MS Abstracts 01c-2g4

IFN-ß is effective in reducing relapses and Magnetic Resonance Imaging (MRI) lesions in Multiple Sclerosis (MS). It is assumed that individual therapeutic responses vary, but methods to identify IFN-ß responsiveness have not been validated.

Our objective was to evaluate methods to classify IFN-ß responder status using relapses and MRI lesions. Data was analyzed from 172 patients who were followed up in a placebo-controlled clinical trial of IFN-ß-1a for 2 years.

Patients were classified as responders or nonresponders using:

1. The number of relapses during the 2-year trial
2. The number of new T₂ lesions after 2 years
3. The number of Gadolinium-enhancing lesions at year 1 and year 2 on study

Outcomes included 2-year change in the Expanded Disability Status Scale, Multiple Sclerosis Functional Composite, and Brain Parenchymal Fraction.

We found that subgroups with high on-study relapse numbers had more disease progression, differences between responder subgroups were similar in the IFN-ß-1a and placebo arms.

In contrast, subgroups with high numbers of new MRI lesions had significantly more disease progression only in the IFN-ß-1a arm. Baseline characteristics failed to account for differential outcome.

New MRI lesion activity during IFN-ß-1a treatment correlates with poor response to IFN-ß-1a.

MRI classification may facilitate rational therapeutic decisions, better clinical trial designs, and studies correlating biomarkers with therapeutic response.

Activated Myelin-specific T-Cells are thought to mediate inflammatory tissue damage in Multiple Sclerosis (MS).

Applying a large panel of Myelin Antigens, we demonstrate the direct ex vivo detection of viable IFN-γ/TNF- producing CD4⁺/CD69⁺ T-Cells 6 hours after Antigenic challenge.

By IntraCellular Flow Cytometry in 3/33 MS patients and 2/26 healthy controls with calculated frequencies of (mean +/- SEM): 0.031% +/- 0.002% versus 0.037% +/- 0.029%.

By comparison, the recently developed IL-7 modified proliferation assay revealed:

1. A higher number of individuals showing Myelin reactivity (17/37 MS patients and 12/24 healthy individuals)
   

2. A significant difference in the response to Myelin Basic Protein (MBP) between the two groups in a longitudinal analysis, indicating a higher activity of Myelin-specific T-Cells in MS patients

Our data provide new perspectives in detecting pathogenetically relevant T-Cells, but clearly demonstrate the different conclusions which must be drawn from various approaches concerning the quantification of AutoReactive T-Cells.

The ImmunoModulatory Cytokine Interferon-beta (IFN-ß) is used in the treatment of AutoImmune Diseases such as Multiple Sclerosis. However, the effect of IFN-ß on Neuronal functions is currently unknown.

IntraCellular recordings were conducted on SomatoSensory Neurons of NeoCortical layers 2/3 and 5 exposed to IFN-ß. The excitability of Neurons was increased by IFN-ß (10 - 10,000 U/ml) in two kinetically distinct, putatively independent manners.

Firstly, IFN-ß reversibly influenced the SubThreshold Membrane Response by raising the membrane resistance RM 2.5-fold and the membrane time constant tau 1.7-fold dose-dependently.

The effect required permanent exposure to IFN-ß, and was reduced in magnitude if the ExtraCellular K⁺ was lowered.

However, the Membrane Response to IFN-ß in the SubThreshold range was prevented by ZD7288 (a specific blocker of Ih) but not by Ni⁺, Carbachol or BiCuculline, pointing to a dependence on an intact Ih.

Secondly, IFN-ß enhanced the rate of Action Potential firing. This effect was observed even after 5 min exposure to IFN-ß and showed no reversibility (up to 210 min).

Current-discharge (F-I) curves revealed a shift (prevented by BiCuculline), as well as an increase in slope (prevented by Carbachol and Ni⁺).

Layer specificity was not observed with any of the described effects. In conclusion, IFN-ß influences the Neuronal excitability in NeoCortical Pyramidal Neurons in vitro, especially under conditions of slightly increased ExtraCellular K⁺.

Our blocker experiments indicate that various Ionic Conductances with different Voltage-dependencies may cause different influences on Sub- and Supra-Threshold behavior, suggesting a more general IntraCellular process induced by IFN-ß.

