MS Abstracts 04a-2g5

Background
The presence of Abzymes (Abzs) in human Sera is a specific feature of different Autoimmune pathologies.

We have shown that IgGs of patients with Multiple Sclerosis (MS) specifically hydrolyze human Myelin Basic Protein (hMBP). However, the presence of hMBP-hydrolyzing MS IgMs and IgAs in patients with MS has not been studied.

Material/Methods
Homogeneous IgM and IgA fractions were isolated from human Sera by affinity chromatography on different adsorbents. The Ab-dependent hydrolysis of hMBP was analyzed using SDS-PAGE.

Results
We present evidence showing that MS IgMs and IgAs (but not Abs from the Sera of healthy individuals) catalyze the hydrolysis of hMBP.

Specific enzymatic activities of IgMs and sIgAs from Sera of any single patient were usually significantly higher than those of IgGs.

Specific inhibitors of Acidic and Thiol proteases demonstrated a weak effect on ProteoLytic activity of IgGs and IgMs. However, specific inhibitors of Serine proteases (AEBSF, PMSF, and Benzamidine) significantly inhibited ProteoLytic activity.

IgMs and IgAs hydrolyze specifically both human and pig MBP but not many other tested proteins. Although the biological function of this ProteoLytic activity is not known, it is clear that MBP-hydrolyzing Abs may play an important role in MS pathogenesis.

Conclusions
The findings display the generation by the Immune Systems of individual MS patients of a variety of PolyClonal IgGs, IgMs, and IgAs with different ProteoLytic properties, which hydrolyze MBP, the major protein component of the Myelin-ProteoLipid shell of Axons and a well-known MS AutoAntigen.

Early signs of inflammatory DeMyelination include entry of Fibrin(Ogen) into the Central Nervous System (CNS), which is normally excluded by the Blood-Brain Barrier, and up-regulation of components of the PlasMinogen Activator System.

Using mice deficient in tissue-type PlasMinogen Activator (tPA(-/-)) and Urokinase PlasMinogen Activator Receptor (uPAR(-/-)), we investigated the involvement of the PA System on the clinical and pathological features of Experimental Allergic Encephalomyelitis, an animal model of Multiple Sclerosis.

tPA(-/-) mice suffered an early and a more severe acute disease characterized by incomplete recovery when compared to wild-type controls, with significantly higher CNS levels of PlasMinogen Activator Inhibitor-1.

This correlated with Fibrin accumulation, which co-localized with NonPhosphorylated NeuroFilament on thickened Axons in Experimental Allergic Encephalomyelitis tissue.

In contrast, uPAR(-/-) mice had a delayed, less acute disease reflected in delayed infiltration of inflammatory cells.

These animals developed chronic disease as a result of steadily increased inflammation, increased levels of Urokinase-type PlasMinogen Activator (uPA), and greater degree of DeMyelination.

Thus, the PlasMinogen Activator System can modulate both inflammatory and degenerative events in the CNS through the respective effects of tPA and uPAR on FibrinoLysis and cell adhesion/migration, manipulation of which may have therapeutic implications for Multiple Sclerosis.

There are few longitudinal studies of Cognition in patients with Multiple Sclerosis, and the results of these studies remain inconclusive. No serial NeuroPsychological data of an exclusively Primary/Progressive series are available.

Cross-sectional analyzes have revealed significant correlations between Cognition and Magnetic Resonance Imaging (MRI) parameters in Primary/Progressive Multiple Sclerosis (PPMS).

This study investigated Cognitive and MRI change in 99 PPMS patients from five European centers for 2 years. They were assessed at 12 month intervals using the Brief Repeatable Battery, a reasoning test, and a measure of Depression.

The MRI parameters of T₁ HypoIntensity load, T₂ lesion load, and partial Brain Volume were also calculated at each time point.

There were no significant differences between the mean Cognitive scores of the patients at year 0 and year 2. However, one-third of the patients demonstrated absolute Cognitive decline on individual test scores.

Results indicated that initial Cognitive status on entry into the study was a good predictor of Cognitive ability at 2 years.

There was only a small number of significant correlations between changes in Cognition and changes on MRI, notably T₁ HypoIntensity load with the two Attentional Tasks (r = -0.266, P = 0.017; r = -0.303, P = 0.012).

It is probable that multiple factors underlie this weak relation between the Cognitive and MRI measures.

