MS Abstracts 03b-2g1

Transgenic mice expressing IL-3 and IFN-alpha under the regulatory control of the GFAP Gene promoter (GFAP-IL3 and GFAP-IFN-alpha mice) exhibit a Cytokine-specific, late-onset Chronic-Progressive Neurological Disorder.

Which resemble many of the features of human diseases such as Multiple Sclerosis, Aicardi-Goutieres Syndrome, and some Viral Encephalopathies including HIV LeukoEncephalopathy.

In this report we show that the MetalloThionein-I+II (MT-I+II) isoforms were upregulated in the Brain of both GFAP-IL3 and GFAP-IFN-alpha mice in accordance with the site and amount of expression of the Cytokines.

In the GFAP-IL3 mice, in situ hybridization analysis for MT-I RNA and RadioImmunoassay results for MT-I+II protein revealed that a significant upregulation was observed in the Cerebellum and Medulla plus Pons at the two ages studied, 1-3 and 6-10 months.

Increased MT-I RNA levels occurred in the Purkinje and Granular layers of the Cerebellum, as well as in its White Matter Tracts.

In contrast to the Cerebellum and BrainStem, MT-I+II were downregulated by IL-3 in the Hippocampus and the remaining Brain in the older mice.

In situ hybridization for MT-III RNA revealed a modest increase in the Cerebellum, which was confirmed by ImmunoHistoChemistry.

MT-III ImmunoReactivity was present in cells that were mainly round or amoeboid Monocytes/Macrophages and in Astrocytes.

MT-I+II induction was more generalized in the GFAP-IFN-alpha (GIFN12 and GIFN39 lines) mice, with significant increases in the Cerebellum, Thalamus, Hippocampus, and Cortex.

In the high expressor line GIFN39, MT-III RNA levels were significantly increased in the Cerebellum (Purkinje, Granular, and molecular layers), Thalamus, and Hippocampus (CA2/CA3 and especially Lacunosum moleculare layers).

Reactive Astrocytes, activated rod-like Microglia, and Macrophages, but not the PeriVenular infiltrating cells, were identified as the cellular sources of the MT-I+II and MT-III proteins.

The pattern of expression of the different MT isoforms in these transgenic mice differed substantially, demonstrating unique effects associated with the expression of each Cytokine.

The results indicate that the MT expression in the CNS is significantly affected by the Cytokine-induced inflammatory response and support a major role of these proteins during CNS injury.

Copyright 2001 Academic Press.

Background
During the relapse of Multiple Sclerosis, the activation of T-Cells, AutoReactive to Myelin Antigens in blood, enhanced and maintained as a result of anomalous mechanisms of their earlier elimination, leads on Para- and Autocrine basis to the activation of Antigen- non-specific cells of Immune System.

In consequence, activated cells secrete a range of proinflammatory Cytokines and display activation Antigen expression on their surface, which results in Blood-Brain Barrier damage.

The differentiation of Lymphocytes into Effector Cells in blood during MS relapse is to increase the number of cells supporting inflammatory reactions and simultaneously to reduce the number of cells which play a role of Suppressors.

Fas Antigen is present among activation Antigens found on T-Cells. Once this Antigen has been combined with the Ligand, it transmits Apoptic signal to the cell.

The presence of Fas Antigen on activated peripheral blood T-Cells may enable us to estimate their activation and it may also indicate a potential to eliminate those cells from blood.

The aim of the study was to provide a quantitative assessment of the subpopulations of CD3⁺, CD4⁺ and CD8⁺ Lymphocytes in peripheral blood.

And to investigate Fas Antigen expression on these subsets in patients with Relapsing/Remitting Multiple Sclerosis, in relation to clinical activation of the disease.

Material And Methods
Thirty-five patients participated in the study, including 14 patients finding themselves in clinical relapse of the disease and 21 patients in the state of remission. Additionally, 21 healthy subjects were included.

Quantitative assessment of individual subpopulations and Fas co-expression was carried out with the use of MonoClonal Antigen anti CD3⁺, CD4⁺ and CD8⁺ as well as anti CD95 AntiBodies, and flow cytometer Pas/Dako Galaxy.

Results
The differences in the percentage of particular Lymphocytes between 3 groups proved insignificant.

Patients in the relapse of the disease showed significantly greater Fas expression on subpopulations CD3⁺ and CD4⁺ when compared to the results obtained from remission patients and control subjects. This difference was not observed for Fas expression on subset CD8⁺.

Conclusions
The investigation of Fas receptor expression may be useful in order to monitor clinical course of the disease, which is characterized by the periods of exacerbation and remission.

AutoImmune Diseases such as Multiple Sclerosis (MS) are characterized by the loss of Tolerance to self-determinants, activation of AutoReactive Lymphocytes and subsequent damage to single or multiple organs.

The mechanisms by which AutoImmune responses are triggered, and how activation of AutoReactive Lymphocytes is initiated and maintained, are not fully understood.

Therapeutic approaches in AutoImmune Diseases have so far concentrated on Antigens and T-Cells.

Given the exceptional capacity of Dendritic Cells (DCs) to induce Immunity in vivo, recent reports of the first successful clinical trials based on vaccination of Tumor patients with autologous blood DCs pulsed in vitro with Tumor Antigen come as no surprise.

The recent identification of Tolerogenic subsets of DCs and their generation in culture may allow a novel approach to induce Tolerance in AutoImmune Diseases.

By selective in vitro manipulation of DCs and their subsequent reinfusion, DC-mediated Tolerance has been achieved in animal models of human AutoImmune Diseases.

