.9. 2003

Objective: To investigate the locomotive function, electronic physiology changes, and expression of dystrophin in Duchenne muscular dystrophy mice(mdx and dko) with bone marrow stem cells transplantation.
Methods: The bone marrow stem cells of C57BL/6

4-to-5 weeks age

were isolated and cultured in vitro for 3 days, then the stem cells about 5.0X106 injected intravenously into the mdx mice and dko mic

7-to-8 weeks age

, respectively. Before injecting, 10 mdx mice and 10 dko mice were preconditioned with 7 Gy gamma ray to decrease the immune reaction. The clinical features of the graft verse host disease (GVHD). in the transplanted mice were assessed and investigated after bone marrow stem cell transplatation..12 weeks after being transplanted, the locomotion function, muscle electrophysiologic features(EMG) and dystrophin expression of the mdx mice and dko mice were investigated..
Results: Three month after bone marrow stem cells transplanting, we find that (1) there were more than 10% of muscle cells expression dystrophin protein in each transplanted mice(mdx and dko); (2) the electromyography showed almost normal waves in the transplantation mdx mice and dko mice; (3)the locomotive function improved obviously in transplantation dko mice; (4)the life span of the transplantation dko mice were more than 200 days( the life span of control of dko mice were less than 140 days).
Conclusions: Allogenic bone marrow stem cells transplantation can improve the electromyographic items, dystrophin expression in muscles of mdx and dko mice. Further more, the stem cell transplantation can improve the locomotive function and prolong the life span of dko mice.

2) Y. Zhu, B. Chen, H. Gong, J. Pan, W. Zhang. EXPRESSION OF TRANSFERRIN RECEPTOR IN DUCHENNE MUSCULAR DYSTROPHY TISSUES. Program No. 80.2. 2003

Duchenne muscular dystrophy (DMD) is an X-linked, fatal disease caused by mutations of the gene encoding the cytoskeletal protein dystrophin. Skeletal muscle of DMD patients is characterized by an ongoing process of degeneration and regeneration. Transferrin receptor (TfR, CD71), an integral membrane glycoprotein, mediates cellular uptake of iron. In most tissues, TfR expression is correlated positively with proliferation and regulated at the post-transcriptional level. To determine if TfR is involved in muscular regeneration in DMD patients, we examined the expression of TfR in muscle samples of 42 patients with DMD, and 10 from normal volunteers. Immunohistochemical staining of TfR was positive with variable intensity in normal control and DMD patients. In normal control, TfR was faintly expressed on the surface of muscle fibers. In DMD patients, strong TfR reaction was detected. In some muscular fibers from DMD patients, TfR was found strongly expressed inside the fibers. Further detection with Enzyme histochemistry and acridine orange (AO) fluorescence techniques showed that these fibers with strong TfR expression were regenerative IIc type fibers. The increase of TfR expression in regenerative fibers suggests that the high uptake of iron may promote the capacity of muscle regeneration and thus may delay the deterioration of DMD patients.

3) C.G. Carlson. STEADY STATE CALCIUM AND RESTING CALCIUM INFLUX IN NONDYSTROPHIC AND MDX MYOTUBES EXPOSED TO PREDNISONE OR PREDNISOLONE. Program No. 413.10. 2003

Certain glucorticoids, such as prednisone, prednisolone and deflazacort, increase muscle strength and slow the progress of Duchenne muscular dystrophy (Fenichel et al., Neurology, 41, 1874, 1991) by one or more mechanisms that have not yet been fully characterized. Evidence from 45calcium uptake studies in the C2C12 muscle cell line suggests that one mechanism may involve decreases in calcium influx following one or more days of exposure to these agents beginning at the period of myoblast fusion (Passaquin et al., Brit. J. of Pharmacol., 1998). Recent evidence from this laboratory obtained in FURA-loaded cultured lens epithelial cells indicates that some steroids, however, may induce very rapid (seconds to minutes) effects on resting calcium influx measured by the manganese quench technique (Samadi et al., Pfluegers Arch. Europ. J. Physiol.,444, 700-709, 2002). To determine whether glucorticoids exert a unique action on resting calcium influx in dystrophic muscle, studies have begun to characterize any acute, nongenomic effects of prednisone or prednisolone on steady state calcium levels and resting calcium influx in cultured nondystrophic and mdx myotubes. Initial results indicate that neither prednisone (10 microM, 100 microM) nor prednisolone (100 microM) has immediate effects on steady state calcium levels in nondystrophic myotubes, but that prednisolone may induce a slight decrease in manganese quench rate in mdx myotubes at relatively early stages in culture (culture day 12-14). These results further demonstrate the utility of the manganese quench technique in assessing whether short or long term exposure to glucorticoids induces a beneficial reduction in resting calcium influx in dystrophic skeletal muscle.

Lisa S. Krivickas, MD (Spaulding Rehabilitation Hospital, Boston, Massachusetts)

In the past, patients with neuromuscular diseases were advised not to exercise because of the fear that too much exercise might produce "overuse weakness." No controlled studies have demonstrated that the phenomenon of overuse weakness actually exists. Most studies of exercise training in patients with neuromuscular disease, despite methodologic limitations, suggest that strength and aerobic capacity gains can occur in patients with slowly progressive disorders. Four forms of exercise training are relevant to patients with neuromuscular disorders: flexibility, strengthening, aerobic, and balance exercises. The literature regarding these forms of exercise in patients with neuromuscular disorders is summarized in this article, and recommendations are made regarding the direction future research on exercise in this population should take.

