SESSION I: MULTIPLE SCLEROSIS: THE DISEASE

Multiple Sclerosis: General Clinical Information

Noseworthy J, Department of Neurology, Mayo Clinic and Foundation, Rochester, MN, USA

Most multiple sclerosis (MS) patients present with episodic and reversible disturbances of CNS function but many later develop progressive clinical disability. Current opinion holds that MS results from an immune-mediated destruction of CNS myelin and/or oligodendrocytes, axons and possibly neurons. There is clinical, genetic, immunological, radiological and pathological evidence that MS is a heterogeneous disorder. This heterogeneity may explain the extreme variability of the clinical course of untreated MS and the partial response to treatment reported in clinical trials. Acute, often reversible, inflammatory CNS demyelination results in axonal conduction delay and block and the reversible clinical features of a “relapse.” Axonal damage may occur early in the disease course. With time, progressive clinical worsening with corresponding axonal loss and brain and spinal cord atrophy are nearly invariable except, perhaps, in the minority of patients with long-standing, “benign MS.” Irreversible, progressive axonal loss may explain the slow clinical deterioration that typifies progressive MS.

Type one interferons, glatiramer acetate and several non-specific immunosuppressive therapies are partially effective in reducing the frequency (severity?) of clinical relapses. These treatments may reduce short-term clinical disability resulting from repeated clinical relapses. Trials of sufficient duration to demonstrate that early treatment alters the subsequent development of progressive MS (e.g., clinical progression independent of recurring relapses) are lacking. Recent trials in which interferons have been started during the secondary progressive phase of MS demonstrate continued beneficial effects on presumed ongoing inflammatory events (e.g., relapses, new MRI lesion formation) but there is limited benefit on continued clinical worsening and progressive brain atrophy.

We need to know whether inflammation underlies all forms of axonal injury. Are existing or will future anti-inflammatory treatments arrest continued axonal injury or are the factors that determine axonal loss independent of the early inflammatory stages of MS? If MS is a heterogeneous condition with multiple, possibly independent mechanisms of disease predominating in an individual patient, how can we identify the critical mechanisms of injury for a given patient (group of patients) in order to target therapies (trials)? What strategies should we use to identify long-term benefits from our treatments?

Application of cerebral magnetic resonance techniques to observe in vivo pathology and elucidate the natural history of multiple sclerosis

Miller DH, Institute of Neurology, University College of London, UK

E-mail: d.miller@ion.ucl.ac.uk

Serial MR imaging and spectroscopy has been applied in the last 15 years to study the course of multiple sclerosis. MRI is a sensitive technique for identifying focal areas of inflammatory demyelination. Serial studies have provided intriguing new insights into the course of multiple sclerosis. In addition to assisting with diagnosis, improving prognosis, and monitoring treatment, MR studies, in combination with the correlation with pathology and clinical status, has elucidated a number of pathogenic mechanisms. The main observations are:

1. Focal demyelination is associated with an initial phase of break down in the blood brain barrier and inflammation.

2. There is axonal damage and loss in lesions and normal appearing tissues in the brain and spinal cord, and these become more evident in progressive disease.

3. Axonal loss is partly related to inflammation and partly independent.

4. Progressive abnormalities occur in the normal appearing white matter that precede focal lesions, and may contribute to progressive disability.

5. Brain plasticity occurs in response to the pathological process.

There are a number of unresolved issues in MS pathogenesis that new imaging techniques may be able to address. These include:

1. Longitudinal studies from onset to elucidate the relationship of inflammation and neurodegeneration, and to explore pathological heterogeneity.

2. Self specific MR contrast agents to explore cellular pathogenic mechanisms.

3. More specific tools for monitoring remyelination.

The Expanding MS Lesion: novel pathogenetic mechanisms

Raine C, Albert Einstein College of Medicine, Bronx, NY USA 10461-1605

E-mail: raine@aecom.yu.edu

The interphase between the demyelinated plaque of multiple sclerosis (MS) and normal adjacent white matter has intrigued investigators for many years. In some lesions, it is the site of florid inflammatory and/or macrophage activity with ongoing demyelination, in others, it is pathologically silent with gliotic demyelinated CNS parenchyma abutting directly normally myelinated tissue. The understanding of molecular events in this zone will be key in the analysis of the remyelinating potential of the MS lesion since it is in this zone that most remyelination is seen. This presentation will focus on two aspects of lesion growth in MS, one pertaining to the cytotoxic effects of an imbalance in glutamate metabolism upon oligodendrocytes at the lesion edge (glutamate excitotoxicity), and the second upon a selection of molecules, expressed by astrocytes and oligodendrocytes at the lesion margin, which restrict remyelination. The latter experiments support the notion that the re-expression of developmental pathways which hold oligodendrocytes at early stages of development may have a restrictive effect upon myelin repair while at the same time, may retain oligodendrocytes in an immature state until conditions are conducive to remyelination. These findings will be discussed in the context of novel therapeutic approaches for MS.

(Supported by NS 08952, NS 11920 and NMSS RG 1001-J-10).

Heterogeneity of pathogenetic pathways in multiple sclerosis: pathological evidence and clinical implications

Lassmann H, Brain Research Institute, University of Vienna, Austria

E-mail: hans.lassmann@univie.ac.at

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system. The pathology of the inflammatory reaction is consistent with a T-cell mediated immune response, which leads to tissue damage through activated macrophages and microglia. Demyelination with relative preservation of axons is the hallmark of MS pathology, although axons and even neurons in the gray matter are also injured. Recent immunopathological studies from our laboratory suggest that the mechanisms of demyelination and axonal damage in MS are heterogenous between patients. The basic pathology, which is present in all lesions and all patients is an inflammatory response, which is dominated by Class I MHC restricted cytotoxic T-lymphocytes. On this background tissue damage may occur either by the cytotoxic T-cells themselves or by activated macrophages and microglia cells (Pattern I). In some patients this basic pathology is modified by the deposition of immunoglobulins and activated complement at the sites of active demyelination, suggesting an antibody mediated mechanism of demyelination (Pattern II). In other patients active lesions closely resemble those found in acute ischemic white matter diseases and show a profound expression of hypoxia-inducible factor-alpha (Pattern III). Finally, in rare patients the inflammatory process is associated with unusually profound destruction of oligodendrocytes at the lesion borders, suggesting that in these cases the there is an increased susceptibility of the target tissue for immune mediated damage (Pattern IV). These different patterns of demyelination in part segregate with clinical features of the disease and can in part also be differentiated by their MRI presentation and by CSF-markers. It is expected that these studies will lead in the future to subtype-specific therapeutic strategies in multiple sclerosis.

Poster No. I-1

Cortical cerebral metabolism correlates with MRI lesion load and cognitive dysfunction in MS

Blinkenberg M, Rune K, Jensen CV, Ravnborg M, Kyllingsbæk S, Holm S, Paulson OB & Sørensen PS, Neurobiology Research Unit, Copenhagen University Hospital, Copenhagen, Denmark

E-mail: blink@pet.rh.dk

Objective: To study the association between the cortical cerebral metabolic rate of glucose (CMRglc), magnetic resonance imaging (MRI) T2-weighted total lesion area (TLA), cognitive dysfunction and neurological disability in multiple sclerosis (MS).

Methods: Twenty-three patients with clinically definite MS underwent measurements of CMRglc, TLA, motor evoked potentials (MEP), cognitive and neurological disability. CMRglc was calculated using positron emission tomography (PET) and 18-F-deoxyglucose (FDG) and compared to nine normal controls.

Results: Reductions of CMRglc (p<0.01) were found in cortical global and regional lobar measurements. Furthermore, regional CMRglc (rCMRglc) was reduced in dorsolateral prefrontal cortex, orbitofrontal cortex, caudate, putamen, thalamus and hippocampus. Global cortical CMRglc correlated with TLA (k= -0.66; p=0.001), and rCMRglc correlated with regional lesion load in all cerebral lobes (p#0.05). Global cortical CMRglc and cognitive disability was also correlated (k=0.58; p=0.015), and stepwise regression analysis showed a significant association between rCMRglc of the right thalamus and cognitive performance as well as TLA. There was no correlation between CMRglc and neurological disability (Expanded Disability Status Scale (EDSS)) or MEP.

Conclusion: Global and regional cortical CMRglc is reduced significantly in MS patients compared with normal control subjects. Furthermore, the CMRglc reductions correlate with TLA as well as with cognitive dysfunction, which indicates that MRI white matter lesion burden has a deteriorating effect on cortical cerebral neural function.

Poster No. II-5

A genome-wide linkage disequilibrium screen in Scandinavian multiple sclerosis patients shows association with chromosome regions at 1q (D1S1601) and 11q (D11S1986)

Harbo HF*, Datta P*, SpurklandA, RyderLP, SawcerS, ÅkessonE, CeliusEG, ModinH, Sandberg-WollheimM, MyhrKM, AndersenO, HillertJ, Soelberg SørensenP, SvejgaardA, Compston A, Vartdal F & Oturai A, Institute of Immunology, National Hospital, Oslo, Norway. (*These authors contributed equally).

E-mail: h.f.harbo@labmed.uio.no

In order to identify genomic regions containing susceptibility genes for multiple sclerosis (MS) in the genetically homogenous Scandinavian population, we performed two genome-wide screens for linkage disequilibrium, using pooled DNA and a dense map of microsatellite markers provided through the GAMES (Genetic Analysis of Multiple sclerosis in EuropeanS) collaboration. In the first screen, 199 cases were compared with 200 controls; in the second, a further 201 cases were compared with a second set of 200 controls. In each screen, allele image profiles (AIP) generated from cases and controls were compared statistically in order to identify those markers showing the greatest differences. A total of 6000 microsatellite markers were considered in each screen, and usable data were achieved from 4041 markers in the first and 4228 markers in the second screen. Results for both screens were available in the same 3360 markers. Twelve markers showed statistically significant differences between case-control AIPs in both screens, among these the HLA marker D6S2447. When additional AIPs were generated for the most promising of these markers, statistical significance was retained for two markers – the D1S1601 marker (1q42) and the D11S1986 marker (11q23). These two novel genomic regions may contain susceptibility genes for multiple sclerosis.

Poster No. II-7

Studies of Quantitative Trait Loci (QTL´s) on chromosome 15 that control susceptibility to murine models of human inflammatory disporders

Karlsson J, Johannesson M, Holmdahl R & Andersson, Å, Section for Medical Inflammation Research, Lund University, Lund, Sweden.

E-mail: jenny-karlsson@inflam.lu.se

The aim of this study is to identify genes on chromosome 15 conferring susceptibility to the inflammatory diseases experimental autoimmune encephalomyelitis (EAE) and Collagen Induced Arthritis (CIA). These are valuable animal models for Multiple Sclerosis and Rheumatoid Arthritis, respectively. The B10.RIII mouse strain is susceptible to both EAE and CIA while the RIIIS/J strain is resistant to development of disease. The two strains have the same MHC class II haplotype, H-2r, suggesting that non-MHC genes are important for disease induction.

Previously, two eae loci (eae2 and eae3) were identified in a F2 intercross between B10.RIII and RIIIS/J. In a (B10.RIII x RIIIS/J)F1 x B10.RIII backcross EAE experiment we have found a broad peak on chromosome 15, which includes eae2. This peak controls disease severity but the inheritance pattern is different compared to what was found in the F2 intercross experiment. Interestingly, we find suggestive linkages to macrophage phenotypes concerning expression of costimulatory molecules and Fc receptors in the same region.

We have established congenic mice for eae2 on chromosome 15 as well as for eae3 on chromosome 3. In an EAE experiment, the eae2 heterozygous congenic mice had a more severe disease compared to homozygous littermates. This is in line with the results from the F2 intercross. In the backcross experiment however, we find that mice homozygous for B10.RIII genes at eae2 have a more severe disease. These results show the importance of the genetic context and of complex gene interactions. To study the interaction between eae2 and eae3 we have produced double congenic mice and immunized them for CIA. Also in CIA the eae2 heterozygous mice have a more severe disease. At present, we are performing an EAE experiment on the eae2 and eae3 double congenic mice.

Poster No. II-12

A study of 432 sibling-pairs with multiple sclerosis showing suggestive linkage in regions on chromosome 10

*Åkesson E, *Coraddu F, Massacesi L, Marrosu M, Hillert J, Sawcer S & Compston A, Department of Neurology, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden. (*These authors contributed equally).

E-mail: eva.akesson@neurotec.ki.se

The first genome wide screens for linkage in multiple sclerosis were published in 1996 (from America, Canada and UK). Since then, further linkage screens have been completed in populations from other countries including Sardinia, Italy and the Nordic countries (Denmark, Norway, Finland and Sweden). None of these studies has shown regions of unequivocal evidence for linkage, however most of the screens have shown more regions of potential or suggestive linkage than would be expected by chance. Regions showing potential linkage in only one or a few studies may reflect type I errors (false positive findings), or result from differences in ethnicity and genetic background of the populations studied. On the other hand, regions that repeatedly appear as potential linkage should be studied in more detail. In the screens from Sardinia, Italy, the Nordic countries and a second set of sib-pairs from the UK (unpublished data) one such region appeared on the short arm and centromere of chromosome 10. To explore and refine this region, we typed these sibling-pairs from Sardinia, Italy, the Nordic countries and UK (n=432) for a high-density set of 13 microsatellite markers (average distance between markers 5.3 cM) in an area covering 64 cM on the short arm and centromere of chromosome 10. On 10p14-15 we found a peak with MLS 2.0. Potential candidate genes of interest in this region include the alpha-receptors of IL-2 and IL-15. This peak was mainly found in the Northern populations (UK and Nordic countries), while analysis of the Southern populations (Sardinia and Italy) disclosed two peaks; one at 10p13 (MLS 2.0) and one on the long arm of chromosome 10 (MLS 2.1).

SESSION II: GENETICS

Family Studies in Multiple Sclerosis

Ebers GC, Sadovnick AD, Risch N and the Canadian Collaborative Study in Genetic Susceptibility to Multiple Sclerosis, Dept of Clinical Neurology, Radcliffe Infirmary, Oxford, UK

E-mail: mo.collins@clinical-neurology.ox.ac.uk

The genetic epidemiology of multiple sclerosis has been perhaps better studied than in any other autoimmune disorder including many which are considered more common. A large environmental component to this disease is most clearly illustrated by consistent geographic disease gradients which are not easily explained by genetic clines. Familial forms of the disease are dependent on the overall background prevalence and demonstrate a strong interaction between genes and environment. The environmental factor(s) has been relatively intractable and there has been a shortage of good answerable questions. In Canada, which has a high prevalence rate, 25% of patients will have an affected family member. Studies of twins, half-siblings, adoptees, conjugal pairs and their offspring and recurrence risks of consanguineous matings all indicate a major role for genes. Genome-wide searches in affected relative pairs have been disappointing and heterogeneity/complexity is the likely explanation for the ambiguous results to date. One potential way of minimising complexity is the study of families with high recurrence risks. In Canada there are some 500 families with three or more individuals affected. There are some differences in the clinical and genetic characteristics of these families and these will be discussed as a potential strategy to identify both heterogeneity and specific genes.

Genetic Analysis of Multiple sclerosis in EuropeanS (GAMES)

Compston, DAS, University of Cambridge Neurology unit, Addenbrooke's Hospital, Cambridge, UK

E-mail: alastair.compston@medschl.cam.ac.uk

GAMES aims to identify genetic factors conferring susceptibility to multiple sclerosis. The strategy is to screen the entire genome indirectly for linkage and association using markers for susceptibility genes evenly distributed across the genome at a density of c0.5 -10 centiMorgans. Independent identification of the same chromosomal regions, using different genetic tactics, increases confidence in their candidature as susceptibility loci. GAMES provides information on genetic susceptibility in 19 European populations together with a pan-European meta-analysis. Results are made available on a dedicated web-site (http://www.mrc-bsu.cam.ac.uk/MSgenetics) as they are analysed. Linkage analyses included in the GAMES project now provide whole genome screening (using a sub-set of 400 microsatellite markers) in 521 multiplex families from the United Kingdom, Italy, Sardinia, Scandinavia, Turkey and Australia. Associations are detected by comparing >200 cases and >200 controls from all 19 populations (23 cohorts). Using a standard set of 6000 microsatellite markers spaced to provide a 0.5cM map of the genome. DNA is pooled from cases or controls to reduce the typing effort and improve efficiency. The most recent study of Europeans suggests that, overall, linkage disequilibrium is extensive but highly variable across the genome and therefore not uniformally accessible to tests for association. It occurs in blocks of varying size but having sharp boundaries. The power of GAMES to detect associations within blocks is high but the susceptibility genes, which by bad luck, are placed in the boundaries could be missed. Because cases are not typed individually, pooling limits the opportunity for stratified analyses of sub-groups defined, for example, by variations in clinical course, severity or response to treatment.

Search for genetic determinants

Risch N, Department of Genetics, Stanford University Medical Centre, Stanford, CA, U.S.A.

E-mail: pmayberg@leland.staford.edu

Abstract not received

Poster No. I-2

Multiple sclerosis and erythema nodosum

Daskalovska V, Petkovska-Boskova T & Daskalovski Z, Clinic of Neurology, Clinical Centre, Skopje, R. Macedonia

E-mail: verus@mt.net.mk

Multiple sclerosis (MS) is frequently associated with other, mainly, autoimmune diseases, such as: uveitis, thyreoiditis, Chron’s disease, etc. A female patient with MS having registered only two attacks of erythema nodosum, with time distance between them of 3 years will be presented. MS started 16 years ago, and its course is relapse-remitting. She has a low degree of disability (2.0), and has not been ill from other diseases save erythema nodosum in two attacks, with typical clinical picture and satisfactory effect from antibiotic and immunosuppressive therapy. Whether patient’s autoimmunity or genetics associate these two diseases, remains indeterminate, with a possibility that some environmental factor could “complicate” this condition.

Poster No. I-4

Different plasma levels of alpha-2-macroglobulin and activated alpha-2-macroglobulin

in patients with multiple sclerosis.

Jensen, PEH, Neuroimmunology Laboratory, Copenhagen University Hospital, Copenhagen, Denmark

E-mail: pejensen@pet.rh.dk

Measurements of the alpha-2-macroglobulin (a2M) concentration in plasma from 60 MS-patients and 132 controls demonstrated a significantly lower concentration in the patients. When the concentrations of the activated form of a2M was measured in the samples, there was a significant difference between the MS-patients and controls, but in this case the concentrations were highest in the patients. The significant difference between the patients and controls is still valid, when the fractions of activated a2M to total a2M in each person is compared.

The result suggests, that a2M may be important for regulation of the immune system in MS-patients, since a2M is a growth factor, cytokine and hormone regulator. The regulation of these factors may be changed by the presence of various concentrations of the non-activated and activated forms of a2M. a2M is also known as an unspecific protease inhibitor, and the relatively high concentration of activated a2M in MS-patients may suggest raised proteolytic activity in the blood circulation of MS-patients.

