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.

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.

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

Increased frequency of recognition of delipidated versus intact CNS myelin in multiple sclerosis and control subjects.

The proliferative response of mononuclear cells from MS patients and normal control subjects to intact and delipidated myelin membranes was examined. The mean frequency of recognition in both groups of human subjects was greater for delipidated myelin than for intact myelin. Human T cell lines established using intact or delipidated myelin as the antigen were highly heterogeneous in response, and were each able to recognize myelin basic protein and myelin proteolipid protein peptides. However, there was no difference in the frequency of recognition of either form of myelin membrane when MS patients were compared to control subjects. Our results suggest that the presentation of delipidated forms of membrane proteins might enhance the response to myelin antigens in vivo, and be relevant to demyelinating diseases.

Oral administration of myelin basic protein is superior to myelin in suppressing established relapsing experimental autoimmune encephalomyelitis.

Oral administration of a myelin component, myelin basic protein (MBP), induces immunological unresponsiveness to CNS Ags and ameliorates murine relapsing experimental autoimmune encephalomyelitis (REAE). However, a recent clinical trial in which multiple sclerosis patients were treated with repeated doses of oral myelin was unsuccessful in reducing disease exacerbations. Therefore, we directly compared the tolerizing capacity of myelin vs MBP during REAE in B10.PL mice. Oral administration of high doses of myelin, either before disease induction or during REAE, did not provide protection from disease or decrease in vitro T cell responses. In contrast, repeated oral administration of high doses of MBP suppressed established disease and MBP-specific T cell proliferation and cytokine responses. The frequency of IL-2-, IFN-gamma-, and IL-5-secreting MBP-specific T cells declined with MBP feeding, implicating anergy and/or deletion as the mechanism(s) of oral tolerance after high Ag doses. We have previously shown that the dosage and timing of Ag administration are critical parameters in oral tolerance induction. Studies presented here demonstrate that Ag homogeneity is also important, i.e., homogeneous Ag (MBP) is more effective at inducing oral tolerance than heterogeneous Ag (myelin).

T cell recognition of myelin proteolipid protein and myelin proteolipid protein peptides in the peripheral blood of multiple sclerosis and control subjects.

Myelin proteolipid protein (PLP) is a prime candidate autoantigen for multiple sclerosis. In order to define potential immunodominant epitopes, T cell lines (TCL) from the peripheral blood of HLA-DR 15(2) MS patients were established which responded to the intact molecule of PLP. These TCL were then tested in individual proliferation assays with a variety of PLP peptides spanning most of the PLP molecule. Multiple peptides were recognized by TCL from the MS population, with more than one peptide often recognized by lines from the same individual. Three immunodominant peptides were identified which were recognized by the majority of MS patients. Estimated frequency analyses were then performed on the peripheral blood of HLA-DR15(2)-positive MS and control subjects using TCL initiated by the three immunodominant peptides, 40-60, 95-117, and 185-206. TCL from HLA-DR15 MS subjects recognized peptide 95-117 significantly more often than TCL from control subjects.

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.

HPRT mutant T-cell lines from multiple sclerosis patients recognize myelin proteolipid protein peptides.

Mutation of the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in a T-cell is believed to be an indication that the T-cell has been activated and has proliferated in vivo. HPRT mutant T-cell lines were generated from peripheral blood mononuclear cells from patients with MS and control subjects. More lines were isolated from the MS patients than from the control subjects. Using stringent criteria for recognition, none of the lines from MS-affected or control subjects recognized intact myelin basic protein (MBP) or myelin proteolipid protein (PLP) molecules. Using stringent criteria, two of the 10 MS patients harbored mutant lines each recognizing distinct PLP peptides (PLP peptide 40-60 recognized by 3 lines from one patient and PLP peptide 178-191 recognized by 2 lines from the other patient). A single line recognizing PLP peptide 89-106 was derived from 1 of 7 normal controls. HPRT mutant lines recognizing multiple epitopes of PLP which spanned much of the molecule could be isolated from MS patients, and to a lesser extent, normal subjects.

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.

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.

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.

Identification of a second T cell epitope of human proteolipid protein (residues 89-106) recognized by proliferative and cytolytic CD4+ T cells from multiple sclerosis patients.

Research into the pathogenesis of multiple sclerosis (MS) has focused on myelin antigens as potential targets of autoimmune attack. Proteolipid protein (PLP) is the most abundant myelin protein comprising more than 50% of central nervous system myelin. Although PLP is a hydrophobic membrane protein which has made it difficult to study, the use of synthetic peptides based on the PLP sequence provides an alternative method for studying the immunological properties of PLP. Using peripheral blood lymphocytes from MS patients, long-term TCL established in the presence of PLP reacted weakly to PLP in proliferation assays; however, these same lines were much more reactive to synthetic peptides of PLP. Thus, we established short-term T cell lines (TCL) from the peripheral blood lymphocytes (PBL) of MS patients in the presence of five separate synthetic PLP peptides. In 6/7 MS patients, proliferative responses were elicited most often to PLP 40-60 compared to four other PLP peptides (PLP 89-106, 103-120, 125-143, and 139-154) (Pelfrey et al., 1993). Interestingly, however, the magnitude of the proliferative response was greatest in response to PLP 89-106. Characterization of PLP 89-106-responsive TCL from several MS patients, indicated that TCL proliferating to the peptide also lysed PLP 89-106 pulsed autologous targets. The majority of cytolytic PLP 89-106 TCL were CD4+ and MHC class II restricted and the predominant restriction elements were those most commonly found in MS patients. These findings suggest that the use of synthetic peptides represents a viable alternative approach to the study of PLP reactivity in humans. We report here that MS PBL recognize several PLP peptides, with the predominant responses to PLP 40-60 and PLP 89-106. Since these cells have both helper (CD4+) and cytolytic capabilities, it is possible that they may play a role in the pathogenesis or progression of MS.

