MS Abstracts 01c-2g3

Myelin-reactive T-Cells are considered to play an essential role in the pathogenesis of Multiple Sclerosis (MS), an AutoImmune Disease of the Central Nervous System.

We have previously studied the effects of T-Cell Vaccination (TCV), a procedure by which MS patients are immunized with attenuated autologous Myelin Basic Protein (MBP)-reactive T-Cell clones.

Because several Myelin Antigens are described as potential AutoAntigens for MS, T-Cell vaccines incorporating a broad panel of AntiMyelin reactivities may have therapeutic effects.

Previous reports have shown an accumulation of activated T-Cells recognizing multiple Myelin Antigens in the CerebroSpinal Fluid (CSF) of MS patients.

We conducted a pilot clinical trial of TCV with activated CD4⁺ T-Cells derived from CSF in five MS patients (four RR, one CP) to study safety, feasibility and Immune effects of TCV.

CSF Lymphocytes were cultured in the presence of rIL-2 and depleted for CD8⁺ cells. After 5-8 weeks CSF T-Cell lines (TCL) were almost pure TCRalphaß⁺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 immunizations with irradiated CSF vaccines (10 million cells) were administered with an interval of 2 months.

The vaccinations were tolerated well and no toxicity or adverse effects were reported. The data from this small open-label study cannot be used to support efficacy.

However, 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 all patients. Anti-MBP/PLP/MOG reactivities remained low or were reduced in all patients.

Based on these encouraging results, we 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.

While much work has concentrated on focal White Matter (WM) lesions in Multiple Sclerosis, there is growing evidence to suggest that Normal-Appearing WM (NAWM) and Gray Matter (GM) are also involved in the disease process.

This study investigated Multiple Sclerosis disease effects on NAWM and Cortical GM (CGM) metabolite concentrations, and the relationships between these metabolite concentrations and clinical impairment.

Proton Magnetic Resonance Spectroscopic imaging (₁H-MRSI) data acquired using Point Resolved Spectroscopic (PRESS) localization (echo time 30 ms, repetition time 3000 ms, nominal Voxel volume 2.3 ml).

From 27 Relapsing/Remitting Multiple Sclerosis and 29 normal control (NC) subjects were processed using LCModel to estimate metabolite concentrations in millimoles per litre.

₁H-MRSI voxel tissue contents were estimated using SPM99 tissue and semi-automatic lesion segmentations of three-dimensional fast spoiled gradient recall scans acquired during the same scanning session.

NAWM and CGM metabolite concentrations estimated were: Choline-containing compounds (Cho); Creatine and PhosphoCreatine (Cr); myo-Inositol (Ins); N-AcetylAspartate plus N-AcetylAspartyl-Glutamate (tNAA); and Glutamate plus Glutamine (Glx).

CGM data came from 24 of the Multiple Sclerosis (mean age 35.2 years, mean disease duration 1.7 years) and 25 of the NC (mean age 34.9 years) subjects.

NAWM data came from 25 of the Multiple Sclerosis (mean age 35.0 years, mean disease duration 1.7 years) and 28 of the NC (mean age 36.7 years) subjects.

Metabolite concentrations were compared between Multiple Sclerosis and NC subjects using multiple (linear) regression models allowing for age, gender, ₁H-MRSI voxel tissue and CSF contents, and Brain Parenchymal Volume.

At a significance level of P < 0.05, CGM Cho, CGM and NAWM tNAA, and CGM Glx were all significantly reduced, and NAWM Ins was significantly elevated.

Spearman correlations of Multiple Sclerosis Functional Composite Scores with tissue metabolite concentrations were significant for the following: CGM Cr (r(s) = 0.524, P = 0.009), CGM Glx (r(s) = 0.580, P = 0.003) and NAWM Ins (r(s) = -0.559, P = 0.004).

These results indicate that metabolite changes in NAWM and CGM can be detected early in the clinical course of Multiple Sclerosis, and that some of these changes relate to clinical status.

The correlation of clinical impairment with CGM Cr and Glx but not tNAA suggests that it is more closely associated with Neuronal metabolic dysfunction rather than loss in Clinically Early Relapsing/Remitting Multiple Sclerosis.