Poster No. I-5

The problem of pain in multiple sclerosis

Malciene L & Petrikonis, K, Department of Neurology, Kaunas Medical University Hospital, Kaunas, Lithuania

E-mail lmalciene@one.lt

The aim – to establish the prevalence of pain in patients with multiple sclerosis.

Patients and methods. The patients, treated due to MS in the department of neurology in one year period were included in the study. We used original questionnaire, which was prepared joining McGill questionnaire, visual analog pain intensity measuring scale (VAS) and questions about the duration, treatment and adjacent diseases.

Results. We had 47 patients. The age ranged between 22 and 63 years. 13 of them were male, 34 - female. There were no adjacent diseases in the group. 25 (53,2%) patients indicated that they are suffering from pain. More than half of them had backache, 36% had pain in the joints, mostly surrounding pain in the knee and wrist joints. 44% of the patients had face pain and headache. The patients themselves indicated the pain using pain descriptors (according McGill). Sensoric pain descriptors were used in 79% of cases, emotional – in 21%. The pain from slight to moderate (3-6 points) was in 74%, severe pain (8-9 points) was in 8% of the patients. Backache was constant and permanent (more than 1 year), the face pain – mostly unilateral and paroxysmal and had duration less than 6 months.

Conclusions. More than half of the patients with MS suffer from the pain.

The backache and sensoric type of pain prevailed.

Poster No. I-6

Concordance for disease course and age at onset in Scandinavian MS co-affected sib pairs

Oturai A, Ryder LP, Madsen HO, Hillert J, Frederiksen S, Myhr KM, Celius EG, Datta P, Harboe HF, Andersen O, Spurkland A, Andersen O, Svejgaard A & Sørensen PS., Multiple Sclerosis Research Unit, Copenhagen University Hospital, Copenhagen, Denmark

E-mail: Annette@oturai.net

Background: Investigation of co-affected sib pairs is one method to determine the genetic influence on many complex diseases, including MS. Investigation of the clinical concordance in co-affected sib pairs may be a prerequisite to identify genes that modify the clinical outcome. The aim of this study was to assess the genetic influence on clinical parameters among co-affected sib pairs, and to compare familial and sporadic MS.

Material and methods: We identified 136 Caucasian Scandinavian families with MS co-affected sib pairs from Denmark, Norway, and Sweden. Cohen’s kappa coefficient and the Intra-class correlation coefficient were used to assess concordances in sib pairs. Furthermore, clinical features and HLA status were compared among familial (probands of sib pairs) and sporadic MS cases (n=230).

Results: When classifying the disease courses into relapsing-remitting, secondary progressive, and primary progressive MS, we found significant concordance of the disease course (kappa = 0,28; p < 0,0001). After correction for collection bias we found significant concordance of age at onset (r = 0,23; p = 0,0036). Among familial MS (probands of sib pairs) HLA-DR2 carrier patients had younger age of onset (p = 0,003), and slower disease progression (p = 0,025). Regarding sporadic cases, we found no significant differences in clinical features between the HLA-DR2 carrier and non-carrier patients.

Conclusion: Analyses of Scandinavian co-affected sib pairs suggest that disease course and age of onset are partly under genetic control. Furthermore, HLA-DR2 is of importance for age at onset and disease progression in familial cases.

Poster No. I-7

Abnormal endothelial tight junctions and BBB leakage in lesions and normal-appearing white matter in multiple sclerosis

Plumb J, Kirk J, Mirakhur M & McQuaid S, Neuropath. Lab, RGHT & QUB Belfast, UK

E-mail: jonnie_plumb@hotmail.com

Increased blood-brain barrier (BBB) permeability is a feature of new and expanding inflammatory lesions in multiple sclerosis (MS) but the pathological mechanisms involved remain ill-defined. Frozen sections from neuropathologically characterised areas of plaque and normal appearing white matter (NAWM) in 14 cases of MS were graded according to the abundance of oil-red O–positive cells. Using single and double immunofluorescence and confocal scanning laser microscopy the TJ-associated proteins ZO-1 and occludin were examined in relation to: endothelial integrity (Ulex), BBB leakage (fibrinogen), lymphocytic infiltration (LCA) and macrophage/microglial activation (HLA-DR). Blood vessels (>2500, 83% of which were <30 μm diam.) were systematically scanned to acquire image datasets for offline analysis. TJ abnormalities (i.e. beading, interruption or absence of fluorescence, separation or opening of junctions) were frequent in ORO-positive active plaques, affecting 42% of vessels, but less frequent in ORO-negative chronic inactive plaques (23%) or NAWM (13%) and in both normal (2%) and neurological controls (<6%). A similar pattern of variation in frequency of abnormal TJs was found amongst the different tissue categories irrespective of the size of vessel examined. Single vessel analysis and grading of sections dual-labelled for TJ proteins and BBB leakage revealed a trend in both NAWM and inactive lesions for vessels with the most marked TJ abnormality to show increased fibrinogen leakage. This was most pronounced however, in active lesions where 41 % of such vessels showed severe leakage. Abnormal or disrupted TJs, arising during acute inflammatory phases of lesion development and affecting both paracellular and transcellular transendothelial pathways are the probable cause of the BBB leakage revealed by enhancment of lesions on in vivo MRI. The finding of TJ abnormality and BBB leakage in NAWM suggests the possibility of new lesion formation, while in inactive lesions it points to a failure, for unknown reasons, of effective and complete TJ restoration.

Poster No. I-8

Systemic activation of CD8 T cells correlates with disease activity in early multiple sclerosis

Sellebjerg F, Langkilde A, Skou Nicolaisen M, Fenst C & Jensen J, The MS Clinic, Department of Neurology, Glostrup Hospital, Glostrup, Denmark and Danish Research Center for Magnetic Resonance, Hvidovre Hospital, Hvidovre, Denmark.

E-mail: sellebjerg@dadlnet.dk

CD4 T cells are generally considered as the main pathogenic cell type in multiple sclerosis (MS). Several lines of evidence does, however, suggest that CD8 T cells are also involved in the pathogenesis. We studied CD8 T cell activation by flow cytometry in patients with clinically isolated syndromes (CIS) suggestive of a first attack of MS, patients with clinically definite MS (CDMS), and neurological and healthy control subjects (OND and HC, respectively). Patients with CIS had more CD26+ CD8 T cells in blood than did patients with OND (p=0.008) and HC (p=0.0002). Patients with CDMS did, however, have more CD26+ CD8 T cells than did CIS patients. In CIS patients the percentage of CD26+ CD8 T cells in blood correlated with the number of active lesions on Gadolinium-enhanced magnetic resonance imaging (Spearman’s r=0.53, p=0.02) and the Kurtzke EDSS score (Spearman’s r=0.66, p=0.001). CD26+ CD8 T cells produced proinflammatory, type 1 cytokines (TNF-a and IFN-g). These results suggest that systemically activated CD8 T cells with a type 1 cytokine secretion profile are involved in the pathogenesis of MS.

Poster No. I-10

Plasma and CSF levels of 24S-hydroxycholesterol may reflect activity and progression of multiple sclerosis

Hillert J, Huddinge University Hospital, Stockholm, Sweden

E-mail: jan@hillert.se

Background. 24S-hydroxycholesterol (24S-OH-chol) is almost exclusively derived from the brain and the levels of this oxysterol in the circulation may be used as a marker for changes in the turnover of brain cholesterol.

Methods. We assessed plasma and CSF levels of 24S-OH-chol in patients with multiple sclerosis (n=118) at different stages of the disease.

Results. In the oldest groups of patients the levels of 24S-OH-chol were significantly lower than in the controls, possibly reflecting loss of neuronal cells responsible for the synthesis. There was a significant inverse relation between the EDSS-grade of the disease and the plasma cholesterol related levels of 24S-OH-chol. There was a tendency to increased plasma levels of 24S-OH-chol in the younger patients and almost all of the high levels were found in the patients of the 3^rd and 4^thdecades of life. Increased levels were only found in patients with positive cranial MRI.

Conclusion. The possibility is discussed that plasma levels of 24S-OH-chol may add significantly to existing methods used for evaluation of the different phases of multiple sclerosis.

Poster No. I-11

Determining the Presence of Depression in Multiple Sclerosis

Birnbaum G & Morgan S, MS Treatment and Research Center, Minneapolis Clinic of Neurology, Golden Valley, MN, U.S.A.

E-mail: birnb001@tc.umn.edu

Introduction: Mood changes are common in persons with MS and contribute greatly to fatigue and cognitive difficulties. However, similar difficulties can occur in MS due to the underlying disease process. It is clinically important to determine the presence of mood changes such as depression in persons with MS and initiate appropriate treatment. Definitive ascertainment of depression is time consuming and expensive. It would be important to have a relatively easy and reliable way to judge a person’s degree of depression.

Methods: With this goal in mind, we studied 39 individuals sent to an MS specialty clinic for evaluation of either the diagnosis of MS or to evaluate treatment options. 25 patients had MS. 14 patients did not have MS. Prior to the consultation patients were sent a copy of the Beck Depression Inventory-II form which they completed before being seen. Patients then underwent a complete neurologic evaluation and were asked, in the course of the interview, to rate their state of neurologic well-being on a scale of 1 (extremely poor) to 10 (normal). Their neurologic disabilities were measured using the standard EDSS. Correlations between BDI score, self-rating scale, and EDSS were then performed.

Results: There was no correlation between an individual’s BDI score and their EDSS (r=0.1856). In contrast, there was a good correlation between EDSS and the self-rating assessment (r=-0.5209) and an even better correlation between the BDI score and the self-rating assessment (r=-0.5635).

Conclusion: When corrected for disability, a self-assessment rating may be a good indicator of depression that will require further evaluation.

Poster No. II-1

A genome-wide linkage disequilibrium screen in Scandinavian multiple sclerosis patients shows association with chromosome regions at 1q (D1S1601) and 11q (D11S1986)

Datta P*, Harbo HF*, SpurklandA, RyderLP, SawcerS, ÅkessonE, CeliusEG, ModinH, Sandberg-WollheimM, MyhrKM, AndersenO, HillertJ, Soelberg SørensenP, SvejgaardA, CompstonA, VartdalF & OturaiA, Multople Sclerosis Research Unit, Copenhagen University Hospital, Copenhagen, Denmark. (*These authors contributed equally).

E-mail pameli@pet.rh.dk

Poster No. II-2

Apolipoprotein E allele distribution in monosymptomatic optic neuritis, multiple sclerosis and healthy controls

Frederiksen JF, Christiansen M, Andersen PS & Roed H, Department of Neurology, Glostrup Hospital, Glostrup, Denmark

E-mail jettefrederiksen@dadlnet.dk

Introduction: The polymorphic ApolipoproteinE (ApoE) gene maps on chromosome 19 that is one of the chromosomes of interest in the genome screening in clinically definite multiple sclerosis (CDMS). One of the most frequent onset symptoms is optic neuritis (ON).

Materials and methods: Blood from 40 patients with monosymptomatic ON, 280 patients with CDMS, and 90 healthy controls were analysed for the distribution of the three common ApoE alleles coding for the three isoforms E2, E3 and E4. ApoE genotypes were studied by PCR amplification and restriction analysis.

Results and concludion: The Allele distribution and the genotypes did not differ significantly among patients with monosymptomatic ON, CDMS and healthy controls. The result of further patients will be available at the time of the Benzon symposium, enabling to further study the relation between ON and CDMS and the ApoE allele distribution. Subgroup analyses correlating the results to the clinical course (monosymptomatic ON, relapsing remitting MS, primary progressive MS, and secondary progressive MS) of the patient will be presented.

Poster No. II-4

Identifying MHC binding peptides from potential sclerotic autoantigens. A screening system to identify MHC binding peptides

Hansen B Endel & Svejgaard A, Department of Clinical Immunology, University Hospital of Copenhagen, Copenhagen, Denmark.

E-mail: b.hansen@immiku.dk

We have tested a new peptide screening system in order to identify the MHC binding peptides hidden within a protein. Overlapping peptides of proteins were synthesised directly on pins in wells of microtiterplates, and MHC binding were allowed to take place within the wells. The amount of bound MHC molecules were measured as an enzymatic colour reaction recognising a biotin-tag on the MHC molecule itself or on a secondary antibody.

In a preliminary study of the HLA-DQ6 molecule the results indicate that this peptide screening system can identify the peptides within a protein that will bind to the HLA-DQ6 molecule. Furthermore, the developed colour intensity of each well ranks the peptides according to their affinity for the MHC molecules.

This screening system is ideally suited to identify MHC binding peptides among large numbers of overlapping peptides from known potential autoantigens in multiple sclerosis as well as other autoimmune diseases. Moreover, the assay is fast and affordable compared to MHC binding studies with peptides in solution and might help identify T-cell epitopes of autoreaktive T cells.

Poster No. II-6

Genetics of multiple sclerosis.

Jensen T Hummelshøj, Ryder LP, Madsen HO, Oturai A, Sorensen P Soelberg & Svejgaard A, Department of Clinical Immunology, University Hospital of Copenhagen, Copenhagen, Denmark

E-mail. tinahummelshoj@hotmail.com

Eta-1 (Early T-lymphocyte Activator 1), also called osteopontin, is a secreted glycoprotein, which is constitutive expressed in bone, kidney, and nerve cells. The gene is located on the human chromosome 4q13. In 1989, Eta-1 was found to be expressed in activated CD4+ cells as the predominant transcript after bacterial infection. Eta-1 enhances Th1 and inhibits Th2 polarization by inducing macrophages to produce IL-12 and INF-γ, and inhibit their IL-10 expression. Recently Eta-1 was found to be expressed 19-fold in Th1-cells compared to Th2-cells. Sequencing of cDNA libraries of sclerotic patients has resulted in identification of abundance Eta-1 transcript in brain. In MS mouse model (EAE) Eta-1 is also found to be elevated in brain, and Eta-1 deficient mice were resistant to progressive EAE. Therefore, Eta-1 may be a potential candidate gene for development of progressive MS.

We screened the human Eta-1 promoter, spanning from position –2267 to +52, for DNA variation and tested the SNPs for associations to MS. We identified six novel SNPs in position –1776, –1748, –616, –443, –155, and –66. The –1748G/A, –616T/G, –443T/C, and –66T/G SNPs were genotyped in 198 MS patients and 162 controls. No significant associations were found. SNPs in the promoter region of the Eta-1 gene may, however, be important for Eta-1 gene expression. Electro mobility shift assays provided evidence for a difference in the binding of the –66G/T SNP to nuclear proteins from the human THP-1 cell line. To elucidate the mechanisms by which the Eta-1 gene is expressed, we analyzed a promoter region of the Eta-1 gene containing the –443, –155, and –66 SNPs by reporter gene assay. The genotype –443C/–155G/–66T resulted in elevated transcriptional activity. These results suggest that expression of Eta-1 is related to at least three promoter SNPs.

Poster No. II-8

Studies of thymocyte apoptosis in mice susceptible/resistant to experimental autoimmune encephalomyelitis (EAE)

Lonskaya I, Karlsson J & Andersson Å, CMB, Section for Medical Inflammation Research, Lund University, Sweden

E-mail irina-lonskaya@inflam.lu.se

Apoptosis has been suggested to play an important role in development of autoimmune disease. We have compared different modes of apoptosis in mouse strains susceptible (B10.RIII) or resistant (RIIIS/J) to EAE. By in vitro studies of fast and delayed apoptosis in thymocytes from the two mouse strains, we have found that thymocytes from RIIIS/J mice are characterized by faster dynamics of early apoptosis (first 2-3 hours after apoptosis induction) and by slow dynamics of delayed apoptosis (24 hours). The different mode of apoptosis that we can observe is abolished in thymocytes from B10.RIII mice after cycloheximide (inhibitor of protein synthesis) pretreatment. Thymocytes isolated from RIIIS/J mice are significantly less sensitive to prevention of apoptosis by the caspase inhibitor zVADfmk. Taken together, the results suggest that apoptosis in thymocytes from the two mouse strains is differently regulated.

In a genetic linkage study of EAE development in a back-cross between B10.RIII and RIIIS/J, we have found several genetic regions linked to disease development. To find gene regions that control the apoptosis traits, and to investigate if apoptosis is linked to genetic regions that have been suggested to be important for EAE development, apoptosis was studied in mice from an additional back-cross between the two mouse strains. Apoptotic cells are detected by flow cytometry either by staining with AnnexinV and propidium iodide or after staining with acridine orange and propidium iodide. A genome scan with micro-satellite markers covering all chromosomes, followed by linkage analysis, is being performed. In a first linkage analysis we find one region on chromosome 15, previously linked to chronicity of EAE, that show linkage to apoptosis induction in mouse thymocytes in vitro.

Poster No. II-9

Genome-wide linkage screen of a consanguineous multiple sclerosis kinship

Modin M, Masterman T, Thorlacius T, Stefánsson M,

Jónasdóttir A, Hillert J & Gulcher J, Neurotec Department, Karolinska Institutet, Huddinge Hospital, Stockholm, Sweden

E-mail: helena.modin@neurotec.ki.se

Multiple sclerosis (MS), like Alzheimer’s (AD) and Parkinson’s disease (PD), is a common neurological disorder thought to be caused by the interaction of several genes with unknown environmental factors. In both AD and PD, the identification of disease forms inherited in a classic Mendelian fashion—though they account for only a small percentage of all cases—has nonetheless helped investigators elucidate generalizable pathogenetic mechanisms. In the present study, we have characterized a consanguineous family of Middle Eastern origin and performed a whole-genome screen, using microsatellite markers, on nine family members now residing in Sweden: the index case, three of her siblings, and her daughter, all of whom have been diagnosed with definite MS; as well as the parents of the index case (who are first cousins), one of her five healthy siblings, and her husband (who is also her first cousin). Non-parametric linkage analysis was performed on genotyping data using the program ALLEGRO. Based on the presence of consanguinity, the a priori hypothesis was that the disease is transmitted in an autosomal recessive fashion in the pedigree; the analysis was in fact underpowered to detect linkage in the event of any other mode of inheritance. An initially significant LOD score of 3.5 for a locus on the long arm of chromosome 9 decreased to 2.3 after analysis of an additional family member. Four of five affected family members were homozygous for a haplotype spanning 43 cM, while the fifth affected subject and all unaffected family members were heterozygous for the haplotype.

