MS Abstracts 7a-2g1

Multiple Sclerosis (MS) has long been known to be associated with Leber's Hereditary Optic Neuropathy (LHON), a disease caused by Mitochondrial DNA (mtDNA) mutations.

We have investigated the possible involvement of LHON mtDNA point mutations in MS.

The study covered a group of 75 unrelated Caucasian patients, with the Relapse/Remitting or Primary/Progressive form of MS, and a control group of 75 volunteers (matched for age, gender and Ethnic origin).

Mitochondrial DNA from each subject was examined by means of restriction site polymorphism and sequencing techniques.

For 4 Primary LHON mutations (at Nucleotide positions 3460, 4160, 11778 and 14484) and 7 Secondary LHON mutations (at Nucleotide positions 4216, 4917, 5244, 7444, 13708, 15257 and 15812).

None of the Primary LHON mutations were detected in the MS patients or in the controls, whereas the proportion of individuals with Secondary LHON mutations was identical (27 p. cent) in the two groups.

A combination of 2 or 3 homoplasmic mutations, defining mtDNA haplogroups, was found in the majority of cases. Haplogroups J, T and X were not particularly associated with MS.

The frequency of the 13708 mutation alone (haplogroup X), or associated with the 4216 mutation (haplogroup J), was somewhat higher (p=0.059) in the subgroup of MS patients with Optic Neuritis (ON).

ON was the initial symptom in all but one of the patients with haplogroups J or X. No other correlation was found between MS phenotypes and mtDNA genotypes.

Our observations confirm previous reports that neither Primary nor Secondary LHON mutations are involved in the development of MS.

However, MS patients with haplogroups J or X appear to have a moderately higher risk of developing Optic Neuritis.

Thus, a specific mtDNA background may be a predisposing Genetic factor for Optic Nerve damage in MS patients.

We have analyzed a CA repeat polymorphism localized 46-kb upstream of the Fas Ligand Gene in Spanish and American populations.

That include 139 healthy controls and a cohort of 177 unrelated Relapsing/Remitting Multiple Sclerosis (MS) patients.

The MS patients consisted of two groups, one with a family history of MS and one without. The frequency of the 13 CA repeats (Allele B) was lower (p=0.01) in MS patients than in controls, 0.45 and 0.55 respectively.

The odds ratio (BB vs. AB/AA) for MS patients vs. healthy controls was 0.51 (95% CI 0.3-0.9; p=0.01).

The odds ratio (BB vs. AB/AA) for MS patients extracted from multiply affected families vs. healthy controls was 0.22 (95% CI 0.07-0.62; p=0.002).

The HLA DRB1*1501-DQB1*0602 haplotype is associated with B Allele with a relative frequency higher than A allele (0.52 and 0.48 in patients vs. 0.68 and 0.32 in controls).

The results suggest that Chromosomes with B Allele have a Genetic background that reduces susceptibility to MS, particularly in the familial forms.

Objectives
To use DNA arrays to identify differences in Gene expression associated with Relapsing/Remitting (RR) MS.

Methods
Total RNA was isolated from Monocyte depleted peripheral blood MonoNuclear Cells of 15 RR MS patients and 15 age- and sex-matched controls.

The RNA was reverse transcribed to RadioLabeled cDNA and the resultant cDNA was used to probe a DNA array containing over 4000 named human Genes.

The binding of RadioLabeled cDNA to the probes on the array was measured by PhosphorImager.

Results
Of more than 4000 Genes tested, only 34 were significantly different in RR/MS patients from controls. Of these, 25 were significantly increased and 9 significantly decreased in the RR MS patients.

Twelve of these Genes have inflammatory and/or Immunological functions that could be relevant to the MS disease process.

The potentially relevant Genes that were elevated (15% to 28%) were P protein, LCK, cAMP responsive element modulator, IL-7 Receptor, Matrix MetalloProteinase-19, M130 Antigen, and Peptidyl-Prolyl Isomerase.

Those that were significantly decreased (15% to 35%) were SAS TransMembrane 4 superfamily protein, STRL22 (C-C Chemokine receptor 6), AFX protein, DNA fragmentation factor-45 and ImmunoGlobulin gamma 3 (Gm marker).

