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Whether and when auto-reactivity after stroke occurs is still a matter of debate. By using overlapping 15mer peptide pools consisting of myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) we show increased frequencies of immunodominant MOG- and MBP T cell responses in acute ischemic stroke which were associated with reduced frequencies of naïve T cells as well as CD8+ TEMRA cells. Auto-reactive CNS antigen-specific T cells responses as well as alterations of T cell subpopulations normalized in long-term follow up after stroke. Our findings suggest that stroke-induced immunodepression might function as an adaptive mechanism in order to inhibit harmful and long-lasting CNS antigen-specific immune responses.

Current treatments to restore neurological deficits caused by axonal disconnection following central nervous system (CNS) injury are extremely limited. Protein phosphatase 2A (PP2A), one of the main serine-threonine phosphatases in mammalian cells, dephosphorylates collapsin response mediator protein-2 (CRMP2) in the developing CNS. In our study, we found that the major CNS inhibiting substrates, including chondroitin sulfate proteoglycans (CSPGs) and myelin associated glycoproteins (MAG), activated epidermal growth factor receptor (EGFR), but inactivated PP2A and downstream CRMP2. Both EGFR inactivation and PP2A activation promoted axon elongation in vitro in the presence of inhibitory substrates. EGFR blockage by AG1478 selectively attenuated the inactive form of PP2A in pY307 phosphorylation, thus increasing PP2A activity. EGFR activation by EGF attenuated PP2A activity, whereas mutation of Y307 to phenylalanine abolished the effect. Furthermore, PP2A activity was down-regulated immediately after spinal cord injury (SCI) in rats. Chronic application of d-erythro-sphingosine (DES), the PP2A agonist, to spinal cord-lesioned rats enhanced the activity of this phosphatase and dephosphorylated CRMP2 around the lesion. PP2A activation induced significant axon sprouting in the lesioned spinal cord and promoted function recovery after SCI. These findings suggest that PP2A works downstream of EGFR and dephosphorylates CRMP2 after CNS injury. Therefore, therapies targeting PP2A may be effective following SCI.

Thymosin β4 (Tβ4) is a 5K actin binding peptide. Tβ4 improves neurological outcome in a rat model of embolic stroke and research is now focused on optimizing its dose for clinical trials. The purpose of this study was to perform a dose-response study of Tβ4 to determine the optimal dose of neurological improvement in a rat model of embolic stroke.Male Wistar rats were subjected to embolic middle cerebral artery occlusion (MCAo). Rats were divided into 4 groups of 10 animals/group: control, 2, 12 and 18 mg/kg. Tβ4 was administered intraperitoneally 24h after MCAo and then every 3 days for 4 additional doses in a randomized controlled fashion. Neurological tests were performed after MCAo and before treatment and up to 8 weeks after treatment. The rats were sacrificed 56 days after MCAo and lesion volumes measured. Generalized estimating equation was used to compare the treatment effect on long term functional recovery at day 56. A quartic regression model was used for an optimal dose determination.Tβ4 significantly improved neurological outcome at dose of 2 and 12 mg/kg at day 14 and extended to day 56 (p-values0.05). The higher dose of 18 mg/kg did not show significant improvement. The estimated optimal dose of 3.75 mg/kg would provide optimal neurological improvement.This study shown that Tβ4 significantly improved the long term neurological functional recovery at day 56 after MCAo with an optimal dose of 3.75 mg/kg. These results provide preclinical data for human clinical trials.

Multiple sclerosis (MS) is a debilitating neurodegenerative disease characterized by axonal/neuronal damage that may be caused by defective remyelination. Current therapies aim to slow the rate of degeneration, however there are no treatment options that can stop or reverse the myelin sheath damage. Bone marrow mesenchymal stem cells (MSCs) are a potential candidate for the cell implantation-targeted therapeutic strategies, but the pro-remyelination effects of MSCs when directly injected into a demyelinated cord lesion have been questioned. Neurotrophin-3 (NT-3) has been shown to serve a crucial role in the proliferation, differentiation and maturation of oligodendrocyte lineages. Here, we showed that implantation of NT-3 gene-modified MSCs via a recombinant adenoviral vector (Adv) into a region of ethidium bromide (EB)-induced demyelination in the spinal cord resulted in significant improvement of locomotor function and restoration of electrophysiological properties in rats. The morphological basis of this recovery was evidenced by robust myelin basic protein (MBP) expression and the extensive remyelination. AdvNT-3-MSC implants promote the endogenous remyelinating cells to participate directly in myelination, which was confirmed under light and electron microscopy. Our study suggested that genetically modified MSCs could be a potential therapeutic avenue for improving the efficacy of stem cell treatment for neurodegenerative diseases such as MS.

Conflicting data exist on expression of gp145trkB, the high affinity receptor for brain-derived neurotrophic factor (BDNF), on peripheral blood immunocompetent cells in multiple sclerosis (MS). We analyzed expression of gp145trkB by western blotting and flow cytometry in myelin basic protein (MBP)- and ovalbumin (OVA)-T cell lines prepared from 12 patients with relapsing–remitting MS and 12 normal healthy subjects (NHS) and correlated it with activation-induced apoptosis. We found a higher percentage of gp145trkB-expressing MBP-T cells in MS patients than in NHS ( p = 0.011). gp145trkB was mainly expressed by CD8 + T cells to a higher extent in MS patients than in NHS ( p = 0.04). MBP-T cell lines from MS patients showed significantly lower apoptosis intensity than those from NHS ( p = 0.011). We found also a significant negative correlation between gp145trkB expression and apoptosis intensity in MS patients only ( p = 0.02). OVA-T cell lines showed a gp145trkB expression similar to that of MBP-T cell lines, with a higher expression in MS patients than NHS, and similar correlations with apoptosis intensity in MS. These findings suggest that gp145trkB is mainly expressed on T cell lines from MS patients and that the BDNF/gp145trkB axis is involved in the regulation of peripheral T cell apoptosis in MS.

Multiple Sclerosis (MS) is a heterogeneous disorder of the central nervous system (CNS) that begins as an inflammatory autoimmune disorder mediated by auto-reactive lymphocyte followed by microglial activation and chronic degeneration. The etiology of Multiple Sclerosis (MS) is unknown but several data support the hypothesis of possible infectious agents which may act as a trigger for the pathogenic cascade. Human endogenous retrovirus (HERV-W/MSRV), Epstein Barr Virus (EBV) and Mycobacterium avium ss. paratuberculosis (MAP) have been associated to Multiple Sclerosis. In this study, we evaluated the humoral response against different peptides: the human endogenous retrovirus HERV-Wenv 73–88 , MAP106c 121–132 from MAP, EBNA1 400–413 from EBV and the homologous human peptide MBP 85–98 in a cohort of MS patients treated with natalizumab. Results showed a statistically significant difference in the response against the HERV-W peptide in MS patients after two years of natalizumab treatment.

Multiple Sclerosis is an autoimmune disease directed against myelin proteins. The etiology of MS is poorly defined though, with no definitive causative agent yet identified. It has been hypothesized that MS may be a multifactorial disease resulting in the same end product: the destruction of myelin by the immune system. In this report we describe a potential role for heat shock proteins in the pathogenesis of MS. We isolated Hsp70 from the normal appearing white matter of both MS and normal human brain and found this was actively associated with, among other things, immunodominant MBP peptides. Hsp70-MBP peptide complexes prepared in vitro were shown to be highly immunogenic, with adjuvant-like effects stimulating MBP peptide-specific T cell lines to respond to normally sub-optimal concentrations of peptide. This demonstration of a specific interaction between Hsp70 and different MBP peptides, coupled with the adjuvanticity of this association is suggestive of a possible role for Hsp70 in the immunopathology associated with MS.

