Clomethiazole Acute Stroke Study in ischemic stroke (CLASS-I): final results.

BACKGROUND AND PURPOSE: A previous trial (the Clomethiazole Acute Stroke Study) generated the hypothesis that clomethiazole is effective in patients with a major ischemic stroke (total anterior circulation syndrome), and this was tested in the present study. METHODS: A total of 1198 patients with major ischemic stroke and a combination of limb weakness, higher cortical dysfunction, and visual field deficits were randomly assigned to clomethiazole (68 mg/kg IV over 24 hours) or placebo. The study drug was initiated within 12 hours of symptom onset. Functional outcome and neurological recovery were assessed at days 7, 30, and 90, with the proportion of patients with a Barthel Index > or =60 at last follow-up as the primary outcome measure. RESULTS: The patients were randomly assigned equally, and the two treatment groups were well matched for baseline characteristics, including stroke severity (mean National Institutes of Health Stroke Scale score 16.9+/-5.2). Ninety-six percent were classified as total anterior circulation syndrome. The proportion of patients reaching a Barthel Index score of > or =60 was 42% in the clomethiazole-treated group and 46% in the placebo-treated group (odds ratio, 0.81; 95% CI, 0.62 to 1.05; P=0.11). There was no evidence of efficacy on any secondary outcome variables (modified Rankin Score, National Institutes of Health Stroke Scale, Scandinavian Stroke Scale, and 30-day CT infarct volumes) compared with placebo. Subgroup analysis showed a similar lack of treatment effect in patients treated early (<6 hours) and in those treated later (6 to 12 hours). Somnolence was an expected pharmacological effect of clomethiazole, and this occurred during treatment as an adverse event in half of the patients randomly assigned to study drug. CONCLUSIONS: The target population was selected, and sufficient drug was given to produce the expected pharmacological effect in the brain. Clomethiazole does not improve outcome in patients with major ischemic stroke.

Characterization of an expanded glutamine repeat androgen receptor in a neuronal cell culture system.

Spinal and bulbar muscular atrophy (SBMA) is an inherited form of lower motor neuron degeneration caused by expansion of a CAG repeat in the androgen receptor (AR) gene. To study the mechanism by which this mutation causes neuronal pathology, we stably transfected a motor neuron hybrid cell line with human AR cDNAs containing either 24 or 65 repeats (AR24 and AR65, respectively). Both forms of receptor were able to bind ligand and activate transcription of a reporter construct equally well. Likewise, the subcellular localizations of AR24 and AR65 were similar, in both the presence and the absence of ligand. AR24- and AR65-expressing clones were phenotypically indistinguishable. They survived equally well after differentiation and were equally susceptible to damage by oxidative stress. Our studies thus demonstrate that, in a neuronal system, the expanded repeat AR functions like the normal repeat AR in several important ways. Because levels of AR65 expression were consistently lower than levels of AR24 expression, we propose that the loss of function of AR seen in SBMA may be due to decreased levels of receptor expression rather than to a difference in intrinsic properties. The postulated gain of function responsible for neuronal degeneration remains to be determined.

Developmental shift of myosin heavy chain mRNA expression due to neural factor(s) and muscle activity.

The adult ventricular isoform of chicken myosin heavy chain (MHC-V) is transiently expressed in all skeletal muscle primordia analyzed and is completely repressed around embryonic days 10-12, when functional innervation is established. By ribonuclease protection assay, we demonstrated that denervation of the adult anterior latissimus dorsi muscle resulted in reexpression of MHC-V mRNA. In contrast, treatment of primary cultures of fetal breast or leg muscles with embryonic brain extract or conditioned media from glial or neuroblastoma cell lines, but not from a myogenic cell line or primary muscle cell cultures, led to inhibition of MHC-V expression. This inhibitory activity was abolished by heating and increased with protein concentration. The acquisition of both brain inhibitory activity and the competence of myogenic cells to downregulate MHC-V mRNA expression were age dependent. Furthermore, either paralysis of muscle in ovo by curare or contraction arrest of cultured myotubes resulted in persistent expression of MHC-V mRNA. Thus a putative soluble factor(s) of nerve origin as well as muscle activity are involved in the developmental downregulation of MHC-V expression in muscle primordia.

Human T-cell lymphotropic virus type-I infection in the severe combined immunodeficiency mouse.

