Cells secreting AntiBodies against guinea pig Myelin and synthetic Myelin Basic Protein (MBP) Peptides were evaluated in CerebroSpinal Fluid (CSF) and blood from patients with Multiple Sclerosis (MS) and a variety of Other Neurological Diseases (OND).

The Peptides used, reproducing Amino Acid sequences 1-20, 70-89, 108-126, or 157-166 of MBP, were selected on the basis of their hydrophilic and EnCephalitoGenic Properties.

Low numbers of cells secreting IGG AntiBodies against Myelin or each of the MBP Peptides (about 1 per 50,000) were detected in Peripheral Blood, with no difference between MS and OND

In CSF, cells secreting IgG AntiBodies to MBP 70-89 were more frequently (p = 0.007) detected in patients with MS (1/380 IgG-secreting cells on average) than in patients with OND (1/2083 IgG-secreting cells on average).

The frequencies of cells secreting AntiBodies against Myelin or the three other MBP peptides were similar in MS and OND.

Thus, evaluation of B-Cell Immunity at the cellular level indicates that MBP 70-89 is an ImmunoDominant B-Cell Epitope in MS.

It is not clear whether this Intrathecal Anti-MBP 70-89 IgG AntiBody response has any PathoGenetic relevance in MS or is the result of Myelin breakdown.

Multiple Sclerosis (MS) is a disease with unknown cause characterized by inflammation and DeMyelination in the Central Nervous System.

Although an AutoImmune PathoGenesis has been suggested, there are no conclusive data on the number of T-Cells AutoReactive with Myelin Antigens in MS compared to controls.

We showed that T-Lymphocytes secreting Interferon-γ in response to possible target AutoAntigens are severalfold more common among PBL MonoNuclear Cells in patients with MS than in patients with Aseptic Meningitis and Tension Headache.

On average T-Cells reactive with Myelin Basic Protein (MBP), two different MBP peptides, or with ProteoLipid Protein amounted to 2.7-5.2/10(5) PBL from MS patients.

MBP-reactive T-Cells were still more frequent among MonoNuclear Cells isolated from the CerebroSpinal Fluid (CSF; 185/10(5) CSF cells).

We concluded that T-Cells reactive with Myelin AutoAntigens are strongly increased in MS.

This approach to detect them could allow definition of ImmunoDominant T-Cell Epitopes in individual MS patients, and thereby enable further development towards specific ImmunoTherapy.

The PathoGenesis of Multiple Sclerosis (MS), the major Neurological Disease of young adults in the western world, is still poorly understood and no effective therapy to block MS is yet available.

It is generally accepted that Reactive Oxygen species have a major role in the mediation of cell damage and that free Sulfhydryl groups are vital in cellular defense against endogenous or exogenous Oxidants.

Modification of the cellular Oxidant/AntiOxidant balance has been involved in the NeuroPathoGenesis of several diseases, e.g., Stroke, Parkinson's Disease, Alzehimer's Disease and physiological aging.

An increasingly important area of AntiOxidant defense is based on Sulfhydryl chemistry, owing to the role of Sulfhydryl groups in the function of macromolecular structures such as enzymes and cellular membranes.

The chemical composition of human CerebroSpinal Fluid (CSF) is considered to reflect Brain metabolism and in the present study we provided experimental evidence of a decrease in Sulfhydryl groups and increased content of products of Lipid PerOxidation.

Such as, ultraweak ChemIluminescence and LipoSoluble Fluorescence, which we found higher in the CSF and Plasma of MS patients than in controls, pointing out the role of Oxidative Stress in the PathoGenesis of MS.

Objective
To compare the sensitivity and specificity of IsoelEctric Focusing (IEF) with immunofixation, Agarose Gel Electrophoresis (AGE) and the IgG index in detecting Intrathecally synthesized IgG in Multiple Sclerosis (MS) and in Other Nervous System Disorders.

Materials And Methods
CerebroSpinal fluid (CSF) and Serum from 147 patients with various Nervous System Diseases, 20 of whom had MS, were compared with IEF, AGE and the IgG index.

Results
CSF-restricted OligoClonal Bands (OCB) were found in 20 of 20 patients with MS using IEF and in 9 of 20 using AGE. OCB were found in 12 patients with Other Nervous System Disorders (OND) using IEF and 4 using AGE.