Poster No. II-10

Impact of CCR5 polymorphism and HLA on clinical course in MS patients treated by interferon beta

Ryder LP¹, Oturai AB², Madsen HO¹, Soelberg Sørensen P^2,, Svejgaard A¹& Koch-Henriksen N³, Tissue Typing Laboratory¹, Dept. Clinical Immunology, Neurological Dept²., The MS Registry³, Rigshospitalet, Copenhagen

E-mail: lars.p.ryder@mail.tele.dk

Multiple Sclerosis is an immuno-inflammatory disease of the central nervous system (CNS). Normally, the CNS and the immune system are separated by the blood-brain barrier. In MS this barrier is to some degree broken down and certain cells are allowed to pass. Among these cells are the actors that exert their damaging effect on the myelin sheaths.

Chemokines are cytokines with special effect on cellular traffic and homing. The chemokine receptor CCR5 displays a genetic polymorphism: a deletion in the gene occurs with a gene frequency of ten percent in the general population, with no deleterious effect in the affected individuals. CCR5 may influence cell traffic through the blood-brain barrier.

Interferon beta is a rather new treatment in MS. A nationwide survey of all MS patients treated with interferon beta has been established. Further, their CCR5 polymorphism has been determined and clinical data have been recorded. This data set comprising 436 patients has been analysed with regard to the relation between the genetic marker and clinical course.

The mean age at onset and the mean progression index were not significantly different between the carriers and non-carriers of the mutation.

Neither the time from IFN treatment to first relapse nor the time to worsening in disability evaluated as one step in the EDSS score were significantly different.

Poster No. II-11

The T cell regulator gene SH2D2A contributes to the genetic susceptibility of multiple sclerosis

Spurkland A, Dai K-Z, Sundvold V, HarboHF, Granum S, Lea T & VartdalF in collaboration with the Nordic study group on MS genetics, Institute of Anatomy and Institute of Immunology, University of Oslo, Oslo, Norway

E-mail: anne.spurkland@labmed.uio.no

We have cloned an immunoregulatory gene (called SH2D2A) which encodes the T cell specific adapter protein (TSAd). TSAd expression is induced upon activation of T cells. We have found that TSAd is involved in the control of early signal transduction events in T cells.The SH2D2A gene is located close to the CD1 cluster on 1q21, a chromosomal region which contributes to the genetic susceptibility of autoimmune diseases in the mouse. A polymorphic dinucleotide repeat in the promoter region of the SH2D2A gene displayed association to MS both in a case-control association study among 313 Norwegian MS patients and 277 controls, and in a transmission disequilibrium analysis of 146 Scandinavian families with at least two affected sibs. No linkage or association of MS to four genetic markers flanking the SH2D2A gene was observed. After activation of naive CD4+ T cells, T cells homozygous for MS associated SH2D2A alleles displayed lower level of TSAd ex vivo than T cells carrying at least one allele, which was not associated to MS.

Recently, we have obtained evidence that TSAd interacts directly with Lck, the first kinase to become activated after triggering of TCR. When TSAd is expressed in Jurkat T cells together with the hyperactive Lck mutant Y505F, the massive phoshotyrosine level of cytoplasmic proteins caused by Lck Y505F is reverted to normal. This indicates that TSAd may be a potent modulator of Lck activity. Since TSAd is expressed only after the T cell has been activated, TSAd may play a role in regulating the continued activation of the cell. As the level of TSAd expression is influenced by the MS associated polymorphism in the SH2D2A promoter region, this may also be a mechanism by which the SH2D2A gene contributes to the susceptibility of MS.

Poster No. II-13

Collagen induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE) map to the same congenic fragment on mouse chromosome 3

Johannesson M, Cook AD, Andersson Å, Jirholt J & Holmdahl R, Medical Inflammation Research, BMC, Lund, Sweden.

E-mail: martina-johannesson@inflam.lu.se

Rheumatoid arthritis (RA) and multiple sclerosis (MS) are common diseases believed to be dependent on autoimmune mechanisms. In spite of large efforts worldwide we do not yet understand why certain individuals are susceptible and why these diseases progress chronically. To understand the basic mechanisms we are trying to identify genes involved in those diseases. A direct analysis of human diseases is a very difficult task because of genetic heterogeneity, polygenicity, phenotypic heterogeneity and environmental influences. In animal models those factors can be controlled and the biological role of candidate genes can be tested.

CIA and EAE in mice are the most commonly used models for RA and MS. A locus on chromosome 3 controlling the severity of both diseases has earlier been identified (denoted eae3 and cia5). We have now confirmed, by the production of novel congenic strains, that both cia5 and eae3 are in the same defined region. For both diseases, RIIIS/J genes on B10.RIII background make the ensuing disease less severe. In EAE, disease onset and chronicity was also affected. In CIA, the genes within the fragment controls effector mechanisms since the antibody responses were not affected.

It is still possible that several different genes within the fragment are associated with the observed influences on EAE and CIA and it is likely that the fragment contains a cluster of genetic polymorphisms of importance for both arthritis and encephalomyelitis in the mouse as well as in other species.

Poster No. II-14

Novel Quantitative Trait Loci controlling development of Experimental Autoimmune Encephalomyelitis (EAE) and relative numbers of lymphocyte sub-populations

Karlsson J, Zhao X, Lonskaya I, & Andersson Å., Medical Inflammation Research, CMB, Lund University, Lund, Sweden.

E-mail asa.andersson@inflam.lu.se

EAE can be induced in genetically susceptible mouse strains and serves as an animal model for Multiple Sclerosis (MS). At present, 26 eae loci controlling different traits of EAE development have been reported in mouse. The B10.RIII mouse strain (H-2^r) develops chronic EAE upon immunization with the myelin basic protein (MBP) 89-101 peptide. In a previously reported F2 intercross between B10.RIII and the RIIIS/J (H-2^r) strain, which is resistant to EAE induction, two loci, eae2 and eae3, were linked to development of EAE. In the present study, EAE development was studied in a (B10.RIIIxRIIIS/J) x B10.RIII back-cross gene segregation experiment. In this cross we could observe development of chronic disease similar to the parental B10.RIII strain and, in addition, we recorded mice with acute, monophasic disease and a number of individual mice with a remitting relapsing type of disease. A complete genome scan followed by linkage analysis with disease phenotypes and immunological sub-phenotypes was performed for the 401 mice. In the linkage analysis, two loci showed strong linkage to disease development; one locus on chromosome 5, which has not previously been reported in mouse and another locus on chromosome 11, which spans a region encompassing at least three loci previously reported in studies of EAE in crosses with different mouse strains. Both loci control development and severity of chronic disease. Furthermore, on both chromosomes we find linkages to sub-phenotypes concerning the relative numbers of different lymphocyte sub-populations in the same regions as the linkages to the disease traits. We are at present studying mice congenic for the interesting regions.

Poster No. II-15

Association studies of Notch4 and Tumour necrosis factor alpha in relation to MS and HLA

Duvefelt K, Division of Neurology, Karolinska Institute, R54, Stockholm, Sweden

E-mail: kristina.duvefelt@neurotec.ki.se

Multiple sclerosis (MS) is known to be associated with a specific HLA DR-DQ haplotype (DR15,DQ6 or HLA-DRB1*1501,DRB5*0101,DQA1*0102,DQB1*0602). We have previously reported that the associated haplotype extends to HLA-B, more than 1 cM telomeric to DRB1, and described an independent association with HLA-A alleles. However, due to a complex situation with extensive linkage disequilibria, it is still unclear whether classical HLA genes are responsible or whether the associations may be due to other polymorphic genes in this gene-dense region.

We investigated if the NOTCH4 and the Tumor necrosis factor-alpha (TNFalpha) genes which are located between the HLA DRB1 gene and the HLA-A gene are of importance for MS.

The NOTCH4 gene was investigated prompted by the report of a strong genetic association with NOTCH4 in schizophrenia. One promoter SNP marker and one intragenic trinucleotide repeat marker were investigated in 181 MS patients and 180 controls also typed for HLA-DRB and HLA-A. An association was observed (OR=3,4 P=0,0269) with the C allele of the SNP at –25 bp. However, two-locus analysis revealed that this association is clearly secondary to the classical DR15,DQ6 association. The HLA-A association did not extend to the Notch4 gene.

TNFalpha is a pluripotent proinflammatory cytokine thought to play an important role in the inflammatory process of MS. In the promoter region of the TNFalpha gene two SNPs at positions -308 and -238 were studied in the same dataset as the NOTCH4 gene. No association between these TNFalpha gene polymorphisms and MS was found.

We conclude that alleles of the Notch4 and TNFalpha genes are unlikely to be of importance for the susceptibility to MS although often carried on the same haplotype as DR15,DQ6.

SESSION III: PATHOGENESIS

A functional and structural basis for T cell receptor crossreactivity in multiple sclerosis.

Jacobsen H^1,3*, Lang H^2*, Ikemizu S², Andersson C^1,3, Harlos K², Madsen L³, Hjorth P⁴, Sondergaard L⁵, Svejgaard A³, Wucherpfennig K⁶, Stuart DI², Bell JI², Jones EY² & Fugger L¹, ¹Aarhus University Hospital, Denmark. ²University of Oxford, UK. ³Copenhagen University Hospital, Denmark. ⁴University of Aarhus, Denmark.⁵University of Copenhagen, Denmark. ⁶Dana-Farber Cancer Institute, USA (^*contributed equally to this work).

E-mail: fugger@inet.uni2.dk

Both genetic and environmental factors are important in the pathogenesis of multiple sclerosis (MS), but the only defined risk factors are certain major histocompatibility complex (MHC) class II alleles, and in particular those in the DR2 haplotype. This haplotype contains three different MS associated MHC class II alleles DRB1*1501, DRB5*0101 and DQB1*0602 which are in strong linkage disequilibrium, making it difficult to determine which is the principal MS risk gene. Here we show by functional and structural studies that the DRB1 and DRB5 loci together may influence susceptibility to MS. We demonstrate that a cross-reactive T cell receptor derived from an MS patient recognizes a peptide from myelin basic protein (MBP), an autoantigen in MS, in the context of the HLA-HLA-DRB1*1501 molecule and also a peptide derived from Epstein-Barr virus (EBV), in the context of the HLA-DRB5*0101 molecule. The crystal structure determination of the HLA-DRB5*0101: EBV peptide complex reveals a remarkable degree of structural equivalence to the HLA-DRB1*1501: MBP peptide complex at the surface presented for T cell receptor recognition, providing a unique structurally validated example of true molecular mimicry involving MHC molecules. The structural details suggest an explanation for the preponderance of class II associations in MHC associated diseases.

Immune responses in the brain

Wekerle, H, Max-Planck-Institute for Neurobiology, D-82152 Martinsried, Germany

E-mail: hwekerle@neuro.mpg.de

Immune reactivity within the brain are efficient, and at the same time tuned to avoid bystander damage within the fragile tissue. We propose that this is achieved by two major regulatory checkpoints:

1. Under normal conditions, the CNS parenchyma is a milieu hostile to immune reactions to unfold. Neuronal activity suppresses expression of “pro-immune” gene expression (e.g. MHC molecules and cytokines). These gene are, however inducible either following an overwhelming pro-inflammatory stimulus, such as is the case in the initiation of EAE, or following loss of neuronal function (like in neuronal degeneration).

2. Antigen-specific T lymphocytes can pass through the endothelial BBB only in restricted functional stages. The resting BBB allows passage of a few fully activated T cells, which prime the CNS for a subsequent full immune response. The primed BBB is then permeable for a recently described migratory T cell phenotype characterized by down-regulation of activation markers and upregulation of chemokine receptors.

We shall discuss implications for the pathogenesis of CNS autoimmune diseases and their therapy.

Large Scale Transcriptional and Proteomic Analysis of MS Tissue Yields New Targets for Therapy

Steinman, L, Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, U.S.A.

E-mail: steinman@stanford.edu

I shall review results on large scale transcriptional and proteomic analysis of MS tissue. These analyses yield new targets for potential therapy.

Autoreactive T cells in MS - their role in the induction or relapses andpathogenesis of disease.

Martin R, Neuroimmunology Branch, NINDS, National Institutes of Health, Bethesda, MD, U.S.A.

E.mail: martinr@ninds.nih.gov

Multiple sclerosis (MS) is considered a T cell-mediated autoimmune disease based on the composition of inflammatory brain infiltrates, immunogenetic background, parallels to the animal model, experimental autoimmune encephalomyelitis (EAE), and the response to immunomodulatory treatment. While a number of other factors contribute to the complex disease process and influence the heterogeneous clinical presentation of MS patients, CD4+ autoreactive T cells are important for initiating and probably also for perpetuating the disease. During the last decade, several laboratories including ours have examined the myelin-specific T cell response in MS patients. Many parallels have been described between observations in EAE and MS with respect to antigen specificity and functional phenotype of myelin-specific T cells. The major findings are: 1) Myelin-specific T helper-1 CD4+ T cells appear to mediate EAE and are found more frequently in MS patients as well. 2) Myelin-specific T cells in EAE and MS often respond to similar immunodominant epitopes of MBP, PLP or MOG, and these epitopes are recognized in the context of MS-associated HLA-DR alleles, particularly DRB1*1501 and DRB5*0101. 3) With respect to one immunodominant epitope, i.e.MBP (83-99) or MBP (84-102), transgenic mouse experiments, the detection of HLA-DR2/MBP (84-102) complexes in MS lesions, as well as the inadvertent induction of disease exacerbations in MS patients by an altered peptide ligand of MBP (83-99) all indicate that myelin antigens are encephalitogenic in MS. 4) Finally, studies of molecular mimicry between myelin antigens andinfectious pathogens argue that myelin-specific T cells can be triggered via this mechanism. The ability of T cells to respond to much larger numbers of antigens than previously anticipated suggests, however, that these data need to be interpreted with caution in the context of inducing or perpetuating autoimmune diseases. In conclusion from the above observations there is little doubt that autoreactive T cells play a prominent role in the pathogenesis of MS.

Poster No. III-4

Remyelination is not a simple recapitulation of primary myelination

Finsen, B (1,2) & Peterson, AC (2), (1) Anatomy and Neurobiology, SDU-Odense University, Denmark and (2) Molecular Oncology Group, McGill University, Montreal, Canada.

E-mail: finsen@imbmed.sdu.dk

Myelin Basic Protein (MBP) is essential for the compaction of CNS myelin. To locate and characterize the function of promoter elements regulating MBP gene expression, transgenic mice were derived bearing lacZ reporter constructs controlled by unique combinations of MBP 5' flanking sequences. In accordance with the developmental profile of MBP mRNA expression, different combinations of regulatory elements were found to control MBP expression at specific stages of maturation. Here, we compared transgene expression in oligodendrocytes (OLs) remyelinating demyelinated lesions induced by cuprizone and L-lysolecithine. Based upon the readily detectable levels of ß-galactosidase revealed during primary myelination in all such transgenic mice, we anticipated transgene expression to occur equally in remyelinating OLs. Remyelination was found to be equivalent amongst the lines of mice examined. Unexpectedly, only the larger 9.6 kb constructs expressed in remyelinating OLs while those in which lacZ was controlled by 3 kb of the 5' flanking sequence were silent. This observation suggests that fundamental differences exist in the regulatory mechanisms controlling MBP expression during primary myelination and those involved in the repair of the mature CNS myelin. The regeneration specific differences observed here in the control of MBP, particularly if they extend to further members of the coordinately-regulated myelin gene family, may account for the limited remyelination response seen in demyelinating CNS diseases.

Supported by the Canadian MS society (ACP); the Danish MRC and MS Society, Benthine Lunds Foundation and Odense University (BF).

Poster No. III-6

Apoptosis Mediators CD95, CD95L and cFLIP are upregulated in peripheral blood mononuclear cells in Relapsing-Remitting Multiple Sclerosis

Gomes A*¹, Jönsson G*², Mjörnheim S¹, Olsson T³, Grandien A² & Hillert J¹, ¹Department of Neurology, Karolinska Institute at Huddinge University Hospital, ²The Wenner Gren Institute, Department of Immunology, Stockholm University and ³Centre of Molecular Medicine, the Karolinska Hospital, Solna, Sweden. (*Contributed equally).

E-mail: andreia.gomes@neurotec.ki.se

The elimination of inflammatory cells within the central nervous system (CNS) plays an important role in protecting the CNS from immune mediated damage. Multiple Sclerosis (MS) is a chronic disease involving an inflammatory reaction within the white matter of the CNS and are mediated by T cells, B cells and macrophages. The pathogenesis of MS is characterised by impaired activation-induced cell death (AICD) of activated myelin specific mature T cells. The death receptor CD95 (FAS/APO-1) and its ligand (CD95L) are crucial mediators of AICD.

Oligmerisation of CD95 induces the formation of active caspase-8. The activation of the caspase-8 can be regulated by the cellular FLICE-inhibitory protein (cFLIP). cFLIP interacts with caspase-8 thereby preventing further activation of the caspase cascade leading to apoptosis. The ligation of CD95 may not only mediate apoptosis, but could also induce proliferation, possibly due to high expression of endogenous cFLIP.

In this study we investigated the mRNA expression of the apoptosis mediators CD95, CD95L, caspase-8 and cFLIP in peripheral blood mononuclear cells (PBMNCs) from MS patients using Real-Time PCR.

We show that in PBMNCs there was a significant increase of CD95, CD95L and cFLIP mRNA expression in MS patients. This increase was most prominent in the inflammatory relapsing-remitting phase of the disease. We confirm that this enhanced expression in the periphery was reflected by an equivalent expression in intrathecal cells.

We conclude that elevated CD95, CD95L and cFLIP expression in PBMNCs may contribute to MS disease activity by extending the viability of pathogenic, autoreactive cells and facilitate their migration into the CNS.

Poster No. III-8

CD4 T cell activation correlates with disease stage in early multiple sclerosis

Jensen J, Langkilde A, Fenst C, Skou Nicolaisen M, Roed H & Sellebjerg F, The MS Clinic, Department of Neurology, University of Copenhagen, Glostrup Hospital, 57 Nordre Ringvej, DK-2600 Glostrup, Denmark and Danish Research Center for Magnetic Resonance, University of Copenhagen, Hvidovre Hospital, DK2630 Hvidovre, Denmark.

E-mail: jak@dadlnet.dk

An altered balance between pathogenic and regulatory CD4 T cells might explain why activation of autoreactive T cells results in the development of multiple sclerosis (MS) in a substantial proportion of patients with clinically isolated syndromes (CIS) of the CNS. Patients with CIS or definite MS had a decreased percentage of CD25+ CD4 T cells, and the percentage of CD25+ CD4 T cells in cerebrospinal fluid (CSF) was especially low in definite MS. CD25+ CD4 T cells expressed intracellular CTLA4, and CTLA4+ CD4 T cells were decreased in CIS and definite MS. In CIS patients a higher percentage of CD25+ CD4 T cells in CSF was associated with a lower risk of developing definite MS and lower levels of demyelination. An increased percentage of CD4+CD26+ T cells was observed in blood from patients with CIS and definite MS, and patients with definite MS had an increased percentage of CD4+CD71+ T cells in CSF; measures of T cell activation correlated with magnetic resonance imaging and clinical measures of disease activity. We suggest that the balance between regulatory CD4+CD25+ T cells and pathogenic T cells determines chronicity and disease severity in the initial clinical stages of MS, a prototype Th1-mediated human disease.

