MS Abstracts 9b-2g1

The expression of Major Histocompatibility Complex (MHC) Class I and Class II in the CNS has received considerable interest because of its importance in NeuroDegenerative or Inflammatory Diseases, such as Multiple Sclerosis (MS).

However, at the moment nothing is known about the expression patterns of transcription factors controlling MHC expression in MS lesions.

Here, we performed an extensive ImmunoHistoChemical analysis on MS affected postmortem Brain tissue to determine the cellular localization and distribution of different MHC-controlling transcription factors.

We show that Phagocytic Macrophages in active DeMyelinating MS lesions displayed a moderate to strong ImmunoStaining of the MHC-specific transcription factors RFX and CIITA.

As well as the general transcription factors NF-kappaB, IRF1, STAT1, USF, and CREB, which was congruent with a strongly enhanced expression of HLA-DR, HLA-DQ, HLA-DP, and HLA Class 1.

In the Normal-Appearing White Matter (NAWM), clusters of activated Microglial Cells forming preactive lesions displayed an overall stronger expression level of these transcription factors.

Combined with a strong to intense level of MHC Class 1 and Class II ImmunoStaining.

In general, Astrocytes and Oligodendrocytes either did not express, or weakly expressed, these Transcription Factors, correlating with a lack of MHC Class II and weak MHC Class I expression.

Together, the elevated expression level of transcription factors governing expression of MHC Class 1 and Class II molecules in activated Microglial Cells and Phagocytic Macrophages strongly suggests a general state of Microglial Cell activation in MS lesions.

Copyright 2001 Wiley-Liss, Inc.

Interactions with self-Major Histocompatibility Complex molecules on Dendritic Cells (DCs) are important for the survival of mature CD4⁺ T-Cells.

We have followed the DC-mediated signal from the T-Cell surface to the nucleus and identified a pattern of activation that correlates with increased in vitro survival.

This response is induced exclusively by DCs and is likely associated with a modulation of the T-Cell activation threshold.

We have also found that DC-mediated activation results in Antigen-independent Cytokine Gene expression, which points to a new role for DCs in shaping the Chemokine milieu.

Such Antigen-independent activation of T-Cells may play a role in protective Immunity, but may also induce and perpetuate AutoImmune states such as Multiple Sclerosis.

Many basic aspects of Brain inflammation, recently disclosed in experimental models, are reflected in the pathology of human inflammatory Brain diseases.

Examples include the key role of T-Lymphocytes in Immune surveillance and in the regulation of the inflammatory response.

The essential contributions of Adhesion Molecules, proinflammatory Cytokines, Chemokines, and Proteases in the recruitment of inflammatory cells into the Nervous Tissue.

The modulating effect of Glia Cells on the inflammatory process and the termination of T-Cell-mediated inflammation by Apoptotic cell death.

Despite this progress in our understanding of the PathoGenesis of Brain inflammation, there are still major unresolved questions.

Because of technical constraints, most of our knowledge on Central Nervous System inflammation so far relates to the role of a specific T-Cell subset, the so-called T-Helper-1 Cells.

Other T-Cell subsets, in particular CytoToxic Class I MHC-restricted T-Lymphocytes, however, appear to be of major importance in human disease.

Furthermore, the detailed mechanisms, which are responsible for the profound differences in the patterns of tissue damage in different human inflammatory Brain Diseases, such as Multiple Sclerosis or various forms of Virus Encephalitis, are largely unresolved.

We discuss the open questions to be addressed in the future, which, when answered, may help to design novel therapeutic strategies.

Copyright 2001 Wiley-Liss, Inc.

The Immune status of the Central Nervous System (CNS) is strictly regulated. In the healthy Brain, Immune responses are kept to a minimum.