Biochemical and ultrastructural studies of ceramide galactosyltransferase (CGT) in a CGT-deficient mouse line (cgt-/-) were complemented by nerve conduction velocity (NCV) measurements in motor nerves (sciatic nerve in the hind limbs) of wild type (wt) and cgt-/- mice. Stimulation and recording electrodes were adapted to the small size of developing mice during their myelination period. Motor NCVs in wt mice ranged between 16 and 26 m/s but in cgt-/- mice between 6 and 13 m/s, which corresponds to the conductance of unmyelinated peripheral nerves. These electrophysiologic data provide additional functional parameters to the neuropathology of a new form of a dysmyelinosis.

Experimental autoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease characterised by inflammation and demyelination of the central nervous system and is the best available animal model of multiple sclerosis (MS). Since previous studies have shown that EAE is less severe or is delayed in onset during pregnancy and that administration of the pregnancy hormone early pregnancy factor (EPF) down-regulates EAE, experiments in the present study were designed to explore further the role of EPF in EAE. By using the rosette inhibition test, the standard bioassay for EPF and, by semi-quantitative RT-PCR techniques, we have now shown that inflammatory cells from the spinal cord of rats with EAE can produce and secrete EPF, with production being greatest during recovery from disease. Administration of EPF to rats with EAE resulted in a significant increase in the expression of IL-4 and IL-10 mRNA and a significant decrease in IFN-gamma mRNA expression in spinal cord inflammatory cells. Encephalitogenic MBP-specific T cell lines were prepared from popliteal lymph nodes of rats with EAE. Proliferation assays using these cells demonstrated the ability of exogenous EPF to down-regulate the responses of T lymphocytes to MBP.

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties that has been shown to suppress acute experimental autoimmune encephalomyelitis (EAE) induced with myelin basic protein (MBP) in Lewis rats. EAE is associated with infiltration of the central nervous system (CNS) with inflammatory cells. Spontaneous recovery involves the loss of T lymphocytes from the CNS and the selective apoptosis of Vbeta8.2(+) cells. In the present study, T cell, macrophage (CD11b/c(+)) and B cell (CD45RA(+)) populations in spinal cord and popliteal lymph nodes (LN) of Lewis rats with EAE were quantitated and apoptosis was studied. Rats were treated with EPF or vehicle. Following treatment on day 14 after inoculation with MBP, neither 1 x 100 mug nor 2 x 100 mug doses of EPF affected the total number of cells infiltrating the spinal cord on day 15, although the higher dose caused a decrease in the number of CD5(+) and CD11b/c(+) cells. Treatment with 2 x 100 mug/day from days 10 to 14 decreased the total number of infiltrating cells, and the numbers of CD5(+), CD11b/c(+) and CD45RA(+) cells. Apoptosis was unaffected. No alteration on the number or type of inflammatory cells in the popliteal LN was observed after treatment on days 10-14. However, treatment with EPF from days 0 to 11 increased the total number of T and B cells and CD5(+) T cells found on day 12 in the LN. Similarly, there was an increase in the frequency of MBP-reactive cells in the LN as determined by limiting dilution analysis. These results suggest that EPF treatment reduces the numbers of lymphocytes and macrophages in the CNS, possibly through an effect on cell trafficking. (C) 2003 Elsevier B.V. All rights reserved.

In Lewis rats, treatment with high doses of cyclosporin A (CsA) suppresses clinical signs of experimental autoimmune encephalomyelitis (EAE), although disease occurs when treatment is ceased. Treatment with low doses of CsA causes EAE to take a chronic relapsing course. We have previously shown that CsA treatment causes a decline in the number of T cells and increased inflammatory cell apoptosis in the spinal cord. The present study was undertaken to assess whether CsA therapy also modulates cytokine mRNA expression by inflammatory cells in the spinal cord of rats with EAE, looking for changes that might contribute to the observed effects of CsA on the course of EAE. EAE was induced in Lewis rats by inoculation with myelin basic protein and adjuvants. At the peak of neurological signs, on day 14 after inoculation, rats were given a single intraperitoneal injection of saline, or CsA at a dose of 8, 16, 32 or 64 mg/kg. The next day, rats were sacrificed, the spinal cords removed, inflammatory cells were extracted from the cords, and mRNA isolated from these cells. Expression of cytokine mRNA was assessed by semi-quantitative reverse transcription polymerase chain reaction (PCR) and by quantitative real-time PCR. With both techniques, we found that CsA suppressed the expression of interferon-gamma mRNA and interleukin-2 (IL-2) mRNA. With real-time PCR, we found that CsA caused significantly increased expression of transforming growth factor-beta mRNA. With the different techniques, we observed no consistent pattern of alteration of expression of interleukin-10 or interleukin-4 mRNA. It is possible that these changes in cytokine mRNA expression contribute to the modulation of the clinical course of EAE that is produced by CsA treatment.

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. During pregnancy, it appears in maternal serum within 6-24 h of fertilization, is present for at least the first two-thirds of pregnancy in all species studied and is essential for embryonic survival. It is a homologue of chaperonin 10, a heat shock protein, but, unlike other members of this family, EPF has an extracellular role. As it has the ability to modulate CD4+ T cell-dependent immune responses, its role in treatment of experimental autoimmune encephalomyelitis (EAE) was investigated. EAE is a CD4+ T cell-mediated disease, the best available animal model of multiple sclerosis (MS). Two models of EAE were investigated, acute EAE induced in Lewis rats by inoculation with myelin basic protein (MBP-EAE) and chronic relapsing EAE induced in SJL/J mice by inoculation with myelin proteolipid protein peptide (residues 139-151) (PLP-EAE). EPF, delivered intraperitoneally or orally to rats or intraperitoneally to mice, suppressed clinical signs of disease. Mice with PLP-EAE were also treated with interferon-beta, with and without EPF. Both EPF and IFN-beta suppressed clinical signs of EAE and, when administered together, gave greater suppression than when given separately. These findings suggest that EPF may be a potential candidate for use in treatment of MS and may be of use in combined therapy with IFN-beta. (C) 2000 Elsevier Science B.V. All rights reserved.

Type IV phosphodiesterase inhibitors are able to suppress EAE. To investigate the effects of this therapy in the central nervous system, we serially analyzed from days 7 to 17 postinoculation the gene expression pattern of tumor necrosis factor (TNF), lymphotoxin, interferon-γ, interleukin-1β, the inducible nitric oxide synthase (iNOs), interleukin-10, the vascular cell adhesion molecule-1 (VCAM-1) and the intercellular adhesion molecule-1 (ICAM-1) in the spinal cord of Lewis rats with actively induced EAE, treated with Rolipram. Treated rats had a delayed and milder disease, and reduced numbers of infiltrates in the nervous tissue. The gene expression profile was similar to that of untreated rats, although delayed, with no evidence of IL-10 upregulation during the observation period. The delayed inflammation was not associated with changes in the expression of VCAM-1 and ICAM-1. In peripheral blood mononuclear cells, TNF mRNA levels were decreased and interleukin-10 was unchanged. This therapy did not alter the proliferative ability of T lymphocytes against myelin basic protein. The encephalitogenic potential of splenocytes from treated animals was also unaffected. The high levels of both iNOs mRNA and nitric oxide (NO) found before the appearance of clinical signs, suggests that NO generation might be a contributing factor to the therapeutic benefit achieved by Rolipram in the rat.

