Aminoguanidine, an inhibitor of inducible nitric oxide synthase, ameliorates experimental autoimmune encephalomyelitis in SJL mice.

Previous work from our laboratory localized nitric oxide to the affected spinal cords of mice with experimental autoimmune encephalomyelitis, a prime model for the human disease multiple sclerosis. The present study shows that activated lymphocytes sensitized to the central nervous system encephalitogen, myelin basic protein, can induce nitric oxide production by a murine macrophage cell line. Induction was inhibited by amino-guanidine, a preferential inhibitor of the inducible nitric oxide synthase isoform, and by NG-monomethyl-L-arginine. Aminoguanidine, when administered to mice sensitized to develop experimental autoimmune encephalomyelitis, inhibited disease expression in a dose-related manner. At 400 mg aminoguanidine/kg per day, disease onset was delayed and the mean maximum clinical score was 0.9 +/- 1.2 in aminoguanidine versus 3.9 +/- 0.9 in placebo-treated mice. Histologic scoring of the spinal cords for inflammation, demyelination, and axonal necrosis revealed significantly less pathology in the aminoguanidine-treated group. The present study implicates excessive nitric oxide production in the pathogenesis of murine inflammatory central nervous system demyelination, and perhaps in the human disease multiple sclerosis.

Decreased dependence of myelin basic protein-reactive T cells on CD28-mediated costimulation in multiple sclerosis patients. A marker of activated/memory T cells.

Although multiple sclerosis (MS) patients and healthy individuals have similar frequencies of myelin basic protein (MBP)-specific T cells, the activation state of these cells has not been well characterized. Therefore, we investigated the dependence of MBP-reactive T cells on CD28-mediated costimulation in MS patients, healthy controls, and stroke patients. MBP-reactive T cells from healthy controls and stroke patients failed to proliferate efficiently when costimulation was blocked using anti-CD28, consistent with a naive T cell response. In contrast, MBP-specific T cell proliferation was not inhibited, or was only partially inhibited when CD28-mediated costimulation was blocked in MS patients. Blockade of CD28 failed to inhibit tetanus toxoid-specific T cell proliferation in both the controls and MS patients, demonstrating that memory cells are not dependent on CD28-mediated costimulation. Limiting dilution analysis indicated that the frequency of MBP-reactive T cells was significantly decreased in healthy controls compared with MS patients when CD28-mediated costimulation was blocked. These data suggest that MBP-reactive T cells are more likely to have been activated in vivo and/or differentiated into memory T cells in MS patients compared with controls, indicating that these cells may be participating in the pathogenesis of MS.

Long-term inhibition of murine experimental autoimmune encephalomyelitis using CTLA-4-Fc supports a key role for CD28 costimulation.

T cell activation involves not only recognition of antigen presented by the MHC, but also nonspecific interactions termed "costimulation." The costimulatory molecules B7-1 and B7-2 are ligands on antigen-presenting cells for the CD28 and CTLA-4 receptors on T cells. Previously, a fusion protein consisting of human CTLA-4 linked to human Fc was shown to bind B7-1 and B7-2 with high avidity and to prevent specific T cell activation. Here we investigated the effects of a recombinant fusion protein consisting of the extracellular domain of human CTLA-4 bound to mouse IgG2a Fc (CTLA-4-Fc) upon experimental autoimmune encephalomyelitis, a T cell-mediated disease that serves as a model for multiple sclerosis. CTLA-4-Fc prevented experimental autoimmune encephalomyelitis in 26 of 28 CTLA-4-Fc-treated mice (median maximum score 0), whereas 28 of 30 mice treated with control mouse IgG2a developed disease (median maximum score 2.75). Less inflammation and virtually no demyelination or axonal loss occurred in CTLA-4-Fc-treated compared with control-treated mice. Activated splenocytes from CTLA-4-Fc-treated mice were able to transfer disease adoptively to naive recipients. These results indicate a key role for the B7/CD28 system in the development of actively induced murine experimental autoimmune encephalomyelitis, suggesting an area of investigation with therapeutic potential for multiple sclerosis.

Pain in multiple sclerosis.

We reviewed 317 patients with multiple sclerosis (MS) and found that the incidence of clinically significant pain, excluding headache and paresthesia, was 28.8%. Successful treatment requires recognition of the pathophysiology of the pain syndromes encountered in MS. Antidepressant drugs have been of particular value in the treatment of chronic pain in these patients.

Oligoclonal band number as a marker for prognosis in multiple sclerosis.

