MS Abstracts 02c-2g5

There are two distinct subtypes of Multiple Sclerosis in Asians, Optic-Spinal (OS-Multiple Sclerosis) and conventional (C-Multiple Sclerosis).

In OS-Multiple Sclerosis, selective and severe involvement of the Optic Nerves and Spinal Cord is characteristic, though its mechanisms are unknown.

The present study aimed to find out possible differences in the Cytokine/Chemokine profiles in CSF between OS-Multiple Sclerosis and C-Multiple Sclerosis and to delineate the relationships between these profiles and NeuroImaging and Pathological features.

Sixteen Cytokines/Chemokines, namely InterLeukin-1beta (IL-1ß), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17, Interferon-gamma (IFN-), Tumour Necrosis Factor-alpha (TNF-), Granulocyte Colony-Stimulating Factor (G-CSF), Monocyte ChemoAttractant Protein-1 (MCP-1) and Macrophage Inflammatory Protein-1beta (MIP-1ß), were measured simultaneously in CSF supernatants from 40 patients.

With Relapsing/Remitting Multiple Sclerosis (20 OS-Multiple Sclerosis and 20 C-Multiple Sclerosis) at relapse and 19 control patients with SpinoCerebellar Degeneration (SCD), together with IntraCellular production of IFN-gamma and IL-4 in CSF CD4⁺ T-Cells.

In CSF supernatants relative to controls, IL-17, MIP-1ß, IL-1ß and IL-13 were only significantly increased in OS-Multiple Sclerosis patients, while TNF- was only significantly increased in C-Multiple Sclerosis patients, using a cut-off level of 1 pg/ml.

IL-8 was significantly elevated in both OS-Multiple Sclerosis and C-Multiple Sclerosis patients.

MCP-1 was significantly decreased in both OS-Multiple Sclerosis and C-Multiple Sclerosis patients, while IL-7 was only significantly decreased in C-Multiple Sclerosis patients.

IL-17, IL-8 and IL-5 were significantly higher in OS-Multiple Sclerosis patients than in C-Multiple Sclerosis patients.

The increases in IL-17 and IL-8 in OS-Multiple Sclerosis were still significant even after exclusion of the patients undergoing various ImmunoModulatory Therapies.

Assays of IntraCellular Cytokine production revealed that both the IFN-+IL-4- T-Cell percentage and IntraCellular IFN-/IL-4 ratio in CSF cells were significantly greater in C-Multiple Sclerosis patients than in controls.

Contrarily, OS-Multiple Sclerosis patients showed not only a significantly greater percentage of IFN-+IL-4- T-Cells than controls but also a significantly higher percentage of IFN--IL-4+ T-Cells than C-Multiple Sclerosis patients.

Among the Cytokines elevated in Multiple Sclerosis, only IL-8 showed a significant positive correlation with the Expanded Disability Status Scale of Kurtzke score.

Both the length of the Spinal Cord lesions on MRI and the CSF/Serum Albumin ratio had a significant positive correlation with IL-8 and IL-17 in Multiple Sclerosis, in which the Spinal Cord lesions were significantly longer in OS-Multiple Sclerosis than in C-Multiple Sclerosis.

Three of six Spinal Cord specimens from autopsied OS-Multiple Sclerosis cases demonstrated numerous MyeloPerOxidase-positive Neutrophils infiltrating necrotic lesions.

These findings strongly suggest that in OS-Multiple Sclerosis, in addition to the Th1 Cell upregulation seen in C-Multiple Sclerosis, Intrathecal activation of the IL-17/IL-8 axis inducing heavy Neutrophil infiltration contributes to extensive Spinal Cord lesion formation.

Subsequent to DeMyelination in Multiple Sclerosis, Myelin repair occurs but, as lesions age, the ability to ReMyelinate diminishes. Molecular pathways underlying Oligodendrocyte behaviour during CNS ReMyelination remain to be elucidated.

In this study, we report for the first time constitutive expression of the CXC/alpha Chemokine Receptors, CXCR1, CXCR2 and CXCR3, on Oligodendrocytes in normal adult human CNS tissue, the levels of which were upregulated in Multiple Sclerosis and Other Neurological Diseases (OND).