Intoxication by Karwinskia humboldtiana presents a neurological picture similar to that for Guillain-Barré syndrome or other polyradiculoneuropathies. Clinical diagnosis in poisoned humans may be difficult if no evidence of previous fruit ingestion is available. We present our experience in the clinical diagnosis of Karwinskia humboldtiana polyneuropathy, as confirmed by toxin detection in blood. We designed an open trial at the Pediatric Neurology service and included all cases with acute ascending paralysis that were admitted to our hospital in the last two years. In all cases, we performed hematological, immunological and biochemical profiles, CSF analysis including immunological studies, oligoclonal bands and myelin basic protein determinations. Electrodiagnostic studies were performed, including motor conduction velocities, distal latencies, F-wave latency and compound muscle action potential (CAMP) amplitude. The presence of Karwinskia humboldtiana toxins in blood were determined by thin layer chromatography. In six cases, T-514 Karwinskia humboldtiana toxin was detected. These cases had a symmetric motor polyneuropathy with the absence of tendon reflexes and no sensory signs or cranial nerve involvement. Only one patient required assisted ventilation due to bulbar paralysis. In two of these cases, a sural nerve biopsy revealed a segmental demyelination with swelling and phagocytic chambers in Schwann cells and without lymphocytic infiltration. All six cases survived, with complete recovery in five. We conclude that this intoxication is common in Mexico. The availability of toxin detection in blood samples allows the clinician to establish an accurate diagnosis and should be included in the study of children with polyradiculoneuropathy, especially in countries where this poisonous plant grows.

We report on the case of a 70-year-old man with primary macroglobulinemia who showed cranial polyneuropathy and extensive radiculoneuropathy. His serum contained an IgM lambda monoclonal antibody which reacted with both a high molecular weight protein in grey matter and purified myelin basic protein (MBP) on immunoblotting. In an immunohistochemical study, strong immunoreactivity was detected only in the cytoplasm of neurons and weak immunoreactivity was detected in myelin. These findings suggest that this antibody may be related to the pathogenesis of neuronal damage in patients with macroglobulinemia.

The classic myelin basic proteins (MBPs) are a family of small membrane-associated proteins that are specifically expressed in myelin-forming cells. MBPs are extremely abundant in compact myelin, accounting for 30–40% of the total protein content in the CNS and 10–20% in the PNS. Initial molecular studies on the structure of the MBP gene identified the gene as ~32 kb in length and comprised of seven exons [1,2]. The MBP gene was found to map to chromosome 18 in the mouse and human and, by alternative RNA splicing, code for all of the known MBP isoforms [1,3–6]. Later, it was determined in both mice and humans that the MBP gene and its myelin-specific promoter were actually part of a much larger transcription unit [7,8]. This larger gene includes five additional exons upstream of the myelin-specific transcription unit that are alternatively spliced to form a separate family of MBP-related (golli) polypeptides. The MBP gene is now known to consist of 11 exons and three promoter regions that span over ~105 kb in mouse and ~180 kb in humans [9]. In the mouse gene, there are three transcription start sites that are active at various pre and postnatal developmental time points. The first transcription start site activates expression of the golli–MBP messenger ribonucleic acids (mRNAs) in neurons and oligodendrocytes (OLs) in the nervous system as well as T-cells and macrophages in the thymus. Unlike the classic MBPs, the golli–MBPs are not components of the myelin sheath but instead are localized primarily within the cell bodies and processes [10]. Transcription start sites 2 and 3 generate the bclassicQ MBP isoforms. There are six different classic MBP isoforms that have similar amino acid sequences but range in molecular mass from 14 to 21.5 kDa [1,2,11]. The MBP proteins are positively charged molecules that are believed to play a key role in the formation of compact myelin by bringing together the cytoplasmic surfaces of the myelin membrane, presumably

To study T cell apoptosis during spontaneous recovery from experimental autoimmune encephalomyelitis (EAE), we extracted lymphocytes from the spinal cords of Lewis rats with EAE induced by inoculation with myelin basic protein (MBP) and adjuvants. Using flow cytometry we assessed the numbers of CD5+ and TCR alpha beta + lymphocytes, as well as V beta 8.2+ lymphocytes, which constitute the predominant encephalitogenic MBP-reactive cells in Lewis rats. Rats developed neurological signs of disease 10-12 days after inoculation. The peak of disease was on day 14 after inoculation and was followed by clinical recovery. The numbers of CD5+, TCR alpha beta + and V beta 8.2+ cells obtained from the spinal cord were greatest on day 13. During spontaneous clinical recovery, there was a decline in the numbers of all the cells studied, with a selective loss of V beta 8.2+ cells from the CD5+ and TCR alpha beta + populations. To determine whether the decline in lymphocyte numbers was due to apoptosis, we used simultaneous surface labelling and propidium iodide staining of the DNA of the cells extracted from the spinal cord. From day 14 onwards, there was selective enrichment of V beta 8.2+ cells in the apoptotic population, and the percentage of V beta 8.2+ cells undergoing apoptosis was greater than the percentages of CD5+ and TCR alpha beta + cells undergoing apoptosis. These findings indicate that recovery from acute EAE is associated with the selective apoptosis, in the central nervous system, of these disease-relevant cells. The findings in this study of actively induced EAE are similar to those of our previous study of EAE induced by transfer of encephalitogenic MBP-specific T cells (Z. Tabi et al., Eur. J. Immunol. 24: 2609-2617, 1994) and further support the hypothesis that selective apoptosis of autoreactive T cells in the central nervous system is of primary importance in spontaneous recovery from EAE.

Oral administration of acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR and complete Freund's adjuvant (CFA) results in the prevention of experimental autoimmune myasthenia gravis (EAMG), and decreased serum levels of anti-AChR antibodies. Using an ELISPOT assay, we have now determined numbers of cells in the popliteal, inguinal and mesenteric lymph nodes, spleen and thymus secreting anti-AChR IgG antibodies. Except for mesenteric lymph nodes, a marked diminution of such cells was detected in these lymphoid organs in rats orally tolerized with AChR compared to buffer-fed or vehicle-fed control rats with EAMG. Of note is that, after AChR feeding, the B cell response to AChR in thymus was diminished to the same low level as in CFA-injected, buffer-fed control rats. The relative affinity of serum anti-AChR IgG antibodies measured by KSCN-ELISA was lower in the orally tolerized rats compared to buffer-fed or vehicle-fed rats. The observations showed that oral administration of AChR, besides preventing clinical EAMG, also counteracts the development of AChR-specific B cells, especially those with high affinity antibody production, in most lymphoid organs.

Encephalitis/encephalopathy is a rare, but severe, complication of pertussis. Here, we report a case of an unimmunized 7-year-old boy with confirmed pertussis complicated by acute encephalitis/encephalopathy. Eighteen days after the onset of pertussis, generalized seizures began. Magnetic resonance imaging (MRI) indicated that marked demyelination without cytotoxic edema may have occurred to the patient. Notably, this is the first report to show precise MRI findings of pertussis-associated encephalitis/encephalopathy. Markedly increased myelin basic protein levels in the cerebrospinal fluid were consistent with the MRI findings. There was no evidence of direct invasion of the causative bacterium or its products into the central nervous system. The levels of interleukin-6 and -10 in the cerebrospinal fluid were higher than those in serum. Taken together, we conclude that indirect immune-mediated mechanisms may have contributed to the pathogenesis of the encephalitis/encephalopathy.

