Murray B. Bornstein

A pilot trial of Cop 1 in exacerbating-remitting multiple sclerosis.

Cop 1 is a random polymer (molecular weight, 14,000 to 23,000) simulating myelin basic protein. It is synthesized by polymerizing L-alanine, L-glutamic acid, L-lysine, and L-tyrosine. It suppresses but does not induce experimental allergic encephalomyelitis, an animal model of multiple sclerosis. It is not toxic in animals. In a double-blind, randomized, placebo-controlled pilot trial, we studied 50 patients with the exacerbating-remitting form of multiple sclerosis, who self-injected either 20 mg of Cop 1 dissolved in 1 ml of saline or saline alone daily for two years. Six of 23 patients in the placebo group (26 percent) and 14 of 25 patients in the Cop 1 group (56 percent) had no exacerbations (P = 0.045). There were 62 exacerbations in the placebo group and 16 in the Cop 1 group, yielding two-year averages of 2.7 and 0.6 per patient, respectively. Among patients who were less disabled on entry (Kurtzke disability score, 0 to 2), there were 2.7 exacerbations in the placebo group and 0.3 in the Cop 1 group over two years. Among patients who were more affected (Kurtzke disability score, 3 to 6), there was an average of 2.7 exacerbations in the placebo group and 1.0 in the Cop 1 group. Over two years, less disabled patients taking Cop 1 improved an average of 0.5 Kurtzke units; those taking placebo worsened an average of 1.2 Kurtzke units. More disabled patients worsened by 0.3 (Cop 1 group) and 0.4 (placebo group) unit. Irritation at injection sites and rare, transient vasomotor responses were observed as side effects. These results suggest that Cop 1 may be beneficial in patients with the exacerbating-remitting form of multiple sclerosis, but we emphasize that the study is a preliminary one and our data require confirmation by a more extensive clinical trial.

Δ9-Tetrahydrocannabinol: a novel treatment for experimental autoimmune encephalomyelitis ☆

Since multiple sclerosis (MS) is believed to be an immune-mediated disease, it follows that its therapies should be directed towards modulating the immune system. Current MS treatments, which include the use of exogenous steroids that are immunosuppressive, do not meet therapeutic objectives. Δ9-Tetrahydrocannabinol (THC), an active component of marijuana, has been shown to be immunosuppressive. To test THCs ability to suppress an immune-mediated disease, experimental autoimmune encephalomyelitis (EAE), the laboratory model of MS, was used. Lewis rats and strain 13 guinea pigs were administered THC either before inoculation for EAE or treated with THC after injection. Control animals received placebo. The effect of dose, in addition to the timing of treatment, was also investigated. All animals treated with placebo developed severe clinical EAE 10–12 days post-injection (d.p.i.) and more than 98% died by 15 d.p.i. THC-treated animals had either no clinical signs or mild signs with delayed onset (13–15 d.p.i.) with survival greater than 95%. Examination of central nervous system tissue revealed a marked reduction of inflammation in the THC-treated animals. Therefore, as THC has been shown to inhibit both clinical and histologic EAE, it may prove to be a new and relatively innocuous agent for the treatment of immune-mediated diseases.

Cerebroside antibody inhibits sulfatide synthesis and myelination and demyelinates in cord tissue cultures.

Antiserum to cerebroside was prepared in rabbits by injection of cerebroside together with bovine serum albumin in complete Freunds adjuvant. When applied to cultures of embryo mouse spinal cord at explantation, this antiserum inhibited sulfatide synthesis and myelination; when applied to myelinated cultures it inhibited sulfatide synthesis and produced demyelination. Complement fixation assays also show antibody to cerebroside in serums from rabbits with experimental allergic encephalomyelitis induced by injection of whole white matter. Absorption of such serum with cerebroside abolishes the inhibiting and demyelinating activities.

