MS Abstracts 5c-2g

T-Cells with specificity for self-Ags are normally present in the peripheral blood, and, upon activation, may target tissue Ags and become involved in the PathoGenesis of AutoImmune processes.

In Multiple Sclerosis, a DeMyelinating Disease of the CNS, it is postulated that Inflammatory damage is initiated by CD4⁺ T-Cells reactive to Myelin Ags.

To investigate the potential Naive vs Memory origin of circulating Myelin-reactive cells, we have generated Myelin Basic Protein (MBP)- and Tetanus Toxoid-specific T-Cell clones from CD45RA⁺/RO- and CD45RO⁺/RA- CD4⁺ T-Cell subsets from the peripheral blood of Multiple Sclerosis patients and controls.

Our results show that:

1. The response to MBP, different from that to TT, predominantly emerges from the CD45RA⁺ subset;

2. The reactivity to ImmunoDominant MBP Epitopes mostly resides in the CD45RA⁺ subset;

3. In each individual, the recognition of single MBP Epitopes is skewed to either subset, with no overlap in the Ag fine specificity; and

4. In spite of a lower expression of costimulatory and Adhesion Molecules, CD45RA⁺ subset-derived clones recognize Epitopes with higher functional Ag avidity.

These findings point to a central role of the naive CD45RA⁺ T-Cell subset as the source for ImmunoDominant, potentially pathogenic effector CD4⁺ T-Cell responses in humans.

The objective of this study was to characterize and compare the Proton Magnetic Resonance spectrum of a Voxel (volume element), containing lesions or Normal-Appearing White Matter (NAWM), in Primary/Progressive (P/P) and Secondary/Progressive (S/P) Multiple Sclerosis (MS) patients.

Single-Voxel Proton Magnetic Resonance Spectroscopy and Magnetic Resonance Imaging were performed in 35 MS patients (17 P/P and 18 S/P) and 17 controls.

Spectra from an 8 ml Voxel located in the Parieto-Occipital region were obtained with a Spin-Echo pulse sequence (1600 ms/135 ms/256, TR/TE/acquisitions).

Resonance areas due to N-AcetylAspartate (NAA), Creatine/PhosphoCreatine (Cr) and Choline compounds (Cho) were determined, and results expressed in terms of area in arbitrary units or as metabolite ratios.

With respect to the control group, there were significant reductions in NAA and NAA/Cho ratio in the P/P-lesion, S/P-lesion, P/P-NAWM and S/P-NAWM groups. There were no significant differences between the P/P-NAWM and S/P-NAWM groups.

These results support the existence of metabolic changes in the White Matter of P/P and S/P patients and suggest that there is Neuronal damage and/or loss in both clinical courses.

Finally, characterization of the Parieto-Occipital region showed no significant differences in the Spectral pattern of NAWM between P/P and S/P clinical courses of MS.

Copyright 2000 John Wiley & Sons, Ltd.

Based on the good results of experimental transplantation in animal models of Multiple Sclerosis and of other AutoImmne Diseases, we have treated 24 patients suffering from Chronic/Progressive Multiple Sclerosis with high-dose ChemoTherapy (BEAM regimen) followed by autologous blood Stem Cell rescue and AntiThymocyte Globulin.

Blood Stem Cells were mobilized with Cyclophosphamide at 4g/m2 and G- (or GM-) CSF. In 9 cases, additional CD34⁺ cell-selection of the graft was performed.

Here we update previously published results of this novel treatment, mainly with regard to clinical efficacy, as the median follow-up time has reached 40 months (range, 21-51).

Infections were the principal toxicity early after the procedure, with death of a patient from Aspergillosis 65 days post Stem Cell infusion.

No serious late events occurred apart from a case of AutoImmune Thyroiditis that developed 11 months after transplant in a patient who had received a CD34⁺ cell-depleted graft.

Mild and transient NeuroToxicity was observed in 10 patients (42%), most probably associated with fever and infections.