Mast cells (MC) have been implicated in the pathogenesis of experimental allergic encephalomyelitis (EAE). In order to further evaluate their role, several morphological and functional studies were performed. Semiquantitative counts of histological sections showed a significant reduction in MC numbers in EAE brains. In addition, a higher proportion of EAE MC (about 50–70%) appeared degranulated compared with about 20% degranulation in controls. Central nervous system (CNS) MC exhibited staining properties of connective tissue MC and about 98% of them, both in diseased and control rats, were located in the thalamus. They were not present in the spinal cord and did not relate to EAE lesions. In vitro incubation of peritoneal MC (of connective tissue phenotype) with either MBP, or with neuropeptides such as substance P or bradykinin resulted in release of β-hexosaminidase and histamine. The latter responses were similar in both EAE and control rats. It is suggested that the decrease in number and in granular content of CNS MC in EAE may reflect prior in vivo activation. The fact that MC were activated by MBP and by neuropeptides in vitro suggests a possible mechanism of MC activation in EAE.

To evaluate systemic T cell activation and reactivity against putative autoantigens in multiple sclerosis (MS), numbers of interleukin-2 (IL-2) secreting cells were determined in peripheral blood of 32 patients with MS, 7 patients with acute aseptic meningitis (AM) and 12 patients with tension headache (TH). Numbers of IL-2 secreting cells were higher in MS patients compared to patients with AM + TH after stimulation with myelin proteolipid protein (PLP), myelin associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG), but not after stimulation with myelin basic protein (MBP). In response to MAG, MOG and acetylcholine receptor (AChR) the frequencies of IL-2 secreting cells were higher in patients with MS than TH, while there were no differences between AM and TH to any of the tested antigens. Between patients with MS and AM there was no difference regarding frequency of IL-2 secreting cells in response to any of the tested antigens except MAG to which the response was higher in MS patients. Six of 10 MS patients had IL-2 secreting cells in response to all four myelin antigens (MBP + PLP + MAG + MOG) or to three antigens, while this broad reactivity was not found in any control patient. There was no correlation between numbers of IL-2 secreting cells in MS patients and clinical variables, including exacerbation versus remission, disability and duration of disease. The results suggest that the systemic T cell response in patients with MS is directed to several antigens.

To study whether nervous tissue trauma provokes myelin antigen autoreactive T and B cell responses in humans we examined consecutive blood samples from 7 patients with polyneuropathy undergoing diagnostic sural nerve biopsy and 8 control patients undergoing other types of minor surgery. The antigen-specific T cells were assessed by enumerating cells secreting interferongamma (IFN-γ) in response to the myelin components P 0 , P 2 , myelin basic protein (MBP) and myelin associated glycoprotein (MAG), and to 4 selected MBP peptides. B cell mediated immunity was assessed by counting numbers of cells secreting antibodies directed against the myelin proteins. On day 7 after biopsy, there were 3-10-fold increased numbers of T and B cells reactive with P 0 , P 2 , MBP and MAG in blood of polyneuropathy patients compared to controls, while levels of cells recognizing purified protein derivate or responding to phytohemagglutinin (PHA) did not differ significantly. Comparison of prebiopsy levels on day 0 with post-biopsy levels on day 7 in the polyneuropathy patients revealed a significant increase in T cells recognizing P 0 , P 2 and MAG, and in B cells secreting IgG antibodies against P 0 and P 2 . On day 14 after nerve biopsy these differences were no longer seen. We suggest that in patients with polyneuropathy, sural nerve biopsy with the ensuing wallerian degeneration and myelin breakdown causes transiently increased levels of circulating myelin autoreactive T and B cells. It remains to be determined if this has a physiological role in nerve trauma responses and/or affects the clinicopathological course of the peripheral neuropathy.

Quantification of the peripheral nerve myelin glycoprotein P0 and antibodies to P0 is difficult due to insolubility of P0 in physiological solutions. We have overcome this problem by using the water-soluble recombinant form of the extracellular domain of P0 (P0-ED) and describe newly developed assays which allow detection and quantitation of P0 and antibodies to P0, in serum and cerebrospinal fluid (CSF). These sensitive and specific assays based on the ELISA technique were used to study humoral immune responses to P0 during experimental autoimmune (“allergic”) neuritis (EAN). In order to establish these tests, monoclonal antibodies to different epitopes of rodent and human P0-ED were produced. A two-antibody sandwich-ELISA allowing quantitation of P0 (lower detection limit of 0.5 ng/ml or 30 fmol/ml) and an antibody-capture ELISA (lower detection limit 1 ng specific antibody/ml) to detect antibodies to P0 in serum and CSF were developed. EAN was induced in rats by active immunization with bovine myelin or the neuritogenic protein P2 or by adoptive transfer using P2 specific CD4 positive T cells. Serum and CSF were assayed for the presence of P0-ED and antibodies to P0-ED or P2. Antibodies to P0-ED were detected during active myelin-induced EAN, but not during P2-induced or adoptive transfer EAN. The anti-P0-ED antibodies in the CSF showed a correlation with disease activity. In contrast, in the same model antibodies to P2 persisted long after the disease ceased. No soluble P0-like fragments could be found in serum or CSF during any of the three types of EAN. We conclude that P0 may be a B-cell epitope in EAN. These findings warrant a screen for antibodies to P0-ED in human immune neuropathies.

Previous research has demonstrated that free (F) and bound (B) anti-myelin basic protein (anti-MBP) can be detected in the cerebrospinal fluid (CSF) of patients with active multiple sclerosis (MS). The purpose of this report was to determine whether the immunoglobulin G (IgG) isolated from central nervous system (CNS) tissue of MS patients contains anti-MBP. IgG was detected in free and bound hydrosoluble protein extracts obtained from the brain, spinal cord and optic nerves of a patient with clinically definite and neuropathologically confirmed MS. IgG was purified from free protein extracts from brain and spinal cord by Protein G-Sepharose affinity chromatography. Anti-MBP was detected by a solid phase radioimmunoassay (RIA) in all free and bound protein extracts. Anti-MBP was isolated from purified IgG from brain and spinal cord by MBP-Sepharose affinity chromatography. Free anti-MBP in the context of whole protein extracts, within purified IgG or as purified antibody as well as tissue-bound anti-MBP in the context of whole protein extracts was completely neutralized by human MBP (h-MBP) and synthetic peptide No. 56 (residues 75-95 of h-MBP) and did not react with synthetic peptide No. 41 (residues 35-58 of h-MBP). Anti-MBP which has previously been detected in the CSF of MS patients with active disease is also present as free antibody in the extracellular space of MS-central nervous system tissue and in a smaller proportion as tissue-bound antibody.

Neuromyelitis optica (NMO) is characterized by severe optic neuritis and longitudinally extended, transverse myelitis. There have been long controversial whether NMO is a variant of multiple sclerosis (MS) or a different disease. However, since the discovery of an NMO-specific autoantibody to aquaporin 4 (AQP4), a dominant water channel in the central nervous system densely expressed on foot processes of astrocytes, the clinical distinction between NMO and MS has become clear, and now AQP4 antibody status is critically important for neurologists in deciding on treatment strategy. Moreover, pathological studies demonstrated an extensive loss of immunoreactivities to AQP4 and glial fibrillary acidic protein (GFAP) with relative preservation of the staining of myelin basic protein in acute NMO lesions, which is not seen in MS. In fact, the GFAP levels in the cerebrospinal fluid during acute exacerbation of NMO are remarkably elevated, while the values in MS are not different from those in controls. In addition, recent experimental studies conducted in vitro and in vivo have shown that AQP4 antibody is pathogenic. These findings strongly suggest that AQP4 antibody has diagnostic, therapeutic and pathogenetic implications, and that severe astrocytic damage mediated by AQP4 antibody distinguishes NMO from MS.