A placebo-controlled, double-blind, randomized, two-center, pilot trial of Cop 1 in chronic progressive multiple sclerosis

We found Cop 1 to be effective and relatively safe in a previous (exacerbating-remitting) clinical trial. This current trial involves 106 chronic-progressive patients. The major end point, confirmed progression of 1.0 or 1.5 units (depending on baseline disability) on the Kurtzke Expanded Disability Status Scale, was observed in nine (17.6%) treated and 14 (25.5%) control patients. The differences between the overall survival curves were not significant. Progression rates at 12 and 24 months were higher for the placebo group (p = 0.088) with 2-year probabilities of progressing of 20.4% for Cop 1 and 29.5% for placebo. We found a significant difference at 24 months between placebo and Cop 1 at one but not the other center. Two-year progression rates for two secondary end points, unconfirmed progression, and progression of 0.5 EDSS units, (p = 0.03) are significant.

Demyelination in vitro

Myelinated cultures of mouse spinal cord have been exposed to sera raised in rabbits against whole white matter (anti-WM), myelin basic protein (anti-MBP) and galactocerebroside (anti-GC), the major glycolipid of CNS myelin, to determine which factor in central nervous system (CNS) tissue in vitro is the target of serum demyelinating and myelin swelling antibodies. The sera were tested by radioimmunoassay for activity against MBP and against GC and were also specifically absorbed with MBP, GC and control antigens. Studies were also performed with and without active complement. The findings show that demyelination and myelin swelling in vitro are caused by antibodies against GC and not against MBP. Ultrastructurally, the effects of anti-WM and anti-GC sera with and without complement were indistinguishable. This study demonstrates that GC is a major target in antibody-mediated demyelination.

Synthesis of serotonin by neurons of the myenteric plexus in situ and in organotypic tissue culture.

Previous studies have suggested that serotonin is a neurotransmitter in the mammalian myenteric plexus. The present study was done to test this hypothesis by determining whether neurons of the myenteric plexus synthesize 5-HT from L-trypophan. Isolated strips of longitudinal muscle with attached myenteric plexus took up L-tryptophan by a saturable transport mechanism Km 4.7 × 10−5M; Vmax 6.7 nmoles/g/min). These strips also synthesized tritiated serotonin ([3H]5-HT) and [3H]5-hydroxy-indoleacetic acid from L-[3H]tryptophan. This synthesis of [3H]5-HT was antagonized by prior treatment of guinea pigs with p-chlorophenylalanine. The drug treatment did not interfere with tryptophan uptake as did inclusion of p-chlorophenylalanine with L-[3H]tryptophan in vitro and thus probably decreased synthesis of [3H]5-HT by inhibiting tryptophan hydroxylase. Organotypic tissue cultures of intestine, lacking mucosa, argentaffin cells or mast cells also converted L-[3H]tryptophan to [3H]5-HT and [3H]5-hydroxyindoleacetic acid. Examination of the myenteric plexus from chemically sympathectomized (6-hydrpxydopamine) animals given L-tryptophan and a monoamine oxidase inhibitor for formaldehyde-induced histofluorescence revealed a diffuse yellow fluorophore in the myenteric plexus with the spectral characteristics of 5-HT. Pretreatment with p-chorophenylalanine prevented development of the diffuse fluorescence induced by L-tryptophan but revealed clusters of fluorescent neuronal cell bodies. The fluorescence of these cell bodies also had the spectral characteristics of the 5-HT fluorophore. Organotypic tissue cultures of intestine, grown for 3 weeks, and then dried and exposed to gaseous formaldehyde contained groups of fluorescent neuronal cell bodies. Their fluorescence was enhanced by adding L-tryptophan and an inhibitor of monoamine oxidase to the culture medium. This fluorescence also had the spectral characteristics of 5-HT. We conclude that the myenteric plexus synthesizes 5-HT from L-tryptophan. The responsible neurons survive in culture and are therefore intrinsic to the gut itself. These observations support the hypothesis that 5-HT is a transmitter of neurons in the mammalian myenteric plexus.

