MS Abstracts 03b-2g6

Dysferlin is a muscle protein involved in cell membrane repair and its deficiency is associated with Muscular Dystrophy. We describe that Dysferlin is also expressed in leaky Endothelial Cells.

In the normal Central Nervous System (CNS), Dysferlin is only present in Endothelial Cells of CircumVentricular Organs.

In the inflamed CNS of patients with Multiple Sclerosis (MS) or in animals with Experimental Autoimmune Encephalomyelitis, Dysferlin reactivity is induced in Endothelial Cells and the expression is associated with Vascular leakage of Serum proteins.

In MS, Dysferlin expression in Endothelial Cells is not restricted to vessels with inflammatory cuffs but is also present in noninflamed vessels.

In addition, many blood vessels with PeriVascular inflammatory infiltrates lack Dysferlin expression in inactive lesions or in the Normal-Appearing White Matter.

In vitro, Dysferlin can be induced in Endothelial Cells by stimulation with Tumour Necrosis Factor-.

Hence, Dysferlin is not only a marker for leaky Brain vessels, but also reveals dissociation of PerivVascular inflammatory infiltrates and Blood-Brain Barrier disturbance in Multiple Sclerosis.

Variation in Major Histocompatibility Complex genes on Chromosome 6p21.3, specifically the Human Leukocyte Antigen HLA-DR2 or DRB1*1501-DQB1*0602 extended haplotype, confers risk for Multiple Sclerosis (MS).

Previous studies of DRB1 variation and both MS susceptibility and phenotypic expression have lacked statistical power to detect modest genotypic influences, and have demonstrated conflicting results.

Results derived from analyses of 1339 MS families indicate DRB1 variation influences MS susceptibility in a complex manner.

DRB1*15 was strongly associated in families (P=7.8x10(-31)), and a dominant DRB1*15 dose effect was confirmed (OR=7.5, 95% CI=4.4-13.0, P < 0.0001).

A modest dose effect was also detected for DRB1*03; however, in contrast to DRB1*15, this risk was recessive (OR=1.8, 95% CI=1.1-2.9, P=0.03).

Strong evidence for under-transmission of DRB1*14 (P=5.7x10(-6)) even after accounting for DRB1*15 (P=0.03) was present, confirming a protective effect.

In addition, a high risk DRB1*15 genotype bearing DRB1*08 was identified (OR=7.7, 95% CI=4.1-14.4, P < 0.0001), providing additional evidence for trans DRB1 allelic interactions in MS.

Further, a significant DRB1*15 association observed in Primary/Progressive MS families (P=0.0004), similar to Relapsing/Remitting MS families, suggests that DRB1-related mechanisms are contributing to both phenotypes.

In contrast, results obtained from 2201 MS cases argue convincingly that DRB1*15 genotypes do not modulate age of onset, or significantly influence disease severity measured using expanded disease disability score and disease duration.

These results contribute substantially to our understanding of the DRB1 locus and MS.

And underscore the importance of using large sample sizes to detect modest genetic effects, particularly in studies of genotype-phenotype relationships.

Multiple Sclerosis (MS) is postulated to be a T-Cell-mediated Autoimmune Disease characterized clinically by a Relapsing/Remitting (RR) stage followed by a Secondary/Progressive (SP) phase.

The Progressive phase is felt to be secondary to Neuronal Degenerative changes triggered by inflammation.

The status of the Innate Immune System and its relationship to the stages of MS is not well understood.

Dendritic Cells (DCs) are professional APCs that are central cells of the Innate Immune System and have the unique capacity to induce primary Immune Responses.

We investigated circulating Myeloid DCs isolated directly from the blood to determine whether there were abnormalities in Myeloid DCs in MS and whether they were related to disease stage.

We found that SP-MS subjects had an increased percentage of DCs expressing CD80, a decreased percentage expressing PD-L1, and an increased percentage producing IL-12 and TNF- compared with RR-MS or controls.

A higher percentage of DCs from both RR and SP-MS patients expressed CD40 compared with controls.

We then investigated the polarization effect of DCs from MS patients on naive T-Cells taken from Cord blood using a MLR assay.

Whereas DCs from RR-MS induced higher levels of Th1 (IFN-γ, TNF-) and Th2 (IL-4, IL-13) Cytokines compared with controls, DCs from SP-MS only induced a polarized Th1 response.

These results demonstrate abnormalities of DCs in MS and may explain the Immunologic basis for the different stages and clinical patterns of MS.

