MS Abstracts 02a-2g3

Background
Studies have suggested that T₂ lesion activity is prominent in early Relapsing/Remitting Multiple Sclerosis.

Whereas Brain Atrophy, while seen early, appears more evident in later Progressive disease. The temporal relation between these processes remains unclear.

Objective
To explore the association between changing Brain lesion loads and subsequent tissue Atrophy in Multiple Sclerosis.

Methods
28 subjects with clinically Probable or Definite Multiple Sclerosis (mean age 46.0 years; 17 female and 11 male) were followed for 14 years after first onset of symptoms.

T₂ Lesion Loads were estimated soon after symptom onset and at around five, 10, and 14 years later.

Disease related Atrophy was estimated at the 14 year follow up by comparing Brain Tissue Volumes (proportional to Total IntraCranial Volumes).

Determined in the Multiple Sclerosis group with data from 29 normal control subjects (mean age 36.7 years; 16 female, 13 male) using multiple linear regression analyzes to allow for differences in age and sex distributions.

Results
Change in Lesion Load in the first five years was more closely correlated to disease related Brain Atrophy at 14 years than later changes in lesion load, although the correlation was only moderate (Spearman correlation = -0.528, p = 0.004).

Conclusions
From this, it appears that early rather than later focal lesion accumulation relates to subsequent Brain Atrophy, but factors unconnected directly with lesion formation probably also play a significant role in determining such Atrophy.

Accumulating data support Axonal Degeneration as the major determinant of irreversible Neurological disability in patients with Multiple Sclerosis (MS).

The extent of Axonal Injury correlates with the degree of inflammation in active MS lesions and occurs at early stages of disease.

Indicating that inflammatory DeMyelination is an important factor behind Axon pathology at the Relapsing/Remitting stage of MS.

Axonal Loss from disease onset can remain clinically silent for many years, and permanent Neurological disability develops when a threshold of Axonal Loss is reached and the CNS compensatory resources are exhausted.

Lack of Myelin-derived Trophic support due to long term DeMyelination may cause continuous Axonal Degeneration in chronic inactive lesions at the Secondary/Progressive stage of MS.

Axonal pathology is not limited to DeMyelinated lesions, but also extends into Normal-Appearing White Matter. The concept of MS as a NeuroDegenerative Disorder has important clinical implications:

• First, proactive AntiInflammatory and ImmunoModulatory treatment should prevent or delay chronic disability since inflammation influences Axonal injury.

• Second, the PathoPhysiological mechanisms underlying Axonal Degeneration in MS need to be clarified in order to develop novel NeuroProtective therapeutics.

• Finally, surrogate markers of Axonal pathology, such as N-AcetylAspartate, can be used to monitor Axonal Dysfunction, Axonal Loss and treatment efficiency in patients with MS.

Background
Recent evidence suggests an altered Glutamate Homeostasis in the Brain of patients with Multiple Sclerosis (MS), as seen in experimental models of MS.

Objective
To test whether the ExcitoToxic insult contributes to the pathological process in MS by measuring Glutamate and Aspartate levels in the CerebroSpinal Fluid of MS patients and control individuals.

Participants
Twenty-five patients with the Relapsing/Remitting form of MS during a stable clinical phase, 30 patients with Relapsing/Remitting MS during relapse, and 25 patients with the Secondary/Progressive form of MS were included in the study.

Data were compared with those of 20 age-matched control subjects without diseases of the Central and Peripheral Nervous Systems.

Methods
Glutamate and Aspartate levels in the CerebroSpinal Fluid were measured by high-performance liquid chromatography.

Results
CerebroSpinal Fluid Glutamate levels were significantly higher in patients assessed during relapse compared with those of the patients with Relapsing/Remitting MS examined during the stable clinical phase and the controls (P< .001).

The levels of Glutamate detected in patients with Relapsing/Remitting MS during the stable phase who had active lesions were significantly higher than in those without NeuroRadiological evidence of disease activity (P< .001).

Significantly higher levels of Glutamate were found in patients with Secondary/Progressive MS with an increase of 1 or more points on the Expanded Disability Status Scale score compared with stable patients with Secondary/Progressive MS and control subjects (P< .001).