In addition, both immature (A2B5+/O4+) and more mature (CNPase+) human Oligodendrocytes in vitro expressed the same three Receptors.

The respective ligands to CXCR1, CXCR2 and CXCR3 [i.e. CXCL8/IL-8, CXCL1/GRO-alpha and CXCL10/IP-10), were absent in CNS tissue from normals and subjects with OND, but were present at high levels on HyperTrophic (reactive) Astrocytes.

At the edge of active (but not silent) Multiple Sclerosis lesions. Astrocytes in vitro could be induced to express Chemokines following stimulation with ProInflammatory Cytokines.

CXCL8 and CXCL1 production by human Astrocytes at both the RNA and protein levels could be induced by InterLeukin-1beta (IL-1ß), while CXCL10 was induced by both IL-1ß and Interferon-gamma.

Since these Cytokines are integral to inflammatory events occurring at the margins of active Multiple Sclerosis lesions, their upregulation in these regions may underlie the dynamics of Chemokine expression observed herein.

The simultaneous expression of different CXC Chemokine Receptors on Oligodendrocytes, and their Ligands on Astrocytes around Multiple Sclerosis lesions, may bespeak novel functional roles for these Immune System molecules in the recruitment of Oligodendrocytes and ReMyelination.

HistoPathological reports of Multiple Sclerosis and its animal models have shown evidence of a link between Axonal injury in active lesions and impaired Glutamate metabolism.

Mature Oligodendrocytes play a role in Glutamate uptake to maintain Glutamate homeostasis but in Multiple Sclerosis White Matter the loss of expression of Glutamate transporters in the lesion vicinity results in ineffective Glutamate removal.

Using a Magnetic Resonance Spectroscopy technique that isolates the Glutamate resonance at 3 T, we compared Glutamate levels between normal subjects and Multiple Sclerosis patients in different Brain areas.

Metabolite concentrations (Glutamate, Glutamine, N-AcetylAspartate, Myo-Inositol, Choline, Creatine) were derived from LCmodel and corrected for T₁ relaxation time.

Glutamate concentrations were found to be elevated in acute lesions (P = 0.02) and Normal-Appearing White Matter (P = 0.03), with no significant elevation in chronic lesions (P = 0.77).

The N-AcetylAspartate level in chronic lesions was significantly lower (P < 0.001) than in acute lesions and Normal-Appearing White Matter. The Choline level in acute lesions was significantly higher (P < 0.001) than in chronic lesions.

Evidence was also found for increased Glial activity in Multiple Sclerosis, with significantly higher (P < 0.001) Myo-Inositol levels in acute lesions compared with control White Matter.

These in vivo results support the hypothesis that altered Glutamate metabolism is present in Brains of Multiple Sclerosis patients.

Inflammation is a prominent feature of several disorders characterized by primary DeMyelination, but it is not clear whether a relationship exists between inflammation and Myelin damage.

We have found that substantial DeMyelination results from the focal inflammatory lesion caused by the injection of LipoPolySaccharide (LPS; 200 ng) directly into the rat Dorsal Funiculus.

Within 24 h, such injections caused a focal inflammatory response consisting of a substantial number of PolymorPhonuclear Cells and ED1-positive and Inducible Nitric Oxide Synthase (INOS)-positive Macrophages/Microglia.

The number of inflammatory cells was substantially reduced by day 7. OX-52-positive T-Cells were less frequently observed but were present in the Meninges at 8 h, reached a maximum in the Dorsal Funiculus at 7 days, and were rare at 14 days.

The inflammation was followed by the appearance of a large lesion of primary DeMyelination that encompassed up to approximately 75% of the cross-sectional area of the Dorsal funiculus.

Treatment with Dexamethasone significantly reduced the number of cells expressing INOS, but did not prevent the DeMyelination. By 28 days the lesions were largely ReMyelinated, usually by Schwann Cells.

These changes were not observed in control, Saline-injected animals. We conclude that the IntraSpinal injection of LPS results in inflammation and subsequently in prominent DeMyelination.