Poster No. III-11

Changes in the Paranodal Region of Myelinated Axons in Chronic Multiple Sclerosis

Wolswijk G & Balesar R, Netherlands Institute for Brain Research, Amsterdam, The Netherlands

E-mail: g.wolswijk@nih.knaw.nl

There is substantial evidence that axons are damaged and lost in the inflammatory demyelinating disease multiple sclerosis (MS). In the present study, we have explored the possibility that changes in the expression of the paranodal protein contactin-associated protein (Caspr), also termed paranodin, may be an early sign of pathological changes in axons. This neuronal protein plays an important role in binding the myelin loops to the axon at the paranodal junction, thereby forming a barrier to limit the free diffusion of ions. Injury-induced changes in the expression of Caspr may thus influence the attachment of the myelin sheath to the axon and saltatory impulse conduction. Triple immunofluorescence and confocal laser scanning microscopy showed that the Caspr-positive structures on axons in control adult human brain and spinal cord tissue were cylindrical and localized to the paranodal region. Axons in the demyelinated centre were generally devoid of Caspr, while reduced numbers of Caspr-positive structures were found in the lesion borders. Although these data suggested that Caspr expression is down regulated following demyelination, additional data indicated that it reappears in the paranodal junction during the remyelination process. Our detailed analysis further showed that many Caspr-positive structures in lesion borders were substantially larger, both in diameter and length, than in control tissue and that the Caspr immunoreactivity sometimes was not concentrated in the paranodal region, but extended for a considerable distance underneath the myelin sheath. The zone around the lesions with enlarged Caspr-positive structures varied in width from < 0.5 mm to > 1.5 mm, irrespective of the relative age of the lesions. The widening of axons at the (para)nodal region may indicate disease-related pathological changes in axons prior to the loss of their myelin sheath and/or their demise.

CD4+CD25+ regulatory T cells in patients with Multiple Sclerosis

Viglietta V, Baecher-Allen C & Hafler DA¹*, Laboratory of Molecular Immunology, Harvard Medical School, Center for Neurologic Disease, Brigham and Women's Hospital, Boston, MA, U.S.A.

E-mail: hafler@cnd.bwh.harvard.edu

Multiple sclerosis is a chronic inflammatory disease characterized by lymphocyte infiltration and demyelination in the central nervous system. Autoreactive T cells capable of recognizing peptides derived from myelin proteins are involved in the pathogenesis of the disease. Populations of regulatory T cells finely modulate these self-reactive T cells. It has been shown that the neonatal thymectomized mice developed multi-organ autoimmune disease due to a loss of a CD4+CD25+ T cell regulatory population in their peripheral lymphoid tissues and that adoptive transfer of this cell population into thymectomized animals prevented autoimmune disease. We have identified a population of CD4+CD25hi regulatory cells in the circulation of normal humans able to effectively inhibit proliferation and cytokine production of CD4+CD25- responders T cells. We hypothesize that a loss of CD4+CD25hi regulatory T cells, either in frequency or function, is responsible for the lack of immunoregulation observed in patients with MS. We are directly examining the number, phenotype, and functional competence of CD4+CD25hi regulatory cells from peripheral blood of MS patients and healthy controls. This study will provide the opportunity to determine whether alterations of these regulatory cells are involved in the induction of autoimmune diseases. A progress report of the analysis will be presented.

SESSION IV: ANIMAL MODELS I

Genetics of EAE

Holmdahl R, Medical Inflammation Research, Lund University, Lund, Sweden

E-mail: rikard.holmdahl@inflam.lu.se

Genetic analysis of EAE may provide insight into which genes are involved and which pathways are operating in encephalomyelitis. However, in similarity with MS the experimental models are also complex polygenic diseases. Thus, there is not on or a few genes that explain the autoimmune pathology but several. The disease is not spontaneous but highly highly dependent on environmental factors, one of the immunization with myelin protein that triggers the pathogenic event s leading to disease. Forward and reversed genetic approaches to dissect the genetic control of EAE will be discussed. Using forward genetic approaches we and others have been identifying several gene regions in different mouse ands rat crosses that control EAE. From these studies it is clear that:

1) EAE is a polygenic disease controlled by different sets of genes in different inbred strains

2) Different species, mouse and rat and also humans, preferentially seem to share many of the identified gene regions.

3) Many gene regions are shared with other inflammatory diseases, in particular arthritis

4) So far the only gene that have been identified and confirmed are MHC class II genes influencing the linkage to the MHC region.

Recent studies on several of the identified gene regions will be presented.

Poster No. III-1

Glial reactions of brain tissue in multiple sclerosis plaques

Bisaga GN, Gajkova ON, Onischenko LS & Chikurov AA, Department of Neurology Military Medical Academy, St.Petersburg, Russia

E-mail: bisaga@mailbox.alkor.ru

Introduction: According to current opinion astrocytes proliferation is suggested to play the main role in the glial scar formation during the maturing of multiple sclerosis (MS) plaque. But not all aspects of this process are clear.

Methods: Two autopsy brains from MS patients saffered of the disease 8 and 15 years were studied by light and electron microscopy. Immunohystochemistry with detection of CD3, CD20 and leucocyte common antigen (LCA) was conducted.

Results: It was found that in plaques myelin fibers were absent. Also only single oligodendrocytes and mikrogliocytes were seen. The number of astrocytes with their processes was gradually decreased from their edge to centre of plaque. That brings to formation of irregular shape multiple microcavities without distinct boundaries.

At the same time in twice more often, than mature astrocytes, we found small lymphocyte-like cells with dark nucleus and narrow rim of cytoplasm. At electron microscopy the nuclei of these cells had dense chromatine, looked in the form of spokes, that is similar to oligodendrocytes apoptosis at EAE. In the narrow rim of cytoplasm we revealed scanty set of organoids. Cytolemma in such cells was found not always. CD3, CD20 and LCA were not discovered.

Conclusion: Thus predominating in MS plaques small cells with dark nucleus, narrow rim of cytoplasm and scanty set of organoids most probably represent to be immature glial cells, being in the stage of apoptosis. Our findings can be useful for better understanding of MS pathogenesis.

Poster No. III-2

Cytokine-induced cell death in human oligodendroglial cell lines: analysis by micro-array technology

Buntinx M, Steels P, Ameloot M, Raus J & Stinissen P Biomedical Research Institute, Limburg University Center and Transnational University Limburg, Diepenbeek, Belgium

In MS, oligodendrocytes (OL) may be under direct attack by the cytokines TNF-a and IFN-g. We studied the cytotoxic effects of TNF-a and IFN-g on the human oligodendroglial cell lines HOG and MO3.13. Our data show that exposure to TNF-a and IFN-g induces apoptosis in both cell lines in a dose-dependent fashion. Stimulation with TNF-a or IFN-g caused a significant reduction in cell viability in both cell lines within 72h, as measured by WST-1 colorimetry. Correspondingly, increased numbers of apoptotic cells were detected by Annexine-V/PI flowcytometry. The synergistic action of both cytokines resulted in enhanced cell cytotoxicity and higher numbers of apoptotic cells. This synergistic effect was more pronounced in the HOG cells. The apoptotic character of cell death was confirmed by Hoechst fluorescence and electron microscopy, showing typical chromatin condensation in dying cells. RNase protection assay revealed that IFN-g specifically activates caspase-1 mRNA in addition to other caspases that are also activated by TNF-a. IFN-g induced an upregulation of MHC-I molecules in both cell lines, while TNF-a only upregulated MHC-I in MO3.13 cells. Remarkably, TNF-a induced an upregulation of Fas expression in MO3.13, whereas IFN-g caused this effect in HOG. In line, we observed an increased sensitivity of IFN-g-primed HOG cells to FasL-induced cell death. Addition of NGF-b could not prevent TNF-a and IFN-g-induced apoptosis. Effects of IFN-b and Il-10 are under investigation. We are currently measuring differential gene expression in treated versus control cells using DNA microarray technology. Preliminary results showed differential expression of 67 genes over 4224 genes tested. This study indicates that oligodendroglial cell lines respond to TNF-a and IFN-g by undergoing apoptosis. The cell lines appear to be good models to study the molecular mechanisms of cytokine-induced cell death, which can contribute to our understanding of OL damage in MS.

Poster No. III-3

IFN-g-induced Ca²⁺-influx in T lymphocytes of multiple sclerosis and rheumatoid arthritis patients: a complementary mechanism for T cell activation?

Buntinx M, Steels P, Ameloot M, Janssen P, Medaer R, Geusens P, Raus J & Stinissen P, Biomedical Research Institute, Limburg University Center and Transnational University Limburg, Diepenbeek, Belgium

E-mail: mieke.buntinx@luc.ac.be

Autoreactive T lymphocytes are considered to play a crucial role in orchestrating a chronic inflammation in the central nervous system (CNS) of multiple sclerosis (MS) patients and in the joints of rheumatoid arthritis (RA) patients. However, it has been suggested that the majority of T cells in the immune infiltrate are nonspecifically recruited into the CNS and into the inflamed joint. In addition, several lines of evidence suggest an important role for interferon-g (IFN-g) in the pathogenesis of MS and RA.

We have studied whether peripheral blood T cells from patients with autoimmune diseases are more susceptible to activation in the presence of IFN-g. The results indicate that IFN-g mediates a sustained elevated [Ca²⁺]_i in T cells of (active) MS and RA patients as compared to healthy controls and patients with common viral infections. No [Ca²⁺]_i increase was observed in Ca²⁺-free medium, excluding an effect of IFN-g on Ca²⁺-release from intracellular stores.

Although the IFN-g-activated Ca²⁺-influx is insufficient to induce T cell proliferation in vitro, our data indicate a significantly augmented proliferation in response to suboptimal doses of PHA in the presence of IFN-g.

This study suggests that the IFN-g-induced Ca²⁺-influx can act as a complementary mechanism in the activation of blood T lymphocytes from MS and RA patients.

Poster No. III-5

CXC chemokine receptors expression in chronic relapsing experimental autoimmune encephalomyelitit

Glabinski A¹, Bielecki B¹, Selmaj K¹ & Ransohoff RM², ¹⁾Department of Neurology, Medical University of Lodz, Lodz, Poland, ²⁾Dept. of Neurosciences, Cleveland Clinic Foundation, Cleveland, OH, USA

E-mail: aglabinski@afazja.am.lodz.pl

Chemokines are potent chemoattractants that regulate migration of inflammatory cells to areas of inflammation. Inflammatory cells respond to chemokine gradient through the chemokine receptors.

The goal of this study was to analyze expression of chemokine receptors belonging to CXC subfamily (CXCR) during different stages of chronic relapsing experimental autoimmune encephalomyelitis (ChREAE) – an experimental model of multiple sclerosis. ChREAE was induced in (SJL/SWR)F1 female mice with 139-151PLP peptide. Expression of CXCR was measured with quantitative Rnase Protection Assay (RPA) in spinal cord and blood. We found significantly increased expression of CXCR2, CXCR3 and CXCR4 in the spinal cord during the first and second disease attacks. The kinetics of this expression in CNS and blood suggest that CXCR2 is expressed by leukocytes migrating from the blood to the CNS, but CXCR4 is expressed mainly by CNS parenchymal cells. Those results support the interpretation that chemokine-chemokine receptor interactions may play an important role in the development of CNS autoimmune inflammation.

Poster No. III-7

A functional and structural basis for T cell receptor crossreactivity in multiple sclerosis

E-mail helle_j@hotmail.com

Poster No. III-9

Studying oligodendroglia in human brain bank material

L. Lyck, I. Dalmau, B. Pakkenberg, K. Kock, H. J. G. Gundersen, H. D. Schrøder and B. Finsen, Anatomy and Neurobiology, University of Southern Denmark, Odense, Denmark

E-mail llyck@health.sdu.dk

Until now quantitative studies on the ontogenesis and differentiation of neural cells during human brain development has focused on the neuronal cell population. The increasing knowledge on the biology of oligodendroglia and the role of these cells and myelin in normal brain function, has made it relevant to create tools for studying the relationship between oligodendroglia and neurons in the human brain. Histological tools based upon immunohistochemical detection of cell type specific markers have in general not been applicable to unbiased stereological quantification and the use of the optical disector. Here we present our strategy to apply specific, robust and standardised immunohistochemical staining techniques to human brain bank material, which due to prolonged fixation presents with low antigenicity, for the use in quantitative studies based upon unbiased stereological methodology and the use of the optical disector. The strategy involves 1) systematical analyses of a spectrum of cell-type and cell-stage specific markers of oligodendroglial cell-lineage, 2) acquisition of full penetration of the histochemical stain, without affecting the specificity or sensitivity or inducing shrinkage of the section, and 3) standardisation and validation of the protocols to yield reproducible staining in tissues of various origins and preparation. On this basis we now present a tested selection of protocols that should be further optimised and standardised for the use in thick sections. This study opens the doors for the use of unbiased stereology for analysis of the cell populations of the human brain in research and pathological diagnostics, and is an ongoing part of a large collaborative project between departments of basic neuroscience and clinical pathology in Odense, Copenhagen and Aarhus with the final goal of performing an exhaustive, quantitative characterisation of the developing and normal adult human brain.

Poster No. III-10

Measles virus receptor usage varies on endothelian cells from different origins

Nichols C, Fleming E & Armstrong, MA, Dept. Microbiology & Immunobiology, Queen's University of Belfast, UK

E-mail: c.nichols@qub.ac.uk

Many viruses from a wide variety of families have been associated with MS. Most prominent of these have been HHV-6 and measles virus. Most recently, significantly increased levels of CD46 in serum and CSF of MS patients have been reported, suggesting a role for complement activation in disease pathogenesis. However, CD46 in addition to its role in complement regulation, is also one of the cellular receptors for measles virus (MV). Other MV receptors include SLAM (Signalling Lymphocytic Activation Molecule) which is found predominantly on T and B cells, and Fc-γRII (CD32).

The aim of this study is to investigate whether the modulation of MV receptors on endothelial cells, and the interaction of endogenous cytokines produced by these cells influence the subsequent viral infectivity.

Primary Human Umbilical Vein endothelial cells (HUVEC), an endothelial cell line derived from liver adenocarcinoma (SKHep-1), an epithelial cell line from bladder carcinoma with endothelial cell characteristics (ECV304) and Vero cells were each infected with the Edmonston measles strain (TC243) at a multiplicity of infection of 0.1.

Flow cytometric analysis at different time points during viral incubation showed that while CD46 was expressed on all of the cells, the levels varied between the different cell types; SLAM levels were significantly upregulated with prolonged exposure to MV on SKHep-1 cells; while Fc-γRII receptor was higher on HUVEC than on the other cell types. Upregulation of SLAM on SKHep-1 may correlate with increased virus infection and exposure to TNFα. This is the first time to our knowledge that SLAM expression has been described on endothelial cells.

Poster No. III-12

Multiple Sclerosis: Deficient Expression Of Transcription Factor Sp3 In Peripheral Blood Mononuclear Cells

Li-yan Qiao, Hua Zhang, Jian Yin, Xue-fei Wei, Hong Wang, *Guang-zhi Liu, Xian-hao Xu

Department of Neurology, Beijing Hospital, Ministry of Health, Beijing 100730,China

E-mail guang-zhi-liu@neurotec.ki.se

Introduction: Sp3 belongs to a family of human transcription, which binds GC/T box elements to either activate or repress transcription. The expression of several genes involved in the control of immune response, have been found to be regulated by Sp3, including Fas, transforming growth factor-beta(TGF-b) and T cell receptor V gene segment, etc. Multiple sclerosis (MS) patients demonstrated increased immune response to a variety of antigens of infectious and host origin, indicating the abnormal alternations in the control mechanism in the process of immune response. it was reported that Sp3 expression was not detected in peripheral blood mononuclear cells (PBMCs) of patients with MS. Therefore, there might be a correlation between Sp3 expression and the control of humoral and cellular immune response of these immune cells in MS.

Objective: The exact expression of transcription factor Sp3 are evaluated in Chinese MS patients, in combination with their clinical manifestations and the relating immunological parameters.

Methods: Poser criteria is adopted to establish the diagnosis of MS. After PBMCs are isolated by Ficoll-Hypague density centrifugation, blood Sp3 expression is measured by reverse -transcriptase polymerase chain reaction (RT-PCR) in 33 cases of MS patients. Expanded disability status scale (EDSS) is used to evaluate disease severity. Meanwhile, the immunological parameters, including serum soluble interleukin-2 receptor (sIL-2R), oligoclonal band (OB), IgG intrathecal synthesizing rate per 24 hours (IgGsyn), IgG index are examined respectively. In addition,26 cases of other neuroimmunological diseases (myasthenia gravis, Gullain-Barre syndrome) patients, 30 cases of non-neuroimmunological diseases(amyotrophic lateral sclerosis , Meige syndrome, cerebrovascular diseases, epilepsy) patients and 30 cases of healthy individuals are included in this study.

Result: The negative rate of Sp3 expression is higher in MS group (44.45%) than the other three groups, including other neuroimmunological diseases (6.7%, P<0.01), non-neuro-immunological diseases (16.67%, P<0.01) and normal controls (10%, P<0.05). In the MS groups, MS patients with negative Sp3 expression had increased level of EDSS, IgGsyn, sIL-2R in comparison with those with positive Sp3 expression. No dynamic changes of Sp3 expression were observed in 4 cases with MS in either acute or remitting stage.

Discussion: The blood Sp3 expression are suppressed in 45 percentage of MS patients, indicating higher deficiency of Sp3 expression in Chinese MS patients, compared to that in Western countries. Furthermore, those MS patients with deficient Sp3 expression tend to lead to more severe features, including both clinical manifestations and immune dysfunction. Therefore, it is indicated that transcription factor Sp3 might play an important role in the abnormal immunoregulatory mechanisms of MS.

SESSION IV: ANIMAL MODELS II

Autoimmune T cell response to myelin antigens: Induction, regulation and tuning
Kuchroo, VK, Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, U.S.A.