15/09/03: Use of Alendronate to treat osteoporosis in boys with muscular dystrophy: a report of 3 cases.

Susan D. Apkon, MD (Children's Hospital and University of Colorado Health Sciences Center, Denver, CO), e-mail: apkon.susan@tchden.org

Archives of Physical Medicine and Rehabilitation - 2003 Academy annual assembly abstracts

Setting: Tertiary care pediatric hospital. Patients: 2 boys with Duchenne’s muscular dystrophy (DMD), ages 11 and 12 years, and 1 boy with Becker’s muscular dystrophy (BMD), age 11 years. All boys are ambulatory for a portion of the day. Case Descriptions: Baseline bone mineral density of the lumbar spine and femoral neck were assessed using dual-energy x-ray absorptiometry (DXA). Results revealed significant osteoporosis at the femoral neck, with a mean z score (SD using ageand sex-matched peers) of –3.73 (range, –3.16 to –4.42). Results at lumbar spine revealed mean z score of –1.14 (range, –1.6 to 0.82). Because all subjects were having frequent falls, parents and physicians felt aggressive treatment was necessary to prevent fractures. Oral alendronate was initiated at 45mg weekly for 6 months. Assessment/Results: All 3 subjects tolerated the medication without side effects. There were no gastrointestinal complaints. Follow-up DXAs performed after 6 months of treatment revealed improvements at the femoral neck in all 3 subjects, with a mean improvement in z score of 0.94 (range, 0.3–1.33) representing an improvement of almost 1 SD. There was improvement at the lumbar spine, with a mean change of 0.28 (range, –0.45 to 1.11). Discussion: Osteoporosis is problematic in boys with DMD and becomes more severe as the disease progresses. The high incidence of fractures in this group of boys is likely due to osteoporosis. This is the first known report on the use of alendronate in children with DMD or BMD in the treatment of osteoporosis. Conclusion: In this small case series, weekly oral alendronate over 6 months was effective in improving bone mineral density in boys with DMD and BMD. A prospective, randomized, double-blinded study is underway.

10/09/03:(Article in Press: Neuromuscular Disorders - 2003) Detection of glucocorticoid-like activity in traditional Chinese medicine used for the treatment of Duchenne muscular dystrophy

Anecdotal reports of positive influence of certain traditional Chinese medicines on the progression of neuromuscular diseases in general and Duchenne muscular dystrophy (DMD) in particular has raised interest in patient support groups and clinical experts alike. However, clinical signs of steroid-specific side effects in patients treated with a particular form of Chinese medicine raised the concern that they may contain glucocorticoids, which in turn could also explain the mild beneficial effects seen in some of the patients. We have extracted and fractionated capsules containing pulverized Chinese medicine that had been used for the treatment of DMD patients and analyzed their content for glucocorticoid-like activity using promoter–reporter assays. We demonstrate that extracts from this Chinese medicine activate a prototype glucocorticoid-response element, increase the level of utrophin protein in human muscle cells and activate the utrophin promoter A. Based on our bioassays we conclude that this particular Chinese medicine used for the treatment of muscular dystrophy patients contains glucocorticoids as one of its active ingredients.

08/09/03: (Article in Press: Neuromuscular Disorders - 2003) Looking under every rock: Duchenne muscular dystrophy and traditional Chinese medicine

J. Andoni Urtizbereaa, Qi-Shi Fan, Elizabeth Vroom, Dominique Recan, Jean-Claude Kaplan

Traditional Chinese medicine has been advocated to alleviate symptoms in Duchenne muscular dystrophy. To investigate this hypothesis, a pilot study was carried out in Beijing on 10 DMD boys treated with various regimens, including pills, decoctions, massages and acupuncture at various stages of their disease course. Despite the limited scientific impact of such a study, it seems as if the benefit, if any, is minimal. Moreover, some indirect clinical clues such as the cushingoid appearance found in a few patients suggest these drugs may also contain corticosteroids to some extent.

08/09/03: (Article in Press: Neuromuscular Disorders - 2003) The Duchenne muscular dystrophy population in Denmark, 1977–2001: prevalence, incidence and survival in relation to the introduction of ventilator use

Mechanical ventilation of patients with Duchenne muscular dystrophy continues to be a subject of study. The purpose was to estimate prevalence, incidence, mortality and use of mechanical ventilation in the total Duchenne muscular dystrophy population in Denmark between 1977 and 2001 and further, to reconstruct the introduction of mechanical ventilation to assess the role of the patient organization. Study objects were collected from five sources and verifiable cases identified. Negotiations between health authorities and the patient organization constituted main empirical data for the reconstruction. While overall incidence remained stable at 2.0 per 105, prevalence rose from 3.1 to 5.5 per 105, mortality fell from 4.7 to 2.6 per 100 years at risk and prevalence of Duchenne muscular dystrophy ventilator users rose from 0.9 to 43.4 per 100. We conclude that survival of Duchenne muscular dystrophy patients has increased and ventilator use is probably a main reason. The patient organization exercised a key role but acted upon preconditions created by other players.