The natural course of disease in multiple sclerosis varies. Multiple sclerosis that is clinically apparent but causes minimal disability over time has been labeled benign multiple sclerosis. The ability to predict the subsequent clinical course of multiple sclerosis on the basis of clinical and other supportive data at presentation would be invaluable. In this article we report our findings based on a retrospective analysis of 1800 patients diagnosed as having multiple sclerosis, of which 44 patients met our inclusion criteria. There was a suggestion that a low or absent number of oligoclonal bands in the cerebrospinal fluid at the time of diagnosis predicts a better prognosis. However, quantification of oligoclonal bands in cerebrospinal fluid remains an insensitive prognostic indicator and must not be used to influence decisions regarding therapeutic options.

Amyloidosis with plasma cell dyscrasia. An overlooked caused of adult onset sensorimotor neuropathy.

In ten previously undiagnosed patients, we have found erstwhile-"primary" nonhereditary amyloidosis as an overlooked cause of a predominately sensory, painful, and hyperesthetic distal neuropathy occurring in middle-age and older patients. These symptoms, associated with orthostatic hypotension, diarrhea or constipation, cardiac abnormality, and male impotence are virtually diagnostic (in the absence of diabetes mellitus). Tissue diagnosis is quickly made by crystal-violet metachromasia of amyloid in fresh-frozen sections of a muscle biopsy specimen. Immunoglobulin and bone marrow evidence of plasma cell dyscrasia in eight of the ten patients suggests that the neuropathy in this form of amyloidosis is actually secondary to a plasma-cell-originating dysproteinemia. Therapy with melphalan and prednisone was not of benefit.

Cerebellar degeneration with Hodgkin disease. An immunological study.

A 21-year-old woman with subacute cerebellar degeneration was found to have Hodgkin lymphoma. Radiation therapy for the lymphoma halted the progression of her neurological disease. Using an immunofluorescent technique, we found the patient's serum to have antibodies to cerebellar Purkinje cells.

Acute aphasia in multiple sclerosis.

Acute aphasia is rare in multiple sclerosis. We describe 3 patients with multiple sclerosis who had acute exacerbations presenting as aphasias. One patient had a mixed transcortical aphasia, 1 had a transcortical motor aphasia, and 1 had a Broca aphasia. Magnetic resonance imaging scans of the brain with contrast enhancement revealed new white matter lesions in the left hemisphere in all 3 patients. Two of the 3 patients had a good response to treatment with methylprednisolone sodium succinate. Arch Neurol. 2000;57:1207-1209

The effect of pharmacologic acetylcholine receptor on fibrillation and myotonia in rat skeletal muscle.

Myotonic discharges in rats given 20, 25-diazacholesterol hydrochloride and fibrillation discharges in denervated rat muscle both were silenced by procaine hydrochloride, tetrodotoxin or ischemia, or potassium chloride (after initial activation). They both were activated by succinylcholine, but only the fibrillations were silenced by alpha-bungarotoxin or atropine sulfate. It is hypothesized that fibrillations and diazacholesterol-induced myotonia are mediated through mechanisms involving ionic channels, that both can be produced by activation of the junctional/nonjunctional acetylcholine receptors (or some mechanism coupled to the receptors), but that an unfettered alpha-bungarotoxin-binding portion of the acetylcholine-receptor molecule and an unblocked atropine-binding site are obligatory only for production of fibrillations.

Prolonged effects of large-dose methylprednisolone infusion in multiple sclerosis.

Three daily 1-gm doses of methylprednisolone were administered to 12 patients with multiple sclerosis. Granulocytes, lymphocytes, T and B lymphocytes, lymphocyte transformation in response to several mitogens and one antigen, plasma cortisol, and serum IgM and IgA all returned to baseline within a few days. However, total white blood count, unstimulated lymphocyte incorporation of 3H-thymidine, hematocrit, serum IgG, and cerebrospinal fluid IgG synthesis remained altered 1 week after the infusion. The relationship of these changes to any clinical effects is unknown.

Thymectomy in multiple sclerosis: a 3-year follow-up.

We studied 34 MS patients who were treated experimentally by thymectomy with or without 1 year of azathioprine therapy. After 3 years, there was no evidence of benefit. Relapsing-remitting patients had done as well or better clinically than controls, and the chronic progressive group did less well statistically than controls.

Interleukin-2 binding proteins in sera from normal subjects and multiple sclerosis patients.

We previously reported elevations of interleukin 2 (IL-2) in the serum of patients with chronic progressive MS. Using gel chromatography, protein A sepharose affinity chromatography, and ELISAs for IL-2 and the IL-2 soluble receptor, we now demonstrate that this cytokine is bound to serum proteins. These serum proteins include antibodies to IL-2, soluble IL-2 receptors, and high-molecular-weight proteins. Using a CTLL cell assay, a serum fraction corresponding to IgG antibodies to IL-2 inhibited the activity of this cytokine. Thus, we present evidence for potential immunomodulation of a pivotal cytokine in MS by serum proteins.