Eighteen patients (18/23; 78%) responded to the treatment, i.e., they were improved or stabilized, while five patients progressed, of which 4 had Primary/Progressive disease.

Of those improved or stabilized (18), 9 patients have maintained stable condition whereas 9 developed relapses or they slowly resumed progression, although their Disability scores have not gotten worse than they were before transplantation.

The probability of progression-free survival (compared to entry status) at 3 years is 92% for patients with Secondary/Progressive disease and 39% for the Primary/Progressive type.

CD34⁺ cell-selection did not seem to yield better results except for a delay in progression or in relapse after transplantation.

These results appear better than those achieved by any other treatment of Progressive Multiple Sclerosis, including ß-Interferon.

But, they need to be confirmed by other open or controlled studies in view of the well-known difficulty of judging objectively the effect of a treatment in patients with this disease.

The authors compare the Spinal Cord Magnetization Transfer ratio (MTR) of Multiple Sclerosis (MS) patients to healthy volunteers, relate MTR to Cord Atrophy, and relate these and other Magnetic Resonance (MR) imaging parameters to Disability.

Sixty-five patients with MS (14 Relapsing/Remitting [RR], 34 Secondary/Progressive [S/P], and 17 Primary/Progressive [P/P] MS), and 9 healthy volunteers were studied using MR at 1.0 T. Disability of the patients was assessed using the Expanded Disability Status Scale (EDSS).

Magnetic Resonance parameters were upper Spinal Cord MTR, number of Focal Spinal lesions, presence of diffuse abnormalities, and Spinal Cord Cross-Sectional Area (CSA).

Correlations were assessed using Spearman's rank correlation coefficient (r). Magnetization Transfer ratio was higher in the controls (median, 33%; range, 30%-38%) than in patients with MS (median, 30%; range, 16-36; p < 0.05).

In patients with MS EDSS correlated with Spinal Cord MTR, albeit weakly (r = -0.25, p < 0.05). Correlation between EDSS and Spinal Cord CSA was better (SRCC = -0.40, p < 0.01).

No correlation was found between MTR and CSA (r = 0.1, p = 0.4). Combining MTR with Spinal Cord CSA improved correlation with EDSS (r = -0.46, p < 0.001), suggesting an independent correlation between Disability and these 2 MR parameters.

Expanded Disability Status Scale scores were higher in patients who had diffuse Spinal Cord abnormality regardless of focal lesions (median, 6; range, 1.5-7.5) than in patients without diffuse abnormalities (median, 3.5; range, 0-8; p < 0.01).

CSA was lower in patients with diffuse Spinal Cord abnormalities (median, 62; range, 46-89 mm2) than in patients without diffuse abnormalities (median, 73; range, 47-89 mm2; p < 0.01).

MTR was slightly lower in patients with diffuse Spinal Cord abnormalities (median, 29; range, 21%-33%) than in patients without diffuse abnormalities (median, 31; range, 16-36; t-test, p < 0.05).

Tumor Necrosis Factor-alpha (TNF-) appears to take part in the PathoGenesis of Multiple Sclerosis and to contribute to the degeneration of Oligodendrocytes as well as Neurons.

TNF- is produced by Microglia and Astrocytes, which also produce Hormones and Cytokines that influence its biological activity.

Thus, in mixed cultures the effects of exogenous TNF- might be modified by products of Astrocytes and Microglia. The effects of TNF- in Oligodendrocyte enriched cultures are reported below.

We prepared the cultures by shaking Oligodendrocytes off Primary mixed Glial Cell cultures from Brains of 2-day-old rats at 7 days in vitro and plating them (0 days post-shake, DPS).

Platelet-Derived Growth Factor and Fibroblast Growth Factor were included in the media at 1-5 DPS in order to encourage proliferation.

At 2 DPS media were added with no TNF- (controls) or 1000, 2000 or 5000 U/ml of TNF-, and at 5 DPS media were replaced with fresh Serum-free media.