The correlation of clinical impairment with a raised NAWM Ins may indicate that Glial proliferation also relates to function at this stage of the disease.

A 56-year-old woman with a 40-year history of Multiple Sclerosis (MS) developed Paroxysmal attacks of Girdle Sensation in the Th5-6 Dermatomes.

The attacks lasted 20-60 minutes and occured up to three times per week. T₂-weighted MR imaging of the Spinal Cord showed high intensity area from Th5 to Th8.

ElEctroCardiography, EchoCardiography and laboratory findings did not indicate Ischemic Heart Disease; therefore, the Paroxysms were attributed to the Spinal Cord lesions. Attacks were successfully suppressed by Acetazolamide 250 mg/day.

Although Carbamazepine is frequently used to treat Paroxysmal attacks in MS, we would like to suggest that Acetazolamide may also be beneficial in some patients with Paroxysmal Symptoms.

Multiple Sclerosis (MS) is likely an AutoImmune Disorder, although this remains unproven. ImmunoTherapeutic treatments have been shown to be helpful, especially in Relapsing forms of the illness.

But the treatments are incomplete, and many patients continue to worsen over time, even with standard therapy. ImmunoTherapies presently available appear to have their greatest effect when used early in the course of the illness.

In Relapsing/Remitting Multiple Sclerosis (RRMS), there is overwhelming Class I data from large clinical trials that supports the use of Interferon-beta-1a (IFN-ß-1a), IFN-ß-1b, and Glatiramer Acetate.

Comparative data are limited, and results published in different trials support the idea that treatment outcomes with the various drugs are more similar than different. Decisions about treatment choice should be tailored to the needs of the individual patient.

With the exception of a small number of patients with Benign MS, all RRMS patients should be treated with one of the Interferons or Glatiramer Acetate.

There are Class I data consistent with the idea that higher dose or more frequent administration of IFN-ß is associated with better clinical outcome and reduced progression of changes on brain MRI scans.

The duration of this effect is not clear, and higher dose with more frequent administration is associated with higher cost, more side effects, and greater production of Interferon AntiBodies.

Interferon AntiBodies possibly reduce efficacy of IFN-ß in RRMS and Secondary/Progressive Multiple Sclerosis (SPMS).

Clinically Isolated Syndromes (CIS) of DeMyelination in the Central Nervous System can be reliably diagnosed, and the risk of further episodes of DeMyelination is consistent with the diagnosis of RRMS stratified by use of Brain MRI scans.

Patients at high risk of developing RRMS after CIS achieve significant benefit after treatment with IFN-ß-1a, and initiation of therapy after CIS should be given strong consideration.

There are no similar data for IFN-ß-1b or Glatiramer Acetate, but logic would dictate a similar response with these agents.

In SPMS, there are Class I data that treatment with IFN-ß-1a or IFN-ß-1b has a significant effect on progression of Brain MRI lesions, but clinical outcomes are less clearly affected.

It is justifiable to treat SPMS patients with IFN-ß. Mitoxantrone may be effective in slowing progression of SPMS, and its risks are moderate. It should be used in patients with SPMS, but potential long-term risks must be discussed with the patient in detail.

Results of treatment of SPMS in advanced cases (Extended Disability Status Score greater than 6.5, or restricted to wheelchair) is mostly unknown.

These patients are at high risk of developing infections, especially if they use indwelling catheters, and the use of agents that induce ImmunoSuppression may be risky.

There are no effective therapies for Primary/Progressive Multiple Sclerosis (PPMS). Although PPMS patients are frequently treated with one or more therapeutic agents, there is no medical justification for this now.

Axonal Injury has been proposed as the basis of permanent deficits in the Inflammatory, DeMyelinating Disease, Multiple Sclerosis. However, reports on the degree of injury are highly variable, and the responsible mechanisms are poorly understood.

We examined the relationships among long-term DeMyelination, Inflammation, Axonal Injury, and Motor Function in a model of Multiple Sclerosis, in which mice develop chronic, Immune-mediated DeMyelination of the Spinal Cord resulting from persistent infection with Theiler's Virus.