Conclusions
The RR/MS disease effect was relatively restricted and most of the mRNAs tested were not different from the normal controls.

However, there were significant differences identified in the expression of a subset of mRNAs, including 13 with Inflammatory/Immune functions that could be relevant to MS.

The systematic use of DNA arrays can provide insight into the dynamic cellular pathways involved in MS PathoGenesis and its phenotypic heterogeneity.

Chronic Relapsing/Remitting Experimental AutoImmune EncephaloMyelitis (CREAE) induced with Myelin Oligodendrocyte Glycoprotein Peptides 35-55 (MOG(35-55)) in NOD mice was successfully treated with Brain-derived Gangliosides (GA).

The GA treatment suppressed the development and severity of CREAE, both clinically and histologically.

Spleen Cells from the GA-treated mice displayed markedly inhibited levels of MOG(35-55) specific proliferation and Interferon-gamma production.

Delayed-type hypersensitivity reactions to MOG(35-55) were suppressed by the GA treatment.

GA modulate various T-Cell effector functions in CREAE and may be an effective therapeutic agent for AutoImmune DeMyelinating Diseases such as Multiple Sclerosis.

The CXCR3 Chemokine Receptor, expressed on activated T-Lymphocytes, is seen within the Central Nervous System (CNS) in inflammatory conditions where a T-Cell response is prominent.

However, the distribution of CXCR3 in Parenchymal CNS cells is unknown.

Using a MonoClonal AntiBody against CXCR3 and post-mortem tissue of patients with and without CNS pathology, we have determined its expression pattern.

CXCR3 was found in subpopulations of cells morphologically consistent with Astrocytes, particularly reactive Astrocytes, and in Cerebellar Purkinje cells.

It was also detected in Arterial Endothelial and smooth muscle cells, particularly in areas associated with AtheroSclerotic plaques.

CXCR3-positive Astrocytes were particularly prominent in the CNS of HIV-positive patients, in patients with Multiple Sclerosis (MS), in Ischaemic Infarcts and in Astrocytic Neoplasms.

ImmunoFluorescence studies of mixed adult primary Glial cultures and fetal Glial cultures also showed expression of CXCR3 in Astrocytes.

CXCR3 mRNA was detected in Purkinje Cells by In Situ Hybridization with a CXCR3-specific probe.

Thus, the predominant expression of CXCR3 in reactive Astrocytes may indicate that it plays a role in the development of reactive Gliosis in a variety of Infectious, Inflammatory, Vascular and Neoplastic processes in the CNS.

The relationship between CXCR3 expression in Astrocytes to its expression in Purkinje Cells, Endothelial Cells and smooth muscle cells is yet to be determined.

The Etiology of Multiple Sclerosis (MS) remains unknown.

Epidemiological, clinical and pathological data support the theory that MS is a complex Disease/Syndrome with many factors affecting its development and progression.

It may be appropriate to regard MS as a syndrome with differing clinical and pathological features occurring along a spectrum.

Patients with MS are more likely to have an affected relative than are individuals without MS, which suggests that there is a Genetic component to this illness.

Despite this Genetic susceptibility, 85% of MS patients do not have an affected relative and only 1 in 3 Monozygotic (identical) twins develops MS if the other twin already has it.

These data strongly suggest that environmental factors influence the development of MS. Many putative infectious agents have been proposed to be involved in the Etiology of MS.

Although research into identifying MS-causative agents dates back for more than 5 decades, no agent has yet emerged with any consensus as the cause of MS.

This controversy is due to a number of factors. Including lack of specificity of an agent to MS, lack of reproducibility in other laboratories, inappropriate controls, laboratory contamination and lack of a standard and easily reproducible assay system.

Chlamydia Pneumoniae is a recently described pathogen that may have a role in the PathoGenesis of MS. C. Pneumoniae is an IntraCellular Bacterial organism that is infectious to humans.

It has recently been detected in the CerebroSpinal Fluid (CSF) of MS patients but not in that of patients with Other Neurological Diseases.

There is also a case report of a patient with CNS C. Pneumoniae infection and rapidly Progressive MS responding to AntiMicrobial therapy directed against this pathogen.

