MS Abstracts 02a-2g5

Background
Intrathecal IgG Synthesis (ITGS), in conjunction with Magnetic Resonance Imaging, can help in the early diagnosis of Multiple Sclerosis (MS).

Recently, we developed a new OligoClonal IgG Band (OCGB) test for ITGS detection that is more sensitive and easier to interpret than previously described methods.

Objective & Design
To assess the accuracy of a new OCGB detection test in the diagnosis of MS. A prospective observational study.

Setting & Patients
A hospital Neurology department, a total of 385 patients with various Neurologic Disorders.

Main Outcome Measures
The sensitivity and specificity of the OCGB detection test for MS diagnosis.

Results
Intrathecal IgG synthesis was found in 127 patients with MS (96.2%), 18 (35.3%) with Central Nervous System infections, and 1 with Motor Neuron Disease.

Two patterns reflected ITGS. One pattern, showing OCGBs restricted to CerebroSpinal Fluid, was predominantly found in MS.

The other pattern, with OCGBs in serum and additional bands in CerebroSpinal Fluid, was mostly found in Central Nervous System infections.

No patients with other Inflammatory Neurologic Diseases showed ITGS. These patients frequently displayed a mirror pattern, with identical bands in Serum and CerebroSpinal Fluid.

Considering all patients, the sensitivity for the diagnosis of MS was 96.2%, and the specificity was 92.5%.

Excluding infections, which usually do not present a differential diagnosis problem with MS, the sensitivity was still 96.2%, and the specificity increased to 99.5%.

Conclusion
The accuracy of this OCGB method reinforces the value of CerebroSpinal Fluid studies in the early differential diagnosis of MS.

Background
Interferon-ß therapy has been shown to reduce the rate of clinical relapse and the frequency of Magnetic Resonance Imaging-defined T2- weighted lesions in patients with Multiple Sclerosis (MS).

When given early, Interferon-ß also reduces the rate of development of Brain Atrophy and improves Axonal integrity.

Nerve Growth Factor (NGF) can retard the severity and course of Experimental Allergic Encephalomyelitis.

Objective
To determine whether Interferon-ß effects on patients with MS could be related to modulation of NeuroTrophin production within the Central Nervous System.

Design
We studied NeuroTrophin production by human Glial and Brain Endothelial Cells in response to coculture with MS patient-derived Lymphocytes.

And, correlated levels of NGF secretion with clinical and Magnetic Resonance Imaging-defined markers of disease.

Results
We demonstrate that production of NGF by human Brain MicroVascular Endothelial Cells is triggered by interaction with T-Lymphocytes derived from MS patients.

No such response was observed using human adult Microglia or human fetal Astrocytes.

Nerve Growth Factor production by Endothelial Cells was potentiated by pretreating Lymphocytes with Interferon-ß in vitro, and by using Lymphocytes derived from MS patients treated with Interferon-ß in vivo.

By using this assay, we show that levels of NGF induced by Lymphocytes from MS patients inversely correlate with Magnetic Resonance Imaging measures of Brain Atrophy and Axonal injury.

Conclusion
These findings suggest that Interferon-ß-mediated production of NGF at the level of the Blood-Brain Barrier.

Whether acting as an ImmunoModulator or directly on Neural Cells, is another potential mechanism contributing to the Magnetic Resonance Imaging-defined effect of Interferon-ß on Brain Atrophy when given early in the course of MS.

Background
Abnormalities in Normal-Appearing Brain tissues may contribute to disability in Primary/Progressive Multiple Sclerosis (PPMS), where few lesions are seen on conventional imaging.

Objectives
To evaluate the mechanisms underlying disease progression in the early phase of PPMS by measuring metabolite concentrations in Normal-Appearing White Matter (NAWM) and Cortical Gray Matter (CGM) and to assess their relationship with clinical outcomes.

Design
Case-control study.

Setting
Tertiary referral hospital. Patients Forty-three consecutive patients within 5 years of onset of PPMS and 44 healthy control subjects.

Main Outcome Measures
Concentrations of Choline-containing compounds, Phosphocreatine, Myo-Inositol, total N-Acetyl-Aspartate (tNAA), and Glutamate-Glutamine were estimated using proton Magnetic Resonance Spectroscopic Imaging.

Brain Parenchymal, White Matter and Gray Matter Fractions and proton density and Gadolinium-enhancing lesion loads were calculated. The Expanded Disability Status Scale and Multiple Sclerosis Functional Composite scores were recorded.