E-mail: kuchroo@cnd.bwh.harvard.edu

T cells that can respond to self antigens are present in peripheral immune repertoire of all healthy individuals. Recently we have found that the unmanipulated SJL mice that are highly susceptible to EAE also maintain a very high frequency of T cells responding to an encephalitogenic epitope of PLP 139-151 in the peripheral repertoire. This is not due to lack of expression of myelin antigens in the thymus resulting in escape of the PLP reactive T cells from central tolerance, but due to expression of a splice variant of PLP named DM20 which lacks the residues 116-150. Inspite of this high frequency, the PLP 139-151 reactive cells remain undifferentiated in the periphery and do not induce spontaneous EAE. This may be because these autoreactive cells are kept under check by an alternate PLP 139-151 specific non-pathogenic repertoire and thus the pathogenic and protective repertoires maintain a balance in healthy individuals. Transgenic mice generated from the TcRs of the pathogenic PLP 139-151 specific T cells result in the induction of fulminant spontaneous EAE in the vast majority of the transgenic mice. However, the transgenic mice that are generated from the TcRs of the alternate PLP 139-151repertoire, do not develop EAE. We have also recently generated TcR transgenic mice expressing TcRs specific for another myelin antigen, myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide in the C57Bl/6 mice. Whereas only limited a number of the MOG TcR transgenic mice develop spontaneous EAE (<5%), a large proportion (>35%) of the MOG-TcR transgenic mice develop optic neuritis without clinical and histological EAE. Thus, whereas PLP 139-51 specific TcR transgenic mice develop spontaneous EAE, the MOG-specific TcR transgenic mice predominantly develop optic neuritis, suggesting a role for different myelin antigens together with genetic backgrounds in the development of different disease phenotypes in MS.

Genetics and pathogenesis of rat neuroinflammation; MOG induced EAE and experimental neurodegeneration

Olsson T, Neuroimmunology Unit, Department of Medicine, Karolinska Hospital, Stockholm

E-mail: tomas.olsson@cmm.ki.se

Details in steps leading to autoaggressive T and B cell mediated damage to the CNS are continuously dissected. However, genetically regulated bottlenecks which allow disease in one individual, but not another are still largely unknown. Such disease regulating genes, or pathogenetic pathways defined by them, would be ideal targets for new therapeutic strategies. A search for disease regulating genome regions (quantitative trait Loci-QTLs) can be done in animal crosses which requires no preformed hypotheses. Striking advantages with disease gene mapping studies in experimental animals as compared to humans are that genetic heterogeneity can be reduced and variation in environmental influences minimized. The experimental models offer possibilities to study disease mechanisms that are shared between species, which can lead to disease. To reveal pathways of importance for human disease, the used models should mimic multiple sclerosis (MS) as closely as possible. This is the case with myelin oligodendrocyte glycoprotein (MOG) induced rat experimental autoimmune encephalomyelitis (EAE). In total, we have mapped 12 QTLs regulating MS like chronic EAE.

Collective conclusions from these studies are that: 1) Some of the regions co-localize with genome regions regulating also other organ specific- inflammatory diseases such as experimental arthritis and experimental autoimmune neuritis (EAN), a model for the Guillain Barre´syndrom. 2) There is a genetic heterogeneity in that different strain combinations reveal different genome regions regulating disease. This means that different individuals are predisposed for autoimmune disease due to different genetic polymorphisms. 3) By synteny, relevance of the genes can be studied in materials of human disease by association analysis. Preliminary evidence suggests that the genes defined experimentally also have impact on human disease.

Using a CNS damage paradigm, we have demonstrated that; (i) phenotypic differences in the inflammatory response to mechanical CNS injury are regulated mainly by non-major histocompatibility (MHC) genes, (ii) different aspects of the inflammatory response are regulated by independently and (iii) that the proneness to mount inflammatory responses in the CNS only partially overlap with the susceptibility for EAE. A locus regulating neuron death has been mapped. An intriguing possibility is that this type of genetic regulation of inflammatory responses is also important for the evolution of pathology in a broader collection of CNS diseases, such as neurodegenerative and cerebrovascular diseases and trauma.

Congenic rat strains and advanced intercross lines allow further fine mapping and disclosure of the pathogenetic steps involved.

MBP-Specific TCR Transgenic Mouse Models: Multiple Mechanisms of Tolerance, Immunoregulation and Autoimmune Disease

Goverman J, University of Washington, Seattle, U.S.A.

E-mail: goverman@u.washington.edu

Myelin basic protein (MBP) is a major component of the myelin sheath that is believed to be targeted in the autoimmune disease multiple sclerosis. My laboratory investigates how immune tolerance to MBP is maintained and how it breaks down in an effort to understand basic mechanisms involved in the pathogenesis of MS. We identified immunogenic regions in MBP that are recognized by either CD4⁺ or CD8⁺ T cells and generated TCR transgenic mice to study the fate of these T cells in vivo. In B10.PL mice, we established CD4⁺ TCR transgenic mice specific either for MBP1-11 or MBP121-140. CD4⁺ MBP1-11-specific T cells largely escape tolerance while CD4⁺ MBP121-140-specific T cells undergo substantial tolerance induction in vivo that occurs in both the thymus and the periphery. Interestingly, tolerance is mediated by bone marrow-derived antigen-presenting cells that constitutively process and present exogenous MBP in both the CNS and periphery. In the absence of regulatory T cells, MBP121-140-specific T cells mediate a severe and widespread autoimmune disease reflecting the continuous presentation of MBP epitopes in the CNS and periphery. We have also identified CD8⁺ MBP79-87-specific cytotoxic T cells in C3H mice and generated two TCR transgenic lines expressing different TCRs specific for this epitope. One model exhibits multiple mechanisms of central and peripheral tolerance while the other TCR transgenic model ignores its self-antigen in vivo. The implications of these disparate fates of MBP-specific T cells in vivo for autoimmune disease, as well as the different types of autoimmune disease observed in these models, will be discussed.

Interferon-gamma plays an essential role in differential inflammatory localization of encephalitogenic T cells

Wensky AK & Lafaille JJ, Skirball Institute for Biomolecular Medicine, New York University Medical Center, New York, NY, U.S.A.

E-mail: lafaille@saturn.med.nyu.edu

Myelin basic protein (MBP)-specific CD4⁺ naïve T cells primed in vitro with MBP in the presence of blocking anti-IFN-g antibodies during the first three days of culture, are capable of transferring a severe form of experimental autoimmune encephalomyelitis (EAE). However the clinical aspects of the disease are unique, in that the animals display head-tilt, rolling on their back, spinning, and have great difficulty in maintaining the right position. We refer to this disease as “non-classical” EAE. Examination of the various organs of the central nervous system (CNS) by histology or FACS revealed that the inflammatory infiltrate predominated in the brainstem and cerebellum in animals which displayed non-classical EAE, whereas, as expected, predominated in the spinal cord in animals afflicted with classical EAE, which can be induced by MBP-specific T cells primed without IFN-g blocking. Thus, both classical and non-classical EAE can be induced by MBP-specific T cell receptor (TCR) transgenic T cells that are initially identical, but are primed with MBP under slightly different conditions.

A spontaneous model for non-classical EAE was developed utilizing MBP-specific TCR transgenic mice crossed into a dual RAG-1 and IFN-g deficient background. These mice reproducibly develop a severe, non-classical form of EAE with 100% incidence with a predominant cellular infiltrate found in the brainstem and cerebellum as opposed to the spinal cord.

Vertigo is a known occurrence in some multiple sclerosis (MS) patients and is often accompanied by brainstem inflammation. At least with regard to some clinical and histological aspects, non-classical EAE may serve as a model for inflammation-triggered vertigo.

Our data supports a model whereby T lymphocytes primed in an IFN-g-poor microenvironment acquire the property to migrate into cerebellum/brainstem whereas T lymphocytes with identical antigen specificity but primed in the presence of IFN-g preferentially migrate to the spinal cord.

Poster No. IV-4

Experimental autoimmune encephalomyelitis; a new multiple sclerosis model for therapy development

‘t Hart B, Biomedical Primate Research Centre, GJ, The Netherlands

E-Mail: hart@bprc.nl

Immunization of common marmosets with human myelin in complete adjuvant evokes in 100% of the animals that in its clinical, radiological and neuropathological presentation chronic MS1,2. Myelin/ oligodendrocyte glycoprotein (MOG) is a key antigen in the initiation and progression of the disease. In each animal EAE is initiated by the Caja-DR*W1201-restricted activation of CD4+ T-cells specific for the encephalitogenic MOG peptide 14-36. Note that Caja represents the common marmoset equivalent of the HLA. Progression of the disease is associated with progressive broadening of the T-cell reactivity with other MOG-peptides, which are presented by polymorphic Caja-DR molecules. Anti-MOG antibodies, in particular those binding to conformational epitopes are critical for demyelination. Due to its proximity to MS this new non-human primate model is highly suitable to test the effectivity of new anti-MS therapies, especially the ones that do work insufficiently in rodents, such as most biotechnology-derived products. An important aspect of the model for therapy development is the possibility to visualize brain white matter lesions with magnetic resonance imaging (MRI). We have implemented a broad panel of qualitative (T2, post-contrast T1) and quantitative (T1, T2 and MTR maps) NMR parameters to monitor the formation of brain white matter lesions the formation of various lesion subtypes can be monitored. Serial MR imaging at two weeks intervals is technically feasible and can be used to determine the effect of new experimental therapeutics on already existing lesions and on the formation of new lesions. I intend to highlight the power of the model using results from recent antibody-based immunotherapy trials.

Poster No. IV-7

Therapeutic potential of IFN-g-modified dendritic cells (IFN-g-DC) in acute and chronic experimental allergic encephalomyelitis (EAE)

Link, H, Yu-Min Huang, Xing-Chen Wu, Bjelke B, Bao-Guo Xiao. Div. Neurology, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden

E-mail: hans.link@neurotec.ki.se

DC are antigen-presenting cells specialized to regulate immune responses. DC activate T cells, and may also tolerise T cells to antigens, thereby minimizing autoimmune responses. We induced acute EAE in Lewis rats by immunization with MBP peptide 68-86, and chronic-relapsing EAE in SJL/J and B6 mice with MOG peptide 35-55, or PLP peptide 139-151, plus FCA. During incipient EAE (day 5 p.i. in rats, day 7 in mice), healthy rat/mouse splenic DC that had been exposed in vitro to IFN-g (IFN-g-DC), were injected subcutaneously (1x10⁶ IFNg-DC per animal). Severity of clinical signs of EAE was dramatically reduced in all IFN-g-DC-treated animals, showing normal magnetic resonance imaging (MRI) of the spinal cord and brain. In contrast, the EAE rats receiving PBS or naive DC had severe clinical signs with multiple and extensive MRI lesions in the spinal cord and brain. IFN-g-DC showed no change of MHC class II, CD80 or CD86 expression, or of antigen presentation capacity, suggesting that IFN-g does not promote DC maturation, as compared with LPS-induced DC maturation. IFN-g-DC-mediated protection from EAE was accompanied by increased proliferation, IFN-g-, IL-10- and NO-production. IFN-g-DC induced apoptosis of MBP-reactive CD4⁺ T cells in vitro. IFN-g-DC-treated animals showed increased frequencies of apoptotic cells among blood mononuclear cells and in spinal cord sections, whereas macrophage and CD4⁺ T cell infiltrates within the central nervous system were dramatically reduced. IFN-g-DC may eliminate EAE by enhanced apoptosis among autoreactive T cells and/or by IL-10-producing regulatory cells. This approach may represent a novel possibility of individualised immunotherapy using autologous, in vitro modified DC as a complement to conventional therapy in multiple sclerosis and other diseases with an autoimmune background.

Poster No. IV-13

Prevention of experimental autoimmune encephalomyelitis by a nonapeptide totally unrelated to the encephalitogen

Weissert R, de Graaf KL, Herrmann M, Barth S, Fleckenstein B, Olsson T, Jung G, Melms A & Wiesmüller KH, Department of Neurology, University of Tübingen, Germany

E-mail: robert.weissert@uni-tuebingen.de

Combinatorial nonapeptide libraries were used to create a complete activity pattern containing favorable and unfavorable amino acid residues for binding to the rat DR-like MHC molecule RT1.Dⁿ. By combining amino acids defined as favorable in sequence positions 1 to 9 several individual acetylated nonapeptide amides were created which revealed high affinity for the RT1.Dⁿ allele. One of these high affinity binders (Ac-FWFLDNAPL-NH₂) successfully inhibited experimental autoimmune encephalomyelitis after co-immunization with the extracellular domain of myelin-oligodendrocyte-glycoprotein (MOG 1-125) and its encephalitogenic core peptide MOG 91-108 (SDEGGYTCFFRDHSYQEE) in LEW.1N (RT1ⁿ) rats. Ac-FWFLDNAPL-NH₂had strong immunomodulatory capacities by raising strong CD4⁺ T cell responses. These results demonstrate that it is possible to prevent EAE with a rationally designed RT1.Dⁿ nonapeptide ligand structurally totally unrelated to the encephalitogen. The search for immunomodulatory high-affinity MHC binders could be a successful approach for prevention of MHC-linked autoimmune diseases in humans.

Poster No. IV-14

CD1 gene regulates chronicity of CNS inflammation in experimental autoimmune encephalomyelitis (EAE)

Teige A, Teige I, Lavasani S, Holmdahl R, Grusby M & Issazadeh-Navikas S, Section for Medical Inflammation Research, DBC, University of Lund, Lund, Sweden

E-mail: shahram.lavasani@inflam.lu.se

Mouse CD1 is a molecule with antigen presenting functions expressed on hematopoietic cells. It has been implicated that CD1 is playing a regulatory role in tolerance and autoimmunity via CD1 dependent NK T cells. Here, we aim to investigate the function of CD1 in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), by utilizing CD1 knock out (ko) mice. We report that CD1 deficient mice have higher susceptibility to EAE and develop a much more severe and chronic EAE compared to wild type (wt) mice. The histopathological investigation reveals that immune cell infiltration in CNS is also significantly increased in CD1 ko mice which correlates well with significantly higher degree of demyelination observed in CD1 ko mice compared to wt. This was not dependent on higher capacity of autoreactive T cells in CD1 ko mice to proliferate to CNS antigens. However, in the initial stage of disease, encephalitogenic T cells in CD1 ko mice produced elevated amount of IFN-g and IL-4 compared to wt. Moreover, there were no differences in production of these cytokines in CNS, indicating that the role of CD1 molecule in down-regulation of neuroinflammation and consecutive clinical symptoms is not attributed to the capacity of NK1.1+ T cells to produce high amount of IL-4. Interestingly, we found that a significant number of cells in CNS of wt mice produced TGF-b, a cytokine with potential anti-inflammatory properties, prior to clinical remission. Moreover, passive transfer of EAE with encephalitogenic T cell line caused same degree of diseases in both CD1 ko and wt mice indicating that CD1-restricted NK T cells need activation through immunization to exert their regulatory function. These findings strongly suggest that CD1 and CD1 dependent cells are playing a major role in EAE in preventing a pathogenic chronic inflammation directed to self in CNS.

SESSION V: TOLERANCE I

Inhibition of Inflammation: Activating and Inhibitory Receptor-Mediated Regulation of Microglia and Myeloid Cell Function

Sedgwick JD, Cherwinski H, Phillips J, Murphy C & Joyce-Shaikh B,. DNAX Research Inc., Palo Alto, California, USA.

E-mail: jon.sedgwick@dnax.org

Through in vitro analyses, cells of the myeloid lineage, including natural killer (NK) cells and macrophages, are known now to be regulated through cell-cell interactions that trigger matched sets of activating and inhibitory receptors. More than forty distinct receptors have been cloned, many falling structurally within the immunoglobulin superfamily of integral membrane proteins. The last three years has seen the first in vivo data emerge that signalling through these receptors is essential for normal myeloid cell regulation in the peripheral immune system. Disruption of signalling through SIRP-a, TREM-1 and FcgRIIB, for example, all either activating or inhibitory receptors expressed on myeloid cells, lead to inhibition or potentiation of cellular function with consequent effects on inflammatory disease processes. These regulatory pathways also extend to the central nervous system (CNS). Thus, we have demonstrated recently that the myeloid inhibitory receptor CD200R interacting with the neuronally expressed ligand CD200 (OX2), strongly influences activities of microglia, the resident CNS macrophage. CD200 is expressed broadly in neural and lymphoid tissues, but is normally not expressed by cells of the myeloid lineage. The CD200R is restricted substantially to the myeloid lineage, including on resting ramified microglia in the adult CNS. Ongoing studies indicate that other receptor-ligand pairs with similar functions to CD200-CD200R exist within the CNS. The role of activating and inhibitory receptors in CNS inflammatory responses will be discussed as well as the implications of these findings for the study of microglia in vitro, removed from the influence of an environment rich in the expression of ligands delivering activating or inhibitory signals.

Molecular Mechanisms of Initiation and Immunoregulation of Virus-Induced Autoimmune Disease

Miller, SD, Olson, JK & Croxford, JL, Northwestern University Medical School, Dept. Microbiology-Immunology, Chicago, IL, U.S.A.

E-mail: s-d-miller@northwestern.edu

Epidemiologic evidence strongly indicates an infectious etiology for multiple sclerosis (MS), an autoimmune disease characterized by CD4⁺ T cell responses to myelin proteins. Theiler’s murine encephalomyelitis virus (TMEV) induces a chronic, CD4⁺ T cell-mediated demyelinating disease with a clinical course and histopathology similar to chronic-progressive MS. Infection of SJL mice with wildtype TMEV leads to a demyelinating disease initiated by virus-specific CD4⁺ T cells targeting virus persisting in CNS macrophages/microglia, while chronic disease is associated with the activation of myelin epitope-specific CD4⁺ autoreactive T cells via epitope spreading. To examine the feasibility of virus-encoded mimic epitopes to initiate immune-mediated demyelination, a non-persisting variant strain of TMEV was engineered to encode a 30-mer peptide encompassing the immunodominant myelin PLP139-151 epitope. SJL mice infected with TMEV encoding either the native PLP139-151 determinant or a mimic epitope normally expressed by the Haemophilus influenzae protease IV protein, sharing only 6/13 amino acids with PLP139-151, developed an early-onset demyelinating disease mediated by PLP139-151-specific Th1 cells. Disease can be induced following virus infection by multiple peripheral routes, as well as intracerebrally, indicating that the primary infection does not have to be in the CNS target organ. The autoimmune nature of this early-onset demyelinating disease is shown by the fact that induction of tolerance to the native mouse PLP139-151 epitope prevents clinical disease and associated PLP139-151-specific T cell responses in animals infected with TMEV encoding either the native epitope or the bacterial mimic epitope without affecting T cell reactivity to virus epitopes. These studies provide conclusive evidence that myelin-specific autoreactive T cells can be induced either by epitope spreading or molecular mimicry following virus infection.

Poster No. IV-1

Mast cells may induce demyelination after disruption of the blood brain barrier

Baloyannis SJ & Costa V, 1^st Department of Neurology, Aristotelian University, Thessaloniki, Greece

E-mail: grmps464@attglobal.net

Intravenous injection of mast cells, collected from the peritoneal cavity of rodents, may provoke extensive demyelination following disruption of the blood brain barrier induced by mild heat lesion on the skull. The present study describes the type and the rate of demyelination following mast cell administration in light and electron microscopy.