Tumor Necrosis Factor-alpha (TNF-) and LymphoToxin-alpha (LT-) are upregulated in and around Multiple Sclerosis plaques and are proposed to play a role during chronic inflammation in DeMyelinating Disease.

Despite the perceived detrimental role of these Cytokines, human clinical trials inhibiting TNF- signaling has led to worsening of symptoms in Multiple Sclerosis (MS) patients.

Our laboratory has verified a role for TNF- in the exacerbation of DeMyelination but, more importantly, has demonstrated a novel role for TNF- in reparative ReMyelination in a Cuprizone-induced DeMyelination model.

This may explain the worsening of symptoms experienced by MS patients. In view of the cross-talk in TNF family signaling, the aim of this study is to understand the role of LT- in DeMyelination and ReMyelination in hopes of improving therapeutic strategies for MS.

Using the same model, we show that mice lacking LT- exhibit a delay in DeMyelination that is greater than that exhibited by TNF- null mice. In this model, LT- is expressed primarily by Astroglia.

The delay in DeMyelination is accompanied by a delay in the loss of mature GSTpi-positive Oligodendrocytes in LT-(-/-) mice compared with wild-type mice.

LT-(-/-) mice have decreased numbers of Microglia at the site of insult during DeMyelination, although the number of Astrocytes present is similar between strains.

In contrast to TNF- the lack of LT- did not alter the time course of ReMyelination, or the number of mature Oligodendrocytes during the ReMyelination phase.

Since LT- is detrimental in inflammation and DeMyelination, but not necessary for ReMyelination and repair, inhibiting LT- signaling may represent a promising strategy to treat MS.

Copyright 2004 Wiley-Liss, Inc.

Prompted by our recent observations of increased MMP-8 and MMP-9 with simultaneous downregulation of Tissue Inhibitor of MetalloProteinase-2 (TIMP-2) and TIMP-3 mRNA levels in the Central Nervous System (CNS) of mice with severe Experimental Autoimmune Encephalomyelitis (EAE), we used Semliki Forest Virus (SFV) to transfer and express recombinant murine TIMP-1-3 genes in the CNS.

TIMP-1, TIMP-2 and TIMP-3 expression was confirmed in cultured cells and in the CNS of infected mice. Following IntraPeritoneal infection with 10(6) Plaque-Forming Units (PFU) of SFV-TIMP, focal TIMP protein expression was achieved throughout the Brain.

Although already treatment with empty vector inhibited development of EAE to some extent, the expression of TIMP-2 by the virus significantly enhanced the inhibition.

TIMP-3-administered mice also had lower disease grade, but the inhibition was not statistically significant. In contrast, SFV-TIMP-1 had no effect, similar to co-infection with TIMP-2 and TIMP-3.

We found TIMP-2 expression also by non-infected CNS-resident cells surrounding the Virus-positive areas, suggesting a bystander TIMP-2 induction.

These data strengthen the view that Matrix MetalloProteinases are involved in the pathogenesis of EAE and provide clear evidence that Virus-mediated delivery of their protein inhibitors can be effective in preventing the clinical disease.

TIMPs might be candidates for novel treatment regimens in CNS AutoImmune Disorders, such as Multiple Sclerosis.

The three currently available Interferon-ß products for the treatment of patients with Relapsing/Remitting Multiple Sclerosis (RRMS) are administered according to different regimens.

Placebo-controlled clinical trials support the efficacy of both alternate-day Interferon-ß-1b (Betaferon) and once-a-week Interferon-ß-1a (Avonex), but benefits to patients are probably dependent on the regimen used.

Once-weekly administration, perceived to have fewer adverse events and greater convenience, may improve compliance, whereas frequent administration might enhance efficacy.

However, more frequent administration is also associated with an increase in NeutrAlizing AntiBody (NAB) production, relative to once weekly treatment.

The issue of NABs is complex, and their clinical relevance, if any, has yet to be fully assessed. Pharmacological evidence suggests that the effects of Interferon-beta on a number of biological markers is maximized when administered every 48 hours.

This might arise as a result of sustained activity in the intracellular molecular signalling pathways regulating beta Interferon-induced Gene expression.

Some evidence suggests that the increase in biological effect at higher more frequent doses is mirrored by improvements in clinical and MRI outcome measures.

Two recent comparative studies demonstrated significantly better clinical and Magnetic Resonance Imaging outcomes in patients with RRMS receiving alternate-day high-dose Interferon-ß-1b (250 micro g subcutaneously).