Conclusions
NeuroToxic events occur in MS patients, and they can be responsible for Oligodendrocyte and Neuronal Cell death in patients with this DeMyelinating Disease.

The manipulation of Glutamate-altered Homeostasis or antagonizing Glutamate Receptor-mediated ExcitoToxicity may have therapeutic implications in MS patients.

In this review, we have tried to summarize the current knowledge on the distribution of important molecular components of InterCellular Junctions-both Tight Junctions (TJs) and Adherens Junctions (AJs)-at the level of UltraStructure.

For this purpose, Immunogold procedure was applied to ultrathin sections of Brain samples obtained from mice, rats, and humans and embedded in hydrophilic resin Lowicryl K4M.

The results of our observations performed with transmission Electron Microscopy (EM) are discussed and compared with findings of other authors.

Although the main structures responsible for the barrier and fence functions of the Blood-Brain Barrier (BBB) and Blood-CSF Barrier are TJs.

Present between Endothelial Cells (ECs) of Brain Capillaries and Epithelial Cells of the Choroid Plexus.

Their functional characteristics (e.g. tightness of the Barrier evaluated by electrical resistance) differ significantly.

Therefore, our main attention is focused on the presence and distribution of both intrinsic, i.e. integral membrane (TransMembrane), molecules such as Occludin, Cadherin, and Junctional Adhesion Molecule (JAM) in TJs, and Cadherins in AJs.

As well as peripheral molecules of both types of Junctions, e.g. Zonula Occludens (ZO) Proteins and Catenins.

The latter group of molecules connects TransMembrane proteins with the cell CytoSkeleton.

A close spatial association of the TJ proteins with those of AJs indicates that both Junctional types are intermingled in the BBB type of Endothelium.

One of most important purposes of this work is to find out the junction-associated molecules that can serve as sensitive markers of normal or disturbed function of Brain Barriers.

Understanding the structural-functional relations between molecular components of junctional complexes in physiological and experimental conditions of both barriers can provide important information.

About the Etiology of various pathological conditions of the Central Nervous System and also help to elaborate new therapeutic approaches.

To evaluate biochemical changes in contrast-enhancing Multiple Sclerosis (MS) lesions, we examined 14 patients with Relapsing/Remitting MS at acute clinical exacerbation with the help of contrast-enhanced Magnetic Resonance Imaging (MRI) and 1H Magnetic Resonance Spectroscopic Imaging (1H MRSI).

Using a 1.5-tesla MR system (Magnetom Vision, Siemens, Germany), we followed 29 contrast-enhancing and 24 nonenhancing MS lesions as well as Normal-Appearing White Matter (NAWM) before and during high-dose MethylPrednisolone (HDMP) therapy.

Metabolite ratios of N-AcetylAspartate (NAA), Choline (Cho), Creatine (Cr), and Lactate (Lac) were calculated. A transient decrease in contrast enhancement under HDMP therapy was observed.

Both groups of MS lesions showed significantly decreased NAA to Cr ratios compared to NAWM with no changes in time.

Baseline 1H MRSI revealed significantly increased Cho to Cr ratios in the contrast-enhancing MS lesions (1.13 +/- 0.25) compared to the NonEnhancing MS lesions (0.85 +/- 0.26, P < 0.001) and NAWM (0.97 +/- 0.22, P = 0.015).

Both the contrast-enhancing and the nonenhancing MS lesions exhibited a significant increase in Cho to Cr ratios from the second to the third 1H MRSI.

We identified resonances of Lactate in both groups of MS lesions and NAWM without any significant group differences or changes over time.

1H MRSI provides additional information that help to estimate Macrophages' activity, cell membrane activation, and Neuronal impairment within MS lesions.

We believe that combined contrast-enhanced MRI and 1H MRSI may help to further investigate inflammatory processes within active MS lesions and should be employed more frequently to the research on therapy effects in MS.

Objective
Wallerian Degeneration in Normal-Appearing White Matter in early Relapsing/Remitting Multiple Sclerosis (RRMS), and its correlation with the number of relapses and disease duration.

Background
Recent pathological studies have demonstrated Wallerian Degeneration in Normal-Appearing White Matter (NAWM) in Multiple Sclerosis (MS), in established RRMS, and in chronic MS.