Identification of an N-terminally acetylated encephalitogenic epitope in myelin proteolipid apoprotein for the Lewis rat.

Proteolipid apoprotein (PLP) is a major component of the central nervous system myelin. As such, it is capable of inducing experimental allergic encephalomyelitis (EAE) in many subhuman species. On the basis of a putative MHC class II binding motif in Lewis rats (RT-1B1) recently identified in our laboratory, the present study identifies one pathogenic T cell epitope of PLP for the Lewis rat, located in the area between amino acid residues 217 and 240. Four overlapping synthetic peptides derived from this region were tested for their antigenicity and encephalitogenicity. Although the longer peptides could not induce EAE in the Lewis rats in their "theoretically" native form after immunization, they were endowed with encephalitogenic ability when modified by N-terminal acetylation. All animals immunized with N-acetylated peptides PLP 217-233 and PLP 224-240 developed inflammation in the lower spinal cord, but with very low incidence of clinical EAE (1 of 12). In contrast, none of the animals immunized with nonacetylated peptides developed either clinical or histologic EAE. Mild inflammation of the spinal cord was also found in two of four rats immunized with N-acetylated peptide PLP 220-234. The animals immunized with the decapeptide, N-acetylated PLP 224-233, did not develop inflammation of the spinal cord. Despite the low incidence of clinical disease, it was possible to generate vigorous T cell lines against all the peptides synthesized from this region of PLP.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Fine-specificity differences in the recognition of an encephalitogenic peptide by T helper 1 and 2 cells.

The lymphokine production of two T-cell clones, which both recognize epitopes within the encephalitogenic 139-151 sequence of myelin proteolipid protein, was examined after stimulation with immobilized antibodies to the CD3 moiety of the T-cell-receptor complex. Clone A1 produced interleukin (IL)-2 and interferon (IFN)-gamma, but no IL-4, while clone D5 produced IL-4, but no IL-2 or IFN-gamma. A1 therefore belongs to the T-helper type 1 (Th1) subset, while D5 is a Th2 clone. In addition, the Th1 clone induced severe experimental allergic encephalomyelitis (EAE), while the Th2 clone did not induce any signs of EAE. Synthetic peptides were used to demonstrate that these clones recognized slightly different epitopes within the 139-151 sequence. Histidine 139 was shown to be optimal for the stimulation of the Th2 clone, while the presence of this residue inhibited the stimulation of the Th1 clone. Th2 cells specific for an encephalitogenic peptide may be important in the regulation of encephalitogenic Th1 cells.

Identification of a novel T cell epitope of human proteolipid protein (residues 40-60) recognized by proliferative and cytolytic CD4+ T cells from multiple sclerosis patients.

Research into the pathogenesis of multiple sclerosis (MS) has focused on myelin antigens as potential targets of autoimmune attack. Proteolipid protein (PLP), which makes up more than 50% of central nervous system myelin, is a hydrophobic membrane protein with many properties that historically have made it difficult to study. The use of synthetic peptides based on the PLP sequence provides an alternative method for studying the immunological properties of PLP. Using peripheral blood lymphocytes from MS patients, long-term TCL established in the presence of PLP reacted weakly to PLP in proliferation assays; however, these same lines were much more reactive to synthetic peptides of PLP. Thus, we established short-term T cell lines (TCL) from the peripheral blood lymphocytes (PBL) of MS patients in the presence of five separate synthetic PLP peptides. In six out of seven MS patients, proliferative responses were elicited most often to PLP 40-60 compared to four other PLP peptides (PLP 89-106, 103-120, 125-143, and 139-154). Characterization of PLP 40-60-responsive TCL from a single MS patient, MS1, indicated that six out of seven TCL proliferating to the peptide also lysed PLP 40-60 pulsed autologous targets. All cytolytic PLP 40-60 TCL were CD4+ and MHC class II restricted and further analysis of MS1 TCL showed that the PLP 40-60 TCL were restricted by DR4 whereas the MBP TCL from MS1 were restricted by DR6. These findings suggest that difficulties in examining the immune response to PLP have been due to the poor response generated in vitro using the whole molecule and that the use of synthetic peptides may represent an alternative approach to the study of PLP. These results also suggest that MS PBL recognize several PLP peptides, with the predominant response to PLP 40-60. Since these cells phenotypically resemble T cells known to mediate experimental autoimmune encephalomyelitis, it is possible that they may play a role in the pathogenesis of 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.

Bacterial toxin superantigens activate human T lymphocytes reactive with myelin autoantigens.

Some bacteria that are common human pathogens produce protein toxins that are potent activators of human T lymphocytes expressing certain types of T-cell receptors. In this study we examined the ability of staphylococcal toxins to stimulate human T lymphocytes that also recognized the myelin autoantigens myelin basic protein and proteolipid protein. T-cell populations responding to myelin basic protein or proteolipid protein were isolated from 4 subjects including 1 individual with multiple sclerosis. All myelin antigen-specific T cells responded in proliferation studies to at least one of the nine superantigenic toxins used in this study. The superantigenic toxins were up to 7 x 10(5)-fold more potent in proliferation assays than the myelin antigens to which the T cells were initially sensitized. In addition, cytotoxic, myelin basic protein-reactive T lymphocytes lysed antigen-presenting cells incubated with superantigenic toxins. These findings demonstrate a mechanism by which some bacterial infections might produce activation of myelin basic protein- and proteolipid protein-reactive T lymphocytes and perhaps contribute to demyelinating disease in humans.