We induced mild heat lesion on the skull in 21 Lewy rats. In 7 of them mast cells were injected, collected from the peritoneal cavity of the same animals, enriched with the factor 48/80.In 7 of the animals only mast cells were injected and in 7 of them only the factor 48/80 was injected. We started sacrificing the animals every 48 hours following the heat lesion until the 20^th day when we sacrificed the last animal. Samples from the brain located underneath the heat lesion, were processed for light and electron microscopy. Extensive disintegration of the myelin sheath was observed 48hours following the heat lesion in the animals which received mast cells enriched with the factor 48/80. At the same time accumulation of mast cells and microglial cells was seen around the capillaries in the subcortical white matter. By the 72 hours large number of macrophages and astrocytes were accumulated. By the end of the first week oligodendrocytes were seen ensheathing the axons. Limited areas of demyelination followed by microglial activation were seen in the animals who received either mast cells or the factor 48/80 . In conclusion: Extensive demyelination and cellular interactions occur 48-72 hours following the administration of mast cells enriched with the factor 48/80. Animals who received either mast cells alone or the factor 48/80 demonstrated minimal lesions attributed presumably to the disruption of the blood brain barrier.

Poster No. IV-3

Perforant path lesioning induces sprouting of myelinated fibres in the mouse hippocampal formation

Drøjdahl N^1*, Hegelund IV¹, Poulsen FR¹, Wree A²& Finsen B¹, ¹Anatomy and Neurobiology, Institute of Medical Biology, University of Southern Denmark-Odense University, Odense, Denmark, ²Institute of Anatomy, Rostock University, Rostock, Germany

E-mail: ndrojdahl@imbmed.sdu.dk

In comparison to the rat, the anatomy of the mouse hippocampus, and in particular the response to entorhinal cortex lesioning, is less well-characterized. Here we studied the axonal sprouting response after lesioning of the entorhino-dentate perforant path projection in the brain of young adult SJL/J and C57BL/6 mice. The growth of new nervefibres were evaluated in the hippocampus by Timm silver-, acetylcholine esterase- and myelin staining of sections from mice put down 60 days after axonal transection. We found that lesioning led to translaminar sprouting of Timm stained CA3-associated fibre systems into the denervated termination zones of the CA3 and dentate gyrus, from the adjacent non-denervated stratum radiatum of CA3. Differences were seen in the Timm staining pattern of the two strains of mice, while the response to lesioning appeared similar albeit less pronounced than that observed in the rat. We also observed an intensified acetylcholine esterase staining reflective of cholinergic sprouting in the denervated perforant path termination zones, which was particularly prominent in areas with sprouting of Timm stained CA3-associated fibres. Finally, we showed that some of the sprouting fibres within the CA3 were myelinated, due to an increased density of silver impregnated myelinated fibres in this region after lesioning. These results show that the basic characteristics of the response to perforant path lesioning in mice are similar to those in the rat, but suggest that the magnitude of the response in the two species is different.

Poster No. IV-5

The Role of Angiogenesis in Experimental Demyelination

Karlik SJ & Kirk SL, University of Western Ontario, Dept. of Pathology, London, Ontario, Canada

E-mail: skarlik@irus.rri.on.ca

Although a link has been established between several chronic inflammatory processes and angiogenesis, only circumstantial evidence is available to support the existence of angiogenesis in the lesions of multiple sclerosis (MS). The objective of this study was to examine the role of neovascularization in demyelinated spinal cord lesions in chronic-progressive experimental allergic encephalomyelitis (CP-EAE) a model for MS. CP-EAE animals show acute inflammatory disease from d0-20 post-immunization followed by a chronic progressive (CP) course which shows extensive myelitis and demyelination. Spinal cord tissue was blindly evaluated from 64 CP-EAE and 17 non-EAE guinea pigs sacrificed intermittently over 90 days. Sections stained with hematoxylin-eosin were scored for inflammatory reaction in the meninges (M), parenchymal perivascular infiltration (P) and myelitis (E). Demyelination, remyelination and myelin debris (D) were evaluated in solochrome-R-cyanin stained sections. The number of factor VIII+ endothelial cells (F8) as an indication of blood vessels and the area and intensity of staining for vascular endothelial growth factor (VEGF) were measured. We found an increase in the number of F8 stained vessels in both the acute (25±3) and chronic (55±6) phases of disease compared to that of controls (9±1) (Anova on ranks, Dunn’s, P<0.05 on all comparisons). The number of F8 stained vessels correlates with clinical score (0.810) and the other pathological scores: M (0.841), P (0.850), E (0.818) and D (0.772) (Spearman, P<0.01). The highest number of vessels was seen in paralyzed chronic animals with extreme pathological scores. VEGF signals were completely absent in the controls animals but increase during the acute phase of EAE peaking at day 26 post immunization before gradually returning to nearly baseline levels by day 80. These observations implicate angiogenesis as a component of chronic neuroinflammation and demyelination and may suggest alternative therapeutic strategies for multiple sclerosis.

Poster No. IV-6

Anterograde (Wallerian) degeneration promotes leukocyte infiltration in mice during EAE

Ladeby R¹, Babcock A², Jensen MB^1,2, Owens T² & Finsen B¹, ¹University of Southern Denmark, Odense, Denmark; ²McGill University, Montreal, Canada

E-mail: rladeby@health.sdu.dk

Neurodegenerative phenomena have been reported diminish the immune privileged status of the CNS, and facilitate transmigration of activated lymphocytes into the affected areas. To investigate if axonal degeneration may attract autoreactive T-cells during EAE and MS, we used the perforant path (PP) lesion paradigm to simulate the transection of myelinated fibres that takes place in early MS plaques and performed this operation on mice with adoptively transferred MBP-reactive T-cells. PP transection was performed on normal adult female mice and mice committed to adoptively transferred EAE at the first clinical signs of disease. The mice were either perfused and had their brain processed for immunohistochemistry for CD4, CD8, CD11b, CD34, CD45 and CD54 or had their hippocampi microdissected for RT-PCR analysis for the chemokines RANTES and MCP-1. Axonal degeneration resulted in local production of MCP-1 and RANTES and increased vascular ICAM-1 expression. MCP-1 was significantly upregulated at day 2, while RANTES was upregulated at day 5. PP-lesioning of mice with adoptively transferred MBP reactive T-cells resulted in a dense infiltration of CD4+ and to a lesser extent CD8+ lymphocytes into the denervated zones of the fascia dentata starting at day 2 and in increasing numbers at day 4, in comparison to PP-lesioned mice and mice with EAE only, which contained few or no infiltrating lymphocytes in their hippocampi. Based on these results we propose that the axonal degeneration seen in multiple sclerosis may contribute to disease progression by attracting autoreactive T-cells and thereby facilitate the development of new lesions along the degenerating fiber bundles due to intraparenchymal production of chemokines and local activation of the BBB.

Poster No. IV-8

The characterization of Genz-29155: An oral, small molecule immunomodulator that inhibits disease in three models of murine Experimental Allergic Encephalomyelitis (EAE).

Davis J, Gregory J, Hirth B, Sneddon S, Williams J, Vinick F, Nazareno R, Kuchroo V, & Nahill SR

E-mail: sharon.nahill@genzyme.com

Clinical studies demonstrate that modulation of circulating TNF-alpha with either soluble receptor or antibody provides benefit to patients suffering from fistulizing Crohn’s disease, rheumatoid arthritis and psoriasis. Our goal is to find an easily administered, small molecule inhibitor of TNF-to treat these and other autoimmune diseases. Genz-29155 is a novel 657MW small molecule identified in a high-throughput screen designed to find inhibitors of TNF-alpha-induced apoptosis of murine fibroblast cells. Genz-29155 also inhibits TNF-alpha-induced apoptosis of primary human fibroblasts (i.c.50 » 2uM). The compound is effective when administered up to 4 hours after the addition of TNF-alpha suggesting that the activity occurs down stream of the TNF-alpha cell surface receptor. While the compound does inhibit several functions attributed to TNF-alpha (upregulation of IL-6, 8, and MCP-1), Genz-29155 does not inhibit Fas-ligand-induced apoptosis, TNF-alpha induction of VCAM expression by endothelial cells, or NFkB promoter-binding activity. However, the administration of Genz-29155 significantly prolongs survival in a TNF-alpha-dependent murine model of sepsis (6/6 20mg/kg Genz-29155-treated vs. 0/6 vehicle-treated mice surviving 12 hours post induction). These results encouraged us to test Genz-29155 in models of autoimmune disease. Although modest activity is seen in the dinitrobenzenesulfonic acid rat model of Inflammatory Bowel Disease (IBD), the compound shows a significant, reproducible and dose responsive delay of disease onset, reduction of mean maximum severity and improved survival in the SJL-PLP model of murine experimental allergic encephalomyelitis (EAE). Prophylactic and therapeutic dosing with Genz-29155 provides benefit in the relapsing-remitting (PLP induced disease in SJL mice) and chronic progressive (MOG induced disease in NOD mice) models of EAE. In addition, Genz-29155 completely inhibits relapse in the NOD and Biozzi models of relapsing disease. Histopathology of the brain and spinal cord shows a dramatic inhibition of lesion frequency and demyelination in the presence of drug. Preliminary adoptive transfer studies suggest that the compound inhibits both the induction and effector functions of T cells. These results, combined with the observation that long term dosing appears to be well tolerated, encourage us to examine Genz-29155 as a therapeutic candidate for Multiple Sclerosis.

Poster No. IV-9

A synthetic androstene derivative and a natural androstene metabolite inhibit EAE. Candidates for clinical trials in MS?

Offner H, Oregon Health and Science University, Postland VA Medical Center R&D-31, Portland, OR, U.S.A.

E-mail offnerva@ohsu.edu

Experimental allergic encephalomyelitis (EAE), a Th1 polarized demyelinating disease of the central nervous system (CNS), shares many pathological and clinical similarities with multiple sclerosis (MS), and thus represents an attractive animal model for this disease. A number of observations support the idea that fluctuations in sex hormone levels are related to changes in autoimmune disease status. Therefore, the goal of this study was to evaluate suppressive effects of fluasterone (HE2500), a synthetic androstene derivative, and androstenetriol (HE2200), a natural androstene metabolite on EAE. SJL mice were immunized with PLP-139-151 peptide/CFA to induce EAE. Starting on day –7, animals were given daily injections (s.c.) of derivatives (3.0 mg) in vehicle, or vehicle alone for 33 days. Both HE2500 and HE2200 significantly delayed the onset, reduced peak clinical score and cumulative disease index of EAE, and prevented or significantly attenuated relapses. Moreover, T cells from treated mice had significantly reduced PLP-139-151 specific T cell proliferation responses and reduced numbers of TNF-a and IFN-g producing cells in the CNS. In a different strain, B10.PL, daily treatment of mice with HE2500, starting on day 0 completely prevented development of disease. Finally, SJL mice treated with HE2500 at EAE onset showed significantly reduced mean clinical scores. In conclusion, we have shown that HE2500 and HE2200 can both completely prevent the development of EAE as well as ameliorate ongoing disease in two different models of EAE. The mechanism of action appears to involve a decrease in Th1-like immune responses. Since fluasterone is practically devoid of toxic, estrogenic, or androgenic side effects and retains the potent anti-proliferative, anti-inflammatory and immune regulatory activities of its parent molecule, especially in terms of providing benefit in EAE, it appears to be an ideal candidate for clinical trials in MS.

Poster No. IV-10

Metallothionein-I+II (MT-I+II) are antioxidant, neuroprotective factors

Penkowa M, Panum Instittue, Department of Medical Anatomy, Copenhagen, Denmark

E-mail: m.penkowa@mai.ku.dk

We examined the effects of MT-I+II deficiency during experimental autoimmune encephalomyelitis (EAE) in MT-I+II-knock-out (MTKO) mice. Also, we examined the effects of MT-II treatment during EAE.

In MTKO mice, EAE incidence and clinical symptoms are significantly increased relatively to normal mice. During EAE in MTKO mice, the inflammatory response of macrophages and T-lymphocytes was significantly increased, while reactive astrogliosis was decreased. Moreover, during EAE proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-a (TNF- a), as well as oxidative stress levels and apoptosis were significantly increased in MTKO mice relatively to normal mice.

Following MT-II treatment during EAE, disease incidence and clinical symptoms were significantly reduced as were the inflammatory response of macrophages and T cells. Also, proinflammatory cytokines (IL-1, IL-6, TNF- a), oxidative stress levels, and apoptosis during EAE were significantly reduced by MT-II treatment relatively to control treatment.

In addition, MT-II treatment of MTKO mice during EAE could significantly reverse the effects of the MT-I+II deficiency.

Furthermore, we have studied the effects of MT-II treatment upon human monocytes/macrophages derived from multiple sclerosis (MS) patients. Interestingly, in these human cells, the MT-II treatment could reduce significantly the expression of proinflammatory cytokines (IL-1, IL-6, TNF- a) and oxidative stress.

Thus, MT-I+II are likely important factors in the CNS during EAE.

Poster No. IV-12

The new orally active immunoregulator ABR-215062 effectively inhibits development and relapses of experimental autoimmune encephalomyelitis

Runström A, Brunmark C, Ohlsson L, Sparre B & Hedlund G, Active Biotech, Lund, Sweden

E.mail: anna.runstrom@activebiotech.com

A new orally active drug, ABR-215062, was evaluated in experimental autoimmune encephalomyelitis (EAE), which share important immunological and clinical features with Multiple Sclerosis (MS). ABR-215062 was shown to completely inhibit the development of acute EAE. Furthermore, leukocyte infiltration into the CNS was abolished in the ABR-215062 treated animals. By direct comparison, based on dose, ABR-215062 was approximately 20 times more potent than the immunomodulator roquinimex. However, when comparing the exposure of free, non-protein bound drug, the potency of ABR-215062 was more than 100 times that of roquinimex. ABR-215062 also had clear therapeutic effects when given after clinical onset in a chronic-relapsing EAE model. In summary, ABR-215062 was proven to effectively inhibit disease in two models of MS and represents a new orally active, immunoregulatory drug for this severe autoimmune disease.

Poster No. IV-15

Teige I, Treschow A, Teige A, Mattsson R, Navikas V, Leanderson T, Holmdahl R, and Issazadeh-Navikas S, Section for Medical Inflammation Research, University of Lund, I11, BMC, Lund, Sweden

E-mail: ingrid.teige@inflam.lu.se

The basic mechanisms behind beneficial effects of interferon beta (IFN-b) in multiple sclerosis (MS) patients is still obscured. We have investigated the effects of IFN-b gene disruption on the commonly used animal model for MS, EAE. Our results revealed significantly higher susceptibility to EAE with more severe and chronic neurological symptoms and a higher degree of CNS inflammation and demyelination in IFN-b knock out (ko) mice compared to wild type (wt) littermates. However, there was no discrepancy observed between the capacity of T cells to proliferate or produce IFN-g in response to recall antigen. Consequently, we addressed the effect of IFN-b on development of encephalitogenic T cells and initiation phase of EAE by passive transfer of MBP 89-101 reactive T cells to both groups. Interestingly, IFN-b ko mice develop augmented EAE with higher incidence regardless of the source of encephalitogenic T cells. Thus, the anti-inflammatory effect of endogenous IFN-b is predominantly exerted on the effector phase of the disease rather than during the priming of autoreactive T cells. Histopathological studies revealed extensive and persistent inflammation in CNS of IFN-b ko mice with infiltration of T cells producing both IFN-g and IL-4. Interestingly, the microglia cells were activated in the IFN-b ko mice, thus they might be involved in maintenance of local inflammation resulting in augmented demyelination and persistent neurological deficits.

Poster No. IV-16

Glatiramer acetate treatment of MOG-induced EAE in DA rats

Goldschmidt TJ, Gustavsson S & Ferm M, Bioscience, Local Discovery Research Area CNS & Pain Control, AstraZeneca R&D Södertälje, Sweden

E-mail: tom.goldschmidt@astrazenca.com

Glatiramer acetate (GA), one of the compounds currently available for treatment of multiple sclerosis (MS), is approved for subcutaneous injections and was recently tested clinically by the oral route. GA is a standardized random mixture of polypeptides, based on the major amino acid composition of the myelin basic protein (MBP), and thus regarded as an MBP analogue. We whished to test the effect of GA on rat EAE induced with MOG, a different myelin-associated protein. Although GA has previously been reported to be suppressive in several EAE models, we wanted to test GA in MOG_1-125 -induced EAE in the DA rat since this model is considered to be one of the most MS-like in rodents. We treated MOG EAE-induced female DA rats by oral administration or subcutaneous injections of GA (CopaxoneÒ, from TEVA Pharmaceutical Industries Ltd). Preliminary results indicate that subcutaneous, but not oral GA suppresses MOG_1-125 induced EAE. Further investigation is needed to address whether involvement of MBP immunity in the MOG-induced EAE, or a more general mechanism, may explain the GA sensitivity. Nevertheless, along with a MS-like histopathology, reported by other investigators, and a relapsing/remitting disease course, the GA sensitivity adds another feature shared between MOG-EAE in the rat and MS.

Poster No. IV-17

Myelin oligodendrocyte glycoprotein is critical for progressive demyelinating disease in mice

Amor, S, Department of Immunobiology, BPRC, GJ Rijswijk, The Netherlands

E-mail: s.amor@ic.ac.uk

In MS the restricted localisation of lesion in the CNS strongly indicates that the critical target for autoimmune reactions is localised within CNS myelin. Of the known myelin antigens that are pathogenic in experimental systems MOG is the only antigen exclusively localised within the CNS white matter. MOG is a powerful encephalitogen in animals leading to experimental disease that mimics both clinical and histological features of MS more closely than any other model. MOG is not only a relevant target for T cells but also for antibodies since MOG-specific antibodies have been shown to augment demyelination in various models including ABH mice. MOG is also the only known encephalitogenic myelin antigen that is not expressed in the thymus indicting a lack of natural tolerance to this antigen.

To further understand the importance of MOG in both the induction and progression of chronic demyelinating disease we have used CNS tissue from mice with a null mutation in the MOG gene (MOG KO mice) to immunise EAE-susceptible mice. In all strains of mice examined whole CNS tissue or purified myelin from MOG KO mice induced significantly less severe clinical signs and demyelination. The major finding that MOG was critical for the induction of chronic progressive disease. The clinical findings and the extent to which autoimmunity to MOG plays a role in progression of disease (epitope spreading) will be discussed. Furthermore myelin derived from the brain of MOG KO mice was equally as effective in the encephalitogenic potential suggesting differential expression of myelin antigens. These results of these studies will be highly relevant in determining the importance of autoimmune responses to MOG in the induction of and progression of chronic neurological disease such as MS as well as highlighting factors that may determine distribution of lesions within the CNS.