08/09/03: (Article in Press: Molecular Brain Research - 2003) Dystrophin antisense oligonucleotides decrease expression of nNOS in human neurons

Department of Cytomorphology, School of Medicine, Cittadella Universitaria, SS 554 Bivio Sestu, 09042, Monserrato (CA), Italy

Nitric oxide (NO) plays an important role in the pathogenesis of neurodegenerative disease. It has been shown that neuronal NO synthase (nNOS), the enzyme that constitutively produces NO in brain, is a component of the dystrophin-associated protein complex. The absence of dystrophin causes Duchenne muscular dystrophy. Thus, we attempted to study whether or not a decrease of dystrophin expression would induce a modification in nNOS expression in cultured human neurons. Human fetal neuronal cultures were treated with antisense oligonucleotides against different isoforms of dystrophin and the expression of nNOS tested by RT-PCR and immunocytochemistry. Results showed that nNOS mRNAwas significantly decreased by about 35% in neurons treated with brain-specific dystrophin (brain Dp427) antisense, whereas iNOS expression was not affected. Accordingly, a decrease in immunostaining for nNOS was observed in antisense treated neurons compared to controls. Expression of neuronal markers, such as bFGF or synaptophysin, was not affected by the same antisense treatment. Astrocytes were not affected by treatment, as shown by utrophin expression, a dystrophin-like protein that was not modified in pure astrocytic cultures. Thus, we conclude that a decrease of dystrophin in human neurons is associated with a decrease of nNOS expression.

27/08/03 (Article in Press): Enhanced in vivo delivery of antisense oligonucleotides to restore dystrophin expression in adult mdx mouse muscle.

Abstract: The use of antisense oligonucleotides (AOs) to induce exon skipping leading to generation of an in-frame dystrophin protein product could be of benefit in around 70% of Duchenne muscular dystrophy patients. We describe the use of hyaluronidase enhanced electrotransfer to deliver uncomplexed 2P-O- methyl modified phosphorothioate AO to adult dystrophic mouse muscle, resulting in dystrophin expression in 20-30% of fibres in tibialis anterior muscle after a single injection. Although expression was transient, many of the corrected fibres initially showed levels of dystrophin expression well above the 20% of endogenous previously shown to be necessary for phenotypic correction of the dystrophic phenotype.

18/08/03: Body composition and energy expenditure in Duchenne muscular dystrophy

Objective: To investigate the relationship between resting energy expenditure (REE) and body composition in Duchenne Muscular Dystrophy (DMD).
Design: An observational study.
Setting: University Research Centre.
Subjects: Nine Duchenne children (age range 6-12 y), mean relative weight 128%, agreed to undergo the investigation and all of them completed the study;
Interventions: Assessment of body composition (total body fat and skeletal muscle mass) by magnetic resonance imaging and resting energy expenditure by indirect calorimetry.
Main outcome measures: Fat mass (FM; kg and percentage weight), fat-free mass (FFM; kg and percentage weight), muscle mass (kg and percentage weight), resting energy expenditure (kJ/kg body weight and kJ/kg fat-free mass).
Results: In Duchenne children fat mass averages 32% and total skeletal muscle mass 20% of body weight. Resting energy expenditure per kg of body weight falls within the normal range for children of the same age range, while when expressed per kg of FFM is significantly higher than reference values. No relationship was found between REE and total skeletal muscle mass.
Conclusions: Our results do not demonstrate a low REE in DMD boys; on the contrary REE per kg of FFM is higher than normal, probably due to the altered FFM composition. We suggest that the development of obesity in DMD children is not primarily due to a low REE but to other causes such as a reduction in physical activity and or overfeeding.

Muscle injury can occur through diverse mechanisms such as mechanicalinjury, muscular dystrophies, infectious diseases, and biochemicaltoxicities. Several types of skeletal muscle injury fall intothe broad category of sport and exercise induced muscle injury.When exercise involves eccentric muscular contractions, it isassociated with overloading of the contractile elements andconnective tissues—that is, the force requirement of themuscle exceeds the habitual requirements—and can resultin injury to skeletal muscle. It has traditionally been feltthat the events following the initial injury, including inflammation,are necessary for optimal repair. Conclusion: Neutrophils and macrophages play a role in muscle damage afterrepeated eccentric exercise and acute stretch injury. However,contrary to conventional thinking, it is possible that certainaspects of neutrophil function cause damage to healing muscleor delay its regenerative capabilities. Because neutrophilscan release oxygen free radicals during phagocytosis, it ispossible that neutrophil derived oxidants exacerbate pre-existingmuscle injury in vivo by damaging previously uninjured muscle.These findings suggest the possibility that innovative treatmentstrategies directed at specific functions of the neutrophilare theoretically possible to improve recovery from muscle injury.Pharmacological intervention may be better targeted againstspecific aspects of neutrophil function such as free radicalproduction, while maintaining the steps necessary for phagocytosisand removal of cellular debris. This possibility is being investigated.