Elevated titers of anti-myelin basic protein (anti-MBP) are highly associated with acute idiopathic unilateral optic neuritis as well as acute relapses of multiple sclerosis (MS). During acute phases of optic neuritis, free/bound antibody ratios are generally above unity, with high titers of free anti-MBP and relatively low or undetectable values of bound antibody. Three to 5 months after the acute phase when the majority of patients have recovered, free/bound anti-MBP ratios are below unity with low titers of free antibody and relatively higher levels of bound anti-MBP. Anti-MBP purified from cerebrospinal fluid of patients with optic neuritis are neutralized by synthetic peptides of human MBP containing overall amino acid residues 61-106 and do not react with synthetic peptides corresponding to residues 1-60 and 107-170. Anti-MBP may either have multiple epitopes in the region corresponding to residues 61-106 or it may react with a discontinuous epitope in this range. The mechanism of the optic nerve demyelination may be associated with anti-MBP binding in situ to MBP in the 61-106 amino acid region.

Two anti-galactosyl ceramide antibodies (polyclonal Ab142 and a monoclonal antibody) were characterized in terms of their reactivity towards purified lipids and myelin basic protein. Polyclonal Ab142 is a rabbit anti-mouse galactosyl ceramide (Gal C) IgG. Antigenic recognition is dependent on both galactose and ceramide since neither could inhibit galactosyl ceramide binding by more than 10%. MAb-Gal C is a monoclonal antibody raised in mice against galactosyl ceramide. Binding of MAb-Gal C to Gal-C was equally inhibited by ceramide and galactose to approximately 50%, indicating that both groups are important for antibody recognition. MAb-Gal C was also shown to be reactive with the structurally related lipids, sphingomyelin and sulfatide. Polyclonal Ab142, although raised against Gal C, was shown to be 3-fold more reactive with component 8 (C-8) of myelin basic protein than Gal C. On the other hand, the MAb-Gal C which also reactive with C-8 than with Gal C. Neither of these antibodies were reactive with component 1 (C-1) of myelin basic protein. An anti-MBP IgG was shown to be reactive with C-1 and C-8 but unreactive with Gal C. In competitive inhibition ELISA, C-8 was able to compete out 44% and 41% of Gal C binding to polyclonal Ab142 and MAb, respectively. The reverse competition demonstrated that Gal C could inhibit 75% of C-8 binding to both antibodies. D-galactose was unable to inhibit C-8 binding to either antibody, whereas ceramide was as efficient as Gal C. When the anti-citrulline antibody was used, it reacted well with C-8 both in solution and in lipid vesicles. Some reaction was obtained against Gal C in solution but not when Gal C was present in lipid vesicles. We propose that the reactivity of anti-Gal C antibodies towards C-8 of myelin basic protein is related to similarities between the ceramide backbone of Gal C and the ureido group of the citrullinyl residue in C-8.

The Guillain-Barre syndrome (GBS) is the most comassociation with lipids. Circulating antibodies to P0 and P2 mon cause of acute neuromuscular paralysis. It constitutes have been detected in a minority of GBS patients and also a neurologic emergency and is associated with significant in other disease and healthy controls [3]. Compact myelin morbidity and mortality which, even with modern intenalso contains the 22-kDa glycoprotein PMP-22 which sive care approximates 10% [1]. The same clinical phenoaccounts for 5% of total protein. Its neuritogenicity is not type can apparently be produced by different variants and a yet established. classification nowadays commonly used differentiates clasLipids make up about 76% of the total nerve dry weight. sical acute inflammatory demyelinating polyneuropathy The major myelin lipids are cholesterol, galac(AIDP), acute motor and sensory axonal neuropathy tosylceramide (galactocerebroside), galactosylceramide-3(AMSAN), acute motor axonal neuropathy (AMAN) that O-sulfate (sulfatide) and, in smaller amounts, gangliosides occurs in epidemics in China, on the Indian subcontinent and complex neutral glycolipids. Glyco(sphingo)lipids are and in South America. and the Miller–Fisher syndrome. composed of the long-chain aliphatic amine ceramide, GBS is generally thought to result from aberrant immune attached to one or more hexose sugars. The ceramide is responses to peripheral nerve antigens [2]. immersed in the membrane lipid bilayer with the carbohyThe myelin sheath is composed of three major proteins drate structure exposed extracellularly. Glycolipids are (P0, P1 and P2) with molecular weights of 14–30 kDa, highly enriched in neuronal and glial membranes where which make up more than 70% of the total protein content they modulate a diverse array of cellular functions. A [3]. In human PNS myelin, P0 accounts for more than half single hexose linked to ceramide (i.e. monohexosyl certhe total membrane protein: it does not occur in central amide) is termed cerebroside, the most abundant of which nervous system (CNS) myelin. P0 is a 29-kDa, 219 amino in man is galactocerebroside (GalC). Sulfatide (sulfated acid transmembrane glycoprotein with a large extracellular galactocerebroside) is the major component of peripheral domain, a highly hydrophobic membrane-spanning portion nerve myelin. Paragloboside, a neutral glycolipid, which and a basic cytoplasmic domain. It belongs to the immunowhen sialylated on the terminal galactose (i.e. globulin gene superfamily and acts as a homophilic cell sialosylparagloboside. SGP) is the major peripheral nerve adhesion molecule. P0 is thought to be involved in starting glycolipid, also known as LM1. Substitution of the termiand stabilising the compaction of the extracellular opposinal sialic acid on SGP for sulfated glucuronic acid forms tion of the myelin membrane in the PNS. P1 is identical to sulfated glucuronyl paragloboside (SGPG), which has a myelin basic protein (MBP) in the CNS, and the 14-kDa closely related homologue termed SGPLG. GalC, sulfatide, P2 protein containing 131 amino acids is common to both LM1 and SGPG have all been identified as antigens for CNS and PNS but predominates in the latter. It is sulphated glucuronyl lactosaminyl paragloboside autoanconcentrated in the paranodal loops and in Schmidt–Lantibodies in subtypes of autoimmune neuropathy including terman incisures rather than in compact internodal myelin. GBS. The conformation of P0 is highly dependent on its Galactosides are complex glycosphingolipids which contain at least one sialic acid residue (N-acetylneuraminic acid, abbreviated to NeuNAc) attached to the internal *Corresponding author. Tel.: 143-316-385-2981; fax: 143-316-325and/or the terminal galactose of an oligosaccharide core 520. E-mail address: hans.hartung@kfunigraz.ac.at (H.-P. Hartung) composed of up to four sugars. There are four major

Transplantation of neural precursor cells has been proposed as a possible approach for replacing missing or damaged central nervous system myelin. Neonatal and adult myelin-deficient shiverer (shi) mice, bearing a mutation of the myelin basic protein (MBP) gene, have been used extensively as hosts for testing cell engraftment, migration, and myelination, but relatively little progress has been made in reversing shi motor deficits. Here we describe a prenatal cell replacement strategy, showing that embryonic stem cells injected into shi blastocyst embryos can generate chimeric mice with strong and widespread immunoreactive MBP expression throughout the brain and a behavioral (motor) phenotype that appears essentially rescued.

Immunoglobulin G (IgG) was purified by affinity chromatography from the CSF of multiple sclerosis (MS) patients and controls. In MS patients, the IgG fraction contains anti-myelin basic protein (anti-MBP), anti-MBP neutralizing antibody and an antibody which inhibits neutralization of anti-MBP. Anti-MBP was detected in patients with acute relapses, anti-MBP neutralizing antibody was present in patients in clinical remission and the inhibiting antibody was detected in patients with chronically progressing MS. A myelin basic protein antibody cascade could be involved in the mechanism of MS.

