Tomohiro Suga

Mdx respiratory impairment following fibrosis of the diaphragm

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease that causes respiratory or cardiac failure and results in death at about 20 years of age. An animal model of DMD, the mdx mouse, is commonly used to estimate dystrophic pathology. The pathological features of limb muscles are relatively mild, however the diaphragm is severely affected and exhibits a degenerative pattern similar to that observed in human DMD. Although, the muscle strength assay of the dystrophic diaphragm has been used to estimate mdx respiratory impairment, systemic functional assessments compared with histopathological analysis have not been demonstrated. Here, we report a sensitive procedure using whole-body plethysmography to monitor respiratory parameters detected during early respiratory insufficiency in the mdx mouse. The dystrophic changes in the diaphragm lead to respiratory dysfunctions. These methods may be useful to assess the therapeutic approaches for the mdx mouse.

Derlin-1 overexpression ameliorates mutant SOD1-induced endoplasmic reticulum stress by reducing mutant SOD1 accumulation

Unfolded protein responses, including induction of stress sensor kinases, chaperones, and apoptotic mediators, are involved in the familial amyotrophic lateral sclerosis (ALS) model related to mutant Cu/Zn superoxide dismutase (SOD1) and sporadic ALS. We hypothesized that the endoplasmic reticulum-resident factor Derlin-1 plays a pivotal role in the regulation of misfolded proteins evoked by mutant SOD1. We show that Derlin-1 overexpression reduced mutant SOD1-induced cell toxicity and increased cell viability by suppressing the activation of the ER stress pathway factors: immunoglobulin-binding protein, activating transcription factor 6 p50, and C/EBP homologous protein. Interestingly, exogenous Derlin-1 resulted in a decrease in the amount of mutant SOD1, and a lesser decrease in that of wild-type SOD1, in transfected cells. Reduced SOD1 protein expression was observed in the microsomal fraction of wild-type and mutant SOD1 cells. Our results indicate that Derlin-1 regulates the turn over of SOD1 by promoting the proteasomal and autophagosomal degradation of SOD1 protein, but not by decreasing mutant SOD1 mRNA levels. Insights into the effects of Derlin-1 on mutant SOD1 may facilitate advancements in the treatment of motor neuron degeneration associated with ALS.

An inhibitor of transforming growth factor beta type I receptor ameliorates muscle atrophy in a mouse model of caveolin 3-deficient muscular dystrophy

Skeletal muscle expressing Pro104Leu mutant caveolin 3 (CAV3P104L) in mouse becomes atrophied and serves as a model of autosomal dominant limb-girdle muscular dystrophy 1C. We previously found that caveolin 3-deficient muscles showed activated intramuscular transforming growth factor beta (TGF-β) signals. However, the cellular mechanism by which loss of caveolin 3 leads to muscle atrophy is unknown. Recently, several small-molecule inhibitors of TGF-β type I receptor (TβRI) kinase have been developed as molecular-targeting drugs for cancer therapy by suppressing intracellular TGF-β1, -β2, and -β3 signaling. Here, we show that a TβRI kinase inhibitor, Ki26894, restores impaired myoblast differentiation in vitro caused by activin, myostatin, and TGF-β1, as well as CAV3P104L. Oral administration of Ki26894 increased muscle mass and strength in vivo in wild-type mice, and improved muscle atrophy and weakness in the CAV3P104L mice. The inhibitor restored the number of satellite cells, the resident stem cells of adult skeletal muscle, with suppression of the increased phosphorylation of Smad2, an effector, and the upregulation of p21 (also known as Cdkn1a), a target gene of the TGF-β family members in muscle. These data indicate that both TGF-β-dependent reduction in satellite cells and impairment of myoblast differentiation contribute to the cellular mechanism underlying caveolin 3-deficient muscle atrophy. TβRI kinase inhibitors could antagonize the activation of intramuscular anti-myogenic TGF-β signals, thereby providing a novel therapeutic rationale for the alternative use of this type of anticancer drug in reversing muscle atrophy in various clinical settings.