Multiple mechanisms of Tolerance are induced by oral Antigen. Low doses favor active suppression, whereas higher doses favor Clonal anergy/deletion.

Oral Antigen induces T-helper 2 [InterLeukin (IL-4/IL-10)] and Th3 [Transforming Growth Factor-beta (TGF-beta)] T-Cells plus CD4⁺CD25⁺ regulatory cells and latency-associated peptide⁺ T-Cells.

Induction of oral Tolerance is enhanced by IL-4, IL-10, Anti-IL-12, TGF-beta, Cholera Toxin B subunit, Flt-3 Ligand, and Anti-CD40 Ligand.

Oral (and nasal) Antigen administration suppresses animal models of Autoimmune Diseases including Experimental Autoimmune Encephalitis, Uveitis, Thyroiditis, Myasthenia, Arthritis, and Diabetes in the Non-Obese Diabetic (NOD) mouse, plus Non-Autoimmune Diseases such as Asthma, AtheroSclerosis, Graft Rejection, Allergy, Colitis, Stroke, and models of Alzheimer's Disease.

Oral tolerance has been tested in human Autoimmune Diseases including Multiple Sclerosis (MS), Arthritis, Uveitis, and Diabetes and in allergy, contact sensitivity to DiNitroChloroBenzene (DNCB), and Nickel allergy.

Although positive results have been observed in phase II trials, no effect was observed in phase III trials of CII in Rheumatoid Arthritis or oral Myelin and Glatiramer Acetate (GA) in MS.

Large placebo effects were observed, and new trials of oral GA are underway. Oral Insulin has recently been shown to delay onset of Diabetes in at-risk populations, and confirmatory trials of oral Insulin are being planned.

Mucosal Tolerance is an attractive approach for treatment of Autoimmune and Inflammatory Diseases because of lack of toxicity, ease of administration over time, and Antigen-specific mechanisms of action.

The successful application of oral Tolerance for the treatment of human diseases will depend on dose, developing Immune markers to assess Immunologic effects, route (nasal versus oral), formulation, Mucosal adjuvants, combination therapy, and early therapy.

Females are more susceptible than males to Multiple Sclerosis (MS). However, the underlying mechanism behind this gender difference is poorly understood.

Because the presence of NeuroAntigen-primed T-Cells within the CNS is necessary for the development of MS, the present study was undertaken to investigate the activation of Microglia by Myelin Basic Protein (MBP)-primed T-Cells of male, female and castrated male mice.

Interestingly, MBP-primed T-Cells isolated from female and castrated male but not from male mice induced the expression of inducible Nitric Oxide Synthase (iNOS) and ProInflammatory Cytokines (IL-1b, IL-1a, IL-6 and TNF-) in Microglia by cell-cell contact.

Again there was no apparent defect in male Microglia because MBP-primed T-Cells isolated from female and castrated male but not male mice were capable of inducing the production of NO in male primary Microglia.

Inhibition of female T-Cell contact-mediated Microglial expression of ProInflammatory molecules by dominant-negative mutants of p65 and C/EBPb suggest that female MBP-primed T-Cells induce Microglial expression of ProInflammatory molecules through the activation of NF-kB and C/EBPb.

Interestingly, MBP-primed T-Cells of male, female and castrated male mice were able to induce Microglial activation of NF-kB.

However, MBP-primed T-Cells of female and castrated male but not male mice induced Microglial activation of C/EBPb.

These studies suggest that Microglial activation of C/EBPb but not NF-kB by T-Cell:Microglial contact is a gender-specific event and that male MBP-primed T-Cells are not capable of inducing Microglial expression of ProInflammatory molecules due to their inability to induce the activation of C/EBPb in Microglia.

This novel gender-sensitive activation of Microglia by NeuroAntigen-primed T-Cell contact could be one of the mechanisms behind the female-loving nature of MS.

Recent studies of Axon-Glia and Glia-Glia communication have emphasized interactivity and interdependence between Central Nervous System (CNS) components.

Concurrently, data from Imaging, BioChemical, and Morphological studies have changed the view of Multiple Sclerosis (MS) from a NeuroInflammatory condition with primary DeMyelination to one in which cumulative Axonal Damage drives progression.

We therefore studied Axonal Damage in the context of inflammation and Glial responses, from the pre-clinical to onset stage of murine Experimental Autoimmune Encephalomyelitis (EAE), an established MS model.