Or, three-times-weekly high-dose Interferon-ß-1a compared to those receiving once weekly low-dose Interferon ß-1a (30 micro g intramuscularly).

Despite some methodological drawbacks, these studies indicate that the benefits of high-dose frequently administered Interferon-beta on relapse rate are seen soon after beginning treatment. Therefore, it seems appropriate to begin the treatment of RRMS with this dosing regimen.

Magnetic Resonance Imaging (MRI) is a collection of very sensitive and versatile techniques for detecting Multiple Sclerosis (MS) related damage in the Central Nervous System.

Each technique is characterized by a particular combination of sensitivity, tissue and pathological specificity, and technical requirements that enable diverse aspects of MS to be explored.

MRI techniques also offer the possibility of quantitatively assessing the effects of therapeutic interventions, and to correlate these effects to clinical outcomes.

Of special interest are newer MR techniques that correlate more strongly with disability than Gadolinium-enhancement and T₂ lesion load, and this review focuses on T₁ HypoIntense lesions, MR Spectroscopy, and Brain Atrophy as surrogate markers of Axonal Loss, and their application in randomized clinical trials.

Several disease-modifying therapies appear to have differential effects on Inflammation, DeMyelination and Axonal Loss as judged by MRI, illustrating the unique capability of MRI to interrogate the pathophysiology of MS.

At the same time it illustrates the difficulties in understanding the mechanisms leading to Axonal Loss and persistent clinical deficit.

Multiple Sclerosis (MS) is a chronic inflammatory disease of the Central Nervous System, associated with primary destruction of Myelin sheaths. Axons are relatively well preserved, although they too are injured in the development of the lesions.

While inflammation and DeMyelination induce Neurological deficit, which is in part reversible, the destruction of Axons, when past the threshold of compensation, is always accompanied by irreversible clinical deficits.

The mechanisms leading to tissue injury in MS are complex and heterogenous. They involve direct CytoToxicity mediated by T-Lymphocytes, specific AntiBodies and Complement as well as toxic products of Macrophages.

In addition, in a small subset of patients a genetically determined increased susceptibility of the Central Nervous System tissue for Immune mediated damage appears to play a role.

Since the pathogenetic pathways of DeMyelination and tissue damage vary between different MS patients, their identification by ParaClinical Markers is of critical importance for diagnosis and therapeutic management.

See: Complement Abstracts Index

Interferon-ß-1b (Betaseron((R)), Betaferon((R))) is a Non-Glycosylated recombinant human Interferon-ß approved for high-frequency, SubCutaneous (SC) administration in the treatment of Multiple Sclerosis (MS).

Its mechanism of action is unknown, but may involve modulation of the AutoImmune pathogenic processes of MS.

In a randomized, double-blind trial in patients with Relapsing/Remitting MS (RRMS), SC Interferon-ß-1b 250 micro g (8 million International Units [MIU]) every other day reduced the annual relapse rate and increased the proportion of relapse-free patients compared with placebo.

It also reduced relapse severity, hospitalizations, and disease activity assessed by Magnetic Resonance Imaging (MRI), and increased the time to first relapse. Progression of disability showed a trend towards reduction relative to placebo and baseline, but did not reach statistical significance.

SC Interferon-ß-1b 250 micro g every other day was shown in a randomized trial to be superior to intramuscular (IM) Interferon-ß-1a 30 micro g (6 MIU) once weekly with respect to reductions in relapse-related parameters, disability progression and MRI-assessed disease activity.

In patients with Secondary/Progressive MS (SPMS), SC Interferon-ß-1b 250 micro g every other day slowed progression of the disease relative to placebo in one randomized, double-blind trial, but not in another.

In both studies, Interferon-ß-1b 250 micro g recipients had fewer relapses and less MRI-assessed disease activity than placebo recipients. The difference in primary outcome may reflect differences in patient entry criteria.

Interferon-ß-1b is generally well tolerated and the common adverse events (e.g. injection site reactions, Asthenia and an Influenza-Like Symptom Complex) are clinically manageable.

In a randomized trial, the tolerability of SC Interferon-ß-1b 250 micro g every other day was generally similar to that of IM Interferon-ß-1a 30 micro g once weekly, except for higher incidences of injection site reactions and Neutralizing Anti-Interferon-ß AntiBodies with SC Interferon-ß-1b.