Human T-cell lymphotropic virus type-I (HTLV-I) is the etiologic agent of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia (ATL). HAM/TSP and ATL occur infrequently among HTLV-I-infected individuals, and rarely develop in the same individual. To study host and viral factors involved in the induction, tissue tropism, as well as pathogenesis of HAM/TSP, peripheral blood lymphocytes (PBL) from 14 patients with HAM/TSP and from 9 controls were introduced into severe combined immunodeficiency (SCID) mice by intraperitoneal injection. Mice were followed for up to 26 weeks. Human IgG was produced from 2 to 14 weeks after reconstitution in all animals. Thirty-two of 44 mice (72%) showed circulating human antibody against the major viral protein products of HTLV-I. Analysis of viral sequences by polymerase chain reaction (PCR) demonstrated HTLV-I sequences in 21/38 (55%) brains and in 7/17 (41%) spinal cords from HTLV-I-hu SCID mice. No animal had clinical evidence of neurological impairment or pathological findings similar to those seen in HAM/TSP. Seven mice who received PBL from Epstein Barr virus (EBV)-seropositive patients developed an intraperitoneal lymphoma. In 2 mice an infiltration of brain by a lymphoblastic tumor of B/T cell type was observed. By PCR, all the tumors were EBV-positive; HTLV-I sequences were detected in 5 of them. Our study suggests that the HTLV-I-hu-SCID mouse provides a potentially valuable system for studying the production, kinetics, and pathogenicity of anti-HTLV-I antibody, and may help clarify the interaction of EBV and retroviruses in the development of disease.

Functional receptor for GDNF encoded by the c-ret proto-oncogene.

Glial-cell-line-derived neutrophic factor (GDNF) promotes the survival and phenotype of central dopaminergic noradrenergic and motor neurons, as well as various subpopulations of peripheral sensory and sympathetic neurons. GDNF is structurally related to members of the transforming growth factor (TGF)-beta superfamily, several members of which have well-characterized receptor systems; however, GDNF receptors still remain undefined. Here we show that GDNF binds to, and induces tyrosine phosphorylation of, the product of the c-ret proto-oncogene, an orphan receptor tyrosine kinase, in a GDNF-responsive motor-neuron cell line. Ret protein could also bind GDNF and mediate survival and growth responses to GDNF upon transfection into naive fibroblasts. Moreover, high levels of c-ret mRNA expression were found in dopaminergic neurons of the adult substantia nigra, where exogenous GDNF protected Ret-positive neurons from 6-hydroxydopamine-induced cell death. Thus the product of the c-ret proto-oncogene encodes a functional receptor for GDNF that may mediate its neurotrophic effects on motor and dopaminergic neurons.

Amyotrophic lateral sclerosis and viruses.

Amyotrophic lateral sclerosis (ALS) is a disease of unknown etiology. A number of theories have been pursued to explain the cause of ALS, including viral infection. This review examines the evidence implicating viruses in the pathogenesis of ALS, as well as current studies of naturally occurring and experimental models of virus-induced motor neuron disease (MND). The association of viruses and ALS remains to be established. The study of animal models of virus-induced MND may shed light on processes relevant to the etiology of ALS.

Neuromuscular transmission in amyotrophic lateral sclerosis.

The functional and structural characteristics of the neuromuscular junction were studied in anconeus muscle biopsies of 10 patients with amyotrophic lateral sclerosis (ALS). Intracellular recordings revealed decreased amplitudes of miniature endplate potentials (MEPPs). The MEPP frequencies were highly variable in ALS patients but the average MEPP frequency was not different from that of control patients. The mean quantal content of endplate potentials (m), the mean quanta available for immediate release (n), and the mean quantal stores (N) were all decreased. In contrast, the mean probability of quantal release (p) was normal and the mean probability of quantal store release (P) was surprisingly high at the majority of ALS endplates. Histologic evidence of denervation and small or absent nerve terminals were observed in all ALS patients. These functional and structural abnormalities of the neuromuscular junction may explain the fatigability and the electromyographic evidence of impaired neuromuscular transmission often encountered in ALS patients.

Intrafamilial heterogeneity in hereditary motor neuron disease.

Although there are varied inheritance patterns in motor neuron disease (MND), the phenotype of MND is reported to be constant within these families, ie, cases of amyotrophic lateral sclerosis or primary lateral sclerosis do not occur in pedigrees with cases of spinal muscular atrophy. We describe four pedigrees whose members diverged in the phenotype of MND expressed. The intrafamilial variation of phenotype suggests a similar pathogenesis for some of the varied types of familial MND and the need for careful inquiry of family history in all patients with MND.

Neuromuscular transmission as a function of motor unit size in patients with prior poliomyelitis.