An association between C. Pneumoniae in the CSF and MS is now apparent, but its role in the development of MS remains unknown.

Further work exploring the role of C. Pneumoniae in Inflammatory DeMyelination is required. This may be accomplished either by developing an animal model or in a therapeutic trial in patients with MS.

Multiple Sclerosis (MS) is an Inflammatory, DeMyelinating Disease of the Central Nervous System (CNS) characterized by Blood-Brain Barrier (BBB) breakdown.

Disruptions of BBB continuity result in an influx of activated T-Cells and Monocytes, and could contribute to lesion formation in the CNS.

Matrix MetalloProteinases (MMP) are enzymes implicated in BBB disruption, and in degradation of ExtraCellular Matrix proteins and Myelin components.

An imbalance in levels of MMP and Tissue Inhibitors of MMP (TIMP) has been implicated in the PathoGenesis of MS.

Since Monocytes form a major cell population in acute MS lesions and may facilitate their entrance into the CNS by secretion of MMP.

Knowledge on MMP expression by blood Monocytes could be useful to improve our understanding of the PathoGenesis of MS.

In the present study, we examined the expression of MMP-1, -3, -7, -9, -14 and TIMP-1 mRNA by blood Monocytes in patients with MS using In Situ Hybridization.

Levels of MMP-1, -3, -7, -9 and of TIMP-1 mRNA expressing Monocytes were elevated in MS compared to controls, while those of MMP-14 did not differ.

We therefore conclude that MS is associated with elevated levels of MMP and TIMP expressing blood Monocytes that may contribute to MS PathoGenesis.

Copyright 2001 Academic Press.

The aim of the present study was to investigate the relationship between involvement of specific areas of the Brain and the occurrence of Depression and Anxiety in patients with Multiple Sclerosis.

We studied 95 patients (62 women and 33 men, mean age 39.5 years, SD 11.2) with definite MS, 97 patients (65 women and 32 men, mean age 40.7, SD 11.9) suffering from chronic Rheumatoid Diseases and 110 healthy subjects (71 women and 39 men, mean age 40.1, SD 12.7).

The disability, the independence, the Cognitive performances, the Depressive and Anxiety symptoms were assessed.

The diagnosis of Major Depression was made according to the DSM-IV. The patients with Multiple Sclerosis underwent a 1.5 Tesla Magnetic Resonance examination including T1 and T2 weighted images.

Calculation of regional and total lesion loads and Brain Volumes were performed.

The number (%) of subjects with a diagnosis of Major Depression was 18 (18.9) among MS cases, 16 (16.5) among controls with chronic disease (p=NS), and 4 (3.6) among healthy volunteers (p < 0.0001).

The Hamilton Depression and Anxiety rating scales median scores were 5 and 18, respectively in the MS patients, 5 (p= NS) and 14 (p= NS) in the chronic Rheumatoid Diseases controls, and 3 (p= < 0.0001) and 6 (p= < 0.0001) in the healthy controls.

Both severity of Depressive symptoms and diagnosis of Major Depression correlated, albeit weakly, with Right Frontal lesion load (r=0.22, p=0.035, and r=0.23, p=0.026, respectively) and Right Temporal Brain Volume (r=0.22, p=0.005 and r=0.22, p=0.036, respectively).

The severity of Depression was related significantly also with total Temporal Brain Volume (r=0.26, p=0.012), Right Hemisphere Brain Volume (r=0.25, p=0.015), disability (r=0.30, p=0.003) and independence of MS cases (r=-0.26, p=0.01).

The Anxiety did not correlate significantly with any of the measures of regional and total lesion loads and Brain Volume or with any of the considered clinical variables.

The similar frequency of Depression and severity of Depressive symptoms in MS patients and in chronic disease patients.

The significant difference in this respect with the normal controls, and the significant correlation between Depression and the disability measures would suggest a Psychological reaction to the impact of the disease.

But the relationship between Depression and the alterations in the Frontal and Temporal Lobes of the Right Hemisphere supports, on the contrary, the causative role of organic Brain damage.

The lack of any significant association between symptoms of Anxiety and either MRI abnormalities or clinical variables led us to the opinion that Anxiety is a reactive response to the PsychoSocial pressure put on the patients.