In Multiple Sclerosis, the relationship between conventional MRI findings and the clinical evolution of the disease is weak. Magnetization Transfer (MT) MRI can provide markers reflecting the more disabling features of Multiple Sclerosis pathology.

The aim of the present study was to assess the value of MT MRI quantities and their short-term changes in predicting the long-term accumulation of disability in Multiple Sclerosis patients.

Conventional and MT MRI scans of the Brain were obtained at baseline and after 12 months in 73 patients, who were followed prospectively with clinical visits for a median period of 8 years.

At baseline and at 12 months, T₂-HyperIntense and T₁-HypoIntense lesion volume, normalized Brain Volume [with Gray (GM) and White Matter (WM) fractions] and average lesion MT Ratio (MTR) were measured.

At the two time points, metrics derived from the MTR Histograms of the Whole-Brain Parenchyma, GM and Normal-Appearing WM were also computed.

A multivariate analysis, adjusted for follow-up duration, was performed to establish which variables were significant predictors of long-term Neurological deterioration.

At the end of follow-up, 44 patients (60%) showed a significant disability worsening.

A multivariable model included baseline GM MTR Histogram peak height [P = 0.029, Odds Ratio (OR) = 0.97], and average lesion MTR percentage change after 12 months (P = 0.016, OR = 0.88) as independent predictors of disability worsening at 8 years (r(2) = 0.28).

The discriminating ability of such a model in predicting the individual patients' outcome was 66%.

MT MRI provides useful prognostic markers for the prediction of the long-term evolution of Multiple Sclerosis.

This study also suggests that GM damage is one of the key factors associated with disability accumulation in this 'White Matter' condition.

Treatment approaches are rare for Chronic/Progressive patients with Multiple Sclerosis (MS).

Objective was to evaluate the clinical benefit of repeated Intrathecal application of the sustained release Steroid Triamcinolone Acetonide or the administration of Mitoxantrone (MIX) in 2 similar cohorts of Chronic/Progressive patients with MS in an open-label fashion.

Expanded Disability Status Scale scores significantly decreased after the first 6 IntraSpinal Triamcinolone Acetonide injections, which were performed every third day, and then remained stable.

Walking distance significantly increased and did not reduce until the end of the 1-year-long trial period.

Mitoxantrone treatment did not improve the Expanded Disability Status Scale score; however, no further significant deterioration appeared.

Walking distance did not significantly decrease. Both treatment regimens were safe; the patients experienced nearly no adverse effects.

Triamcinolone Acetonide application provided a clinical benefit, whereas MIX administration prevented further worsening of MS symptoms.

We stress that only specialists with a broad experience in IntraSpinal Triamcinolone Acetonide application and MIX administration should perform both kinds of therapy only after a careful information and risk-benefit evaluation in cooperation with the patient.

Future trials will show the efficacy of combination of both treatment approaches in Chronic/Progressive patients with MS.

Statins, inhibitors of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase, are widely prescribed for their Cholesterol-lowering properties to reduce Atherogenesis and CardioVascular morbidity.

Over recent years, Statins have also been shown to exert pleiotropic ImmunoModulatory effects that might be of therapeutic benefit in Autoimmune Disorders.

The primary mechanism by which Statins alter Immune function appears to be mediated through the inhibition of post-translational protein prenylation of small GTP-binding proteins and is largely independent of lipid-lowering.

In Experimental Autoimmune Encephalomyelitis (EAE), the mouse model for Multiple Sclerosis (MS), Statins prevent or reverse paralysis and were recently shown to exert synergistic benefit when combined with agents approved for MS therapy.

Based primarily upon the beneficial effects in EAE, Statins are now being tested in patients in MS clinical trials.

Our previous results revealed that Igs in lesions and single chain variable fragment Abs (scFv-Abs) generated from clonal B-Cells in the CerebroSpinal Fluid (CSF) from patients with Multiple Sclerosis (MS) bind to Axons in MS Brains.

To study the Axonal Ags involved in MS, we identified the GlycoLytic Enzymes, TriosePhosphate Isomerase (TPI) and GAPDH, using Igs from the CSF and scFv-Abs generated from clonal B-Cells in the CSF and in lesions from MS patients.

Elevated levels of CSF-Abs to TPI were observed in patients with MS (46%), Clinically Isolated Syndrome (CIS) suggestive of MS (40%), Other Inflammatory Neurological Diseases (OIND; 29%), and Other Noninflammatory Neurological Diseases (ONIND; 31%).

Levels of GAPDH-reactive Abs were elevated in MS patients (60%), in patients with CIS (10%), OIND (14%), and ONIND (8%).