In contrast, in a variety of Inflammatory and NeuroDegenerative Diseases, including Multiple Sclerosis, Infections, Trauma, Stroke, Neoplasia, and Alzheimer's Disease, Glial Cells such as Microglia gain Antigen-Presenting capacity through the expression of Major Histocompatibility Complex (MHC) molecules.

Further, ProInflammatory Cytokines, such as Tumor Necrosis Factor-alpha (TNF-), InterLeukin-1beta (IL-1ß), and Interferon-gamma (IFN-γ), as well as Chemokines, are synthesized by resident Brain cells and T-Lymphocytes invade the affected Brain tissue.

The proinflammatory Cytokines stimulate Microglial MHC expression in the lesioned CNS areas only. However, the induction of Brain Immunity is strongly CounterRegulated in intact CNS areas.

For instance, recent work demonstrated that Microglia are kept in a quiescent state in the intact CNS by local interactions between the Microglia Receptor CD200 and its Ligand, which is expressed on Neurons.

Work done in our laboratory showed that Neurons suppressed MHC expression in surrounding Glial Cells, in particular Microglia and Astrocytes.

This control of MHC expression by Neurons was dependent on their electrical activity.

In Brain tissue with intact Neurons, the MHC Class II inducibility of Microglia and Astrocytes by the ProInflammatory Cytokine IFN-γ was reduced.

Paralysis of Neuronal electric activity by NeuroToxins restored the induction of MHC molecules on Microglia and Astrocytes.

Loss of Neurons or their physiological activity would render the impaired CNS areas recognizable by invading T-Lymphocytes.

Thus, Immunity in the CNS is inhibited by the local MicroEnvironment, in particular by physiologically active Neurons, to prevent unwanted Immune mediated damage of Neurons.

Copyright 2001 Wiley-Liss, Inc.

Theiler's Murine EncephaloMyelitis Virus-induced DeMyelinating Disease has been extensively studied as an attractive infectious model for human Multiple Sclerosis.

Virus-specific inflammatory Th1 cell responses followed by AutoImmune responses to Myelin Antigens play a crucial role in the pathogenic processes leading to DeMyelination.

AntiBody and CytoToxic T-Cells (CTL) responses to Virus appears to be primarily protective from DeMyelinating Disease.

Although the role of Th1 and CTL responses in the induction of DeMyelinating Disease is controversial, assessment of Cytokines produced locally in the Central Nervous System (CNS) during the course of disease.

And the effects of altered inflammatory Cytokine levels strongly support the importance of Th1 responses in this Virus-induced DeMyelinating disease.

Induction of various Chemokines and Cytokines in different Glial and Antigen Presenting Cells upon Viral infection appears to be an important initiation mechanism for inflammatory Th1 responses in the CNS.

Coupled with the initial inflammatory responses, Viral persistence in the CNS may be a critical factor for sustaining inflammatory responses and consequent Immune-mediated DeMyelinating Disease.

MRI is the most powerful imaging technique in managing patients with suspected or confirmed Multiple Sclerosis (MS).

However, conventional MRI variables show nonspecific abnormalities weakly correlated with clinical progression of the disease. New techniques, now routinely available, offer better characterization of the PathoPhysiology.

We combined conventional MRI, including lesion load, contrast enhancement and "black holes" with Magnetization Transfer and Diffusion-weighted imaging and localized Proton MR Spectroscopy (MRS) to study their relationship with disability, course and duration of MS.

The variables that were the most significantly linked to the course of the disease (Relapsing/Remitting versus Secondary/Progressive) were lesion load, mean overall Magnetization Transfer Ratio and Apparent Diffusion Coefficient (MGADC).

The percentage of ADC in (PADCIMD), and out of (PAD-COMD) modal distribution, and the ratio N-AcetylAspartate and Creatine-containing compounds on MRS of the Centrum Semiovale.

MGADC and PADCIMD were the independent factors most related to disability and duration of disease.

Combining MRI techniques is clinically relevant and feasible for studies of MS and may be applied to other diseases of the Central Nervous System.