Poster No. IV-19

Stereological estimation of microglial cell numbers in mouse CNS

Wirenfeldt M*, Dalmau I, and Finsen B, Anatomy and Neurobiology, University of Southern Denmark, Odense, Denmark

E-mail: mwnielsen@health.sdu.dk

Stereology is an efficient way to obtain an unbiased and precise estimate of the total number of cells in a defined region. The optical fractionator technique is not affected by injury-induced shrinkage or expansion of the tissue. This technique requires relatively thick sections (20 µm or more after mounting) for unbiased positioning of the optical disector, and the unequivocal identification of labelled cells throughout the section thickness. We have optimised our immunohistochemical protocol for visualisation of Mac-1⁺ microglia in thick vibratome sections (70 µm) for stereological counting within the murine hippocampus, and compared the staining results to those obtained with enzyme histochemical NDPase- and tomato lectin staining of microglia. The optimized tissue processing and staining technique gives sections of high quality with a mean section thickness above 21 µm and with excellent rendition of Mac-1⁺ microglia through the entire height of the section. Similarly, the NDPase-staining gave an excellent visualization of microglia although with this thickness the intensity of the staining is too high to distinguish single cells. This problem can be solved by reducing the section thickness. With our current method the tomato lectin is not well suited for stereology. The perspectives of optimizing the staining protocols for stereological counting of microglial cells, improves the possibility of studying the dynamics of the microglial cell population in response to injury. Supported by The Hede Nielsen Foundation, The Lundbeck Foundation, The Novo Nordic Foundation, and The Danish MRC.

SESSION V: TOLERANCE II

Oral tolerance

Weiner HL, Center for Neurologic Diseases, Boston, MA, U.S.A.

E-mail: hweiner@rics.bwh.harvard.edu

Oral tolerance is a long recognized method to induce peripheral immune tolerance. Multiple mechanisms of tolerance are induced by oral antigen administration. Low doses of oral antigen favor active suppression, whereas higher doses favor clonal anergy and deletion. Oral antigen preferentially generates Th2 (IL-4/IL-10) and Th3 (TGF-beta) regulatory T cells which suppress Th1 type autoimmune diseases such as EAE. In addition, oral antigen has been shown to induce CD4+CD25+ regulatory cells that act via the secretion of IL-10 and TGF-beta. Because Th3 type regulatory T cells generated by oral antigen are triggered in an antigen-specific fashion but suppress in an antigen non-specific fashion by secretion of cytokines such as TGF-beta, they mediate "bystander suppression" when they encounter the fed autoantigen at the target organ. Thus it may not be necessary to identify the target autoantigen to suppress an organ specific autoimmune disease such as MS via oral tolerance, only to orally administer a protein capable of inducing regulatory cells that secrete suppressive cytokines. Induction of oral tolerance is enhanced by IL-4, IL-10, anti-IL-12, TGF-beta, Cholera Toxin B subunit, Flt-3 ligand, anti-CD40 ligand, and interferon tau. Oral (and nasal) tolerance has been used successfully to treat animal models of autoimmune diseases including EAE, uveitis, thyroiditis, myasthenia; collagen- and adjuvant- induced arthritis; and diabetes in the NOD mouse. Other disease processes have been successfully treated by mucosal antigen including asthma, atherosclerosis, graft rejection, allergy, colitis, stroke and models of Alzheimer’s disease. Oral tolerance has been tested in human autoimmune diseases including MS, arthritis, uveitis, and diabetes. It has also been tested in allergy, contact sensitivity to DNCB, nickel allergy,. Although positive results have been observed in phase II trials, no effect was observed in a large phase III trial of CII in rheumatoid arthritis or oral myelin and glatiramer acetate (GA) in multiple sclerosis. Large placebo effects were observed in these trials. In the trial of GA in MS, no immunologic effects were observed in patients even though prominent immunologic effects are observed with injectable GA, suggesting that the formulation or dose may have been incorrect. New trials of oral GA are being planned. Oral (and nasal) tolerance remains an attractive approach for treatment of MS because of its lack of toxicity, ease of administration over the long term, and antigen specific mechanism of action. The successful application of oral tolerance for the treatment of human diseases will depend on dose, developing immune markers to assess immunologic effects, route (nasal vs. oral), formulation, mucosal adjuvants, combination therapy and early therapy.

Immune Regulation in MS and EAE

Anderton, S., Harber, M., Massey, E., Mazza, G., Nicolson, K., Sunstedt, A., Streeter, H., Wraith, D*, Department of Pathology and Microbiology, University of Bristol, UK

E-mail: d.c.wraith@bris.ac.uk

Autoimmune diseases, such as MS, arise because T cells escape tolerance induction in the thymus. Recent studies have revealed three mechanisms by which T cells specific for self-antigens may escape deletion despite apparent expression of the antigen in the thymus. These include alternative RNA splicing, destruction by antigen processing and low avidity interactions. Autoreactive T cells thus form a measurable part of the T cell repertoire in both MS patients and healthy individuals. Why then is autoimmune demyelinating disease not more common? Recent work from this and other laboratories has revealed the role played by regulatory T cells in suppressing autoimmune disease. Natural, CD25+ve cells originate in the thymus, at least in rodents, and appear to function in a cell-cell contact dependent fashion. In addition, various groups have shown that regulatory cells can be induced by administration of specific antigen. Oral administration of intact antigen leads to the activation of Th2 and TGF-beta producing Th3 cells whereas peptide administration can induce IL-10 producing Tr1 cells. IL-10 producing Tr1 cells suppress the responses of naïve T cells in vitro and in vivo and are responsible for protecting mice from EAE, following soluble peptide administration. These cells are both functionally and phenotypically distinct from the natural, thymus-derived CD25+ve regulators. Our laboratory has helped to clarify both the cellular and the molecular basis for induction of Tr1 regulatory cells by soluble peptide antigens. In particular, the peptides must mimic ‘naturally processed’ antigens when they bind to MHC. It should now be possible to design and test peptides for the induction of regulatory cells and treatment of autoimmune conditions such as MS.

*Supported by grants from the Wellcome Trust and Multiple Sclerosis Society of Great Britain and Northern Ireland.

SESSION VI: OTHER IMMUNE THERAPIES

Currently approved immunomodulators: Status report

Wolinsky, JS, The University of Texas Health Science Center at Houston, Houston, Texas, U.S.A.

E-mail: jswolinsky@aol.com

The availability of immunomodulatory drugs with demonstrated near-term efficacy (over 2 to 3 years of controlled therapy) varies regionally, based on a variety of regulatory and accessibility factors. Where generally available, these agents have had considerable uptake and impact on the management of multiple sclerosis (MS). Three classes of disease modifying agents are currently licensed within the United States. Two classes are strictly immunomodulators, the beta interferons (interferon beta-1b (Betas(f)eron^®) and interferon beta-1a (Avonex^® and Rebif^®)), and glatiramer acetate. The third class, mitoxantrone, is more appropriately considered as a general immunosuppressant. An extensive body of controlled data on the beta interferons suggests that they are adequately tolerated and effective in rapidly attenuating short-term measures of clinical and MRI-monitored disease in patients with relapsing MS. However, their clinical benefit to patients in the more progressive phases of the disease is less obvious. Glatiramer acetate is well tolerated and also attenuates short-term measures of clinical and MRI-monitored disease in relapsing patients. It is under assessment in primary progressive MS for its benefits on progression. While not formally tested as such, mitoxantrone currently is used more for stabilization of patients who appear unresponsive to the available immunomodulators. Current needs include the capacity to predict or reliably identify poor responders, assuming that the response to these drugs is truly heterogeneous, and novel trial designs that reliably screen potential new therapeutic compounds or drug combinations in the face of partially effective treatments.

Myelin Repair with the Use of Human Monoclonal Antibodies

Rodriguez M, Bieber A, Warrington A, Ciric B, Van Keulen V, Mitsunaga Y, Howe C, & Larry Pease L, Mayo Clinic, Rochester, U.S.A.

E-mail: rodriguez.moses@mayo.edu

Our laboratory has been developing novel feasible approaches to enhance myelin repair in multiple sclerosis. We have shown that an autoimmune response directed antigens expressed on the surface of oligodendrocytes can enhance myelin repair in three experimental models of demyelination (virus-induced demyelination, lysolecithin-induced demyelination and autoimmune demyelination). To date, six mouse monoclonal antibodies and two human monoclonal antibodies have been shown to induce repair. All of these antibodies share the same phenotype: (1) IgM class, (2) bind to antigens on the surface of oligodendrocytes (3) have germline immunoglobulin nucleotide sequences with rare somatic mutations, (4) bind to antigens primarily consisting of lipids, and (5) induce influx of calcium into glial cells within seconds to minutes of applying monoclonal antibodies to purified cultures. Two human monoclonal antibodies identified as Lym 22 and Lym 46 are cloned and sequenced. Molecular tools are available to express these antibodies for large-scale manufacturing. The data support the hypothesis that these antibodies function to promote remyelination is by directly activating oligodendrocytes to induce a remyelination program.

Poster No. V-3

TRC-specific T cells possess TREG activity

Vandenbark AA, Postland VA Medical Center R&D 31, Portland, OR, U.S.A.

E-mail: vandenba@ohsu.edu

TCR-specific T cells constitute an important natural regulatory mechanism for inhibiting inflammatory Th1 responses that likely contribute to autoimmune diseases such as multiple sclerosis (MS). Recently, there has been much interest in naturally occurring CD4+CD25+ Treg cells that prevent onset of autoimmune diseases. Upon specific activation through the TCR, these cells can inhibit other T cells non-specifically, primarily through cell-cell contact. However, the specificity of Treg cells is largely unknown. We hypothesized that TCR-specific T cells might constitute an important subset of Treg cells that may be deficient or missing in MS patients. To address this hypothesis, we evaluated the frequency and Treg activity of TCR-specific T cells in MS patients undergoing vaccination with a cocktail of 3 TCR peptides. We found that MS patients had reduced or deficient responses to a set of 116 TCR peptides representing the entire AV and BV gene repertoire, compared to healthy controls that had about 8% of their T cells specific for TCR determinants. Upon vaccination with 3 of these peptides, MS patients developed robust anti-TCR responses to the injected peptides, and T cell lines specific for these peptides demonstrated potent Treg activity, assessed by inhibition of activation of CD4+CD25- T cells by anti-CD3/CD28 antibodies. In some MS patients with very strong responses to the TCR vaccine, Treg activity was also detected, in contrast to a complete lack of Treg activity in MS patients that did not respond to a weaker formulation of the TCR vaccine or those receiving adjuvant alone. These results demonstrate for the first time that TCR-reactive T cells possess potent non-specific Treg activity, and that vaccination with TCR peptides can at least partially restore Treg activity in MS patients.

Poster No. V-4

Role of antigen presenting cells in self-tolerance and genetic resistance of EAE

Waldner H & Kuchroo VK, Center for Neurologic Diseases, Harvard Medical School, Boston, MA 02115, USA.

E-mail: hwaldner@rics.bwh.harvard.edu

B10.S mice share the same major histocompatibility complex (MHC) with SJL mice but show relative resistance to induction of experimental autoimmune encephalomyelitis (EAE) after immunization with myelin antigens including proteolipid protein (PLP) peptide 139-151 compared to SJL mice.

To study the mechanisms of self-tolerance in B10.S mice, we have generated transgenic mice that express the rearranged T cell receptor (TCR) genes from a PLP 139-151 specific T cell clone. Transgenic T cells were positively selected in the thymus and represented the vast majority of T cells in the peripheral lymphatic organs of these mice. Furthermore, transgenic T cells were not anergic since purified T cells responded to their antigen PLP139-151 in vitro by proliferation and IL-2 secretion. Antigen-specific proliferation of B10.S transgenic T cells was also demonstrated in vivo using fluorescent dye labeling. A small group of transgenic B10.S mice developed spontaneous EAE, which was in significant contrast to their SJL counterparts and suggested that antigen presenting cells (APC) may contribute to the control of self-tolerance in this autoimmune model. The expression of key molecules in APCs from B10.S and SJL mice and protocols to overcome the tolerance to PLP139-151 in transgenic B10.S mice will be discussed.

Poster No. VI-4

Treatment of experimental autoimmune encephalomyelitis with intravenous immunoglobulin

Humle Jørgensen S, Laursen H & Soelberg Sørensen S, MS Research Unit, Copenhagen University Hospital, Copenhagen, Denmark.

E-mail: shumle@nru.dk

Objective: Clinical trials have shown that intravenous administration of polyclonal immunoglobulin (IVIg) has the potential to reduce the disease activity in multiple sclerosis (MS). However, the mechanisms by which IVIg may interfere with the pathophysiology of MS are not yet fully understood. In the present study we evaluated IVIg treatment of experimental autoimmune encephalomyelitis (EAE), the primary animal model for human MS. The objectives of the study were to assess the effects of IVIg on: 1) the incidence and severity of active EAE, and 2) the EAE pathology in the CNS.

Methods: EAE was induced in rats by immunization with Freund’s adjuvant and a spinal cord homogenate. Control rats were immunized with saline. At day 0 and 1 post immunization, animals were treated with infusions of polyclonal IVIg (1 g/kg) or placebo (10% maltose). The animals were weighed and observed daily, and the clinical disease severity graded on a scale 0-6. During the acute attack at day 11, the rats were sacrificed and the brain and spinal cord dissected and cut for histological examinations. The tissue sections were blinded and scored for inflammation and demyelination.

Results and Conclusion: Treatment with IVIg significantly suppressed the development of EAE as measured by incidence (placebo 92%, IVIg 50%), day of onset, weight loss and maximal average EAE score (placebo 2.6, IVIg 0.7). In the placebo group, the development of active disease was associated with severe inflammation and demyelination in the spinal cord, brain stem and cerebellum. When animals were treated with IVIg, the pathological changes in the central nervous system were significantly reduced as measured by the average histological score (placebo 8.8, IVIg 5.3). In conclusion, IVIg treatment of EAE did not simply ameliorate the clinical symptoms of experimental autoimmune disease but had a protective effect against the pathological changes in the CNS.

Poster No. VI-6

Further evidence for very long term efficacy in interferon beta1b in relapsing remitting patients

Rice GPA, Kirk S, Nicolle E, Kremenchutzky M & Karlik SJ, University of Western Ontario, Department of Neurology, London, Ontario, Canada

E-mail: grice@uwo.ca

In our 12-year data, interferon beta 1b treated RR patients showed a highly significant decrease in MRI T2 lesion burden and individual lesions compared to untreated RR controls imaged in the same epoch. The purpose of this study was to analyze the gadolinium enhancement patterns of this cohort and quantify the black hole lesions on the 12-year T1-weighted MRI scans. There were no T1-weighted entry scans available. The hypointense lesions seen on pre-contrast T1-weighted images were manually outlined and divided into two groups: conventional “black holes” and “grey holes” where the signal intensities were between black and the signal from normal appearing white matter. The number of enhancing lesions was counted on the post-contrast T1 weighted scans. We had scans available for 28 treated patients and 17 controls matched for disease at the onset of interferon treatment. For total and individual measurements, the interferon-treated patients showed much less T1 lesion burden.

	n	Black holes (cc) Median	Grey holes (cc) Median	Total T1 abnormities (cc) Median
Betaferon	28	1.722	0.536	2.50
Control	17	4.463	2.413	7.91
p-value MW-U		0.009	0.002	0.002

There were 4 gadolinium-enhancing lesions and 12 ring-enhancing lesion in the control group compared to 6 and 10, respectively, in the interferon-treated group (Chi squared, n.s.). There appears to be agreement that “black hole” abnormalities, which do not revert, represent the final end product of the MS pathological process. In this study, we observed a significantly lower T1 burden in the patients treated for up to 12 years, suggesting a lower accumulation of end-stage disease burden compared to controls. However, no difference in the incidence of gadolinium enhancement was observed. Although ring-enhancing lesions might be expected in older lesions, these patients have quite inactive disease.

Novel synthetic amino acid copolymers that inhibit autoantigen-specific T cell responses and suppress experimental autoimmune encephalomyelitis

Fridkis-Hareli M¹, Santambrogio L², Stern JNH¹, Fugger L³, Brosnan C⁴, & Strominger JL^1,2, ¹Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, U.S.A.; the ²Department of Cancer Immunology and AIDS, Dana Farber Cancer Institute, Boston, MA 02115, U.S.A.; the ³Department of Clinical Immunology, Aarhus University Hospital, Skejby Sygehus, DK-8200, Aarhus N, Denmark; and the ⁴Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461, U.S.A.

E-mail: jlstrom@fas.harvard.edu

Copolymer 1 (Cop 1, Copaxone®), a random amino acid copolymer of Y, E, A and K, reduces the frequency of relapses by 30% in relapsing-remitting multiple sclerosis (MS) patients. In the present study novel random four-amino acid copolymers, whose design was based on the nature of the anchor residues of the immunodominant epitope of myelin basic protein (MBP) 85-99 and of the binding pockets of MS-associated HLA-DR2 (DRB1*1501), have been synthesized by solid phase chemistry. Poly (Y, F, A, K) (YFAK) inhibited binding of the biotinylated MBP 86-100 epitope to HLA-DR2 molecules more efficiently than either unlabeled MBP 85-99 or any other copolymer including Cop 1. Moreover, YFAK and poly (F, A, K) (FAK) were much more effective than Cop 1 in inhibition of MBP 85-99-specific HLA-DR2-restricted T cell clones. Most importantly, these novel copolymers suppressed EAE, induced in the susceptible SJL/J (H-2^s) strain of mice with the encephalitogenic epitope PLP 139-151, more efficiently than Cop 1. Thus, random synthetic copolymers designed according to the binding motif of the human immunodominant epitope MBP 85-99 and the binding pockets of HLA-DR2 might be more beneficial than Cop 1 in treatment of MS.

Poster No. V-1

T Cell Vaccination (TCV) for Secondary Chronic Progressive Multiple Sclerosis (CPMS), Associated to b Interferon (b IFN) and Total Plasma Exchange (TPX) Pre-Treatment

Moviglia GA, Iraola N, Varela G, Bertoloto R, Hirsch J, Maffeis A, Varela O, Palleros CA,López AM & Schuster GS,. Regina Mater Foundation, Buenos Aires. Argentina.