The coexistence of both AutoAntiBodies was detected in 10 MS patients (29%), and 1 CIS patient (3%), but not in patients with OIND/ONIND.

Two scFv-Abs generated from the CSF and from lesions of a MS Brain showed ImmunoReactivity to TPI and GAPDH, respectively. The findings suggest that TPI and GAPDH may be candidate Ags for an Autoimmune response to Neurons and Axons in MS.

In this study, we employed a sensitive activity assay system to measure CerebroSpinal Fluid (CSF) and Serum levels of active Matrix MetalloProteinase-9 (MMP-9).

In 37 Relapsing/Remitting (RR), 15 Secondary/Progressive (SP) and nine Primary/Progressive (PP) Multiple Sclerosis (MS) patients, grouped according to clinical and Magnetic Resonance Imaging (MRI) evidence of disease activity.

We also studied, as Neurological controls, 48 patients with Other Inflammatory Neurological Disorders (OIND) and 48 with Non-Inflammatory Neurological Disorders (NIND).

To assess active MMP-9/TIMP-1 circuit, CSF and Serum levels of MMP-9 Tissue Inhibitor TIMP-1 were quantified by ELISA in the same patient population.

CSF mean levels of active MMP-9, CSF active MMP-9/TIMP-1 ratios and Intrathecal active MMP-9 synthesis, as indicated by specific index, were more elevated in MS than in NIND (P < 0.05, < 0.02 and < 0.02, respectively).

Serum active MMP-9/TIMP-1 ratio was higher in MS (P < 0.01) and OIND (P < 0.02) than in NIND, and serum TIMP-1 concentrations were lower in MS than in NIND (P< 0.05).

More importantly, Serum active MMP-9 mean levels, Serum active MMP-9/TIMP-1 ratio and Intrathecal production of active MMP-9 were increased in MS patients with clinical (P < 0.001, < 0.001 and < 0.05, respectively) and MRI (P < 0.001, < 0.001 and < 0.02, respectively) disease activity.

Whereas, CSF mean concentrations of active MMP-9 and CSF active MMP-9/TIMP-1 ratio were enhanced only in MS patients with MRI evidence of disease activity (P < 0.02 and < 0.01, respectively).

Altogether, these findings suggest that a shift in MMP-9/TIMP-1 balance towards proteolytic activity of MMP-9 could be relevant in MS Immune dysregulation.

In addition, our results indicate that CSF and Serum levels of active MMP-9 may represent a potential surrogate biomarker for monitoring MS disease activity.

In particular, Serum active MMP-9/TIMP-1 ratio seems to be a very appropriate indicator of ongoing MS inflammation, since it is easily measurable.

Purpose
Focal inflammatory/DeMyelinating lesions are thought to be the source of Wallerian Degeneration or other injury to local, transiting fiber tracts in the Brain or Spinal Cord in Multiple Sclerosis (MS).

A methodology is established to isolate connections between focal DeMyelinating lesions and intersecting fibers to permit explicit analyzes of the pathology of secondary fiber injury distant from the focal lesion.

Materials And Methods
A strategy is described and feasibility demonstrated in three patients with a Clinically Isolated Syndrome and positive MRI (at high risk for MS).

The strategy utilizes streamtube Diffusion Tractography to identify Neuronal fibers that intersect a focal lesion and pass through a region of interest, in this case the Corpus Callosum, where distal (to focal lesion) interrogation can be accomplished.

Results
A sizeable fraction of the Normal-Appearing White Matter (NAWM) in the early stages of disease can be defined in the Corpus Callosum, which is distinctive in that this tissue connects to distant DeMyelinating lesions.

Conclusion
The new class of tissue called Fibers-At-Risk for degeneration (FAR) can be identified and interrogated by a variety of quantitative MRI methodologies to better understand Neuronal Degeneration in MS.

J. Magn. Reson. Imaging 2006. Published 2006 Wiley-Liss, Inc.

Saltatory Conduction in the Nervous System is enabled through the intimate association between the leading edge of the Myelin sheath and the Axonal membrane to demarcate the Node of Ranvier.

The 186 kDa Neuron specific isoform of the Adhesion Molecule Neurofascin (Nfasc186) is required for the clustering of Voltage gated Na⁺ channels at the Node, whilst the 155 kDa Glial specific isoform (Nfasc155) is required for the assembly of correct ParaNodal Junctions.