Adult-onset neurological degeneration in a patient with cockayne syndrome and a null mutation in the CSB Gene

TO THE EDITOR Cockayne syndrome (CS) is characterized by various clinical manifestations such as postnatal growth failure and progressive neurological dysfunction, but it conveys no predisposition to skin cancer. CS can be caused by mutation in two genes, CKN1/CSA and ERCC6/ CSB. CSA and CSB protein play a key role in transcription-coupled DNA repair, which removes UV-induced DNA lesions only in the actively transcribed DNA strand (Hanawalt, 2002). CS was classified into three clinical subtypes: classical, severe, and mild CS (Nance and Berry, 1992). The clinical diagnosis of classical CS requires the presence of growth failure, neurological dysfunction, and at least three of the following: photosensitivity, pigmentary retinopathy, sensorineural hearing loss, dental caries, and cachectic dwarfism. Mild CS patients have some of the clinical signs of CS but lack one or more of the criteria, and their symptoms are mild or late in onset. Mild CS patients also have normal reproductive capacity. As clinical symptoms of CS are not associated with cellular UV hypersensitivity (Lehmann, 2003), some recent investigations have focused on activities of CSB, other than transcriptioncoupled DNA repair, that might be responsible for CS characteristics. CSB interacts with many proteins and thus has many possible functions (Licht et al., 2003). Importantly, cells from patients accumulate oxidative DNA lesions after X-ray exposure (Tuo et al., 2001), and the repair deficiency of oxidative lesions is corrected by expression of formamidopyrimidine DNA glycosylase from Escherichia coli (Ropolo et al., 2007). Some CS patients rarely show slower course than usual, resulting in survival into adulthood (Rapin et al., 2006). We present herein a patient with CS, named KPSX6, who was a 49-year-old Japanese woman with a history of freckling and dry skin in sun-exposed areas, which had developed since her teens (Figure 1). Although she showed normal mental development, she had growth retardation similar to that of her five siblings. Four siblings had no skin lesions, but her sister had pigmentation. Her parents were consanguineous. She had two daughters who were normal. When she was 47 years old, her family noted dementia, ataxia, and hearing loss, which had progressed over 2 years. Physical examination revealed a remarkably small body (height, 131.7 cm; weight, 33.1 kg) with an aged face and scoliosis. Her skin was dry and pigmented on the neck, arms, and legs. However, there were no skin cancer lesions, and her hair was normal. On neurological examination, the deep tendon reflexes were indicative of asthenia. Her muscles showed hypotonus but no atrophy, and she had difficulty in walking. Sensory examination revealed no abnormalities, and the cranial nerve examination was normal. She had difficulty with hearing and speech. She exhibited intracranial calcification on computed tomography and cerebellar atrophy on magnetic resonance imaging. There were no findings on ophthalmologic examination. The minimal erythema dose

Long-Term Outcome of Polymyositis Treated with High Single-Dose Alternate-Day Prednisolone Therapy

Background: We previously reported no difference in the efficacies of high-dose alternate-day (ADT) and daily-dose (DDT) prednisolone therapies in myositis patients, but that the incidence of side effects was lower in the former. The aim of the present study was to compare the long-term outcomes of both treatments in polymyositis patients. Methods: We compared clinical courses, efficacies, adverse reactions, and outcomes of 115 consecutive, biopsy-proven polymyositis patients treated between 1970 and 2008 with ADT (32 patients) or DDT (83 patients). Results: Mean onset ages, disease severity, incidences of malignancy, and response rates did not differ between the ADT and DDT groups. Adverse reactions (incidence of diabetes) were significantly higher in the DDT group. In this group, the incidences of hyperlipidemia, infection, hypertension, and psychiatric symptoms were also slightly higher, but not significantly so. The 20-year survival rate of the ADT group (68%) was significantly higher (p = 0.0112) than that of the DDT group (37%). Conclusion: ADT might be useful as an initial treatment option for polymyositis.

Rescue From Respiratory Dysfunction by Transduction of Full-length Dystrophin to Diaphragm via the Peritoneal Cavity in Utrophin/Dystrophin Double Knockout Mice

Duchenne muscular dystrophy (DMD) is an inherited severe muscle wasting disorder with, thus far, no effective therapy. DMD causes respiratory and cardiac failure as well as muscle wastage. Among the various symptoms, respiratory insufficiency is a major cause of death in DMD patients at about 20 years of age. So, naturally, the improvement of respiratory function will extend the patients life. We report here, for the first time, a sensitive procedure using whole-body plethysmography to monitor respiratory parameters detected in the utrophin/dystrophin double knockout mouse (dko mouse), showing quite similar systemic symptoms to human DMD including restrictive ventilatory impairment. Furthermore, we show that a highly efficient dystrophin-transduction to the dkos diaphragm--achieved by simple intraperitoneal injection of a helper-dependent adenovirus vector (HDAdv) containing the full-length dystrophin expression cassette--provided beneficial results. In spite of dystrophin expression only in the diaphragm, this focal gene transfer could result in the rescue from ventilatory impairment (increased tidal volume (TV) and improvement of compensatory hyperpnea). Our result suggests that a DMD patients mortal ventilatory impairment may be improved via technically easy means through the intraperitoneal injection of HDAdv.