15/07/03:IMPORTANT - Ultrasound Tissue Characterization Detects Preclinical Myocardial Structural Changes in Children Affected by Duchenne Muscular Dystrophy

Vincenzo Giglio, Vincenzo Pasceri, Loredana Messano, Fortunato Mangiola, Luciano Pasquini, Antonio Dello Russo, Antonello Damiani, Massimiliano Mirabella, Giuliana Galluzzi, Pietro Tonali, Enzo Ricci

OBJECTIVES Our goal was to identify early changes in myocardial physical properties in children with Duchenne muscular dystrophy (DMD).

BACKGROUND Duchenne muscular dystrophy (DMD) is caused by the absence of dystrophin, which triggers complex molecular and biological events in skeletal and cardiac muscle tissues. Although about 30% of patients display overt signs of cardiomyopathy in the late stage of the disease, it is unknown whether changes in myocardial physical properties can be detected in the early (preclinical) stages of the disease.

METHODS We performed an ultrasonic tissue characterization (UTC) analysis of myocardium in DMD with normal systolic myocardial function and no signs of cardiomyopathy. Both the cyclic variation of integrated backscatter (cvIBS) and the calibrated integrated backscatter (cIBS) were assessed in 8 myocardial regions of 20 DMD, age 7 + 2 years (range 4 to 10 years), and in 20 age-matched healthy controls.

RESULTS We found large differences in the UTC data between DMD and controls; the mean valueof cvIBS was 4.4 + 1.5 dB versus 8.8 + 0.8 dB, whereas the mean value of cIBS was 36.4 + 7.1 dB versus 26.9 + 2.0 dB (p 10+6 for both). In DMD, all eight sampled segments showed cIBS mean values to be significantly higher and cvIBS mean values to be significantly lower than those in the controls. Finally, interindividual differences were greater in DMD than in controls for both parameters.

CONCLUSIONS The myocardium in DMD displays UTC features different from those in healthy controls. These results show that lack of dystrophin is commonly associated with changes in myocardial features well before the onset of changes of systolic function and overt cardiomyopathy.

PARTS OF THE EDITORIAL OF THIS ARTICLE: (Jeffrey A. Towbin, Houston, Texas)

In most centers, individuals affected with DMD obtain echocardiograms after symptoms of heart failure begin. Unfortunately, this approach may lead to irreversibly progressive disease, ending in death. Hence, the concept of early referral and a noninvasive imaging approach that identifies early disease could be a more useful methodology with better long-term outcomes. Clearly, new approaches that enable early, presymptomatic diagnostic capability would be useful. Giglio et al. (10) took this one step further, seeking early detection of myocardial physical property changes in children with no signs or symptoms of cardiac involvement, including normal electrocardiograms and normal LV size and systolic function indexes. Patients and controls displayed significantly different UTC parameters, and interindividual differences were larger in DMD patients than in controls as well. Based on their results, the investigators (10) suggest that this method demonstrates subtle involvement of the myocardium early in DMD and could be potentially useful and reliable in assessing myocardial changes over time. We have been studying children through the use of echocardiography and cardiac magnetic resonance imaging; the children are evaluated starting at 10 years of age and are evaluated longitudinally on a yearly basis, with therapeutic intervention using angiotensin-converting enzyme inhibitors and beta-blockers at the earliest signs of myocardial disease. In a high percentage of cases, both reverse remodeling and normalization of LV size and systolic function rapidly occur using this approach. It is possible that earlier use of these agents would be beneficial as these patients are "destined" to develop cardiac disease. Perhaps UTC analysis will answer this question. The potential improvement in the care of DMD patients attributable to the development of UTC analysis is exciting.

15/07/03: Antifibrotic effects of suramin in injured skeletal muscle after laceration NEW

Yi-Sheng Chan,1,2,3 Yong Li,1,2 William Foster,1 Takashi Horaguchi,1,2 George Somogyi,4 Freddie H. Fu,2 and Johnny Huard1,2,5

Muscle injuries are very common in traumatology and sports medicine. Although muscle tissue can regenerate postinjury, the healing process is slow and often incomplete; complete recovery after skeletal muscle injury is hindered by fibrosis. Our studies have shown that decreased fibrosis could improve muscle healing. Suramin has been found to inhibit transforming growth factor (TGF)- beta1 expression by competitively binding to the growth factor receptor. We conducted a series of tests to determine the antifibrotic effects of suramin on muscle laceration injuries. Our results demonstrate that suramin (50 g/ml) can effectively decrease fibroblast proliferation and fibrotic-protein expression (alfa-smooth muscle actin) in vitro. In vivo, direct injection of suramin (2.5 mg) into injured murine muscle resulted in effective inhibition of muscle fibrosis and enhanced muscle regeneration, which led to efficient functional muscle recovery. These results support our hypothesis that prevention of fibrosis could enhance muscle regeneration, thereby facilitating more efficient muscle healing. This study could significantly contribute to the development of strategies to promote efficient muscle healing and functional recovery.