Cultures were fixed with 4% ParaFormaldehyde at 5, 7, 9 and 12 DPS and ImmunoStained.

Oligodendrocyte Progenitors were not reduced in numbers immediately after the incubation with TNF- (i.e. at 5 DPS).

However, after an additional 4 days in culture fewer Progenitors remained in the cultures that had been treated with TNF- than in the untreated cultures.

In the absence of the Growth Factors there were fewer Progenitors, but their numbers also were reduced by TNF-.

Maturation to the Myelin Basic Protein (MBP)-positive stage was inhibited by about 36% at 9 DPS by 1000-2000 U/ml of TNF-, while numbers of O4+/MBP- Precursors were unaffected.

It is interesting that the steady-state number of O4-positive Precursors was unchanged by TNF- at 9 DPS, when there were reductions in the numbers of A2B5-positive Progenitors and MBP-positive mature Oligodendrocytes.

That observation suggests that the rates of proliferation, death and maturation are controlled by multiple factors, with a particularly vulnerable time at the maturation to the MBP-positive stage.

At 5000 U/ml TNF - the specific effect on maturation was overtaken by CytoToxicity.

These data and a summary of the literature suggest that inhibition of MBP expression is sensitive to lower TNF- concentrations and incubation times than is cell survival.

Specific effects on numbers of MBP-positive cells, morphology and MBP expression occur at 1000-2000 U/ml for 48-72 h or at up to 10000 U/ml for</=24 h, and the deficits remain after removal of the TNF-.

InterLeukin-7 (IL-7), originally described as a B-Cell growth factor, has recently been found to play a critical role in T and B-Lymphocyte development and function.

This study evaluated the effects of IL-7 on Myelin specific T-Cells. IL-7 strongly enhanced proliferation of ProteoLipid Protein (PLP) 139-151 specific T-Cells in association with elevated secretion of the T-Cell Growth Factor IL-2.

Co-stimulation with IL-7 preferentially increased the levels of ProInflammatory Cytokines secreted by PLP 139-151 specific T-Cells and adoptive transfer of these cells into naive recipients induced a profound enhancement of Experimental AutoImmune EncephaloMyelitis, an animal model for the human disease Multiple Sclerosis.

These results suggest that IL-7 may be a critical co-stimulatory factor that enhances the ExtraThymic expansion of Inflammatory T-Cells and may play an important role in the PathoGenesis of a number of Inflammatory AutoImmune Disorders.

Although Multiple Sclerosis (MS) is an Inflammatory DeMyelinating Disease, there can be substantial Axonal injury and loss.

We therefore hypothesized that adaptive Cortical changes may contribute to limiting functional Impairment, particularly in the early stages of the disease.

To test our hypothesis, we used functional Magnetic Resonance Imaging (MRI) to characterize the localization and volumes of activation in the Motor Cortex during simple Flexion-Extension finger movements.

There were differences in the patterns of Cortical activation with movement between the 12 MS patients and the 12 normal controls.

All patients showed greater relative Supplementary Motor Area activation than did the normal controls.

The relative Hemispheric lateralization of SensoriMotor Cortex (SMC) activation decreased in direct proportion to the total Cerebral T₂-weighted MRI HyperIntense lesion load.

This appeared to be due primarily to increases in IpsiLateral (Same Side) SMC activation with increasing lesion load in White Matter of the Hemisphere ContraLateral (Opposite Side) to the limb moved.

The center of activation in the ContraLateral SMC was shifted a mean of 8.8 mm posterior in patients relative to controls, providing additional evidence for Cortical adaptive responses to injury.

The magnitude of this posterior shift in the SMC activation increased with greater T2 lesion loads.

These observations demonstrate that Cortical recruitment for simple finger movements can change both quantitatively and qualitatively in the SMCs of MS patients, suggesting that Cortical reorganization or "unmasking" of latent pathways can contribute to functional recovery.