Including Experimental AutoImmune EncephaloMyelitis in Lewis rats and SJL/J mice and spontaneous diabetes in NOD mice.

In vitro observations of human blood DCs are promising for DC-based treatment of MS and other diseases with an AutoImmune component.

Data from animal models and human materials suggest that DC-based ImmunoTherapy could be beneficial at least as a complement to conventional therapy.

Molecular-biological approaches to Tolerogenic DCs could provide a rationale for designing ImmunoTherapeutic strategies in AutoImmune Diseases.

Atrophy of the Spinal Cord is known to occur in Multiple Sclerosis but the cause and the timing of its onset are not clear. Recent evidence suggests that Atrophy may start to occur early in the disease.

The aim was to determine whether Atrophy of the Spinal Cord could be detected in vivo using MRI techniques, in patients presenting with a Clinically Isolated Syndrome, which in many cases is the earliest clinical stage of Multiple Sclerosis.

The cross sectional area of the Spinal Cord was measured in 43 patients presenting with a Clinically Isolated Syndrome and 15 matched controls.

T₂ weighted imaging of the Brain was also performed to determine the number and volume of high signal lesions consistent with disseminated DeMyelination. Both patients and controls were restudied after 1 year.

The Spinal Cord Area was significantly smaller in the 74% of patients with an abnormal Brain MRI at presentation than in controls (mean areas 73.9 mm(2) and 78.1 mm(2) respectively, p=0.03).

No significant difference was found in the Spinal Cord area between controls and patients with normal baseline Brain imaging. The annual rate of change in patients did not differ significantly from controls.

In conclusion, the finding of a smaller Cord area in the subgroup of patients with Clinically Isolated Syndrome with the highest risk of developing Multiple Sclerosis.

That is, with an abnormal Brain MRI, suggests that Atrophy has developed in some patients with Multiple Sclerosis even before their first clinical symptoms.

However, the lack of a detectable change in Cord Area over 1 year of follow up contrasts strikingly with the results of an earlier study of patients with Relapsing/Remitting Multiple Sclerosis, suggesting that the rate of Atrophy increases as the disease becomes more established.

Knowledge concerning the relationship between Multiple Sclerosis and Epilepsy is reviewed. Epidemiological studies have established that Epileptic Seizures are more frequent in Multiple Sclerosis than predicted by chance.

Partial Epilepsies with focal Seizures often with atypical symptoms and with or without secondary generalization are the usual pattern.

In the survey special emphasis is laid on the direct correlation between Paroxysmal phenomena and plaques now demonstrable by modern imaging techniques.

These images have shown that Epileptic Seizures can be caused by Cortical and SubCortical lesions and by their accompanying Edema.

We extend the review to NonEpileptic Paroxysmal Symptoms, such as Tonic Spasm, which may be confused with Epileptic Seizures.

As far as they are supported by data, recommendations for diagnosis and therapy are given. Open questions are identified and issues for further research are suggested.

Objective
A new parametric simulation procedure based on the Negative Binomial (NB) model was used to evaluate the sample sizes needed to achieve optimal statistical powers for parallel groups (with (PGB) and without (PG) a baseline correction scan).

It was also used for baseline versus treatment (BVT) design clinical trials in Relapsing/Remitting (RR) and Secondary/Progressive (SP) Multiple Sclerosis (MS), when using the number of new enhancing lesions seen on monthly MRI of the Brain as the measure of outcome.

Methods
MRI data obtained from 120 untreated patients with RRMS selected for the presence of MRI activity at baseline, 66 untreated and unselected patients with RRMS, and 81 untreated and unselected patients with SPMS were fitted using an NB distribution.

All these patients were scanned monthly for at least 6 months and were all from the placebo arms of three large scale clinical trials and one natural history study.

The statistical powers were calculated for durations of follow up of 3 and 6 months.

Results
The frequency of new enhancing lesions in patients with SPMS was lower, but not significantly different, from that seen in unselected patients with RRMS.

As expected, enhancement was more frequent in patients with RRMS selected for MRI activity at baseline than in the other two patient groups.

As a consequence, the estimated sample sizes needed to detect treatment efficacy in selected patients with RRMS were smaller than those of unselected patients with RRMS and those with SPMS.

Baseline correction was also seen to reduce the sample sizes of PG design trials. An increased number of scans reduced the sample sizes needed to perform BVT trials, whereas the gain in power was less evident in PG and PGB trials.

Conclusion
This study provides reliable estimates of the sample sizes needed to perform MRI monitored clinical trials in the major MS clinical phenotypes, which should be useful for planning future studies.

Multiple Sclerosis (MS) is a chronic AutoImmune Disease triggered by unknown environmental factors in Genetically susceptible hosts. MS risk was linked to high rates of Cow Milk Protein (CMP) consumption, reminiscent of a similar association in AutoImmune Diabetes.

A recent rodent study showed that Immune responses to the CMP, Butyrophilin, can lead to Encephalitis through antigenic mimicry with Myelin Oligodendrocyte Glycoprotein.

In this study, we show abnormal T-Cell Immunity to several other CMPs in MS patients comparable to that in Diabetics. Limited Epitope mapping with the milk protein BSA identified one specific Epitope, BSA(193), which was targeted by most MS but not Diabetes patients.

BSA(193) was Encephalitogenic in SJL/J mice subjected to a standard protocol for the induction of Experimental AutoImmune Encephalitis.

These data extend the possible, Immunological basis for the association of MS risk, CMP, and CNS AutoImmunity.

To pinpoint the same Peptide, BSA(193), in Encephalitis-prone humans and rodents may imply a common endogenous Ligand, targeted through antigenic mimicry.