Although the treatment of multiple sclerosis has made significant strides in the last decade, the therapeutic enhancement of repair has yet to make the successful translation from laboratory to clinic. Nevertheless, advances in the biology of stem and precursor cells, particularly in relation to myelin damage, make this a realistic proposition during the next decade. Replacing lost myelin (remyelination) is currently thought to be an important clinical objective because of the role it might play in slowing or preventing axonal degeneration. Stem/precursor cell-based strategies for enhancing remyelination can be divided into those in which cells are transplanted into a patient (exogenous or cell therapies) and those in which the patient's own stem/precursor cells are mobilised to more efficiently engage in healing areas of demyelination (endogenous or pharmacological therapies). While the two approaches tend to be regarded separately they are not mutually exclusive. This article focuses on the endogenous approach and reviews the nature and nomenclature of the stem and precursor cells present within the adult CNS that engage in remyelination and that are therefore potential targets for pharmacological manipulation.

Multiple sclerosis (MS) is the prototypic inflammatory demyelinating disorder of the central nervous system (CNS). MS is a clinically heterogeneous entity; symptoms may manifest in a relapsing–remitting or progressive fashion, the course may be benign or rapidly incapacitating, and the neuraxis may be involved in a widespread fashion; or the disease may predominantly affect spinal cord and optic nerves [1]. Heterogeneity is also evident in structural studies of MS lesions, revealing that diverse mechanisms— including antibody mediation, cellular mechanisms, and perhaps a primary oligodendroglial apoptosis—may underlie demyelination in different individuals (Fig. 1). Autoantibodies against myelin constituents, including myelin oligodendrocyte glycoprotein (MOG) [2] and myelin basic protein (MBP), have been implicated in tissue damage in some multiple sclerosis (MS) patients. Such antibodies have been identified in situ in actively demyelinating lesions and have also been identified against surface structures of oligodendrocytes. Berger et al. [3] reported that IgM antibodies specific to MOG and MBP can be detected by Western blot in the serum of patients with initial attacks of demyelination (e.g. clinically isolated syndromes [CIS]); furthermore, the presence of these antibodies, and especially those against MOG, predict evolution of CIS to chronic MS. These data, which still have not been fully confirmed, suggest that an easily measured antibody test might represent a biomarker for early MS. They also add support to the concept that therapies, which target humoral pathways, might prove useful in MS. Compelling data indicate that susceptibility to MS is inherited. The MS-prone genotype results from multiple independent or interacting polymorphic genes, each exerting a small or at most moderate effect to the overall risk. It is also likely that genetic heterogeneity exists, meaning that specific genes influence susceptibility and pathogenesis in some individuals but not in others. Concordance in families for early and late clinical features has been observed as well, indicating that in addition to susceptibility, genes influence disease severity or other aspects of the clinical phenotype. Hence, some genes may be involved in the initial pathogenic events, while others could influence the development and progression of the disease. The strongest and most consistently replicated evidence for an MS susceptibility gene has been localized to the major histocompatibility complex (MHC) superlocus on chromosome 6p21.3. Studies in multi-case families confirm the known association with the HLA class II DR2 haplotype (HLA-DRB1*1501

This brief overview summarizes some of the ways that the structure, location and function of proteins in myelin sheaths affect the pathological abnormalities brought on by immunological, viral, genetic or other insults occurring in myelin disorders. The composition of compact myelin membranes is novel in comparison to other biological membranes, because of its high lipid content and the presence of several major proteins that are relatively specific for myelin [1,2]. The overall morphological structures of compact central nervous system (CNS) and peripheral nervous system (PNS) myelin are similar, even though they are formed by different cell types, that is, oligodendrocytes and Schwann cells, respectively. On the other hand, there are morphological differences in their periodicity and relationship to the myelin-forming glial cells, quantitative differences in lipid composition, and important qualitative differences in protein composition. These differences in protein composition are likely to be especially important for determining the pathologies that characterize disorders of myelin formation and degeneration in the CNS and PNS. Proteolipid protein (PLP) is a very hydrophobic tetraspan protein that accounts for over half the total protein in CNS myelin [1,3], whereas the amount of PLP in the PNS is minimal. By contrast, the type 1 transmembrane P0 glycoprotein accounts for over half the protein in PNS myelin [1,4,5]. Compact PNS myelin also contains a 22kDa tetraspan glycoprotein, peripheral myelin protein-22 (PMP-22), which accounts for less than 5% of the total and is absent in CNS myelin [1,4,6]. Myelin basic protein (MBP) is a prominent, positively charged, extrinsic membrane protein of both CNS and PNS myelin, which accounts for about 30% of the total in CNS myelin, but only 5–15%

ABCA2, which belongs to the A subclass of the ATP-binding cassette (ABC) transporter superfamily, is predominantly expressed in the cytoplasm of oligodendrocytes and Schwann cells, the myelin-forming cells in brain and peripheral nerve, respectively. Here, we demonstrate by immunoblot and immunohistochemistry that ABCA2 is expressed in benign vestibular schwannomas, which contain neither axons nor compact myelin. The expression patterns of ABCA2 in combination with other markers showed phenotypic heterogeneity in schwannomas. The majority of cells in fascicular Antoni type A areas coexpressed ABCA2, Ca 2+ -binding protein S100β, and p75 nerve growth factor receptor. In contrast, considerably varied expression levels of ABCA2 and p75 were more prominent in hypocellular type B areas than in type A areas. These data suggest that ABCA2 may be useful as a marker for cellular characterization of schwannomas.

The present paper compares the genetic polymorphism of a part of the myelin basic protein (MBP) gene in 64 Danish MS patients with that of 57 normal controls. PCR analysis, using primers flanking the 5' area from 479 to 1812 bp upstream the initiator methionine in the MBP gene, revealed that genetic susceptibility to MS is linked to polymorphism in the part of the MBP gene studied. Thus we found three different band patterns i.e. a homozygote with a 1445 bp long fragment, a homozygote with a fragment 1318 bp long and a heterozygote with both bands present. 59% of 64 patients with MS were homozygous for 1.445 kb allele, versus 40% of 57 control subjects, 18% of the control subjects were homozygous for the 1.318 kb, while only 0.7% of the MS patients possessed this allele. The differences between incidence of the three band pattern in the MS and the control group were significant at 1% level. Validation analysis furthermore support, the view that the 1445 bp PCR fragment is associated with MS.

An autoimmune response to one or more myelin-protein components is thought to be part of the pathogenesis of multiple sclerosis (MS). The immunodominant-autoantibody epitope may be localized on a linear peptide segment, on a conformation-sensitive epitope, or on an epitope resulting from post-translational modifications. Primary, secondary, and tertiary structures of myelin proteins may determine the specific site for binding of autoantibodies. A myelin protein-specific autoantibody can bind to either a linear or conformational epitope, whereas all of the T cell epitopes are linear. At present, the conformational epitopes of myelin proteins have not been identified; most of the methods used to identify the myelin-protein epitopes corresponding to the pathogenesis of multiple sclerosis are involved in the linear epitope mapping. Polymorphism or mutations may cause inappropriate expression of the myelin proteins with alterations to their linear and/or conformational epitopes, and make them susceptible to autoantibody binding, especially if these changes occur at the surface of the protein. This review focuses on the specificity of autoantibodies to the epitopes of myelin proteins and correlates this to the structures of proteins. Factors that influence the expression of myelin-protein epitopes such as the alpha-helical or beta-sheet structure of the protein, the tri-proline site, and the post-translational modifications as well as physicochemical properties of amino acid changed are included.