Muscle Fiber Type-Predominant Promoter Activity in Lentiviral-Mediated Transgenic Mouse

Variations in gene promoter/enhancer activity in different muscle fiber types after gene transduction was noticed previously, but poorly analyzed. The murine stem cell virus (MSCV) promoter drives strong, stable gene expression in hematopoietic stem cells and several other cells, including cerebellar Purkinje cells, but it has not been studied in muscle. We injected a lentiviral vector carrying an MSCV-EGFP cassette (LvMSCV-EGFP) into tibialis anterior muscles and observed strong EGFP expression in muscle fibers, primary cultured myoblasts, and myotubes isolated from injected muscles. We also generated lentiviral-mediated transgenic mice carrying the MSCV-EGFP cassette and detected transgene expression in striated muscles. LvMSCV-EGFP transgenic mice showed fiber type-dependent variations in expression: highest in types I and IIA, intermediate in type IID/X, and lowest in type IIB fibers. The soleus and diaphragm muscles, consisting mainly of types I and IIA, are most severely affected in the mdx mouse model of muscular dystrophy. Further analysis of this promoter may have the potential to achieve certain gene expression in severely affected muscles of mdx mice. The Lv-mediated transgenic mouse may prove a useful tool for assessing the enhancer/promoter activities of a variety of different regulatory cassettes.

Drastic therapy for listerial brain abscess involving combined hyperbaric oxygen therapy and antimicrobial agents.

Background Listeria monocytogenes (L. monocytogenes) is a rare causative pathogen of brain abscess that is often found in immunocompromised patients. Although patients with supratentorial listerial abscesses showed a longer survival with surgical drainage, the standard therapy for patients with subtentorial lesions has not been established. Case Report We report herein a patient with supra- and subtentorial brain abscesses caused by L. monocytogenes infection. These abscesses did not respond to antibiotics, and his symptoms gradually worsened. Drainage was not indicated for subtentorial lesions, and the patient was additionally treated with hyperbaric oxygen therapy, which dramatically reduced the volume of abscesses and improved the symptoms. Conclusions This is the first report of drastic therapy for a patient with listerial brain abscesses involving combined antibiotics and hyperbaric oxygen therapy. The findings suggest that hyperbaric oxygen therapy is a good option for treating patients with deep-seated listerial abscesses and for who surgical drainage is not indicated.

Muscle biopsy findings predictive of malignancy in rare infiltrative dermatomyositis

OBJECTIVE The characteristic pathological muscular findings of polymyositis (PM) and dermatomyositis (DM) have been shown to reflect their different pathogeneses. Here, we characterized the muscle biopsy findings of PM and DM patients with or without malignancy. METHODS We evaluated the muscle biopsy findings of 215 consecutive PM and DM patients admitted to our hospital between 1970 and 2009. Pathology of the lesion biopsy sections was classified into 3 types: endomysial infiltration-type, perivascular infiltration-type, and rare-infiltrative-type. RESULTS There was no difference between the muscle pathology of PM patients with and without malignancy. However, the incidence of rare-infiltrative type muscle pathology in DM patients with malignancy was significantly higher than in those without such tumors (p=0.0345). CONCLUSION The incidence of rare-infiltrative type muscle pathology may be a predictive marker of DM with malignancy.

P3.10 Lentiviral vector mediated delivery of full-length dystrophin for gene therapy of muscular dystrophy

including 960 mg/kg/injection, with no adverse effects. Findings were generally limited to the kidney, and were generally reversible, as shown in the 28 day recovery groups. No evidence of kidney function change was detected. In cynomolgus monkeys, AVI-4658 was also well tolerated at all doses including 320 mg/kg/injection, with no adverse effects. Findings were similar to those seen in the mouse studies. Conclusion: AVI-4658, the first PMO for DMD, was extremely well tolerated at all doses in dystrophic mice, normal mice and primates. In addition, AVI-4225, which restores dystrophin in mdx mice, also led to no adverse effects. Based on this preclinical package, and encouraging safety and dystrophin expression results from a concurrent UK clinical study, US clinical studies are anticipated.