The authors report an intense downregulation of Transforming Growth Factor-beta1 (TGF-ß1) Serum levels 4 weeks from start of Interferon-beta-1a (IFN-ß-1a) treatment.

At 44 microg/week in 271 patients with Relapsing/Remitting MS, which was still present after 1 year (p < 0.001). In line with previous data, InterLeukin-10 Serum levels did not vary significantly.

These results indicate that the ImmunoModulatory effects of IFN-ß might not be restricted to the postulated anti-inflammatory mechanisms and address the role of TGF-ß in the PathoGenesis of MS.

Objective
To determine levels of Serum Interferon-beta (IFN-ß) Neutralizing AntiBody (NAb) and Neopterin-an IFN biologic response marker in patients with MS treated with Betaseron or Avonex.

Background
Controversy exists over the relative Immunogenicity of IFN-ß-1a and IFN-ß-1b and the reasons for any such difference.

To determine the role of patient profile and test methodology in IFN-ß, NAb levels need to be measured blindly and simultaneously in a predefined closely matched MS patient cohort.

Methods
Serum NAb and Neopterin levels were measured in closely matched patients on Avonex (n = 98) or Betaseron (n = 64).

NAb were determined by Athena Diagnostics and Serum Neopterin levels by Covance Laboratories using a competitive binding RadioImmunoassay.

Results
More patients taking Betaseron (22%) than Avonex (7%) had elevated Titers of NAb (p = 0.008).

Mean Serum Neopterin levels were lower in patients with high as compared to low NAb titers (p = 0.0002).

No difference in mean Neopterin levels was found comparing the total Betaseron group to the Avonex group.

However, in the subset of patients with low NAb Titers, mean Neopterin levels were higher in the Betaseron than in the Avonex group (p = 0.027).

A random cross-sectional sampling of patients on Avonex showed a decrease in Neopterin levels over time between weekly doses.

Conclusion
NAb are more commonly found with Betaseron than Avonex.

More studies are needed to determine the correlation among Serum Neopterin levels, other biologic response markers, NAb, and disease activity in patients with MS being treated with IFN-ß.

Background
The PRISMS study demonstrated significant clinical and MRI benefit at 2 years for Interferon-ß-1a (Rebif^®), 22 and 44 mcg thrice weekly (tiw), compared with placebo in Relapsing/Remitting MS. Years 3 and 4 extension study results are reported.

Methods
Patients initially receiving placebo were randomized to blinded Interferon-ß-1a, 22 or 44 mcg tiw (n = 172; crossover group); others continued blinded treatment with their originally assigned dose, 22 mcg (Rx22 group) or 44 mcg (Rx44 group) tiw (n = 167 per group).

Patients had 3- to 6-month clinical and annual MRI assessments.

Results
Relapse rates for 4 years were 1.02 (crossover), 0.80 (Rx22, p < 0.001), and 0.72 (Rx44, p < 0.001); the dose effect approached significance (p = 0.069; risk ratio, 0.88; 95% CI, 0.76-1.01).

Crossover groups showed reductions in relapse count, MRI activity, and lesion-burden accumulation with Interferon-ß-1a compared with their placebo period (p < 0.001 both doses).

Time to sustained disability progression was prolonged by 18 months in the Rx44 group compared with the crossover group (p = 0.047).

Rx22 and Rx44 reduced new T₁ lesion number and lesion burden compared with crossover (p < 0.001); Rx44 was superior to Rx22 on several clinical and MRI outcomes.

Persistent Neutralizing AntiBodies developed in 14.3% (Rx44) and 23.7% (Rx22) of patients and were associated with reduced efficacy.

Conclusions
Clinical and MRI benefit continued for both doses up to 4 years, with evidence of dose response.

Outcomes were consistently better for patients treated for 4 years than for patients in crossover groups. Efficacy decreased with Neutralizing AntibBody formation.