We report three major findings:
• The first evidence of Axonal injury before significant T-Cell entry into the Parenchyma
• Coincidence of the earliest manifestation of Axonal Damage and Astrocytic responses
• An association between accumulation of Axonal and Astrocytic changes and specific forms of MS

These data demonstrate the relationship between the initiation of Axonal injury and early inflammation.

Significantly, we show that, in common with a growing number of NeuroDegenerative conditions, the pathology of murine EAE is characterized by early active contribution from Astrocytes.

This marks a change in the understanding of the role of Astrocytes in MS pathogenesis and has important implications for the development of NeuroProtective strategies.

(c) 2005 Wiley-Liss, Inc.

To reevaluate whether an association exists between the clinical course of Multiple Sclerosis (MS) and the activation of Memory T-Cells, we investigated the phenotype of T-Cells in peripheral blood and CerebroSpinal Fluid (CSF) of patients with MS using five-color flow cytometry.

A cross-sectional study with 39 Relapsing/Remitting MS patients demonstrated that the percentage of CD25⁺CD45RO⁺CD4⁺CD3⁺ cells was significantly increased in peripheral blood as well as in CSF of active MS patients compared with inactive MS patients.

A longitudinal study with 11 Relapsing/Remitting MS patients also showed a higher percentage of CD25⁺CD45RO⁺CD4⁺CD3⁺ cells in peripheral blood at the phase of exacerbation than during remission.

On the other hand, regardless of the disease activity, the percentage of CD25⁺CD45RO⁺CD8⁺CD3⁺ cells in peripheral blood was significantly higher in patients with MS than in healthy control subjects.

A lower percentage of CD25⁺CD45RO⁺CD8⁺CD3⁺ cells in CSF was observed in active MS patients compared with inactive MS patients.

These results suggest that the activation of Memory CD4⁺ T-Cells is associated with the exacerbation of MS and activation of Memory CD8⁺ T-Cells reflects systemic Immunological dysregulation in MS patients.

Transient as well as continuous activation of T-Cells by recall Antigens may be involved in the disease course of MS.

Objectives
The aims were to investigate the frequency of Intrathecal synthesis of specific AntiBodies against Measles (M), Rubella (R) and Varicella Zoster (Z) Viruses (MRZ reaction) as a diagnostic marker between Multiple Sclerosis (MS) and NeuroBorreliosis (NB) groups and to postulate the most typical CerebroSpinal Fluid (CSF) variables profile of these entities.

Methods
Three cohorts of patients were investigated: MS (n = 42), NB (n = 27) and Other Neurological Diseases (OND) (n = 15).

Measles, Rubella, Varicella Zoster and Borrelia-specific IgG AntiBodies were measured by ELISA, Q(alb) (CSF/Serum Albumin ratio) as a marker of Blood-CSF Barrier function and specific AntiBody Indices (AI) were calculated according to relevant formulae.

IgG OligoClonal Bands (OB) were detected by isoelectric focusing and immunoenzymatic staining.

Results
Eighty-eight percent of MS patients had positive MRZ reaction and 26.2% had positive Anti-Borrelia AI.

Eighty-nine percent of NB patients had positive Anti-Borrelia AI and two patients had individually Anti-Measles and Rubella positive AI.

MS-CSF variables profile included the presence of IgG OB in 81%, elevated Q(alb) in 31% and normal cell count in 66.7%, Of NB patients IgG OB were positive in 74%, elevated Q(alb) in 81.5% and normal cell count in 7.4%.

Conclusion
MRZ reaction was proved as statistically significant marker in differential diagnosis between MS and NB.

Typical CSF variables profile of these two entities is highly supportive, especially when MRZ is included.

Sex Hormones play a central role as modulators of Immune responses and AutoImmune Diseases.

We hypothesized that suppression of MS disease during pregnancy may be mediated by Sex Steroid Hormones via regulation of CoStimulatory molecules such as CD40L or CD80/CD86 (B7-1/B7-2).

We tested two sex Hormones that are implicated in Immune suppression during pregnancy: Estriol and Progesterone. We also examined whether this regulation is gender-specific or disease-related.

PBMC from untreated Relapsing/Remitting Multiple Sclerosis (RR MS) patients and controls were examined for expression of T-Cell and Monocyte CoStimulatory molecules following Mitogen stimulation in the presence or absence of Sex Hormones.

In the absence of Hormones, we confirmed that mitogen stimulation induced significantly more CD40L on the surface of CD4⁺ T-Cells in MS patients compared to controls, and we extend these findings by showing there were no gender differences in induction of CD40L.