Prospective serial analysis of interleukin-2 and soluble interleukin-2 receptor in relapsing-remitting multiple sclerosis.

We performed a longitudinal analysis of serum interleukin-2 (IL-2) and soluble IL-2 (sIL-2R) concentrations in 60 patients with relapsing-remitting (R-R) multiple sclerosis (MS) as well as in 33 age- and sex-matched normal controls. Overall, we found that serum IL-2 levels remained low (less than 10 U/ml) and did not change appreciably over time; however, marked fluctuations in sIL-2R levels were observed in both the patient and control groups. Using patients as their own controls, we calculated an interrelapse (disease stable) mean sIL-2R concentration as a baseline for comparison with relapse values; sIL-2R levels greater than the 90th percentile of the Student's t distribution of stable values were defined as "peaks." There were a total of 27 sIL-2R peaks, eight (30%) of which correlated with clinical relapses but were potentially predictive of only 18% (8/45) of all the recorded clinical relapses. There was no difference in disease severity (Expanded Disability Status Scale) score between peak-correlated and noncorrelated relapses. Our data suggest that despite reports of elevated levels of IL-2 and sIL-2R in MS, neither may be a useful marker for predicting clinical disease activity in R-R MS.

High-titer selective serum anti-beta-tubulin antibodies in chronic inflammatory demyelinating polyneuropathy.

Although chronic inflammatory demyelinating polyneuropathy (CIDP) is presumed to be an autoimmune disorder, no neural antigen has been recognized as an immune target. We found that serum IgM from a patient with CIDP and an IgM paraprotein reacted with a 53-kd protein by Western blot analysis. Amino acid sequence analysis identified this protein as beta-tubulin. We then studied sera from 70 CIDP patients, 35 Guillain-Barré syndrome (GBS) patients, and 483 disease (amyotrophic lateral sclerosis, Alzheimer's disease, multiple sclerosis, diabetes, and other polyneuropathies) and normal controls for selective high-titer anti-beta-tubulin using ELISA methodology. Forty-two percent (30/70) of patients with CIDP had selective high titer IgM reactivity against beta-tubulin; 23% (16/70) had selective high-titer IgG reactivity against beta-tubulin. Overall, 57% of CIDP patients, 20% of GBS patients, and 2% of control patients had selective, high serum IgM or IgG anti-beta-tubulin reactivity. Selective high-titer serum anti-beta-tubulin antibodies occur in a majority of patients with CIDP but are rare in other chronic neuropathies or CNS disorders.

Morphologic and immunologic studies in experimental autoimmune myasthenia gravis and myasthenia gravis.

An indirect immunoperoxidase technique was used to study by light microscopy the binding of serum from experimental autoimmune myasthenia gravis (EAMG) rabbits to junctionally and extrajunctionally located acetylcholine receptors (AChRs) in human and rat muscles. Binding was restricted to junctional AChR. Alpha bungarotoxin (a-BGT) partially blocked the binding of EAMG serum, while myasthenia gravis serum, carbamylcholine, decamethonium, and tubocurarine did not. A radioimmunoassay showed significant binding of antibodies in EAMG sera to 125l AChR. This binding was not inhibited by a-BGT, nor by carbamylcholine, decamethonium, or tubocurarine. Sera from 10 myasthenia gravis patients did not contain antibodies binding to the 125l AChR. We suggest that EAMG in rabbits induced by Torpedo AChR differs serologically from myasthenia gravis in patients, probably owing to antigenic differences between Torpedo and human AChR, and that antigenic differences also exist between junctional and extrajunctional receptors.

B cells and antibodies in CNS demyelinating disease.

There is much evidence to implicate B cells, plasma cells, and their products in the pathogenesis of MS. Despite unequivocal evidence that the animal model for MS, EAE, is initiated by myelin-specific T cells, there is accumulating evidence of a role for B cells, plasma cells, and their products in EAE pathogenesis. The role(s) played by B cells, plasma cells, and antibodies in CNS inflammatory demyelinating diseases are likely to be multifactorial and complex, involving distinct and perhaps opposing roles for B cells versus antibody.

Serum cytokine levels in chronic progressive multiple sclerosis: interleukin-2 levels parallel tumor necrosis factor-alpha levels.

Serum levels of the cytokines interleukin-1 alpha (IL-I alpha), IL-1 beta, IL-2, IL-4, IL-6, tumor necrosis factor-alpha (TNF-alpha) and the soluble IL-2 receptor were measured in chronic progressive multiple sclerosis patients (CPMS) and normal, inflammatory, and noninflammatory disease controls. Serum IL-2 levels displayed the most consistent abnormalities in the group of tests for the CPMS group, and were the only cytokine levels to achieve significance in statistical group analyses. However, several patients with CPMS had normal serum IL-2 levels. An incidental finding was a statistical correlation between serum IL-2 and TNF-alpha levels among all groups tested. This finding was supported on analysis of serial serum samples from CPMS patients. These results suggest a linkage of IL-2 and TNF-alpha production, especially in pathological conditions.