The availability of a group of multiple sclerosis (MS) patients at the University of Maryland, who had participated in the pivotal Copaxone trial in the early 1990s, provided an opportunity to examine the long-term immunologic effects of Glatiramer acetate (GA) treatment in MS. Forty-eight GA-reactive T-cell lines (TCL) were generated from 10 MS patients who have been receiving GA treatment for 6-9 years. Proliferative responses, cytokine production, and cross-reactivity with myelin basic protein (MBP) and the MBP immunodominant peptide 83-99 were compared to responses obtained from 10 MS patients who were tested pretreatment and after a shorter period of treatment ranging from 1 to 10 months. The results indicate that while long-term treatment with GA results in a 2.9-fold decrease in the estimated precursor frequency of GA-reactive T-cells, the sustained response to GA remains Th2-biased and in part cross-reactive with MBP and MBP (83-99) as measured by proliferation and cytokine release assays. The results indicate that despite a drop in the precursor frequency of GA-reactive T-cells with long-term treatment, the sustained response remains predominantly Th2-biased and cross-reactive with MBP, which is consistent with the anti-inflammatory effects of the drug and bystander suppression.

The effects of oxalyldihydrazone (cuprizone) on weanling rodents provided an early protocol for toxic demyelination in vivo, in which degeneration of oligodendrocytes preceded disruption of the myelin sheath, and in which remyelination could take place. We administered cuprizone to oligodendrocyte-enriched glial-cell cultures and to mixed glial-cell cultures from neonatal rat brains. The cultures were treated with cuprizone for 1 h and allowed to continue differentiating on subsequent days. Treated cultures and respective control cultures were fixed with 4% paraformaldehyde (w/v) and immunostained with double immunofluorescence. MAbO4 was used to mark precursors and mature oligodendrocytes, and anti-myelin basic protein (MBP) to mark mature oligodendrocytes (O4+/MBP+), as distinguished from precursors, which were O4+/MBP-. Cell counts suggested that cuprizone inhibited the maturation of oligodendrocytes without diminishing the numbers of precursors, and appeared to affect the mitochondria in those cells.

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.

Background: The pathogenesis of multiple sclerosis (MS) appears to involve autoimmune phenomena in the central nervous system. Activation of the complement system is suggested to be involved in the pathogenesis. Patients and methods: Cerebrospinal fluid (CSF) and plasma samples from 65 patients with acute optic neuritis (ON) as a possible first symptom of MS (n=18), ON (n=16) or other attacks of clinically definite MS (n=15), and neurological control subjects (n=16) were studied. Activation of the initial part of the complement activation cascade was assessed by measuring activation of the C3 molecule; terminal activation of the complement cascade was assessed by measuring the terminal complement complex (TCC). Demyelination was estimated by the CSF concentration of myelin basic protein and neurological disability was assessed with the Kurtzke expanded disability status scale (EDSS) score. Results: Activation of the initial part of the complement activation cascade occurred in each of the three groups of patients with demyelinating disease, but was not correlated to demyelination or disability. Increased concentrations of TCC were detected in patients with attacks of MS other than ON. The CSF concentrations of TCC, myelin basic protein (MBP) and neurological disability correlated significantly. The strongest correlation was between neurological disability and the CSF concentration of TCC (r=0.55, P=0.003). Interpretation: Full activation of the complement cascade during attacks of MS may be restricted to patients with more advanced disease and is significantly correlated to the degree of neurological disability. This suggests that specific treatment with agents that inhibit complement activation may interfere with mechanisms involved in the pathogenesis of neurological disability in patients with MS.

Oral tolerization with acetylcholine receptor (AChR) and myelin basic protein (MBP) prior to immunization with AChR+MBP+ complete Freund's adjuvant (CFA) alleviated clinical signs of experimental autoimmune myasthenia gravis (EAMG)+experimental allergic encephalomyelitis (EAE) and AChR- or MBP-specific T and B cell responses. Tolerance induced via the nasal route needs much less tolerogen and may still be as effective as oral tolerance induction. We now immunized Lewis rats with AChR+MBP+bovine peripheral nerve myelin (BPM)+CFA, which resulted in a multiphasic clinical picture with a combination of clinical signs of the EAMG+EAE+experimental allergic neuritis (EAN), accompanied by massive macrophage infiltrations in sections of muscle, spinal cord and sciatic nerve, and strong T and B cell responses to AChR, MBP and BPM in lymphoid organs. Nasal administration of microg doses of AChR+MBP+BPM prior to immunization with a mixture of these antigens+CFA effectively suppressed the incidence and severity of clinical disease, reduced macrophage infiltrations in sections of muscle, spinal cord and sciatic nerve, and down-regulated autoreactive T cell responses to the three antigens in lymphoid organs. Numbers of AChR-, MBP-, BPM-reactive Th1 type of cytokine interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha mRNA expression in lymph node cells were markedly suppressed, while transforming growth factor-beta (TGF-beta) mRNA expression was upregulated from nasally tolerized rats, suggesting an active suppression mechanism may act partly in the induction of tolerance. The results implicate the possibility to establish multiple autoantigen-based vaccination for the prevention of autoimmune diseases in humans.

Peptide-based tolerance induction may be useful for antigen-specific immunotherapy of human autoimmune diseases. Induction of tolerance to myelin basic protein (MBP) was examined in a Phase I clinical trial in multiple sclerosis (MS) patients with chronic progressive disease using a peptide that is immunodominant for MBP specific T cells and B cells. Tolerance induction was monitored by quantification of MBP specific autoantibodies in cerebrospinal fluid (CSF). The route of peptide administration was important since only intravenous but not intrathecal or subcutaneous injection induced tolerance to MBP. Following a single intravenous injection of a peptide containing epitope P85VVHFFKNIVTP96, MBP autoantibodies were undetectable for three to four months. Tolerance was more prolonged following a second injection since autoantibodies were low or undetectable after one year in the majority of patients. Duration of tolerance to MBP depended on MHC class II haplotypes of patients; tolerance was long-lived in all patients with disease associated HLA-DR2. No neurological or systemic side effects were observed, regardless of the route of peptide administration. These data demonstrate that intravenous administration of a soluble peptide can result in long-lasting tolerance to an autoantigen in humans.

Following acute central nervous system myelin injury, immunoreactive myelin basic protein (MBP) has been detected in the cerebrospinal fluid, blood and urine. In order to clarify the fate of MBP in the circulation, distribution and degradation of intravenously injected bovine MBP was followed in anaestethized rats for 5 to 240 min by using 125I-labelled MBP. Five minutes after injection of a dose of 60–400 ng of MBP, 44% of the label was recovered in the liver, 6.3% in the kidneys, 4.7% in the lungs and 15% in the blood circulation, the corresponding figures at a dose of 0.8 mg being 51, 7.4, 0.8 and 22%. The liver discarded the label fastest, 3% of the dose remaining 4 h after injection. The amount of label in urine increased simultaneously, the recovery at 4 h being 5.5% of the lower and 4.2% of the higher MBP dose. The percentage of total dose of the label per gram of tissue at 5 min (= distribution percentage, DP-5) was 3–4% in the liver and kidney and 1.6% in the spleen. The label content in the pancreas was increased at 15–60 min, compared to the DP-5 of 0.3% with a two-fold maximum at 30 min. The duodenum concentrated MBP in a similar manner as the pancreas but not as extensively. The DP-5 of 0.1% in the thymus was concentrated two-fold with a maximum at 60 min. A slight concentration occured in the heart. The DP-5 of 0.03% in muscle, testis and brain was concentrated 3-fold at 60 min, 3.6-fold at 60–240 min and 2-fold at 30–60 min in the aforementioned tissues, respectively. In spite of degradation of the label in tissues, the distribution of high molecular weight (HMW = TCA-precipitable) MBP was similar. Other experiments showed that the kidney, lung and duodenum contained most of the HMW MBP at 20 h. Upon continuous release of MBP, the pancrease, thymus, duodenum, muscle and testis would thus cumulatively concentrate MBP, and the kidney, lung and duodenum would be quantitatively most affected. MBP was previously shown to enter into cells of pancreative islets and to stimulate insulin and glucagon release. It could have biological effects in other tissues as well. These effects could explain some peripheral symptoms present in neurological disorders.