Experimental Allergic Encephalomyelitis: An Ultrastructural Study of Experimental Demyelination in Vitro

Myelinated cultures of rat spinal cord were exposed to the demyelinating action of 25 per cent serum from rabbits suffering from experimental allergic enceplmlomyelitis (EAE). The fine structural changes occurring during the first few hours of exposure were observed. Myelin changes began within 15 minutes of exposure, and by 6 hours all fibers were demyelinated. Axons remained unaffected in the majority of cases. Myelin degeneration occurred in two ways, either by interlamellar swelling resulting in the formation of large vacuoles around denuded axons, or by a melting process in which the myelin became smudged and dissociated from the unaffected axon. The latter was often accompanied by the formation of a whorl of astroglial processes around the affected fiber. The phagocytic activity of astroglia in this process is noted. Non-specific dilatation of cellular elements also occurred. Oligodendroglia appeared selectively damaged and showed degeneration. These changes are compared with patterns already described in E.A.E. and multiple selerosis.

Maturation of cultured embryonic CNS tissues during chronic exposure to agents which prevent bioelectric activity

Summary Fetal rodent cerebral cortex and spinal cord tissues were explanted at stages prior to the onset of synaptic function. The culture media contained chemical agents which produced sustained depression of the characteristic bbioelectric excitability of the neurons. Addition of xylocaine (50 μg/ml) or a 10-fold increase in Mg 2+ concentration were effective in blocking all complex bioelectric activities—or, at least, in markedly raising the threshold for triggering such synaptically mediated phenomena. They did not, however, appear to interfere with the characteristic morphologic growth and development of the tissues in vitro , as observed by light microscopy. Representative CNS cultures tested at various intervals during chronic exposure (5–30 days) to these blocking agents showed no spontaneous, or evoked, complex bioelectric activity, although simple action potentials could be triggered, in some cases, at unusually high stimulus intensities. These CNS tissues have, therefore, been cultured under conditions which apparently prevent all spontaneous, as well as fortuitously evoked, bioelectric discharges during the critical period when synaptic networks normally form in control cultures. Nevertheless, within minutes after removing the blocking agent from the bathing fluid, the first electric stimulus applied to such a virginal explant often evoked a complex bioelectric response similar to those seen in mature control explants. The mimicry of these responses to patterns characteristic of organized multisynaptic networks, in situ , suggests that ontogenetic development of some types of complex interneuronal CNS functions may be programmed to occur independent of prior bioelectric excitation of the cellular elements composing the system. Moreover, after endogenous formation of such a neuronal cell assembly it can be maintained in a quiescent state for at least several weeks and, yet, remain organized with characteristic bioelectric excitability.

Plasmapheresis in multiple sclerosis Preliminary findings

In seven of eight patients with progressive multiple sclerosis subjected to long-term plasmapheresis in combination with azathioprine and pulsed prednisone therapy, we found modest improvement of neurologic function. There was no change in auditory and visual evoked responses or serum demyelinating activity. In six of seven patients, cerebrospinal fluid IgG content decreased. Three additional patients in acute, severe exacerbation refractory to prednisone therapy made a substantial recovery, which commenced with plasmapheresis therapy. In two of them, the onset of clinical improvement after plasmapheresis was corroborated by decreased latency or increased amplitude of somatosensory evoked potentials. These results suggest that blood-borne factors, possibly autoantibodies, may play a role in the pathogenesis of the disease. The lesions may be at least partially reversible with plasmapheresis therapy, but a controlled trial is necessary to confirm these preliminary findings.

Organotypic Bioelectric Activity in Cultured Reaggregates of Dissociated Rodent Brain Cells

Complex repetitive-spike or slow-wave discharges can be evoked, and can also occur spontaneously, in small clusters of neurons which reaggregate in vitro after dissociation of cerebral cortex, brainstem, or spinal cord from the fetal mouse. Even after random dispersion in culture, these cells still form functional synaptic networks with bioelectric discharge patterns and pharmacologic sensitivities characteristic of the organ (that is, organotypic).