15/07/03: IGF-I gene transfer by electroporation promotes regeneration in a muscle injury model

Takahashi, T 1; Ishida, K 1; Itoh, K 1; Konishi, Y 2; Yagyu, K-I 2; Tominaga, A 2,3; Miyazaki, J-I 4; Yamamoto, H 1

The goal of this study was to determine whether insulin-like growth factor-I (IGF-I) gene delivery by electroporation promotes repair after muscle injury. An injury-repair model was created using mice in which a hamstring muscle was cut and sutured. A total of 50 [mu]g of IGF-I DNA or green fluorescent protein (GFP) DNA (both in pCAGGS) was injected into the lesion and introduced into muscle cells by electrostimulation using an electric pulse generator. The number of regenerating muscle fibers in the IGF-I DNA group was significantly more than that in the GFP DNA group at 2 weeks after injection. The diameter of regenerating muscle fibers from the IGF-I DNA group was larger than that of the GFP DNA group at 4 weeks after injection. There was no significant difference in the serum IGF-I concentration between the IGF-I DNA group and the GFP DNA group at 1, 2, and 4 weeks after injection. However, muscle IGF-I concentration in the IGF-I DNA injection group was significantly greater than that in the GFP DNA injection group at 2 weeks after injection. These results demonstrated that the effects of enhanced IGF-I production were local and limited to the injected area. The ratio (injected/uninjected; intact) of the amplitude of compound muscle action potentials (CMAP) in the IGF-I DNA injection group was greater than that in the GFP DNA injection group at 4 weeks after injection and of the control group. In conclusion, IGF-I gene transfer by electroporation proved to be a simple, safe, inexpensive, and effective method to promote the regeneration of injured muscles in our injury model.

12/07/03: Evaluation of cardiac disease in adult female carriers in familial dystrophinopathies (abstract presented on The International Congress of Genetics, July 6-11, Melbourne, Australia)

ROBYN OTWAY¹, Jian Janet Liu¹, Guanglan Guo¹, Jasmine Hessell¹, Peter Macdonald², Anne Keogh², Christopher Hayward², Diane Fatkin¹

¹Sr Bernice Research Programme in Inherited Heart Diseases, Victor Chang Cardiac Research Institute, Darlinghurst, NSW Australia; ²Cardiac Transplant Unit, St Vincent’s Hospital, Darlinghurst, NSW Australia

Dystrophinopathies are a group of X-linked myopathies, including Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD) and X-linked Dilated Cardiomyopathy (XLDCM) caused by mutations in the DMD gene, that encodes the large cytoskeletal protein, dystrophin. In males, dystrophinopathies exhibit variable skeletal muscle and cardiac involvement. Clinically evident DCM has been reported to occur in 0-26% of female carriers of DMD and BMD. There is a paucity of data regarding cardiac disease in female carriers with XLDCM. Traditionally, cardiac disease in female carriers has been presumed to be relatively mild and occur in later adult life. We performed clinical and genetic evaluation of 10 adult members of a 4-generation kindred with an atypical BMD/XLDCM phenotype. Sequence analysis of the DMD gene showed a 5’ splice site mutation, exon 1+1G>T. Five females (71%), aged 21 to 64, were carriers for this mutation. Cardiac investigations (ECG, echocardiogram) were performed in all female carriers. One individual, aged 63 years, presented with symptomatic DCM. Two individuals, aged 60 and 42 years, respectively, had structurally normal hearts. In 2 female carriers, aged 39 and 21 years, asymptomatic echocardiographic abnormalities (mild left ventricular dilation with normal or borderline contractile function) were identified. These data indicate that the severity and age of onset of cardiac disease in female carriers with DMD mutations is variable. Regular cardiac screening of all female carriers is warranted from late adolescence. Elucidation of the pathophysiological basis for variable phenotypic expression in female carriers may facilitate rationalisation of cardiac screening strategies.

Ottawa team discovers 'switch' that tells muscles to repair selves

29/06/03: Expression of Dystrophin Driven by the 1.35-kb MCK Promoter Ameliorates Muscular Dystrophy in Fast, but Not in Slow Muscles of Transgenic Mdx Mice

MOLECULAR THERAPY Vol. 8, No. 1, July 2003

Patrick Dunant,1 Nancy Larochelle,1,2 Christian Thirion,1 Rolf Stucka,1 Daniel Ursu,3 Basil J. Petrof,4 Eckhard Wolf,1 and Hanns Lochmu¨ller1,*

E-mail: hanns@lmb.uni-muenchen.de

Successful gene therapy of Duchenne muscular dystrophy may require the lifelong expression of a therapeutic gene in all affected muscles. The most promising gene delivery vehicles, viral vectors, suffer from several limitations, including immunogenicity, loss of therapeutic gene expression, and a limited packaging capacity. Therefore, various efforts were previously undertaken to use small therapeutic genes and to place them under the control of a strong and muscle-specific promoter. Here we report the effects of a minidystrophin (6.3 kb) under the control of a short muscle-specific promoter (MCK 1.35 kb) over most of the lifetime (4–20 months) of a transgenic mouse model. Dystrophin expression remained stable and musclespecific at all ages. The dystrophic phenotype was greatly ameliorated and, most importantly, muscle function in limb muscles was significantly improved not only in young adult but also in aged mice compared to nontransgenic littermates. Dystrophin expression was strong in fasttwitch skeletal muscles such as tibialis anterior and extensor digitorum longus, but weak or absent in heart, diaphragm, and slow-twitch muscles. Additionally, expression was strong in glycolytic but weak in oxidative fibers of fast-twitch muscles. This study may have important implications for the design of future gene therapy trials for muscular dystrophy.