In order to understand the relationship between these vital structures and how they are affected in Multiple Sclerosis we have examined the expression of Nfasc155 and Nfasc186 in areas of Inflammation, DeMyelination and ReMyelination from post-mortem Brains.

Fourteen cases of NeuroPathologically confirmed Multiple Sclerosis (8 female and 6 male; post-mortem delay 7-24 h; age 37-77 years; and disease duration 15-40 years), comprising 20 tissue blocks with 32 DeMyelinating or ReMyelinating lesions, were used in this study.

A significant early alteration in Nfasc155⁺ ParaNodal structures occurs within and adjacent to actively DeMyelinating White Matter lesions that are associated with damaged Axons.

Shaker-type Kv1.2 channels, normally located distally to the ParaNode, overlapped with the disrupted Nfasc155⁺ structures.

In the absence of Nfasc155, Kv1.2 channels abutted normally clustered Nfasc186⁺ Nodes, indicating that complete disruption of the ParaNodal structure and movement of Kv1.2 channels precede alterations at the Node itself.

Within areas of partial ReMyelination, a number of atypical triple-Nfasc155⁺ structures were noted that may represent transient Oligodendrocyte-Axonal contacts during the process of Myelin repair or aberrant interactions.

Within shadow plaques discretely clustered Nav⁺, Nfasc186⁺ and Nfasc155⁺ domains indicated the restoration of normal Nodal architecture.

The alterations in Oligodendrocyte Nfasc155 expression that accompany inflammation and DeMyelination suggest an ongoing disruption to the Axonal-Oligodendrocyte complex.

Within newly forming as well as established lesions in Multiple Sclerosis, resulting in destruction of the Nfasc186⁺/Nav⁺ Nodal complex vital to successful fast NeuroTransmission in the CNS.

Background And Purpose
Multiple Sclerosis (MS) disease processes in Normal-Appearing White Matter (NAWM) may be different close to MR-visible lesions than farther from these lesions.

We aimed to investigate the relationship of NAWM changes to the distance to the lesions.

Methods
We measured B(1)-corrected T₁ and Magnetization Transfer Ratio (MTR) maps in 63 patients with MS (11 Primary/Progressive, 34 Relapsing/Remitting, 18 Secondary/Progressive).

We used Histogram analyses to assess the global properties of lesions, of 4 consecutive 1-mm pixel layers of NAWM around the lesions, and of distant NAWM located at least 4-mm from lesions in all directions.

In 22 healthy controls, we measured White Matter MTR and T₁ Histograms. Histogram parameters were statistically analyzed by using a linear mixed model.

Results
The first and second NAWM pixel layers around the lesions had a significantly lower MTR Histogram peak position than distant NAWM.

Whereas T₁ Histogram peak position was similar between all types of NAWM.

Furthermore, MTR Histograms of distant NAWM were statistically indistinguishable from those of control White Matter.

Whereas T₁ Histograms of distant NAWM had significantly decreased peak height for Relapsing/Remitting MS and Secondary/Progressive MS and significantly increased peak position for Secondary/Progressive MS.

Conclusion
Our results may suggest that Axonal damage and DeMyelination in NAWM mainly arise as a secondary result of visible lesions, with the largest effect close to these lesions.

NAWM disease farther from the lesions may be mainly characterized by subtle Blood-Brain Barrier damage, with leakage of Fibrinogen into the Parenchyma and microplaque formation, processes that are detected with T₁ but not with MTR.

Although the diagnosis of Multiple Sclerosis relies on the demonstration of disease dissemination in space and time, the exclusion of other Neurological Disorders is also essential.

The limited specificity of abnormalities disclosed by MRI may increase the likelihood of diagnosis of Multiple Sclerosis in patients affected by other disorders.

The available criteria for diagnosis of Multiple Sclerosis have not taken advantage of the potential of MRI to detect features "not suggestive" of Multiple Sclerosis.

Recognition of such features in the work-up of patients suspected of having Multiple Sclerosis may reduce the likelihood of a false positive diagnosis of the disorder in some, while suggesting the correct alternative diagnosis in other patients.

On the basis of this, a workshop of the European MAGNIMS (Magnetic Resonance Network in Multiple Sclerosis) was held to define a series of MRI red flags in the setting of clinically suspected Multiple Sclerosis that is derived from evidence-based findings and educated guesses.

The presence of such red flags should alert clinicians to reconsider the differential diagnosis more extensively.

In this review we will report on the conclusions of this international consensus, which should represent a first step beyond the concept of "no better explanation", and inform future diagnostic criteria for Multiple Sclerosis.