However, the presence of Wallerian Degeneration early in the disease and its correlation with relapse and with disease duration has not been studied.

Methods
We performed Proton Magnetic Resonance Spectroscopic Imaging in 21 MS patients, and 4 healthy controls, age and gender matched, aged under 45 years, with a maximum of 4 years since first bout.

And, an EDSS score of less than 3.0. N-AcetylAspartate (NAA) (an index of Axonal integrity) was measured in the NAWM from the Pons and the Cerebellar Peduncles.

Results
We observed that the NAA levels were abnormally low in the NAWM in the early RRMS patients (p = 0.04, Student's t-test).

The decrease in the NAA concentration correlated with disease duration in the two areas studied (p = 0.03 for Pons and p = 0.04 for Cerebellar Peduncle); and with the number of previous relapses (Pearson's correlation = -0.582, p < 0.002).

Conclusion
Wallerian Degeneration measured by the NAA concentration at Pons and Cerebellar Peduncles is present early in the disease and correlates with the number of relapses and disease duration.

It is well known that Multiple Sclerosis (MS) pathogenesis continues even during periods of clinical silence.

To quantify the metabolic characteristics of this activity we compared the absolute levels of N-AcetylAspartate (NAA), Creatine (Cr), and Choline (Cho) in the Normal-Appearing White Matter (NAWM) between Relapsing/Remitting (RR) MS patients and controls.

Metabolite concentrations were obtained with 3D proton MR Spectroscopy at 1.5 T in a 480 cm(3) Volume-Of-Interest (VOI), centered on the Corpus Callosum of 11 MS patients and 9 matched controls.

Gray/White-Matter/Cerebral-Spinal-Fluid (CSF) volumes were obtained from MRI segmentation.

Patients' average VOI tissue volume (V(T)), 410.8 +/- 24.0 cm(3), and metabolite levels, NAA = 6.33 +/- 0.70, Cr = 4.67 +/- 0.52, Cho = 1.40 +/- 0.17 mM, were different from the controls by -8%, -9%, +22% and +32%.

The Cho level was the only single metric differentiating patients from controls at 100% specificity and > 90% sensitivity.

Diffusely elevated Cho and Cr probably reflect widespread microscopic inflammation, Gliosis, or De- and ReMyelination in the NAWM.

Both metabolites are potential prognostic indicators of current disease activity, preceding NAA decline and Atrophy.

Copyright 2003 Wiley-Liss, Inc.

About two-third of patients with Multiple Sclerosis (MS) accumulate severe disabilities before the age of 50. Disability depends mostly on Axonal Loss even if persistent Conduction Block also may be a contributing factor.

Axonal Loss is predominant in the early phase of the disease and is strictly related to the inflammatory activity.

In this phase the Axonal damage is mostly, if not exclusively, determined by Axonal transection inside the lesions, so that reduction of the lesion rate results in a proportional effect of the irreversible tissue damage.

With disease evolution, the redundancy of the Central Nervous System (CNS) starts to be insufficient to compensate the Axonal Loss resulting from reactivation (or persistence at a low degree) of inflammatory activity inside the lesions.

Secondary Axonal Degeneration of Axons damaged by previous attacks and (possibly) by a primitive Axonal Degeneration. In this phase of the disease, most of the irreversible damage does not depend on new lesions.

As a consequence, the reduction of disease activity produces only marginal efforts on Nervous Tissue degeneration.

Clinical trials demonstrate that the positive effect of ImmunoModulatory and AntiInflammatory therapies is maximal in patients with Clinically Isolated Syndromes.

And, it starts to decrease in Relapsing/Remitting phases and is almost lost in Secondary/Progressive phases. From the practical point of view, early treatment is mandatory.

Multiple Sclerosis (MS) has a wide spectrum of clinical courses, characterized by multifocal Central Nervous System (CNS) damage, postulated to be mediated by CNS Antigen-specific T-Cells.

Dendritic Cells (DC), the most potent Antigen-Presenting Cell, play a pivotal role in the decision between T-Cell activation or Anergy.

MonoClonal AntiBodies to CD1a⁺ (immature DC) and CD83⁺ (mature DC) were used to screen lesions with evidence of recent DeMyelinating activity.