29/06/03: Successful heart transplantation in patients with inherited myopathies associated with end-stage cardiomyopathy

29/06/03: Circular letter from WANDA (World Alliance of Neuromuscular Disorder Association) about myoblast transfer treatment

Fake treatment for Duchenne turns into a police affair

21/06/03: Pharmacokinetics of the Dietary Supplement Creatine [Review Article]

Creatine is a nonessential dietary component that, when supplemented in the diet, has shown physiological benefits in athletes, in animal-based models of disease and in patients with various muscle, neurological and neuromuscular disease. The clinical relevance of creatine supplementation is based primarily on its role in ATP generation, and cells may be able to better handle rapidly changing energy demands with supplementation.

Although the pharmacological outcome measures of creatine have been investigated, the behaviour of creatine in the blood and muscle is still not fully understood. Creatine is most probably actively absorbed from the gastrointestinal tract in a similar way to amino acids and peptides. The distribution of creatine throughout the body is largely determined by the presence of creatine transporters. These transporters not only serve to distribute creatine but serve as a clearance mechanism because of creatine ‘trapping’ by skeletal muscle. Besides the pseudo-irreversible uptake by skeletal muscle, creatine clearance also depends on renal elimination and degradation to creatinine.

Evidence suggests that creatine pharmacokinetics are nonlinear with respect to dose size and frequency. Skeletal muscle, the largest depot of creatine, has a finite capacity to store creatine. As such, when these stores are saturated, both volume of distribution and clearance can decrease, thus leading to complex pharmacokinetic situations. Additionally, other dietary components such as caffeine and carbohydrate can potentially affect pharmacokinetics by their influence on the creatine transporter. Disease and age may also affect the pharmacokinetics, but more information is needed.

Overall, there are very limited pharmacokinetic data available for creatine, and further studies are needed to define absorption characteristics, clearance kinetics and the effect of multiple doses. Additionally, the relationship between plasma creatine and muscle creatine needs to be elucidated to optimise administration regimens.

Muscle cells undergoan unusual developmental program in which several partiallydifferentiated cells called myoblasts fuse to form a multinucleatedmyotube. This nascent myotube undergoes further maturation andgrowth, which requires the addition of nuclei by fusion of moremononucleated myoblasts with myotubes. Valerie Horsley, GracePavlath, and colleagues (Emory University, Atlanta, Georgia)have found that nascent myotubes promote fusion, and thus theirown growth, by secreting a cytokine normally associated withimmune cells.The cross-system cytokine is IL-4, which is required in immunecells for macrophage fusion. Not one to throw away a good thing,Nature evidently coopted the system for muscle cells. As inimmune cells, IL-4 expression in nascent myotubes is drivenby a member of the NFAT transcription factor family. Myotubeslacking either IL-4 or the NFAT factor were smaller and hadfewer nuclei than wild-type cells. Recovery from muscle injurywas also diminished by the lack of IL-4 or the IL4alfa receptor.Myoblasts are the targets of IL-4 action, which may promotefusion by inducing myoblast expression of adhesion moleculessuch as integrins (as in macrophages) or VCAM. Alternatively,IL-4 may act as a chemokine, as it does for osteoblasts, tostimulate migration of myoblasts toward myotubes. Whatever themechanism, stem cell therapies for disorders such as musculardystrophies may be improved by expression of IL-4 to increasethe fusion capacity of the muscle stem cells.

Reference: Horsley, V., et al. 2003. Cell. 113:483–494

Nicole LeBrasseur lebrasn@rockefeller.edu

14/06/03:Photo from manifestation of brazilian DMD parents and boys on a Hospital Equipment Fair: we fight to receive the noninvasive ventilator (BIPAP) and pulmonary assistance free; the Brazilian Health Minister saw and heard this manifestation.

Comments from Terence Partridge (Muscle Cell Biology Group, MRC Clinical Sciences Centre, ICSM, Hammersmith Hospital Site,London) about this article:

Electrical stimulation has been proposed for DMD for a number of reasons. Slow stimulation patterns – 10Hz – is supposed to convert muscle to the ‘slow’ type which is said to be less susceptible to necrosis than fast muscle (though the evidence for the latter point is rather dicey) has been proposed but I do not know if it has been tested in DMD boys. In the genethon, one of the people I am working with believes that denervated segments of muscle fibre in DMD muscle causes the fibres to convert directly to fat. He has some evidence that this may be so. It does not yet overcome my scepticism but we have discussed it and there are ways in which he could test it more stringently. His point is that electrical stimulation might prevent or even reverse the conversion.
My overall feeling is that any non-traumatic way of keeping muscle in physical shape would probably be good – moreover, it would be possible to assess this benefit if there is any by non-invasive methods.