E-mail: regimat@ba.net

Rationale: TCV has shown poor effectiveness for CPMS patients. Some Immune features may explain this fact. There is a polyclonal anti oligodendrocyte disease where new clones are continually generated (an on-going autoimmune process, never remits), because the autoaggressive cells produces themselves the necessary TNFa and gIFN to sustain the aggression. These cells are protected by the blood brain barrier, with poor peripheral expression. Patients worsening their condition when they increase the Th1 leukines production. bIFN treatment switches Th1 to Th2 reaction by intracellular competition with gIFN. It helps to remove some lymphocytes from the MS lesions to send them to the blood stream and partially stabilize the disease progression. TPX removes the Th1 leukines from the plasma and gives temporary disease stability. This report shows the results of the association of bIFN+TPX+TCV. Methods: It was a phase I-II open trial. 40 Patients, with proven diagnosis of CPMS were divided in four treatment groups and followed for 2 to 8 years. Group 1: 13 patients treated with TCV only (ACTRIMS 1997, Moviglia et al), Group 2: 6 patients treated with TPX (6 procedures, 1 blood volume every other day) + TCV; Group 3: 11 patients treated with bIFN (Ares-Serono) +TCV; and Group 4: 10 patients treated with bIFN+TPX+TCV. Immunizations were done by intramuscular way (IM) (29 patients) or intravenous way (IV) (11 patients) for six to 12 times. Results: Average EDSS variation for each group was G1 +0,04; G2 -0,83; G3 -0,32; G4 -1,05 Immune evaluation showed a correlation of clinical results with the development of an anti-idiotype reaction. Additionally we observed that 7/29 IM and 0/11 IV immunized patients had Disease Progression, 16/29 IM and 2/11 IV had Disease Stabilization, and 6/29 IM and 9/11 IV had Disease Improvement Adverse and side-effects: 20/29 IM and 0/11 IV developed local aseptic abscess, 25/29 IM and 11/11 IV flu-like symptoms, with fever around 38ºC. 5/29 IM and 6/11 IV showed transient hypotension that lasted 60 hours. Conclusions: The immune stabilization obtained for the pre treatment of bIFN+TPX associated to the TCV IV immunization improves the effectiveness on CPMS with low toxic and sustained results.

Poster No. V-2

T-cell Vaccination in MS with CSF-derived Activated CD4+ T-cells: Results from a Pilot Study

Stinissen P, Van der Aa A, Medaer R, Hellings N, Gelin G & Raus J, Biomedisch Onderzoeksinstituut (BIOMED), Limburgs Universitair Centrum, Diepenbeek, Belgium

E-mail: niels.hellings@luc.ac.be

We have previously studied the effects of T-cell vaccination (TCV) with autologous MBP-reactive T-cell clones in MS. Vaccines incorporating a broader panel of anti-myelin reactivities may have therapeutic effects in MS patients. Previous reports showed an accumulation of activated anti-PLP and anti-MBP T-cells in CSF of MS patients. We studied the effects of TCV with activated CD4+ T-cells derived from CSF in 5 MS patients (4 RR, 1 CP). CSF lymphocytes were cultured in the presence of rIL-2 and depleted for CD8 cells. After 5-8 wks CSF T-cell lines (TCL) were almost pure TCRab⁺CD4⁺ cells of the Th1/Th0 type. The TCL showed reactivity to MBP, MOG and/or PLP as tested by Elispot and had a restricted clonality. Three immunisations with irradiated CSF vaccines (10 million cells) were administered with an interval of 2 months. The vaccinations were well tolerated. All patients remained clinically stable or had reduced EDSS with no relapses during or after the treatment. Proliferative responses against the CSF vaccine were observed in 3/5 patients. Anti-ergotypic responses were observed in 4/5 patients. Anti-MBP/PLP/MOG reactivities remained low or were reduced in all patients. We have recently initiated a double-blind placebo-controlled trial with 60 MS patients to study the effects of TCV with CSF-derived vaccines in early RR MS patients.

Poster No. V-5

Superantigen-anergized T cells are functionally similar to CD4+CD25+ regulatory T cells

Grundström S*†, Cederbom L†, Scheipers P* & Ivars F†, * Active Biotech Research, Lund, Sweden; † Section for Immunology, Dept. of Cell and Molecular Biology, Lund University, Lund, Sweden.

E-mail: susanna.grundstrom@activebiotech.com

The superantigen staphylococcal enterotoxin A (SEA) polyclonally activates Vb3-expressing T cells both in vivo and in vitro. Repeated exposure to SEA in vivo has been reported to eliminate or anergize the activated T cells. Functional similarities of anergic CD4⁺ T cells isolated from mice immunized with SEA three consecutive times (3xSEA) and ex vivo CD4⁺CD25⁺ regulatory T cells have been described. This prompted us to ask whether the anergic CD4⁺ T cells would suppress CD4 T cell proliferation in vitro in the same way as CD4⁺CD25⁺ T cells. We demonstrate that anergic CD4⁺ T cells as well as CD4⁺CD25⁺ regulatory T cells inhibit both proliferation and IL-2 production of CD4⁺CD25^- responder T cells in a cell contact-dependent way. In addition, both of these T cell populations down-modulate the costimulatory molecules CD80 and CD86 on dendritic cells. We also show that CD4⁺CD25⁺ T cells from 3xSEA treated mice, consistent with their activated phenotype, are much more efficient effector cells than the ex vivo regulatory T cells from untreated mice. We conclude that the anergic CD4⁺ T cells from 3xSEA mice include CD4⁺CD25⁺ T cells that have responded to the 3xSEA immunization, but have not been eliminated perhaps because they are resistant to activation induced cell death.

Further, we have used two different in vivo modelsystems to investigate if the development of anergy in the CD4⁺CD25^- T cell population of 3xSEA treated mice as well as suppression, are dependent on the presence for CD4⁺CD25⁺ naturally regulatory T cells. Interestingly, we found that conventional CD4⁺ T cells can be both anergic and suppressive in the absence of CD4⁺CD25⁺ T cells. It is yet to be determined if the CD4⁺CD25^- and CD4⁺CD25⁺ T cells use different mechanisms of suppression or not.

Poster No. V-6

Generation of IL-10-producing regulatory T cells after tolerogenic antigen exposure in vivo

Sundstedt A*, Massey EJ, Burkhart C, Lowrey PA, Wraith DC, Department of Pathology and Microbiology, University of Bristol, United Kingdom. (*current address: Active Biotech Research, Lund, Sweden).

E-mail: anette.sundstedt@activebiotech.com

Experimental autoimmune encephalomyelitis (EAE) is a CD4⁺ T cell-mediated inflammatory disease with similarities to multiple sclerosis in humans. Intranasal (i.n.) administration of a myelin basic protein (MBP)-derived peptide can induce tolerance in the responding T cells and protect susceptible mice from EAE. The protective mechanisms involved are not yet fully understood, but the immunoregulatory cytokine interleukin-10 (IL-10) has been implicated to play a major role.

To analyse changes taking place during the induction of tolerance by peptide inhalation, transgenic mice expressing a TCR specific for the immunodominant N-terminal peptide of MBP were treated repeatedly intranasally with the high affinity peptide analogue Ac1-9(4Y). After a transient phase of activation the T cells were pushed into an anergic state, failing to proliferate and produce IL-2 when restimulated with peptide in vitro. Interestingly, analysis of cytokine production in vivo demonstrated a marked shift from IL-2 production in the activation phase to high levels of IL-10 after tolerance induction. This finding indicates that T cells rendered anergic in vivo may become regulatory T cells that are able to influence neighbouring immune responses. Indeed, the tolerant CD4⁺ T cells markedly suppressed T cell proliferation when cultured with naïve CD4⁺ T cells. The nature of these cells and the mechanisms involved in suppression will be discussed.

Poster No. V-7

Re-induction of tolerance in established autoimmune CNS disease with a combination of transient T cell deletion and intravenous antigen

Pryce G, O'Neill JK, Croxford JL, Smith PA, Amor S, Hankey DRJ, Giovannoni G & Baker D, Institute of Neurology, University College London, London, UK

E.mail: d.baker@ion.ucl.ac.uk

Prophylactic autoimmune tolerance may easily be induced by a variety of different means in animal models of autoimmunity, yet therapeutic tolerance in established disease provides a more difficult goal. In a robust, relapsing mouse model of multiple sclerosis (MS) we have found that transient and substantial T cell deletion only delays the eventual return to disease progression. Intravenous-myelin antigen administration likewise induces an unresponsiveness that is readily reversed. However by combining these two approaches in chronic relapsing experimental allergic (EAE) encephalomyelitis in mice we have found that even in later phases of disease, such as after the first, second or even third relapse, disease can be silenced through a combination of a transient-deletion of primed, auto-reactive cells followed by intravenous myelin-antigens, using a variety of different methods. Through the use of recombinant myelin proteins tolerance can be induced without any prior knowledge of the pathogenic epitopes which are likely to be different between humans, as they are between individual animal strains, due to variablity in genetics and disease duration. However through i.v tolerance we were able to define the important peptide epitopes that induce relapsing disease within the ABH mouse strain and demonstrate that relapses can develop independently of epitope spread, thought by some to drive relapsing disease in animals and humans. Independently leukocyte depletion and myelin-antigen delivery have already been attempted and failed in MS. Through a combination of such approaches it may be possible to limit the adverse reactions sometimes associated with myelin-antigen therapy and provide a better chance of success in combating the immunological components of MS.

Poster No. VI-1

Intravenous immunoglobulin treatment efficacy in multiple sclerosis: The role of IgM enreachment

Bisaga GN, Kalinina NM, Davidova NI & Pozdnjakov AV, Department of Neurology Military Medical Academy, All Russian Center of Emergency&Radiation Medicine, St.Petersburg, Russia

E-mail: bisaga@mailbox.alkor.ru

Introduction: IgM suggested to prevent tumor necrose factor (TNFa) releasing from monocytes. So for the the purpose of multiple sclerosis (MS) therapy optimization we have compared intravenous immunoglobulin (IVIG) treatment with the various IgM contents in it.

Methods: We studied 15 MS relapsing-remitting (RR) patients in stable state. I group: 9 patients were treated by IgM enriched IVIG Pentaglobinum (Biotest); II group: 6 patients - by non IgM enriched IVIG (Imbio). The treatment regimen in both groups was: 0.4 g/kg for 3 days and then monthly 0.15-0.20 g/kg for 3-30 months. Ten control RR MS patients, received only symptomatic therapy, were matched with the studied patients for age, disease duration and number of attacks per year. The IL1, IL2, IL4, IL6 and TNFa levels by using ELISA test were measured and MRI with MR-spectroscopy were performed in every 6 months.

Results: The exacerbation rate decreased in the I group from 2.2±0.4 per year before treatment to 0.9±0.3 per year during the treatment, II group – 1,17±0,7 and 0.98±0,4, control - 1,9±0,4 and 1,8±0,5 accordingly. The EDSS score decreased in the I group from a mean of 4.6 to 4.2, II group from 6.1 to 5.9, whereas in the control - increased from 3.8 to 3.9 points. Positive changes of TNFa, IL1, IL2, MRI and MR-spectroscopy were most prominent in the I group and were absent in control.

Conclusion: Clinical effectiveness of IgM enriched IVIG in MS come to total of 67%, non IgM enriched IVIG – 37%, that is very promising for further investigation.

Poster No. VI-2

The risk of treatment failure in secondary progressive multiple sclerosis: A short meta-analysis

Cendrowski WSP, The Neurological Out-Patients Clinic, Warsaw, Poland

E-mail: piotr_cendrowski@netia.pl

Introduction. A considerable proportion of patients with secondary progressive multiple sclerosis (SP MS) fails to respond to the new disease-modifying drugs. Betaferon^® in the NASPMS trial and Rebif^® in the SPECTRIMS study became unable to delay progression of disability.

Aim. To analyse baseline data in three therapeutic trials of IFN beta-1b s.c. or IFN beta-1a s.c. and to calculate the relative risk (rr) of treatment failure which may be associated with baseline variables.

Patients and method. Three cohorts of SP MS patients from the NASPMS, the SPECTRIMS and the EUSPMS trial (1998–2001) were included into this meta-analysis. A comparison of baseline data from these controlled trials was made in order to analyse whether entry variables may be accompanied by treatment failure. Rr of treatment failure associated with six variables was calculated in three cohorts. Treatment failure was defined as lack of progression delay in SP MS patients.

Results. A comparison of baseline data between the NASPMS trial and the EUSPMS study shows no rr increase of treatment failure associated with following variables: age of patients (rr = 1.49), duration of the disease (1.16) and mean EDSS (Expanded Disability Status Scale) > 5.5 at entry (1.81). Prestudy lower annual relapse rate in 2 years was associated with decreased risk of treatment failure (rr = 0.69). Higher rr of treatment failure was found in patients without relapses in 2 years (2.55) and with smaller number of new GdT1 lesions (rr = 2.13). Another comparison of baseline data from the SPECTRIMS trial and the EUSPMS study did not show increased rr of treatment failure concomitant with age of patients (rr = 1.19), duration of the disease (1.11), absence of relapses in the prestudy 2 years (1.05) and EDSS level at entry (1.28).

Conclusions. Increased rr of treatment failure in the NASPMS trial occurred in patients without relapses 2 years prior to the study (rr = 2.55) and in patients with smaller number of new GdT1 lesions (rr = 2.13). No rr increase of treatment failure was found in the SPECTRIMS trial.

Poster No. VI-3

CD1a expression and IL-12p70 production by blood mononuclear cells (MNC) in multiple sclerosis (MS) are reduced more by combination therapy with IFN-b and glatiramer acetate than by monotherapy with IFN-b

Yu-Min Huang, Hussien Y, Sanna A, Söderström M & Link H, Karolinska Institute, Stockholm, Sweden

E-mail: yu-min.huang@neurotec.ki.se

Background: Beneficial but modest effects have been reported using monotherapy with the immunomodulators IFN-b or glatiramer acetate (GA) in MS. Previous in vitro studies showed that IFN-b and GA act differently on APC, but both create a permissive microenvironment for Th2/Th3 cell differentiation. We speculate that combination therapy with IFN-b and GA may have potentials in patients progressing clinically during monotherapy with IFN-b or GA. Aims: To compare in vivo effects of treatment with IFN-b and GA vs. monotherapy with IFN-b alone vs. no treatment, on surface molecules on HLA-DR⁺ blood MNC and on cytokine production by MNC. Materials and methods: A cross-sectional study included 14 treated with IFN-b and GA, 22 with IFN-b alone, 18 untreated and 23 healthy controls (HC). In a follow-up study, 19 MS patients deteriorating clinically despite monotherapy with IFN-b alone were followed before and after addition of GA. Two-colour flow cytometry was used to detect surface molecules on HLA-DR⁺ MNC, and ELISA to measure cytokine production. Results: % of HLA-DR⁺ MNC in MS patients are similar irrespective of treatment compared to HC. But untreated MS patients show highest CD1a⁺ and CD80⁺ cells among HLA-DR⁺ MNC compared to other MS groups and HC. Untreated MS patients have lower IL-10 but higher IFN-g and IL-12p70 compared to those treated with IFN-b + GA and HC. Monotherapy with IFN-b resulted in decrease of CD1a⁺ and IL-10. MS patients treated with IFN-b and GA had lowest CD1a⁺ cells, and IFN-g as well as IL-12p70. Conclusion: The more pronounced reduction of CD1a⁺ cells and IL-12p70 in patients treated with IFN-b and GA is in line with previous in vitro observation that IFN-b plus GA affects APC and cytokines in favour of a Th2/Th3 permissive environment to a larger extent than IFN-b or GA alone.

Poster No. VI-5

Interferon beta inhibits monocyte-dericed dendritic cell maturation

McClurg A, Fleming E, Hawkins SA*, Duddy ME*, McQuaid S⁺ & Armstrong MA, Depts Microbiology & Immunobiology and Medicine*, Queen’s University, Belfast, UK and Dept Pathology⁺ Royal Victoria Hospital Belfast, UK.

E-mail: m.armstrong@qub.ac.uk

Multiple Sclerosis (MS) has a wide range of presentations and a spectrum of clinical courses, which is characterised by multifocal Central Nervous System (CNS) damage, postulated to be mediated by CNS antigen-specific T cells. Dendritic cells (DC) are the most potent antigen presenting cell known and are unique in their ability to initiate primary immune responses. They are thought to play a pivotal role in the decision between T cell activation and anergy.. We have identified mature CD83+ DC in active MS plaque tissue, and have shown in in vitro studies that interferon beta, a disease-modifying drug used in the treatment of MS, can interupt the differentiation pathway of peripheral blood monocyte-derived DC. resulting in a cell which, by electron microscopy, morphological and flow cytometric analyses we have shown to be intermediate in size between a monocyte and a dendritic cell, to express CD14^low, CD1a^low, CD40⁺, HLA-DR⁺, CD16^low, in comparison to immature monocyte derived DC which are CD14^-,CD1a ^high, CD16^-, CD40⁺, HLA-DR⁺, or monocytes which are CD14^high, CD1a^-, CD16^-, CD40^low,HLA-DR⁺. Culture of these intermediate-type cells with different concentrations of LPS, which stimulates immature monocyte-derived DC to mature and upregulate CD83, did not change their phenotype to either mature DC or to macrophages. Therefore, these intermediate type cells may be anergic and unable to potentiate the immune response. The mechanism of action of interferon beta in vivo is still to be discovered. We would suggest that the ability of this drug to disrupt dendritic cell maturation may contribute to its efficacy as a disease-modifying drug in MS.

Poster No. VI-8

The Potential of Cannabinoids in the Treatment of Multiple Sclerosis

Pryce G, Z.Ahmed, Hankey DRJ., Giovannoni G, & Baker D, Institute of Neurology, University College London, London, UK.

E-mail: d.baker@ion.ucl.ac.uk

Although multiple sclerosis (MS) is believed to be an autoimmune disease, the findings that disability progresses in many patients even following anti-immunological treatments has highlighted that chronic neurodegenerative processes are important in the accumulation of disability. Experimental allergic encephalomyelitis (EAE) in mice shows not only a relapsing-remitting paralytic disease progression but also the accumulation of significant axonal loss, which is associated with the development of additional signs, such as spasticity and tremor. These are troublesome signs that reduce quality of life to MS patients. We have found that the cannabinoid system exhibits tonic control of these signs and trials of cannabis extracts in MS are ongoing, however symptom control is largely mediated by cannabinoid receptor (CB1) agonism, which is the receptor that also mediates the well-known psychoactive effects of cannabis. However these may be avoided by inhibitors of endocannabinoid degradation as that may give some localized benefit in symptom control and may provide a novel class of therapeutics for symptom control in MS. The importance of CB1 in symptom control can be definitively shown by examining the effects of cannabinoids during EAE in CB1-deficient mice. However, importantly these mice have also demonstrated that the cannabinoid system can limit the development of axonal loss. Following EAE induction in CB1-deficient mice, there is substantial axonal pathology compared with “wild-type” litter mates which leads to the rapid development of disability. Consistent with this we have found that cannabinoid receptor agonists can induce neuroprotective effects in autoimmune disease of the central nervous system and therefore cannabinoids may be new class of agents which may slow down the progression of MS.