And chronic plaque and Normal-Appearing White Matter (NAWM) tissue sections from 12 MS cases by ImmunoCytoChemistry. No CD1a⁺ Cells were detected in the MS or control CNS tissue blocks investigated.

CD83⁺ Cells were not detected in tissues from any of the control cases or in the majority of PeriVascular Cuffs in the MS tissue sections.

However; in eight of the MS tissue blocks with evidence of recent DeMyelination, and in one block each from chronic plaque and NAWM, small numbers of distinct CD83⁺ Cells were present within occasional PeriVascular Cuffs.

In one area only of MS NAWM were CD83⁺ Cells detected in the tissue Parenchyma, in an area of intense Immunological activity.

DC in MS tissue may originate in the peripheral circulation as Monocytes or immature DC and migrate to areas of plaque in response to signals received from CNS-produced Chemokines.

The Blood-Brain Barrier(BBB) restricts exchanges of soluble factors and cells between the blood and Brain, thus playing a crucial role in maintenance of Cerebral homeostasis.

In Multiple Sclerosis (MS), disruption of the BBB is the initial key step in the development of inflammatory lesions. BBB impairment may occur before the formation of DeMyelinative foci or T-Cell infiltration around small vessels.

However, once the BBB breaks, massive infiltration of T-Cells, augmented expression of Adhesion Molecules on Endothelial Cell surface, and leakage of inflammatory Cytokines and AntiBodies will aggravate the MS lesions.

Although GlucoCorticoids and Type 1 Interferons are now widely used and are known to decrease BBB permeability additively, the development of novel therapeutic strategy focusing on the repairment of BBB integrity should be awaited.

In the initial Relapsing/Remitting phase of the disease, the Inflammatory-DeMyelinating component seems to predominate, whereas the Progressive disease appears to be characterized by a NeuroDegenerative component leading to extensive NeuroAxonal Damage in the MS Brain.

Axon Loss most likely determines the persistent Neurological deficit in Progressive MS. Recent studies pointed out that Axon Damage occurs early in the disease and during lesion development.

Two different phases of Axon degeneration were characterized, the first occurring during active Myelin breakdown and the second in chronic DeMyelinated plaques in which the naked Axon seems more susceptible to further damage.

The exact mechanisms and effector molecules of Axonal degeneration, however, are not yet defined, and an Axon-protective therapy has not yet been established.

AutoReactive T-Cells are involved in DeMyelination, NeuroDegeneration, and the recruitment of Peripheral Macrophages and nonspecific activated T-Cells in AutoImmune Diseases such as Multiple Sclerosis (MS).

The Ligation of CoStimulatory B7 molecules on Microglia with CD28⁺/CTLA-4⁺ on T-Cells is thought to be crucial to the onset and course of MS and its rodent model Experimental AutoImmune Encephalomyelitis (EAE).

It is currently unclear as to how far the nature of infiltrating T-Cells has an impact on the expression of the B7 molecules on Microglia, the resident Antigen-Presenting Cells (APCs) of the Brain.

We studied the expression of B7-1 and B7-2 on Microglia after encounter with preactivated Th1 and Th2 Cells from transgenic mice.

Whose T-Cells express a Receptor (TCR) either specific to Myelin Basic Protein (MBP) or Ovalbumin (OVA) using murine Organotypic Entorhinal-Hippocampal Slice Cultures (OEHSC).

Our main finding was that Th1 Cells downregulate the constitutive expression of B7-2 and induce B7-1 expression while Th2 Cells do not induce this B7-1 upregulation.

The main difference between MBP- and OVA-specific cells was seen in experiments were Th1 Cells had direct contact to APCs but not to Brain tissue.

In contrast to MBP-specific Th1 Cells, OVA-specific Th1 Cells required the addition of Antigen to upregulate B7-1 and downregulate B7-2.

When the cells were allowed to have contact to Brain tissue, no difference was seen in the pattern of B7 regulation between OVA- and MBP-specific T-Cells.

Our data suggest that T-Cells are able to modulate B7 expression on MicroGlial Cells in the Brain independent of Antigen presentation through TCR/MHC-II Ligation but presumably by soluble mediators.

Copyright 2001 Wiley-Liss, Inc.