The mechanisms underlying the DeMyelination are not clear, but it is notable that it typically begins with disruption of the AdAxonal Myelin.

Indeed, there is an early loss of Myelin-Associated GlycoProtein within the lesion, despite the persistence of ProteoLipid Protein.

This combination is a feature of the Pattern III lesion recently described in Multiple Sclerosis (Lucchinetti et al., 2000), and we therefore suggest that LPS-induced DeMyelination may serve as the first experimental model available for the study of this type of Multiple Sclerosis lesion.

In this review, we focus on different pathogenetic mechanisms of CorticoSteroids that induce short- and long-term Brain Volume fluctuations in a variety of systemic conditions and disorders.

As well as on CorticoSteroid-induced ImmunoModulatory, ImmunoSuppressive and Anti-Inflammatory mechanisms that contribute to the slowdown of Brain Atrophy progression in patients with Multiple Sclerosis (MS).

It appears that chronic low-dose treatment with CorticoSteroids may contribute to irreversible loss of Brain Tissue in a variety of Autoimmune Diseases.

This side effect of Steroid therapy is probably mediated by Steroid-induced protein catabolism mechanism.

Evidence is mounting that high-dose CorticoSteroids may induce reversible short-term Brain Volume changes due to loss of IntraCellular water and reduction of abnormal Vascular permeability, without there having been Axonal Loss.

Other Apoptotic and selective inhibiting mechanisms have been proposed to explain the nature of CorticoSteroid-induced Brain volume fluctuations.

It has been shown that chronic use of high dose IntraVenous MethylPrednisolone (IVMP) in patients with MS may limit Brain Atrophy progression over the long-term via different Immunological mechanisms.

Including downregulation of Adhesion Molecule expression on Endothelial Cells, decreased Cytokine and Matrix MetalloProteinase secretion, decreased AutoReactive T-Cell-mediated inflammation and T-Cell Apoptosis induction, Blood-Brain Barrier closure, DeMyelination inhibition and, possibly, ReMyelination promotion.

Studies in nonhuman primates have confirmed that short-term Brain Volume fluctuations may be induced by CorticoSteroid treatment, but that they are inconsistent, potentially reversible and probably dependent upon individual susceptibility to the effects of CorticoSteroids.

Further longitudinal studies are needed to elucidate pathogenetic mechanisms contributing to Brain Volume fluctuations in Autoimmune Diseases and Multiple Sclerosis.

Background
The EVIDENCE (Evidence of Interferon Dose-Response: European North American Comparative Efficacy) Study demonstrated that patients with Multiple Sclerosis (MS) who initiate Interferon-ß-1a therapy with 44 microg 3 times weekly (TIW) were less likely to have a relapse.

Or, activity on Magnetic Resonance Imaging (MRI) compared with those who initiate therapy at a dosage of 30 microg 1 time weekly (QW).

Objective
To determine the effect of changing the dosage from 30 microg QW to 44 microg TIW in this extension of the EVIDENCE Study.

Design/Patients
Patients with Relapsing MS originally randomized to Interferon-ß-1a, 30 microg QW, during the comparative phase of the study changed to 44 microg TIW, whereas patients originally randomized to 44 microg TIW continued that regimen.

Patients were followed up, on average, for an additional 32 weeks.

Main Outcome Measure
The within-patient pretransition to post-transition change in relapse rate.

Results
At the transition visit, 223 (73%) of 306 patients receiving 30 microg QW converted to 44 microg TIW, and 272 (91%) of 299 receiving 44-microg TIW continued the same therapy.

The post-transition annualized relapse rate decreased from 0.64 to 0.32 for patients increasing the dose (P < .001) and from 0.46 to 0.34 for patients continuing 44-microg TIW (P = .03). The change was greater in those increasing dose and frequency (P = .047).

Patients converting to the 44-mug TIW regimen had fewer active lesions on T2-weighted MRI compared with before the transition (P = .02), whereas those continuing the 44-microg TIW regimen had no significant change in ₂ active lesions.