We studied two strains of mice, inbred SJL/J and C57BL/6x129 mice deficient in ß-2-MicroGlobulin and therefore CD8⁺ Lymphocytes. After 8 months of disease, SJL mice had considerably worse Motor Function than beta-ß-MicroGlobulin-deficient mice.

Motor Dysfunction correlated linearly with the extent of DeMyelinated lesions in the Spinal Cord (Lesion Load) within each strain, but no difference in lesion load was present between strains. Also, the extent of ReMyelination did not differ between strains.

Instead, the disparity in Motor Deficits reflected differences in the integrity of Descending Neurons. That is, retrograde labeling of ReticuloSpinal, VestibuloSpinal, and RubroSpinal Neurons, although reduced in all chronically diseased mice, was two to seven times higher in ß2-MicroGlobulin-deficient mice.

The labeling was superior in ß2-MicroGlobulin-deficient mice despite the fact that lesion expanse and therefore the number of Axons traversing lesions were similar in both strains.

Thus, by all criteria Axons were equivalently DeMyelinated in SJL and ß-2-MicroGlobulin-deficient mice, but the extent of Axonal Injury differed significantly.

These results indicate that mechanisms of DeMyelination and Axonal Injury are at least partly separable, and are consistent with the hypothesis that CytoToxic CD8⁺ Lymphocytes may selectively injure DeMyelinated Axons.

Additionally, the data suggest that Axonal injury obligatorily results from chronic Inflammatory DeMyelination and significantly contributes to Neurological Deficits.

Background
In 10-15 % of patients with Multiple Sclerosis (MS), the clinical course is characterized by slow progression in Disability without relapses (Primary/Progressive (PP) MS). The mechanism of disability in this form of MS is poorly understood.

Using Magnetization Transfer Ratio (MTR) imaging, we investigated Normal-Appearing White Matter (NAWM) and Normal-Appearing Gray Matter (NAGM) in PPMS and explored the relationship of MTR measures with disability.

Methods
Thirty patients with PPMS and 30 age matched controls had spin echo based MTR imaging to study lesions and Normal-Appearing Tissues.

The Brain was segmented into NAWM and NAGM using SPM99 with lesions segmented using a semiautomated local thresholding technique.

A 75 % probability threshold for classification of NAWM and NAGM was used to diminish partial volume effects. From normalized Histograms of MTR intensity values, six MTR parameters were measured.

Mean lesion MTR and T₂ lesion volume were also measured. Disability was assessed using Kurtzke's Expanded Disability Status Scale (EDSS).

Results
Compared with controls, patients exhibited a significant reduction in mean NAWM (p = 0.001) and NAGM (p = 0.004) MTR.

Spearman's rank correlation of EDSS with the six MTR parameters in NAWM and NAGM, mean lesion MTR, and T₂ lesion volume, was only significant with mean NAGM MTR (r = -0.41, p = 0.02), the 25th percentile of NAGM MTR intensity (r = -0.37, p = 0.05), and T₂ lesion volume (r = 0.39, p = 0.04).

Multiple regression analysis of the relationship between EDSS and 4 MR parameters representing each tissue type (mean NAWM MTR, mean NAGM MTR, mean lesion MTR, T₂ lesion volume) showed that the association of EDSS with mean NAGM MTR remained significant.

Conclusions
There appear to be significant abnormalities in the NAGM in PP MS. Further investigation of the pathological basis and functional significance of Gray Matter abnormality in PPMS is warranted.

We have studied in vivo effect of Interferon-ß-1a (IFN-ß-1a) (6 MIU once weekly i.m.) on InterLeukin-12 (IL-12).

And, Transforming Growth Factor-beta-1 (TGF-ß-1) Serum levels during 6 months of therapy in group of 20 patients with Relapsing/Remitting Multiple Sclerosis (MS).

IL-12 and TGF-ß-1 concentrations were measured by Enzyme Linked ImmunoabSorbent Assay (ELISA). There was a significant increase of IL-12 levels in MS patients in comparison with control group, suggesting a role of this Cytokine in Immunity of MS.

We have also found a significant increase of TGF-ß-1 levels after 6 months of therapy with IFN-ß-1a, however, there was no in vivo effect of the therapy on IL-12 levels.

The results suggest that IFN-ß-1a may exert its action through up- regulation and increase secretion of TGF-ß-1.