Various therapeutic approaches to Multiple Sclerosis (MS) have been presented, but no specific and effective method has so far been established.

In recent years, Macrophage Migration Inhibitory Factor (MIF) has been re-evaluated as a pluripotent Cytokine involved in a broad spectrum of inflammation and Immune Responses.

During the course of MIF study, increased levels of MIF were observed in the CerebroSpinal Fluids of patients with MS.

In parallel with exacerbation of clinical symptoms, and a number of Lymphocytes strongly expressing MIF infiltrate into the pathogenic lesions.

It is expected that regulation of the action of MIF by an Anti-MIF AntiBody or small molecule inhibitors would be an effective therapeutic method for this DeMyelinating Disease.

There have been considerable advances made recently in the treatment of Multiple Sclerosis (MS). In particular, Interferon-beta (IFN-ß) has been demonstrated in several independent, multicenter clinical trials to lower unequivocally the biological activity of this illness.

The results of these trials have been remarkably consistent, demonstrating a reduction in both disease activity and cumulative disability, using a combination of clinical and Magnetic Resonance Imaging outcome measures.

Nevertheless, the importance of the total weekly IFN-ß dose in the clinical management of individual patients has been controversial.

However, there is considerable information available regarding the effect of IFN-ß dose on the various biochemical and clinical markers that are affected by IFN-ß, which is derived both from pre-clinical studies and multicenter clinical trials.

On balance, convincing evidence is provided to support the notion that there is a clinically relevant dose-response in the use of IFN-ß to treat patients with Relapsing/Remitting MS.

However, many of the clinical trials of IFN-ß in MS have confounded the potential effects of dose with the possible effects of frequency of IFN-ß administration.

As a result, it is possible that the apparent dose-response observed in these clinical trials may be due, in part, to the more frequent dose administration schedule rather than the total weekly dose.

IFN-ß may modify the clinical course of Multiple Sclerosis (MS) but is not curative, and there are also patients whose disease does not respond to IFN-ß as currently administered.

Tests are warranted with a capacity to early discriminate responders from non-responders, thereby altering treatment option for the individual patient.

In vitro effects of IFN-ß on expression of activation-associated cell surface markers and Cytokine production need to be explored in this context.

Here we report on the influence in vitro of IFN-ß on blood MonoNuclear Cells (MNC) prepared from MS patients and healthy controls.

MNC were subjected to short-term culture in the presence of IFN-ß at concentrations of 100 U/ml and 1000 U/ml.

Expression of cell surface molecules CD40, CD69, CD80, CD86, CD95 and HLA-DR was measured by flow cytometry.

IL-10 and IL-12 p40 production in culture supernatants was measured by ELISA.

MNC exposed to IFN-ß in vitro enhanced expression of the co-stimulatory CD80, CD86, the early activation Antigen CD69 and the cell death receptor CD95.

Expression of CD40 and HLA-DR was not influenced. IFN-ß increased IL-10 but suppressed IL-12 p40 production.

In vitro effects of IFN-ß on MNC were similar in MS patients and in healthy subjects, except that IFN-ß-induced augmentation of CD86 and CD69 expression was less pronounced in MS, in particular in untreated MS patients.

Individual MS patients clearly responded differently to IFN-ß in vitro in comparison with the majority of patients in this cross-sectional study.

In conclusion, anti-inflammatory effects of IFN-ß on blood MNC include augmentation of IL-10 production and suppression of IL-12 p40 production, which are accompanied by enhancement of CD69, CD80, CD86 and CD95 expression.

The less pronounced IFN-ß-induced effects on CD86 and CD69 expression in MS vs controls might reflect a defect in ImmunoRegulation in MS.

Larger groups should be evaluated, and follow-up studies performed in MS patients before/during IFN-ß treatment.

In relation to clinical outcome measures to evaluate the usefulness of these markers for possible differentiation between responders and non-responders to IFN-ß treatment.