Patients who converted to high-dose/high-frequency Interferon-ß-1a therapy had increased rates of adverse events and treatment terminations consistent with the initiation of high-dose subcutaneous Interferon therapy.

Conclusions
Patients receiving Interferon-ß-1a improved on clinical and MRI disease measures when they changed from 30 microg QW to 44 microg TIW.

Background
Diffusion-Tensor (DT) Magnetic Resonance Imaging (MRI) has the potential to elucidate some characteristics of tissue microstructure inaccessible to other MRI techniques.

Objective
To investigate whether Normal-Appearing Brain Tissue abnormalities occur in patients with Multiple Sclerosis at the earliest clinical stage and whether their severity is predictive of a short-term disease evolution by using DT MRI.

Design
Forty-five patients and 22 healthy control subjects were studied. All patients had had a Clinically Isolated Syndrome within the 3 months preceding study enrollment and paraclinical evidence of disease dissemination in space.

During a single session, dual-echo, pulsed-gradient spin-echo echo-planar, and post Gadolinium T₁-weighted images of the Brain were obtained from each subject.

In patients, dual-echo and enhanced images were obtained after 3 and 12 months, to detect MRI signs of disease dissemination in time. An on-study Neurological Examination was also conducted to ascertain the occurrence of clinical relapses.

Mean Diffusivity and Fractional Anisotropy maps were derived from DT images.

Normal-Appearing White Matter (NAWM) and Normal-Appearing Gray Matter Mean Diffusivity and Fractional Anisotropy Histograms were produced and analyzed.

Results
During the study period, 29 patients showed MRI evidence of disease dissemination in time.

When compared with healthy controls, patients showed higher average NAWM Mean Diffusivity (P = .01), lower average NAWM Mean Diffusivity peak height (P < .001), and Fractional Anisotropy (P < .001).

The DT MRI characteristics of patients did not differ between those with and those without disease dissemination in time at follow-up.

Conclusions
In patients with Multiple Sclerosis at the earliest clinical stage, the severity of NAWM damage does not predict new lesion formation in the short term.

Suggesting that the "diffuse" component of tissue damage is, at least partially, independent of the "discrete," predominantly inflammatory aspects of the disease since its clinical onset.

Background
We have previously shown that the inducible Kinin B1 Receptor is expressed on T-Lymphocytes during relapses and progression in Multiple Sclerosis.

Objective
To evaluate the correlation between the expression of B1 Receptor on peripheral blood MonoNuclear Cells derived from patients who have Multiple Sclerosis with serial, clinical Magnetic Resonance Imaging and Immunological study-derived measures.

Design
Using frozen samples obtained from a high-frequency Magnetic Resonance Imaging-Immunological study, we analyzed B1 Receptor messenger RNA (mRNA) expression in peripheral blood-derived MonoNuclear Cells serially.

Collected from 6 patients with Multiple Sclerosis and 8 healthy control subjects by semiquantitative radioactive duplex reverse transcriptase-polymerase chain reaction amplification.

Time-course Kinin B1-Actin mRNA ratios were subsequently compared with corresponding clinical Magnetic Resonance Imaging and Immune parameters.

Results
The time-course Kinin B1-Actin mRNA ratio correlated positively with the Expanded Disability Status Scale index (P < .001), occurrence of clinical relapse (P = .02), volume of lesion on T₂-weighted images (P < .003).

And InterLeukin 2 Receptor and Major Histocompatibility Complex Class II expression on CD4⁺ Lymphocytes, but not with Gadolinium-enhancing lesions.

The time-course Kinin B1-Actin mRNA ratios were 5 to 25 times lower in samples derived from healthy controls.

Conclusion
The correlation of Kinin B1 Receptor mRNA levels with dynamic clinical and Magnetic Resonance Imaging measures suggests that expression of this Receptor can serve as an index of disease activity in Multiple Sclerosis.

Using Functional MRI (fMRI), patients with Multiple Sclerosis showed a greater extent of motor activation than controls.

Although functional changes are often interpreted as adaptive and as a contributing factor in limiting the clinical deficit, no longitudinal studies have yet been performed for Multiple Sclerosis.