21/05/03: (IN PRESS: Drug & Market Development) Muscular Dystrophies: Recent Milestones

• This article was been written by G. Thor, PhD and J. Terryberry, BS, who are associated with NeuroConsultants based in San Diego, gautamthor@earthlink.net

• Gene therapy human clinical trials have been reactivated after a 2-year freeze. Several gene therapeutic approaches to study Muscular Dystrophies (MD) – a set of neuromuscular diseases wherein muscle atrophies - are being resumed in clinical and preclinical stages.

• About one in 3,000 boys, independent of ethnic background, is born with this disease, which is caused by a mutation or damage of the Duchenne or Dystrophin gene carried on the X chromosome.

• More than 90% of MD genetic defects have been identified and have led to several components of the Dystrophin Glycoprotein complex that are in turn being used as targets for disease manipulation.

• Genetic tests for detecting alterations in the Dystrophin gene or Creatine Kinase detection by a blood test are available for prenatal screening.

• Prevention of MD by gene transfer in a mouse model and the creation of dog models that mimic Muscular Dystrophies phenotypes are enabling researchers to design effective strategies to study and conquer this set of musculature related diseases.

• Stem cell research has gained ground as mice models prove to be rewarding testing grounds for therapeutic manipulation of Dystrophin and other related polypeptides of the Dystrophin Glycoprotein Complex (DGC).

IMPORTANT IN THIS ARTICLE IS THIS CONCLUSION: Although gene therapeutic approaches offer the most promise for an ultimate cure for DMD, gene therapeutics are not expected to be available for several years or longer, meaning that many patients diagnosed at the present time are not likely to benefit from gene therapy. Since the incidence of DMD in the US is about 100,000 new cases per year, DMD is not an orphan disease. Pharmaceutical companies should therefore be interested in developing drugs for DMD; since Orphan Drug status is not applicable. Furthermore, developing drugs for DMD could be made more attractive to pharmaceutical companies as part of an idea to discover drugs for muscle weakness. Synthetic Dystrophin will not pass through the muscle cell membrane and must be produced or introduced inside the membrane by the muscle cell to be effective. WE NEED SAFE TREATMENTS TO DELAY/STOP THE EVOLUTION OF THE DISEASE.

Summary: Osteoporosis causes significant morbidity for boys with Duchenne muscular dystrophy. Corticosteroid therapy given to prolong mobility may increase the rate of osteoporosis and risk of fracture. This study of 33 boys with Duchenne muscular dystrophy determined retrospectively the incidence of vertebral fractures particularly after initiation of corticosteroids. A latency period of 40 months after commencement of steroids occurred before the first vertebral fracture appeared. However, by 100 months of treatment approximately 75% had sustained a vertebral fracture. Clin Pediatr. 2003;42:353-356

05/05/03: IMPORTANT AND INEDITED: Results of Phase I Clinical Trial of Plasmid-Dystrophin Intramuscular Administration into Duchenne and Becker Dystrophy Patients - that will be presented on the 6th Annual Meeting of American Society of Gene Therapy in June 4-8,2003, in Washington.

Norma B. Romero,1 Olivier Benveniste,2 Aurore Choquel,2 Christine Payan,1 Glenn E. Morris,3 Jean-Gerard Guillet,4 Jean-Claude Kaplan,4 France Leturcq,4 Serge Braun,5 Brigitte Mourot,5 Christine Thioudellet,5 Patrick Squiban,5 Serge Herson,2 Michel Fardeau.1

1Institut de Myologie, Groupe Hospitalier Pitié-Salpêtrière, Paris, France; 2Clinical Medicine, Groupe Hospitalier Pitié-Salpêtrière,Paris, France; 3North East Wales Institute, Wrexham, UnitedKingdom; 4ICGM - INSERM U445, and Groupe Hospitalier Cochin Port-Royal, Paris, France; 5Transgene S.A., Strasbourg, France.

Duchenne dystrophy is a recessive X-linked inherited disorder with deletions or mutations in the dystrophin gene. Clinical onset occurs usually before the age of four years and is fatal (death during the third decade of life). A milder form (Becker dystrophy) is of more variable phenotype, but with abnormalities in the same dystrophin gene. After extensive pre-clinical studies in mdx mice and GRMD dogs we have carried out the first gene therapy phase I clinical trial in both Duchenne and Becker patients. The goal of this study was to provide indications on safety of a full-length human dystrophin-plasmid administration and exogenous dystrophin expression in DMD/BMD patients. Many ethical aspects were considered, including age and sequential inclusion of the patients, and low plasmid dosage. Complete data of each patient was carefully analysed by a steering committee composed of investigators, promoters and external, independent experts, before enrolment of the following patient. Three cohorts of 3 patients, presenting with large deletions, were injected in the radial muscle with either a single injection of 200 µg (cohort 1) and 600 µg (cohort 2) of plasmid, or 2 injections 2 weeks apart with 600 µg plasmid (cohort 3). In all patients, a muscle biopsy was performed in the injected site 3 weeks after the first injection. Each biopsy was serially sectioned and studied for the presence of plasmid (PCR) and for dystrophin expression (nested RT-PCR + immunohistochemistry). The histological aspect of the muscle biopsy and local inflammatory processes were analysed. Besides the usual biochemical / cytological blood parameters (including muscle enzymes), patients were also followed for specific immunological endpoints (anti-DNA and antidystrophin cellular and immunological responses) for 3 months after plasmid injection. Normal dystrophin expression was found in few muscle fibers of 2/3 patients of the first cohort and in 1/3 patients of the second cohort. All the 3 patients of the 3rd cohort displayed significant amounts of weakly dystrophin-stained muscle fibers. Plasmid was detected in the injected muscle sample in all patients. All patients showed perfect tolerance to the plasmid administration. Neither anti-DNA nor anti-dystrophin immune response was found. As demonstrated by MRI, EMG and muscle strength analysis, the procedure did not impair the injected muscle function. These results show for the first time that exogenous dystrophin expression can be obtained in DMD/BMD skeletal muscle in vivo following gene transfer, and without adverse effects. This very cautious approach paves the way for further developments. We are currently working extensively (in collaboration with JA Wolff and Mirus Corp.), on a more ambitious, intra-arterial delivery administration of human full-length dystrophin plasmid, that we intend to evaluate in a next human clinical trial.