Results
In CGM, concentrations of the tNAA (P < .001) and Glutamate-Glutamine (P = .005) were lower in patients with PPMS than in controls.

In NAWM, Myo-Inositol levels were higher (P = .002) and tNAA levels were lower (P = .005) in patients with PPMS than in controls.

The Expanded Disability Status Scale score correlated with the tNAA concentration in CGM (r = -0.44; P = .03) and with Myo-Inositol (r = 0.41; P = .01) and Glutamate-Glutamine concentrations (r = 0.41; P = .01) in NAWM.

Proton density lesion load correlated negatively with CGM tNAA concentration and positively with NAWM Myo-Inositol concentration.

Conclusion
Metabolite changes, which differ in CGM and NAWM occur in early PPMS and are linked with disability.

Background
Diffusion Tensor Magnetic Resonance Imaging (DT MRI) has the potential to provide in vivo information about tissue microstructure.

In Multiple Sclerosis (MS), DT MRI has disclosed the presence of occult structural damage in the Normal-Appearing Brain tissues.

Objective
To investigate whether DT MRI is sensitive to longitudinal changes of Brain damage that may occur beyond the resolution of T₂-weighted images in patients with Relapsing/Remitting MS.

Design
Twenty-six untreated patients with Relapsing/Remitting MS were followed up for 18 months. Dual-echo, DT and postcontrast T₁-weighted MRIs of the Brain were obtained at baseline and then every 3 months.

Mean Diffusivity (D) Histograms of Normal-Appearing Gray (GM) and White Matter were produced.

Total T₂-HyperIntense and T₁-HypoIntense lesion volumes; normalized Whole Brain Tissue, GM, and White Matter volumes; percentage Brain Volume change between the study entry and exit images; average lesion D; and Fractional Anisotropy were also calculated.

Results
During the study period, a significant decrease of normalized Whole Brain Tissue, average lesion Fractional Anisotropy and Normal-Appearing GM D Histogram peak height.

And, a significant increase of average Normal-Appearing GM D and T₂-HyperIntense lesion volumes were observed.

Changes of Normal-Appearing GM Diffusivity were independent of the concomitant changes of normalized Whole Brain tissue and GM volumes.

Conclusions
The DT MRI findings show progressive microstructural changes in the Normal-Appearing GM of patients with untreated Relapsing/Remitting MS.

Such changes do not reflect a concomitant development of Brain Atrophy and confirm the importance of GM pathology in MS.

Summary We have recently reported Brain Atrophy in the early stages of Primary/Progressive Multiple Sclerosis (PPMS), affecting both Gray and White Matter (GM and WM).

However, to date no clinical or radiological predictors of GM and WM Atrophy have been identified.

The aim was to investigate short-term changes in GM and WM volumes and to assess the predictive value of demographic, clinical and radiological variables in order to gain a better understanding of the pathological substrate underlying these changes.

Thirty-one subjects with PPMS within 5 years of symptom onset were studied at baseline and after 1 year.

At baseline, patients underwent Neurological Examination and were scored on the Expanded Disability Status Scale (EDSS) and Multiple Sclerosis Functional Composite.

They had 3D inversion-prepared Fast Spoiled Gradient Recalled (FSPGR), dual-echo and triple-dose post-contrast T₁-weighted spin echo MRI scans. Proton density and enhancing lesion loads were determined.

The 3DFSPGR sequence was repeated after 1 year and Brain Volume changes were calculated using two techniques, SPM99 (statistical parametric mapping) and SIENA (structural image evaluation, using normalization, of Atrophy).

Stepwise linear regression models were applied to baseline variables to identify independent predictors of Atrophy development.

Using SPM99, a decrease in Brain Parenchymal Fraction (-1.03%; P < 0.001) and GM fraction (-1.49%; P < 0.001) was observed.

The number of enhancing lesions independently predicted decrease in Brain Parenchymal Fraction (P = 0.019) and decrease in WM fraction (P = 0.002). No independent predictors of GM fraction decrease were found.

A Mean Brain Volume change of -0.63% (range -4.27% to +1.18%; P = 0.002) was observed using SIENA, which was independently predicted by EDSS (P = 0.004).

Global and GM Atrophy can be detected over a 1-year period in early PPMS.

The former may be predicted by the degree of inflammation, while the latter seems to be independent of it. SIENA and SPM-based methods appear to provide complementary information.

PharmacoTherapeutic options for Multiple Sclerosis (MS)-related Fatigue are limited.