In conclusion, SC Interferon-ß-1b 250 micro g every other day reduces the frequency and severity of relapses and MRI measures of disease activity and may delay the progression of disability in RRMS.

The drug appeared to be more effective than, and as well tolerated as, IM Interferon-ß-1a 30 micro g once weekly. Interferon-ß-1b also has positive effects on relapse rates and disease activity in patients with SPMS, although its effects on disease progression remain uncertain.

The drug is generally well tolerated, and the common adverse events are clinically manageable. Thus, Interferon-ß-1b is a valuable first-line therapy for patients with RRMS and a potentially useful option in those with SPMS.

Human peripheral blood T-Lymphocytes possess two types of K⁺ Channels: the Voltage-gated Kv1.3 and the Calcium-activated IKCa1 Channels.

The use of Peptidyl Inhibitors of Kv1.3 and IKCa1 indicated that these Channels are involved in the maintenance of Membrane Potential and that they play a crucial role in Ca⁺ signaling during T-Cell activation.

Thus, in vitro blockade of Kv1.3 and IKCa1 leads to inhibition of Cytokine production and Lymphocyte proliferation.

These observations prompted several groups of investigators in academia and pharmaceutical companies to characterize the expression of Kv1.3 and IKCa1 in different subsets of human T-Lymphocytes and to evaluate their potential as novel targets for ImmunoSuppression.

Recent in vivo studies showed that chronically activated T-Lymphocytes involved in the pathogenesis of Multiple Sclerosis present unusually high expression of Kv1.3 Channels and that the treatment with selective Kv1.3 Inhibitors can either prevent or ameliorate the symptoms of the disease.

In this model of Multiple Sclerosis, blockade of IKCa1 Channels had no effect alone, but improved the response to Kv1.3 Inhibitors.

In addition, the expression of Kv1.3 and IKCa1 Channels in human cells is very restricted, which makes them attractive targets for a more cell-specific and less harmful action than what is typically obtained with classical ImmunoSuppressants.

Studies using high-throughput toxin displacement, (86)Rb-efflux screening or Membrane Potential assays led to the identification of Non-Peptidyl small molecules with high affinity for Kv1.3 or IKCa1 channels.

Analysis of structure-function relationships in Kv1.3 and IKCa1 Channels helped define the binding sites for Channel Blockers, allowing the design of a new generation of small molecules with selectivity for either Kv1.3 or IKCa1, which could help the development of new drugs for safer treatment of AutoImmune Diseases.

Susceptibility to Multiple Sclerosis (MS) is associated genetically with Human Leucocyte Antigen (HLA) Class II Alleles, including DRB1*1501, DRB5*0101, and DQB1*0602, and it is possible that these Alleles contribute to MS through an enhanced ability to present Encephalitogenic Myelin Peptides to pathogenic T-Cells.

HLA-DRB1*1502, which contains Glycine instead of Valine at position 86 of the P1 Peptide-binding pocket, is apparently not genetically associated with MS.

To identify possible differences between these Alleles in their Antigen-Presenting function, we determined if T-Cell responses to known DRB1*1501-restricted Myelin Peptides might be diminished or absent in Transgenic (Tg) DRB1*1502-expressing mice.

We found that Tg DRB1*1502 mice had moderate to strong T-Cell responses to several Myelin Peptides with favorable DRB1*1501 binding motifs, notably Myelin Oligodendrocyte GlycoProtein (MOG)-35-55 (which was also Encephalitogenic), ProteoLipid Protein (PLP)-95-116, and MOG-194-208, as well as other PLP and MOG Peptides.

These Peptides, with the exception of MOG-194-208, were also Immunogenic in healthy human donors expressing either DRB1*1502 or DRB1*1501. In contrast, the DRB1*1502 mice had weak or absent responses to Peptides with unfavorable DRB1*1501 binding motifs.

Overall, none of the DRB1*1501-restricted Myelin Peptides tested selectively lacked Immunogenicity in association with DRB1*1502.

These results indicate that the difference in risk association with MS of DRB1*1501 versus DRB1*1502 is not due to a lack of Antigen-Presenting by DRB1*1502.

At least for this set of Myelin Peptides, and suggest that other mechanisms involving DRB1*1501 may account for increased susceptibility to MS.

Copyright 2004 Wiley-Liss, Inc.