Sixteen patients with Multiple Sclerosis, two patients with Possible Multiple Sclerosis and nine age-matched controls underwent two fMRI studies with a time interval of 15-26 months.

The Motor task consisted of a self-paced sequential finger opposition movement with the right hand. Patients with Multiple Sclerosis exhibited greater BiLateral activation than controls in both fMRI studies.

At follow-up, patients showed a reduction in functional activity in the IpsiLateral SensoriMotor Cortex and in the ContraLateral Cerebellum. No significant differences between the two fMRI studies were observed in controls.

Activation changes in IpsiLateral Motor Areas correlated inversely with age, extent and progression of T₁ lesion load, and occurrence of a new relapse.

This study may help the understanding of the evolution of Brain plastic changes in Multiple Sclerosis.

Indicating that, in younger patients with a less structural Brain damage and Benign clinical course, the Brain reorganizes its functional activity towards a more lateralized pattern of Brain activation.

The tendency towards a normalization of Brain functional activity is hampered in older patients and in those developing relapses or new irreversible Brain damage.

Axonal degeneration is a prominent pathological feature in Multiple Sclerosis observed over a century ago. The gradual loss of Axons is thought to underlie irreversible clinical deficits in this disease.

The precise mechanisms of Axonopathy are poorly understood, but likely involve excess accumulation of Ca Ions. In healthy fibers, ATP-dependent pumps support homeostasis of Ionic gradients.

When energy supply is limited, either due to inadequate delivery (e.g., Ischemia, Mitochondrial dysfunction) and/or excessive utilization (e.g., Conduction along DeMyelinated Axons), Ion gradients break down, unleashing a variety of aberrant cascades, ultimately leading to Ca overload.

During Na pump dysfunction, Na can enter Axons through non-inactivating Na Channels, promoting Axonal Na overload and depolarization by allowing K egress.

This will gate Voltage-sensitive Ca Channels and stimulate reverse Na-Ca exchange, leading to further Ca entry. Energy failure will also promote Ca release from IntraCellular stores.

NeuroTransmitters such as Glutamate can be released by reverse operation of Na-dependent transporters, in turn activating a variety of Ionotropic and metabotropic receptors, further exacerbating overload of cellular Ca.

Together, this Ca overload will inappropriately stimulate a variety of Ca-dependent Enzyme systems (e.g., Calpains, Phospholipases), leading to structural and functional Axonal Injury.

Pharmacological interruption at key points in these interrelated injury cascades (e.g., at Voltage-gated Na channels or AMPA receptors) may confer significant NeuroProtection to compromized Central Axons and supporting Glia.

Such agents may represent attractive adjuncts to currently available ImmunoModulatory Therapies.

This review on the role of NeuroFilaments as surrogate markers for Axonal degeneration in Neurological Diseases provides a brief background to Protein synthesis, assembly, function and degeneration.

Methodological techniques for quantification are described and a protein nomenclature is proposed.

The relevance for recognizing AntiNeuroFilament AutoAntiBodies is noted. Pathological implications are discussed in view of ImmunoCytoChemical, cell-culture and Genetic findings.

With reference to the present symposium on Multiple Sclerosis, the current literature on body fluid levels of NeuroFilaments in DeMyelinating Disease is summarized.

Axonal Degeneration is a major cause of permanent Neurological Deficit in Multiple Sclerosis (MS).

The mechanisms responsible for the degeneration remain unclear, but evidence suggests that a failure to maintain Axonal Sodium Ion homeostasis may be a key step that underlies at least some of the degeneration.

Sodium Ions can accumulate within Axons due to a series of events, including Impulse activity and exposure to inflammatory factors such as Nitric Oxide.

Recent findings have demonstrated that partial blockade of Sodium Channels can protect Axons from Nitric Oxide-mediated degeneration in vitro, and from the effects of NeuroInflammatory Disease in vivo.

This review describes some of the reasons why Sodium Ions might be expected to accumulate within Axons in MS.

And recent observations suggesting that it is possible to protect Axons from degeneration in NeuroInflammatory Disease by partial Sodium Channel blockade.