The mean IgG index was 0.50 in OND and 0.96 in MS (P< 0.0001). Of 20 MS patients, 9 had an IgG index above the defined cut-off value of 0.72.

Conclusions
IEF is about twice as sensitive as AGE in detecting OCB in MS. IEF is also far superior to the IgG index in determining Intrathecal IgG synthesis.

Introduction
Besides Magnetic Resonance Imaging, the presence of locally produced OligoClonal IgG Bands (OCB) in the CerebroSpinal Fluid (CSF) is the most consistent laboratory abnormality in patients with Multiple Sclerosis (MS).

The most sensitive method for the detection of CSF OCB is IsoelEctric Focusing (IEF). Occasional patients with clinically definite MS lack evidence for Intrathecal IgG synthesis [7, 8].

This study was designed to compare clinical data and Evoked Potential (EP) findings between CSF OCB positive and OCB negative MS patients.

Patients And Methods
The study comprised 22 OCB negative patients with clinically definite MS [11] and 22 OCB positive controls matched for age, disease duration, activity and course of MS.

In both groups clinical assessment was performed by using Expanded Disability Status Scale (EDSS) Score and Progression Rate (PR).

All patients underwent multimodal EP: Visual (VEPs), BrainStem Auditory (BAEPs) and median SomatoSensory (mSEPs). The VEPs were considered abnormal if the P100 latency exceeded 117 ms or inter-Ocular difference greater than 8 ms was detected.

The BAEPs were considered abnormal if waves III or V were absent or the interpeak latencies I-III, III-V, or I-V were increased.

The mSEPs were considered abnormal when N9, N13 and N20 potentials were absent or when increased interpeak latencies were recorded.

The severity of the NeuroPhysiological abnormalities was scored for each modality as follows:
1. normal EP score 0;
2. every other EP abnormality except the absence of one of the main waves, score 1
3. absence of one or more of the main waves, score 2

Results
Both mean EDSS score (4.0 vs. 3.5) and PR (0.6 vs. 0.5) were similar in OCB positive and OCB negative group, (p > 0.05).

In the first group males were predominant, but without statistical significance.

Disease started more often with the BrainStem symptoms in the OCB positive than in OCB negative MS group (p = 0.028), while there was no differences in other initial symptoms between the groups.

The frequency of (multimodal) EP abnormalities was higher in the OCB positive group but the differences were not statistically significant, except for bilateral SEP abnormalities (p = 0.012).

The severity of the AEPs abnormalities was similar in both groups while for the VEPs and SEPs abnormalities were more pronounced in the OCB positive group but not significantly.

Discussion
The male preponderance of OCB negative MS patients in our study is in accordance with previous studies.

This finding could be potentially ascribed to the well known gender-related differences in both Humoral and Cellular Immune Responses.

We found no statistically significant differences in either disability or PR between the two patient groups, although OCB negative MS patients had lower EDSS score and PR than OCB positive cases.

In accordance with these findings, Fukazawa et al. also failed to show differences in disability between OCB negative and positive MS patients. On the other hand, few studies reported that OCB negative MS patients have a better prognosis.

The only clinical difference between two groups of patients that we found was that the disease more often started with BrainStem symptoms in OCB positive MS patients (p = 0.028). OCB positive MS patients had more often bilateral SEPs abnormalities (p = 0.012).

There was no statistically significant differences between two groups of patients in the severity of trimodal EPs abnormalities and the frequency of BAEPs and VEPs abnormalities although OCB negative patients had trend towards less pronounced EP disturbancies.

Conclusion
Our results did not reveal significant difference in clinical and NeuroPhysiological parameters between two groups of patients. However, they indicate a trend towards better prognosis of the disease in OCB negative MS patients.

Locally produced OligoClonal IgG Bands (OCB) are present in the CerebroSpinal Fluid (CSF) of 95% patients with Multiple Sclerosis (MS). The most sensitive method for the detection of OCB is IsoelEctric Focusing (IEF).

Occasional patients with Clinically Definite MS lack evidence for Intrathecal IgG synthesis. This study was designed to compare Brain Magnetic Resonance Imaging (MRI) findings between CSF OCB positive and negative MS patients.

The study comprised 22 OB negative patients with Clinically Definite MS and 22 OCB positive controls matched for age, disease duration, activity and course of MS.

