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Objective: Duchenne and Becker muscular dystrophies (DBMD) are allelic disorders caused by mutations in dystrophin. Adults with DBMD develop life-threatening cardiomyopathy. Inhibition of phosphodiesterase 5 (PDE5) improves cardiac function in mouse models of DBMD. To determine whether the PDE5-inhibitor sildenafil benefits human dystrophinopathy, we conducted a randomized, double-blind, placebo-controlled trial (ClinicalTrials.gov, number NCT01168908)., Methods: Adults with DBMD and cardiomyopathy (ejection fraction ≤ 50%) were randomized to receive sildenafil (20mg 3× daily) or placebo for 6 months. All subjects received an additional 6 months of open-label sildenafil. The primary endpoint was change in left ventricular end-systolic volume (LVESV) on cardiac magnetic resonance imaging. Secondary cardiac endpoints, skeletal muscle function, and quality of life were also assessed., Results: An interim analysis (performed after 15 subjects completed the blinded phase) revealed that 29% (4 of 14) of subjects had a ≥10% increase in LVESV after 6 months of sildenafil compared to 13% (1 of 8) of subjects receiving placebo. Subjects with LVESV > 120ml at baseline were more likely to worsen at 12 months regardless of treatment assignment (p = 0.035). Due to the higher number of subjects worsening on sildenafil, the data and safety monitoring board recommended early termination of the study. There were no statistically significant differences in outcome measures between treatment arms., Interpretation: Due to the small sample size, comparisons between groups must be interpreted with caution. However, this trial suggests that sildenafil is unlikely to improve cardiac function in adults with DBMD., (© 2014 American Neurological Association.)

Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), two related conditions of differing severity. BM is a relatively mild dominantly inherited disorder characterized by proximal weakness and distal joint contractures. UCMD was originally regarded as an exclusively autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. We and others have subsequently modified this model when we described UCMD patients with heterozygous in-frame deletions acting in a dominant-negative way. Here we report 10 unrelated patients with a UCMD clinical phenotype and de novo dominant negative heterozygous splice mutations in COL6A1, COL6A2, and COL6A3 and contrast our findings with four UCMD patients with recessively acting splice mutations and two BM patients with heterozygous splice mutations. We find that the location of the skipped exon relative to the molecular structure of the collagen chain strongly correlates with the clinical phenotype. Analysis by immunohistochemical staining of muscle biopsies and dermal fibroblast cultures, as well as immunoprecipitation to study protein biosynthesis and assembly, suggests different mechanisms each for exon skipping mutations underlying dominant UCMD, dominant BM, and recessive UCMD. We provide further evidence that de novo dominant mutations in severe UCMD occur relatively frequently in all three collagen VI chains and offer biochemical insight into genotype-phenotype correlations within the collagen VI-related disorders by showing that severity of the phenotype depends on the ability of mutant chains to be incorporated in the multimeric structure of collagen VI., ((c) 2008 Wiley-Liss, Inc.)

Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS) that is increasingly recognized as a disease that affects children. Similar to adult-onset MS, children present with visual and sensory complaints, as well as weakness, spasticity, and ataxia. A lumbar puncture can be helpful in diagnosing MS when CSF immunoglobulins and oligoclonal bands are present. White matter demyelinating lesions on MRI are required for the diagnosis; however, children typically have fewer lesions than adults. Many criteria have been proposed to diagnose MS that have been applied to children, mostly above 10 years of age. The recent revisions to the McDonald criteria allow for earlier diagnosis, such as after a clinically isolated event. However, children are more likely than adults to have monosymptomatic illnesses. None of the approved disease-modifying therapies used in adult-onset MS have been approved for pediatrics; however, a few studies have verified their safety and tolerability in children. Although children and adults with MS have similar neurological symptoms, laboratory (cerebrospinal fluid) data, and neuroimaging findings, the clinical course, pathogenesis, and treatment of childhood onset MS require further investigation.

We devised a simple method to isolate mitotically active human Schwann cells from sural nerve biopsy specimens and expand the population in culture. Nerve fascicles were treated with cholera toxin for 7 days in culture before dissociation, which increased the cell yield at least twenty-five-fold over immediated tissue dissociation. Digesting the tissue completely with enzymes in serum-containing medium resulted in the highest cell viability, and released 2 to 6 x 10(4) cells/mg of tissue. Seeding the cells on a poly-L-lysine substrate in a small volume of serum-free medium optimized the plating efficiency. Although Schwann cells comprised 90% of the initial culture population, their numbers declined over time due to a faster mitotic rate of the fibroblasts in the presence of cholera toxin alone. However, treating the cultures with a combination of cholera toxin and forskolin, which act synergistically to elevate cyclic AMP levels, inhibited fibroblast growth without causing Schwann cell toxicity. Adding glial growth factor to the adenyl cyclase activators maximized Schwann cell proliferation, and the population rapidly and selectively expanded. Therefore, it should be possible to generate large numbers of Schwann cells from diseased nerves to study defects in cell function or from normal nerves to study the effects of Schwann cell grafts on neuronal regeneration.

Rapidly dividing transfected Schwann cells were grown on Matrigel, a reconstituted basement membrane gel. Matrigel decreased the proliferation of the cells by 75% when compared to sister cultures that were grown on an untreated plastic substrate. Some transfected cells plated onto a Matrigel substrate formed colonies similar to that observed when the cells were plated on a plastic substrate. Additionally, many cells on Matrigel assembled themselves into fascicles projecting away from the colonies. These fascicles were composed of transfected Schwann cells that had assumed a bipolar appearance reminiscent of quiescent secondary Schwann cells in culture. Transfected cells grown on Matrigel contained approximately 10-fold less glial fibrillary acidic protein when compared to sister cultures grown on an untreated plastic substrate. By indirect immunofluorescence laminin immunoreactivity appeared as globules within the cytoplasm of the cells which were cultured on a plastic substrate. However, cells that were grown on the Matrigel substrate appear to organize laminin in a linear array around themselves. These results demonstrate that the presence of an artificial basement membrane alters the morphology, rate of proliferation, and state of differentiation of a transfected Schwann cell line.

The effects of chronic lead intoxication were studied by examining the optic nerve of mice given lead-containing mother's milk from day 1 to day 21 of life. Biochemical assays for myelin basic protein, 2',3'-cyclic nucleotide phosphodieterase, and cerebroside sulfotransferase showed that the total amount of myelin produced by the lead-exposed animals was decreased. Lead exposure did not alter the number of oligodendroglia or the relationship between axons and myelin sheaths. The hypomyelination was paralleled by a reduction in size of optic axons in the lead-exposed animals. The data suggest that chronic exposure to lead in developing mice results in a primary effect on neurons and that hypomyelination is secondary to reduction in axon size.

The presence of myelin basic protein (MBP)-like material in cerebrospinal fluid (CSF) usually reflects breakdown of central nervous system myelin. Immunoreactive MBP levels were measured in 70 CSF specimens from 66 patients with a variety of peripheral neuropathies. Immunoreactive MBP was present in CSF in 70% (16 of 23) of patients with biopsy-proved chronic demyelinating polyneuropathies, whereas in neuropathies producing primarily axonal damage it was present in only 22% (5 of 23). In Guillain-Barré syndrome, 45% (9 of 20) of patients had immunoreactive MBP in the CSF. We conclude that MBP-like material is present in the CSF of most patients with longstanding demyelinating polyradiculoneuropathies and probably reflects detection of peripheral nervous system P1 protein.