Although Axonal pathology is recognized as one of the major pathological features of Multiple Sclerosis, it is less clear how early in its course it occurs and how it correlates with MRI-visible lesion loads.

To assess this early Axonal pathology, we quantified the concentration of Whole-Brain N-AcetylAspartate (WBNAA) in a group of patients at the earliest clinical stage of the disease and compared the results with those from healthy controls.

Conventional brain MRI and WBNAA using unlocalized Proton Magnetic Resonance Spectroscopy were obtained from 31 patients at presentation with Clinically Isolated Syndromes (CIS) suggestive of Multiple Sclerosis and paraclinical evidence of dissemination in space, and from 16 matched controls.

An additional conventional MRI scan was obtained in all patients 4–6 months later to detect dissemination of lesions in time. The mean WBNAA concentration was significantly lower in patients compared with the controls (P < 0.0001).

It was not significantly different between patients with and without enhancing lesions at the baseline MRI or between patients with and without lesion dissemination in time. No correlation was found between WBNAA concentrations and lesion volumes.

Widespread Axonal pathology, largely independent of MRI-visible inflammation and too extensive to be completely reversible, occurs in patients even at the earliest clinical stage of Multiple Sclerosis.

This finding lessens the validity of the current concept that the Axonal pathology of Multiple Sclerosis is the end-stage result of repeated inflammatory events, and argues strongly in favour of early NeuroProtective intervention.

Objective
To determine the degree of Axonal damage in relationship to signal abnormalities on T₂-weighted high-resolution MRI in Spinal Cord tissue of patients with MS.

Methods
Spinal Cord specimens of nine patients with MS and four controls were imaged at high resolution (4.7 T) in an Axial plane and scored for lesions with increased Signal Intensity (SI).

Histopathologic sections were cut and ImmunoStained with NE14 (NeuroFilament marker) and Luxol fast blue (Myelin stain). For each area, Axonal density and diameter were quantified:

Axonal irregularity, NE14 Axonal staining intensity, and Myelin content were semiquantitatively scored. Included were 209 areas from MS cases and 109 areas from control cases distributed over Lateral, Posterior, and Anterior Columns.

Results
In control cases, no SI changes were found, average density of Axons was 26,989/mm(2), average diameter was 1.1 micro m, and all scores for Axonal irregularity, NE14 staining intensity, and Myelin were normal.

In MS cases, areas with increased SI were found, average Axonal density was 11,807/mm(2) (p < 0.0001), and average Axonal diameter 2.0 micro m (p = 0.001).

Areas with high SI on MRI had:
1. lowest Axonal density
   • average count: 10,504/mm(2); range: 3,433 to 26,325/mm(2)
2. largest diameter
   • average: 2.3 micro m; range: 1.0 to 4.0 micro m
3. highest Axonal irregularity and NE14 staining intensity compared to Normal Appearing Cord Tissue (NACT)

However, NACT of MS cases also had lower Axonal density (14,158/mm(2)) and higher average Axonal diameter (1.6 micro m) than controls.

Conclusions
Marked Axonal loss occurs in MS Spinal Cords, largely independent of the degree of signal abnormality on T₂-weighted MRI.

Objective
To assess:

1. The correlations between Magnetization Transfer Ratio (MTR) Histogram derived measures of the Brain and the Cervical Cord from patients with different Multiple Sclerosis phenotypes
2. The correlation between these metrics and clinical disability

Magnetization Transfer imaging is sensitive to the most destructive aspects of Multiple Sclerosis pathology. Magnetization Transfer Ratio Histogram analysis encompasses the macroscopic and the microscopic lesion burdens.

Methods
Seventy seven patients with Multiple Sclerosis were studied (40 Relapsing/Remitting (RR), 28 Secondary/Progressive (SP), and nine Primary/Progressive (PP)).

For the Brain, we obtained dual echo, T₁ weighted, and Gradient Echo (GE) scans (with and without an MT saturation pulse).

For the Cervical Cord, fast Short Tau Inversion Recovery (STIR) and GE scans (with and without an MT saturation pulse) were obtained.

Brain T₂ and T₁ weighted Lesion Volumes (LVs) were measured. The number and length of Cord lesions on fast STIR scans were assessed.