Thirty patients were randomly assigned to Aspirin (ASA) 1,300 mg/day or placebo in a double-blind crossover study.

Results favored ASA for the main clinical outcomes: Modified Fatigue Impact Scale scores (p = 0.043) and treatment preference (p = 0.012). There were no significant adverse effects.

The results warrant further study and support a role for ASA-influenced mechanisms, perhaps Immunologic, in the generation of MS-related chronic Fatigue.

Functional Magnetic Resonance Imaging (fMRI) using Paced Auditory Serial Addition Test (PASAT) as paradigm was used to study the functional connectivity in 18 patients at the very early stage of Multiple Sclerosis (MS) compared with 18 controls.

To determine the existence of circuitry disturbance inside the Working Memory network and its relationship with White Matter abnormalities assessed by conventional MRI and Magnetization Transfer Ratio (MTR) imaging.

The left BA 45/46 was selected as the seed region to compute correlation maps with other Brain regions.

After obtaining the correlation map for each subject, between-group comparisons were performed using random effect procedure.

Compared with controls, patients did not show any greater functional connectivity between left BA 45/46 and other regions during PASAT.

In contrast, decrease in functional connectivity was observed in patients between left BA 45/46 and left BA 9, right BA 3, and the Anterior Cingulate Cortex (BA 24).

In patients, no correlations were found between altered functional connectivity and clinical data.

However, functional connectivity observed between left BA 45/46 and BA 24 in patients was correlated with the MTR of Normal-Appearing White Matter, and with Brain T₂ lesion load.

Altered functional connectivity is present inside the working memory network of patients at the very early stage of MS and is related to the extent of diffuse White Matter changes.

Abnormalities within Normal-Appearing Gray and White Matter (NAGM and NAWM) occur early in the clinical course of Multiple Sclerosis (MS) and can be detected in-vivo using the Magnetization Transfer Ratio (MTR).

To better characterize the rates of change in both tissues and to ascertain when such changes begin, we serially studied a cohort of minimally disabled, early Relapsing/Remitting MS patients, using NAGM and NAWM MTR Histograms.

Twenty-three patients with clinically definite early Relapsing/Remitting MS (mean disease duration at baseline 1.9 years), and 19 healthy controls were studied.

A Magnetization Transfer Imaging sequence was acquired yearly for two years.

Twenty-one patients and 10 controls completed followup. NAWM and NAGM MTR Histograms were derived and mean MTR calculated.

A hierarchical regression analysis, adjusting for Brain Parenchymal Fraction,was used to assess MTR change over time.

MS NAWM and NAGM MTR were significantly reduced in comparison with controls at baseline and, in patients, both measures decreased further during follow-up: (-0.10pu/year, p = 0.001 and -0.18pu/year, p < 0.001 respectively).

The rate of MTR decrease was significantly greater in NAGM than NAWM (p = 0.004).Under the assumption that such changes are linear, backward extrapolation of the observed rates of change suggested that NAWM abnormality began before symptom onset.

We conclude that increasing MTR abnormalities in NAWM and NAGM are observed early in the course of Relapsing/Remitting MS.

It is now important to investigate whether these measures are predictive of future disability, and consequently, whether MTR could be used as a surrogate marker in therapeutic trials.

Mitoxantrone is an approved drug for patients with worsening Relapsing/Remitting, Secondary/Progressive and Progressive/Relapsing Multiple Sclerosis (MS).

From a cohort of 820 MS patients, 52 (6%) were treated with this drug between December 1991 and April 2003.

Mitoxantrone was administered at a dose of 12 mg/m(2) once a month for three months and then at three-month intervals to reach a total cumulative dose of 144 mg/m(2).

The left Ventricular Ejection Fraction was checked by Radionuclide VentriculoGraphy prior to treatment and every six months.

Treatment was stopped if the Ejection Fraction was below 50% in two consecutive VentriculoGraphies performed one to three months apart. Cardiotoxicity during the course of the treatment was not observed.

However, three patients developed Congestive Heart Failure 24, 39 and 80 months after the last dose of Mitoxantrone. Other Cardiac causes were excluded. Two of these patients had been treated previously with Cyclophosphamide.

All patients first recovered on medical treatment, but two worsened a few months later. One patient remained severely symptomatic in spite of optimal medical treatment.

Although Mitoxantrone is generally well tolerated and reduces progression of disability and clinical exacerbations, our observation of a delayed CardioToxicity makes necessary a long-term follow-up of MS patients treated with this drug.