We studied neuromuscular transmission in 16 patients with prior poliomyelitis by measuring single fiber electromyographic (SFEMG) jitter. This was compared with 3 indirect methods of assessing reinnervation: SFEMG fiber density, macro EMG, and the presence of fiber type grouping on muscle biopsy. In patients with acute poliomyelitis before the age of 10, there was a positive correlation between the extent of neuromuscular transmission impairment, demonstrated by increased SFEMG jitter, and the enlargement of the motor unit, as indicated by increased fiber density, increased macro EMG signals, and fiber type grouping on muscle biopsy. However, there was no correlation between any of these parameters and the presence or absence of new symptoms of weakness. These findings suggest that impaired neuromuscular transmission is most common in patients with prior poliomyelitis whose motor units have been maximally enlarged by axonal sprouting, but is independent of the presence or absence of new symptoms of weakness.

Embryonic mouse spinal cord motor neuron hybrid cells.

Studies of motor neurons are difficult because of limitations in their isolation and culture. One solution is to produce clonal neural hybrid cells that can express motor neuron characteristics; we fused an aminopterin-sensitive and neomycin-resistant mouse neuroblastoma cell line to isolated embryonic mouse spinal cord motor neurons. Several hybrid neuron cell lines expressing high levels of choline acetyltransferase (CHAT) enzyme activity were found. These were cloned and clones with high CHAT activity isolated. The hybrid nature of cloned cells was confirmed by karyotyping and determining glucose phosphate isomerase allozymes. The availability of these embryonic clonal hybrid cells will enable molecular, physiological, and biochemical studies to define motor neuron-specific properties.

Polyclonal IgM anti-GM1 ganglioside antibody in patients with motor neuron disease and variants.

Recent studies reported the presence of anti-ganglioside antibodies in occasional patients with motor neuron disease. We found polyclonal serum IgM anti-GM1 antibodies by an anti-GM1 enzyme-linked immunosorbent assay (ELISA) in 9 (19%) of 48 patients with motor neuron disease. A comparable frequency of IgM anti-GM1 antibodies was found in 4 (10%) of 40 sera from patients with other neurological disease. Three (17%) of 18 sera from the patients with motor neuron disease and 2 (17%) of 12 sera from patients with other neurological diseases had anti-GM1 immunostaining as shown by thin layer chromatography immunoblot. One patient with a lower motor neuron variant of motor neuron disease or motor axonopathy without multifocal conduction block had a markedly elevated polyclonal IgM anti-GM1 ELISA titer (greater than 1:64,000) with prominent immunostaining of GM1, moderate immunostaining of GM2, and weak and inconsistent immunostaining of GD1b by thin layer chromatography immunoblot. Treatment with prednisone resulted in clinical improvement despite increasing anti-GM1 antibody titers. These data indicate that patients with motor neuron disease have measurable levels of anti-ganglioside antibodies as frequently as patients with other neurological diseases. This contrasts with a small subgroup of patients with a lower motor neuron variant of motor neuron disease or motor axonopathy who have markedly elevated levels of serum anti-ganglioside antibodies and a clinical syndrome that is treatable with immunosuppression.

Isoelectric focusing studies of serum and cerebrospinal fluid in patients with antecedent poliomyelitis.

The post-poliomyelitis syndrome (PPS) refers to symptoms of new weakness, fatigue, and pain years after recovery from acute poliomyelitis. Oligoclonal IgG bands have been reported in the cerebrospinal fluid (CSF) from PPS patients, suggesting that the syndrome is immune mediated or caused by persistent viral infection. We studied 15 paired serum and CSF samples and 6 unpaired CSF samples from a total of 21 patients with a prior history of poliomyelitis. Quantitative immune studies failed to show evidence for increased intrathecal IgG production relative to patients with noninflammatory central nervous system (CNS) disease. We found definite oligoclonal IgG bands in the CSF from only 1 patient, who also carried a diagnosis of multiple sclerosis. An isoelectric focusing poliovirus antigen overlay study showed evidence that suggested a CNS-specific antipoliovirus immune response in only 1 patient. Our results fail to support a dysimmune or persistent viral cause for post-poliomyelitis progressive muscular atrophy or PPS.

Sleep apnea in olivopontocerebellar degeneration: treatment with trazodone.

A 37-year-old man with a 19-year history of progressive autosomal-dominant olivopontocerebellar degeneration developed excessive daytime sleepiness and paroxysmal episodes that clinically resembled an ictal or postictal state. Polysomnography showed sleep apnea. Long-term therapy with trazodone resulted in resolution of the paroxysmal episodes, disappearance of daytime sleepiness, and gradual improvement of sleep architecture over several months.