Memantine, a clinically employed drug with N-methyl-D-aspartate (NMDA) receptor antagonistic effects, dose-dependently ameliorates neurological deficits in Lewis rat experimental autoimmune encephalomyelitis (EAE). Interestingly, this therapeutic effect was not due to dampened CNS inflammation, as assessed by immunohistochemical evaluation of spinal cord tissue. Furthermore, numbers of interferon gamma (IFN gamma) mRNA expressing cells were not decreased, as assessed by in situ hybridization. Systemic immunity in terms of numbers of IFN gamma secreting cells in response to immunodominant myelin basic protein (MBP) peptides ex vivo was not reduced, and non-toxic doses of memantine did not affect lymphocyte proliferation or IFN gamma secretion in vitro. Considering these findings, we hypothesize that effector mechanisms responsible for reversible neurological deficits in EAE may involve NMDA receptors, and this highlights neurons as targets during autoimmune neuroinflammation.

Congenital hypomyelination neuropathy (Lyon type) is characterized by a non-progressive clinical course and a histopathological formation of atypical onion-bulb. We have studied the immunohistochemical expression of the major peripheral myelin proteins including P0 protein, myelin basic protein (MBP) and P2 protein in three such patients. No significant difference was observed between the patients and the controls, as to the P0 and MBP staining. In contrast, P2 protein antiserum scarcely stained the patients' nerve fibers except for a few scattered adequately myelinated fibers. Assuming the pathogenetic contribution of the extremely decreased P2 protein to the disease, we investigated P2 protein gene by sequencing all coding regions but failed to detect any change in the nucleotide sequence. Further investigation including the analysis of promoter region of P2 protein gene is needed to elucidate the mechanism of congenital hypomyelination neuropathy.

A double blind Phase 1 clinical research project was conducted in vivo in multiple sclerosis (MS) patients to determine the effect of myelin basic protein (MBP) synthetic peptides on free (F) and bound (B) titers of anti-MBP in cerebrospinal fluid (CSF). Intrathecal administration of peptide MBP75-95, either as a single dose, or as repeated injections for periods up to 10 weeks, produced complete binding-neutralization of F anti-MBP with no change in B levels. A control peptide MBP35-58 had no effect on F and B anti-MBP levels. Intravenous administration of MBP75-95 resulted in significant decline of F and B CSF anti-MBP levels over a period of one month. Administration of MBP synthetic peptides to MS patients either intrathecally or intravenously did not have any adverse neurological effects and systemic complications did not occur. The MBP epitope for MS anti-MBP was further localized to an area between Pro85 and Pro96.

In situ hybridization, RT-PCR and Northern blot analysis as well immunohistochemistry were used to examine the expression of C1q, a subcomponent of the rat complement system, in brains of rats infected with Borna disease virus (BDV) and rats afflicted with experimental allergic encephalomyelitis (EAE) induced by the adoptive transfer of myelin basic protein specific T cells. C1q mRNA, which was not detected in normal brain, became clearly detectable using RT-PCR analysis by d14 post infection (p.i.) with BDV. Maximal levels of C1q mRNA were reached 21 days p.i. when inflammatory reactions in the brain were also at a peak. Similarly, C1q mRNA was elevated when the clinical symptoms of EAE became evident 5 days following cell transfer. C1q positive cells, as identified by immunohistology, were preferentially localized in grey and white matter of the hippocampus and basolateral cortex. The C1q positive cells resembled microglial cells in morphology. The correlation of C1q expression with the development of neurological disease as well as the dramatic increase of C1q within brain regions with inflammatory lesions suggest that local biosynthesis of C1q may play a role in the pathogenesis of Borna virus induced and autoimmune encephalomyelitis.

The concentrations of NG,N′G-dimethylarginine [Me2(sym)Arg] and NG,NG-dimethylarginine [Me2(asym)Arg] were determined in the urine samples from multiple sclerosis (MS) and control subjects, using a highly sensitive HPLC post-column o-phthaldialdehyde derivatization method. The presence of approximately equal amounts of both dimethylarginine isomers, of Arg concentration nearly half of Me2Arg, and of the undetectable amount of NG-monomethylarginine were the characteristic urinary excretion pattern in all human samples studied. The urinary excretion of Me2(asym)Arg and Me2(sym)Arg from MS (n = 9) and control (n = 7) were analyzed: the mean values from the samples were approximately 20% (for all MS) and 33% (for chronic-progressive MS) lower than those from the control for both dimethylarginine-derivatives when compared to the respective compounds. Although there were contrasting trends between controls and MS patients in the relationship of urinary NG-dimethylarginines and myelin basic protein like material (MBPLM), the correlations were not significant. Differences in the ratios of the concentrations of the two dimethyl derivatives, Me 2 (sym)Arg Me 2 (asym)Arg , were not significantly different between MS and control groups. These findings warrant further investigation of possible links between urinary excretion of NG-dimethylarginine and MBPLM in MS. The possible significance of myelin metabolism in relation to urinary NG-dimethylarginines in MS is discussed.

T-cell reactivity to human myelin basic protein (MBP) has been extensively studied using T-cell lines and clones generated from both peripheral blood and cerebrospinal fluid, from normal controls and multiple sclerosis (MS) patients. These studies have largely utilized myelin basic protein isolated from control human adult white matter. In our study, we used MBP reactive T-cell lines as a probe to investigate antigenic differences in a series of MBP preparations isolated from either control human white matter or white matter from the central nervous system (CNS) of MS patients. Autologous peripheral blood derived mononuclear cells were used as antigen presenting cells (APC). Although the majority of T-cells were found to react equally well with all preparations of MBP isolated from both control and MS white matter, we were also able to identify T-cell lines which reacted well with all preparations of MBP isolated from controls but failed to react with MBP isolated from MS white matter. These differences were unlikely to reflect differences in degradation products or excess peptides present in the MS brain since SDS-PAGE and HPLC did not show any difference in the MS samples compared to the controls, and the concentration response curves for a human T-cell clone specific for the 84–102 region of MBP were similar for all the MBP preparations. We did not detect differences in amino acid content amongst MBP preparations although single amino acid substitutions cannot be ruled out. These results raise the possibility that MBP isolated from MS brain may differ in charge microheterogeneity which would affect antigenic determinants. The processing of MBP by different APCs is another variable which could underlie the differences in T-cell reactivity to various MBP preparations.

Proteolipid protein (PLP) is the major protein of central nervous system (CNS) myelin. In some species, intradermal inoculation with PLP and adjuvants causes experimental autoimmune encephalomyelitis (PLP-EAE) characterized by neurological signs of tail and limb weakness and by inflammation and demyelination in the CNS. A previous study found that inoculation of Lewis rats with 100 micrograms of PLP causes PLP-EAE with a low incidence of neurological signs and a highly variable clinical course. In the present study we assessed PLP-EAE produced by inoculation with 1000 micrograms of PLP per rat. Fifty-one of 59 (86%) Lewis rats developed neurological signs 8 to 20 days (mean = 12.0 +/- 2.0) after inoculation with 1000 micrograms of PLP. In such rats, mononuclear cell infiltrates were present in the brain and spinal cord while primary demyelination occurred mainly in the subpial regions of the spinal cord, especially in the dorsal root entry and ventral root exit zones. The histological findings were compared with those in acute EAE induced in the Lewis rat by inoculation with whole CNS tissue or with myelin basic protein: in PLP-EAE, in contrast to these other models, the disease was essentially restricted to the CNS. This form of EAE should be useful in future studies of the consequences of autoimmunity to PLP.