Magnetization Transfer Ratio maps were created from GE images and MTR Histograms of the entire Brain and Cervical Cord were obtained.

Results
Brain T₁ LV, and number and size of Cord lesions were significantly higher and Brain MTR Histogram peak location was significantly lower in patients with SPMS than those with RRMS or PPMS.

Cord MTR Histogram peak location was also significantly lower in patients with SPMS than in those with RRMS.

The univariate correlations between MTR Histogram derived metrics obtained from the Brain and the Cervical Cord were all non-significant, with the exception of that between average Brain MTR and Cord MTR Histogram peak location.

On a multivariable analysis, both increasing Brain T₂ LV and decreasing Cord MTR Histogram peak location values were significantly associated with a higher probability for patients to have SPMS or to have Locomotor Disability.

Conclusions
This study shows that the extent and severity of tissue damage in the Brain and Cervical Cord are both relevant to determine disability in Multiple Sclerosis and that the assessment of Brain and Cord pathology provides complementary information.

Multiple Sclerosis (MS) is an Inflammatory DeMyelinating Disease of the Central Nervous System (CNS).

Cognitive Impairment (CI) may develop at any time during the course of the disease in the presence or absence of Neurological Disability.

On the basis of comprehensive NeuroPsychological studies, there is now a consensus among investigators that 45 percent to 65 percent of MS patients suffer from some degree and form of Cognitive difficulty.

Features of CI include Bradyphrenia; Impaired Attention, Concentration and Abstract Reasoning; Reduced Manual Speed and Dexterity; deficits in Memory Retrieval; and Language Deficits in both the Relapsing/Remitting and Progressive forms of MS.

Impairments in all Cognitive Domains may result from the diffuse spread of microscopic pathology, although a preferential Lobar distribution of plaques can present with a predominant deficit in the corresponding Cognitive function.

Nevertheless, the severity of CI best correlates with total microscopic and macroscopic disease burden of the Brain as defined by recently developed Magnetic Resonance Imaging (MRI) sequences.

A disruption of connecting InterCortical and SubCortical pathways is likely to be the main cause of metabolic and functional abnormalities in Neurons.

However a direct toxic effect of soluble inflammatory products may also compromize Neuronal function and survival.

Early treatment of MS with Interferons and Copaxone can prevent or delay the onset of both Neurological and Cognitive disabilities by reducing the inflammatory activity and damage in the CNS.

Until more powerful NeuroProtective agents become available, simple NeuroPsychological screening and Cognitive rehabilitation for Memory and Language impairments will remain important components in the care of MS patients.

Background
Although in situ pathological studies and in vivo Magnetic Resonance (MR) investigations have shown that Axonal injury can be significant in the early stages of Multiple Sclerosis (MS), diffuse Axonal Injury is generally considered a secondary event.

Cerebral Axonal damage can be specifically assessed in vivo by measuring levels of Brain N-AcetylAspartate (NAA, a specific index of Axonal integrity detected by MR Spectroscopy).

Other new MR measurements such as Magnetization Transfer Ratio (MTR) or computed estimation of Brain Volume can provide less specific indexes of tissue damage.

Objective
To determine whether diffuse Axonal and tissue injury is present in patients with Definite MS who do not show clinically significant disability.

Methods
We measured Brain NAA levels (normalized to Creatine [Cr]), MTR values, and Cerebral volumes in patients with definite MS who had low T₂-weighted MR imaging lesion volumes and no clinical disability, and also in age-matched healthy control subjects.

Results
Values of Central Brain NAA/Cr and MTR in Normal-Appearing White Matter were significantly lower in the MS patients than in controls (P< .001). In contrast, total Brain Volumes were not significantly different between these groups.

Similar results were found for MS patients with early disease (duration, < 3 years) and with a particularly low Cerebral T₂-weighted MR imaging lesion load (< or = 2 cm(3)).

Conclusions
Cerebral NAA/Cr and MTR values are diffusely decreased in MS patients with early disease, low DeMyelinating Lesion Load, and no significant disability.

This suggests that Axonal and/or tissue injury begins very early in the course of MS and might be at least partially independent of Cerebral DeMyelination.