01/05/03:IN PRESS (Neuromuscular Disorders) Correlated NOS-Iµ and myf5 expression by satellite cells in mdx mouse muscle regeneration during NOS manipulation and deflazacort treatment

Judy E. Anderson, Cinthya Vargas
Department of Human Anatomy and Cell Science, Faculty of Medicine, University of Manitoba, 730 William Avenue, Winnipeg, MB, Canada

Abstract

Satellite cells, muscle precursor cells in skeletal muscle, are normally quiescent and become activated by disease or injury. A lack of dystrophin and changes in the expression or activity of neuronal nitric oxide synthase (NOS-I) affect the timing of activation in vivo. Nitric oxide synthase inhibition delays muscle repair in normal mice, and worsens muscular dystrophy in the mdx mouse, a genetic homologue of Duchenne muscular dystrophy. However, the potential role of activation and repair events mediated by nitric oxide in determining the outcome of steroid or other treatments for muscular dystrophy is not clear. We tested the hypothesis that the extent of repair in dystrophic muscles of mdx mice is partly dependent on NOS-Iµ expression and activity. Myotube formation in regenerating muscle was promoted by deflazacort treatment of mdx dystrophic mice (P<0.05), and improved by combination with the nitric oxide synthase substrate, L-arginine, especially in the diaphragm. NOS-Iµ mRNA expression and activity were present in satellite cells and very new myotubes of regenerating and dystrophic muscle. Deflazacort treatment resulted in increased NOS-Iµ expression in regenerating muscles in a strong and specific correlation with myf5 expression (r=0.95, P<0.01), a marker for muscle repair. Nitric oxide synthase inhibition prevented the deflazacort-induced rise in NOS-Iµ and myf5 expression in the diaphragm without affecting the diameter of non-regenerating fibres. These in vivo studies suggest that gains in NOS-Iµ expression and nitric oxide synthase activity in satellite cells can increase the extent and speed of repair, even in the absence of dystrophin in muscle fibres. NOS-Iµ may be a useful therapeutic target to augment the effects of steroidal or other treatments of muscular dystrophy.

01/05/03: IMPACT OF TNF-ALPHA ELIMINATION ON VENTILATION AND DIAPHRAGM CONTRACTILITY IN DYSTROPHIC MICE

Barkley, J1; Spencer, M2; Farkas, G A.1; McCormick, K M. FACSM1; Gosselin, L E. FACSM1

The American College of Sports Medicine Volume 34(5) Supplement 1,May 2002, p S155

Duchenne muscular dystrophy is associated with extensive diaphragm muscle injury and a persistent inflammatory response that leads to muscle degeneration. Ultimately, patients die of respiratory failure by their early twenties. The purpose of this study was to determine if eliminating the inflammatory cytokine tumor necrosis factor-alpha (TNF[alpha]) would improve ventilatory reserve and diaphragm muscle contractility in mdx (dystrophic) mice. For this study mdx mice were crossbred with transgenic. TNF[alpha]-deficient mice to generate TNF[alpha]-deficient mdx (TNF[alpha]-/mdx) mice. At 11–12 months of age, ventilation was measured in awake mice during room air breathing and in response to 7% CO2 using the barometric technique. During room air breathing, neither tidal volume, respiratory rate nor minute ventilation (Ve) differed between TNF[alpha]-/mdx and TNF[alpha]+/mdx mice. In response to 7% CO2, TNF[alpha]-/mdx mice significantly increased Ve whereas Ve did not change in the TNF[alpha]+/mdx group (P < 0.05). In vitro isometric contractile properties of the diaphragm were also measured. Maximal isometric force of the diaphragm muscle was significantly higher in the TNF[alpha]-/mdx group when compared to TNF[alpha]+/mdx group (7.57 N/cm2 vs. 5.56 N/cm2, respectively, P < .05). These data suggest that TNF[alpha] plays a role in ventilatory and diaphragm muscle dysfunction in dystrophic mice.

17/04/03: IN PRESS (Lancet Neurology, 2003, vol. 2, may ) Novel therapies for Duchenne muscular dystrophy: Review

The development of therapeutic strategies that overcome the unique problems posed by Duchenne muscular dystrophy (DMD) has lead to the development of many contemporary approaches to human disease in general. Various treatment approaches have been ex

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