Objectives
To correlate Magnetic Resonance Imaging (MRI) T₂-weighted Lesion Load and measures of White Matter Atrophy in the Brain, to disability in a population-based sample of patients with Multiple Sclerosis (MS).

Material And Methods
A well defined cohort of patients was drawn at random from the general MS population by using the Danish Multiple Sclerosis Reigistry.

A semi-automated local thresholding technique was used to quantify T₂-weighted lesions on MRI.

Whereas manual tracing was applied to measure the Corpus Callosum Brain Ratio (CCR) and the Ventricle Brain Ratio (VBR).

Results
A sample of 86 patients with a mean age of 43.3 years (SD 4.3), mean disease duration of 13.6 years (SD 4.4) and a median Expanded Disability Status Score (EDSS) of 6.0 was identified.

The correlation between Total Lesion Area of the Brain (TLA) and Disability (EDSS) for the whole sample was moderate (Spearman rank correlation coefficient r=0.48, P<0.001).

Also correlations of CCR and VBR to Disability (r=0.32-0.46) were significant.

Conclusions
Correlations of TLA and disability in this study were rather strong.

Hence, T₂-weighted MRI lesion load in the Brain still plays an important role as a surrogate marker of disease and as a secondary outcome measure in Phase III Treatment Trials.

Areas of DeMyelination can be ReMyelinated by transplanting Myelin-forming cells.

Schwann Cells are the naturally ReMyelinating cells of the Peripheral Nervous System and have a number of features that may make them attractive for cell implantation therapies.

In Multiple Sclerosis, in which spontaneous but limited Schwann Cell ReMyelination has been well documented.

Schwann Cells can be expanded in vitro, potentially affording the opportunity of autologous transplantation; and they might also be spared the DeMyelinating process in Multiple Sclerosis.

Although rat, cat, and monkey Schwann Cells have been transplanted into rodent DeMyelinating lesions, the behavior of transplanted human Schwann Cells has not been evaluated.

In this study we examined the consequences of injecting human Schwann Cells into areas of acute DeMyelination in the Spinal Cords of adult rats.

We found that transplants containing significant FibroBlast contamination resulted in deposition of large amounts of Collagen and extensive progressive Axonal degeneration.

However, Schwann Cell preparations that had been purified by positive ImmunoSelection using AntiBodies to human low-affinity Nerve Growth Factor Receptor containing less than 10% FibroBlasts were associated with ReMyelination.

This result indicates that FibroBlast contamination of human Schwann Cells represents a greater problem than would have been appreciated from previous studies.

Multiple Sclerosis is a DeMyelinating Disorder of the Central Nervous System with a putative AutoImmune Etiology in which several Genes are thought to be involved.

Four published genomic screens have confirmed that a Gene influencing MS resides within or close to the HLA Class II region in 6p21.

Still, this locus is likely to confer only a part of the Genetic susceptibility in MS. Further, all four studies identified a number of other regions with possible linkage.

We have investigated eight Chromosomal intervals syntenic to loci of importance for Experimental AutoImmune model diseases in the rat in 74 Swedish MS families.

Possible linkage (a non-parametric linkage NPL score of 1.16 by GENEHUNTER computer package) was observed with markers in 12p13.3. A region syntenic to the rat Oia2 locus which is importance for Oil Induced Arthritis (OIA).

Four markers in the T-Cell Receptor-ß chain Gene region in 7q35 showed possible linkage (highest NPL score of 1.16).

This locus is syntenic to the rat Cia3 locus (Collagen Induced Arthritis).

These two loci at least partially overlap with Chromosomal regions showing indicative evidence for linkage in the previous MS Genomic screens.

Indeed, both Oia2 and Cia3 were recently found to be linked also with Experimental AutoImmune EncephaloMyelitis, a commonly used model for MS.

Markers in 2p12, 3p25, 10q11.23, 17q21-25, 19q13.1, and 22q12-13 failed to provide evidence for linkage.

We conclude that evidence is amounting that 12p13-12 and 7q34-36 may harbor Genes with an importance for MS. The synteny with experimental loci may eventually facilitate their identification.