Background And Objectives
Rate and pattern of progression of Cognitive decline in Multiple Sclerosis (MS) has not been clearly identified.

The present study aimed to identify correlations between Cognitive tests and disease duration, construct longitudinal Cognitive curves, and assess pattern of change over time.

Methods
The NeuroPsychological Screening Battery for Multiple Sclerosis was administered in 150 consecutive MS patients, and tests that correlated with disease duration were identified.

Percentile curves were constructed and the pattern of Cognitive decline over time explored. The Cognitive curves were validated in an additional group of 83 patients with MS.

Results
Three of four measures of the Spatial Recall Test (SPART 7/24), and the Paced Auditory Serial Addition Task for two seconds (PASAT 2'), correlated with disease duration.

These tests were used to construct cross-sectional curves identifying the pattern of Cognitive decline over time in the MS population.

On the basis of this cross-sectional analysis, the earliest Cognitive decline occurred in the SPART 7/24 trials 1-5 between one and three years from onset.

Followed by decline in the SPART delayed recall between three and seven years, and then by decline in the PASAT 2' after seven years from onset.

Conclusions
Verbal Fluency and Verbal Memory appear to be affected earliest in MS.

The pattern of Cognitive decline is further characterized by a decrease in VisuoSpatial Learning, followed by Delayed Recall, and then by Attention and Information Processing Speed.

Cognitive percentile curves can be used to evaluate the pattern of progression and identify patients at increased risk.

Background
Primary/Progressive Multiple Sclerosis (PP-MS) differs from Relapsing/Remitting or Secondary/Progressive MS (RR/SP-MS) in ways suggesting differences in the pathogenic pathways.

Susceptibility to both PP-MS and RR/SP-MS is linked to carriage of the HLA molecule DRB1*1501. Several serologically defined HLA-DR groups (DR1, DR4, DR6, and DR9) occur less often in RR/SP-MS than in controls.

Some or all of the HLA-DR molecules encoded by alleles in these serologically defined groups have a negatively charged Glutamic Acid at residue 71 or 74 of the ß1 chain (ß1(71)/ß1(74)).

Residues at these positions are important in the formation of pocket 4 in the Antigen binding site of the HLA-DR molecule.

Objectives
To investigate whether the presence of Alleles encoding HLA-DR molecules containing Glutamic Acid at ß1(71)/ß1(74) correlates with the course of MS.

Methods
HLA-DR and HLA-DQ alleles and genotypes were analyzed in 121 MS patients (50 with PP-MS) and 109 controls by molecular typing.

Results
Alleles encoding HLA-DR molecules containing a Glutamic Acid at ß1(71)/ß1(74) occurred less often in patients with RR/SP-MS than in those with PP-MS or controls.

In subjects not carrying the DRB1*1501 allele, a much higher proportion of PP-MS patients carried alleles encoding HLA-DR molecules containing a Glutamic Acid at beta1(71)/ß1(74) than did RR/SP-MS patients or controls.

Conclusions
The Amino Acid residues involved in determining the shape and charge of pocket 4 of the HLA-DR ß1 chain could influence the clinical course of MS by determining protection against RR/SP-MS or susceptibility to the development of PP-MS.

Interferon-gamma (IFN-γ) plays a pivotal role in the pathogenesis of Multiple Sclerosis (MS), while IFN-ß may be able to modify the clinical course of the disease, eventually also by counterbalancing IFN-γ-mediated effects.

Catecholamines (CA) exert important effects on the Immune Response, both as transmitters between the Nervous and the Immune System, as well as Autocrine/Paracrine mediators in Immune Cells, and several lines of evidence support their involvement in MS.

In particular, dysregulated production of CA seems to occur in Peripheral Blood MonoNuclear Cells (PBMCs) of MS patients. We assessed the effects of IFN-ß and IFN-γ on endogenous CA in PBMCs.

In cultured PBMCs stimulated with PhytoHaemAgglutinin (PHA), IFN-ß increased CA production and induced CA release in the culture medium.

While IFN-γ decreased both CA production and the expression of mRNA for the CA-synthesizing Enzyme Tyrosine Hydroxylase.

Coincubation with both IFNs prevented the inhibitory effect of IFN-γ, as well as the stimulatory effect of IFN-ß. IFNs are the first physiological compounds shown to affect endogenous CA in PBMCs:

In view of the role of CA-dependent mechanisms in the Immune Response, these findings may help to better understand the mechanisms of action of IFN-ß as an ImmunoModulatory Drug in MS.