Although supra-physiologic doses of Hormones mildly suppressed CD40L expression on activated T-Cells, in vitro exposure to typical pregnancy-related physiologic doses of Estriol or Progesterone showed very little or no suppression of CD40L.

On Monocytes, neither Estriol nor Progesterone significantly altered the expression of CD80/CD86.

These results suggest that physiologic doses of Estriol or Progesterone cannot alter CD40L on T-Cells or CD80/CD86 on Monocytes sufficiently to explain the improvement observed in MS during pregnancy.

Thus, although amelioration of MS and other AutoImmune Diseases during pregnancy is thought to be due to increased sex Hormones, the present results do not support a role for suppression of CoStimulation via Estriol or Progesterone.

In 1995, it was observed that the administration of Statins to Heart transplant patients resulted in fewer episodes of rejection, thus a role for Statins in the treatment of Inflammatory Disease was suggested.

To date, the results of a single, open-label trial in Multiple Sclerosis patients have demonstrated that treatment with one of the Statins, Simvastatin, reduced the number and volume of lesions, as observed using Gadolinium-enhancing Magnetic Resonance Imaging.

While the results of this first study seem promising, the rationale for using a Cholesterol-lowering drug in a DeMyelinating Disease must be addressed, in addition to the potential problems that the side effects of Statins may produce in Multiple Sclerosis patients.

From 1991-2002, we treated 58 patients with Multiple Sclerosis (MS) using the humanized MonoClonal AntiBody, Campath- 1H, which causes prolonged T-Lymphocyte depletion.

Clinical and surrogate markers of inflammation were suppressed. In both the Relapsing/Remitting (RR) and Secondary/Progressive (SP) stages of the illness, Campath-1H reduced the annual relapse rate (from 2.2 to 0.19 and from 0.7 to 0.001 respectively; both p < 0.001).

Remarkably, MRI scans of patients with SP disease, treated with Campath-1H 7 years previously, showed no new lesion formation. However, despite these effects on inflammation, disability was differently affected depending on the phase of the disease.

Patients with SPMS showed sustained accumulation of disability due to uncontrolled progression marked by unrelenting Cerebral Atrophy, attributable to ongoing Axonal loss.

The rate of Cerebral Atrophy was greatest in patients with established Cerebral Atrophy and highest inflammatory lesion burden before treatment (2.3 versus 0.7 ml/year; p = 0.04).

In contrast, patients with RR disease showed an impressive reduction in disability at 6 months after Campath-1H (by a mean of 1.2 EDSS points) perhaps owing to a suppression of on-going inflammation in these patients with unusually active disease.

In addition, there was a further significant, albeit smaller, mean improvement in disability up to 36 months after treatment.

We speculate that this represents the beneficial effects of early rescue of Neurons and Axons from a toxic inflammatory environment, and that prevention of DeMyelination will prevent long-term Axonal Degeneration.

These concepts are currently being tested in a controlled trial comparing Campath-1H and IFN-ß in the treatment of drug-naive patients with early, active RR MS.

Objective
To measure White Matter (WM) and Gray Matter (GM) Atrophy and lesion load in a large population of patients with Multiple Sclerosis (MS) using a fully automated, operator-independent, multiparametric segmentation method.

Methods
The study population consisted of 597 patients with MS and 104 control subjects. The MRI parameters were Abnormal WM fraction (AWM-f), global WM-f (gWM-f), and GM fraction (GM-f).

Results
Significant differences between patients with MS and control subjects included higher AWM-f and reduced gWM-f and GM-f.

MRI data showed significant differences between patients with Relapsing/Remitting and Secondary/Progressive forms of MS. Significant correlations between MRI parameters and between MRI and clinical data were found.

Conclusions
Patients with Multiple Sclerosis have significant Atrophy of both White Matter (WM) and Gray Matter (GM);

Secondary/Progressive patients have significantly more Atrophy of both WM and GM than do Relapsing/Remitting patients and a significantly higher lesion load (abnormal WM fraction);

Lesion load is related to both WM and even more to GM Atrophy;

Lesion load and WM and GM Atrophy are significantly related to Expanded Disability Status Scale score and age at onset (suggesting that the younger the age at disease onset, the worse the lesion load and Brain Atrophy); and

GM Atrophy is the most significant MRI variable in determining the final disability.