Studies on the thymus from patients with multiple sclerosis and myasthenia gravis.

The thymus glands which were excised for therapy (myasthenia gravis; MG) or experimental therapy (multiple sclerosis; MS) were compared to thymic biopsies from patients undergoing cardiac surgery. There was no difference in the weight or total cells of MG and MS thymuses or of the cell density of control, of MG or MS glands. Only 1 of 25 MS thymuses was hyperplastic, as were 2 of 9 of the MG thymuses and none of the controls. Several differences were noted for thymic lymphocyte proliferation to mitogenes in MS patients and to antigens in MS and MG patients. Ms thymuses had a decreased stimulation index to antithymocyte globulin and to optimal concentrations of pokeweed mitogen. Myasthenia gravis thymuses showed a significantly increased stimulation of myelin basic protein. The % B and % T cell counts were normal for the MS patients. No differences were noted in the incidence of mixed lymphocyte reactions between thymocytes and peripheral lymphocytes in the three groups. Fresh thymic lymphocytes did not suppress concanavalin A stimulated lymphocyte proliferation. It is not known if the differences in lymphocyte proliferation between MS, MG, and control thymuses represent a primary or secondary change.

Mediation of inflammation by encephalitogenic cells: interferon gamma induction of nitric oxide synthase and cyclooxygenase 2.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory demyelinating disorder of the central nervous system (CNS) which serves as a prime animal model for the human disease multiple sclerosis. Previous studies from these laboratories demonstrated excess nitric oxide (NO) in the CNS of EAE-affected mice, and amelioration of EAE with a selective inhibitor of the inducible nitric oxide synthase (iNOS). Recent studies from other laboratories have indicated that prostaglandin PGE2 is increased in CNS tissues of EAE-affected rodents and that EAE is prevented by the inhibition of cyclooxygenase activity. The present study investigated the ability of encephalitogenic lymphoid cells to induce NOS and cyclooxygenase (COX-2) in the murine macrophage line, RAW 264.7. In order to mimic the extracellular milieu present in EAE lesions, conditioned medium (CM) of activated EAE-inducer cells was added to this macrophage line. CM caused a time-dependent increase in nitrite, indicating NO production. Reverse-transcriptase PCR demonstrated iNOS mRNA in RAW 264.7 cells, first detected at 3 h, and Western blots confirmed the induction in RAW cells of the 130-kDa iNOS protein. Production of nitrite by CM-exposed RAW 264.7 cells was blocked by inhibitors of NOS (L-N-methylarginine or aminoguanidine) or by antibodies to murine IFN-gamma or IL-1 beta. CM of activated encephalitogenic cells induced production of PGE2 by RAW 264.7 cells, as determined by ELISA, and Western blots identified the presence of the 70-80-kDa inducible COX (COX-2) protein. Induction of COX-2 could be inhibited by antibody to IFN-gamma. Thus, encephalitogenic cells are capable of inducing the expression of the inflammatory enzymes iNOS and COX-2 in a murine macrophage line via the T cell cytokine IFN-gamma, alone or in combination with IL-1 beta.

Immune processing of proteolipid protein by subsets of antigen-presenting spleen cells.

Myelin proteolipid protein (PLP) contains one established antigenic epitope within the 139-151 amino acid sequence, with encephalitogenic activity for SJL mice (PLP139-151). In the current study, the processing and presentation of PLP by subsets of splenic antigen-presenting cells (APC) were examined by comparing their capacity to stimulate PLP-responsive T-cell clones, two of which are specific for PLP139-151, and one which is not specific for this peptide. In order to study whether PLP requires processing before its presentation by APC, PLP-pulsed and fixed APC were shown to stimulate PLP-specific T cells. However, the addition of PLP to unpulsed, fixed APC resulted in the absence of T-cell stimulation, while the ability of these fixed APC to bind antigenic peptide and efficiently present it to T cells, was demonstrated by their ability to use a synthetic peptide for the stimulation of the T cells. In order to study potentially different processing efficiencies among APC subsets, spleen cells were fractionated by adherence to plastic, and their respective APC activities were studied separately. The non-adherent (NAd) APC were unable to stimulate PLP139-151 specific T-cell clones with PLP as antigen. In contrast, a T-cell clone specific for a separate, but unidentified epitope on PLP was stimulated by NAd APC efficiently. In addition, stimulation of PLP139-151-specific T-cell clones by NAd APC did occur when the synthetic peptide instead of intact PLP was used as antigen, indicating a defect in PLP processing by the NAd APC.(ABSTRACT TRUNCATED AT 250 WORDS)