In the both groups clinical assessment was performed by using Expanded Disability Status Scale (EDSS) score. T2 weighted MRI of the Brain was performed on a Siemens Magnetom (1.0T).

Lesions were counted and sized for 15 anatomically defined locations: 7 PeriVentricular (PV) and 8 Non-PeriVentricular (NPV) regions.

An arbitrary scoring system weighted for lesions size was used to estimate total and regional lesion loads:
• 1 point was given for each lesion with a diameter 1-5 mm
• 2 points for one lesion with a diameter 6-10 mm
• 3 points for one over 10 mm
• Confluent lesions scored one extra point

Atrophy were scored as follows:
1. Normal size
2. Mild Atrophy
3. Moderate Atrophy
4. Severe Atrophy

Mean score of total Brain MRI loads was lower in OCB negative than in OCB positive MS patients (44 vs. 50) but the difference was not statistically significant.

Mean PeriVentricular (32 vs. 23), non-PeriVentricular (26 vs. 19) and InfraTentorial (11 vs. 9) scores were higher in OCB positive MS group in comparison with OCB negative patients, but non-significant.

There was no correlation between EDSS score and total MRI lesions load in OCB negative MS patients, while in OCB positive group we detected significant correlation between EDSS score and total MRI lesions load (p = 0.026).

The results of this study demonstrate that by using conventional Brain MRI the extent end severity of the pathological process seems to be similar in OCB negative and OCB positive MS patients.

On the other hand, we found statistically significant correlation between Brain MRI total lesion load and EDSS in the OCB positive MS patients, while this correlation was not detected in OCB negative MS patients.

Differences in Brain MRI findings between OCB positive nad OCB negative MS patients have been already analyzed.

In the first, Zeman et al. reported that OCB negative MS patients have lower total MRI Brain lesion loads in comparison to OCB positive group, but the differences was not statistically significant.

In accordance with these findings, Fukazawa et al. also failed to show differences in the distribution, extent, shape and number of Brain MRI lesions between OCB positive and negative MS patients.

On the other hand, it has been demonstrated that the rate of Intrathecal IgG synthesis apparently correlates with plaque volume in the Brain, as demonstrated on MRI, in MS patients.

However, our results along with those from two above-mentioned previous studies do not support this notion.

In conclusion, trend towards lesser MRI lesion load and lack of its correlation with EDSS in OCB negative MS patients, warrants further investigations.

With new MRI techniques (Magnetic Resonance Spectroscopy and Magnetization Transfer), including the thorough exploration of Normal-Appearing White Matter, in OCB negative MS patients.

Multiple Sclerosis (MS) is an Inflammatory DeMyelinating Disease of the Central Nervous System.

We compared the diagnostic performance of IsoelEctric Focusing (IEF) combined with IgG immunoblotting to High-Resolution Agarose Gel Electrophoresis for the detection of Intrathecal synthesis of IgG due to the MS disease process.

In 20 patients with definite MS, IEF and High-Resolution Agarose Gel Electrophoresis had sensitivities of 90% and 60%, respectively.

In the 51 patients with no evidence of MS, the methods had specificities of 94% and 96%, respectively.

With a prevalence of 15% in this test population, IEF and High-Resolution Agarose Gel Electrophoresis had positive predictive values of 73% and 73% and negative predictive values of 98% and 93%, respectively.

The IEF method improves the sensitivity and the negative predictive value of the OligoClonal Banding assay, the IEF gels are easier to interpret, and the IEF assay requires a smaller CerebroSpinal Fluid volume.

Background
A sensitive method to detect Intrathecal IgG production is important in diagnosing Inflammatory Central Nervous System (CNS) Diseases, including Multiple Sclerosis (MS).

Objective
To compare CerebroSpinal Fluid (CSF) Electrophoresis with IsoElectric Focusing (IEF), ImmunoFixation-Peroxidase Electrophoresis (IFPE) and High-Resolution Agarose Electrophoresis with protein-staining (HRAGE).

Methods
Paired Serum and CSF samples from 307 consecutive patients attending a general Neurology clinic were examined with IEF, IFPE and HRAGE.

Clinical diagnosis was based on review of the patients' medical records after an average of 4 years.

Results
The sensitivity for detecting any Inflammatory (Autoimmune or infectious) CNS Disease (52 patients) was 67% for IEF, 50% for IFPE and 29% for HRAGE.

The sensitivity for detecting MS (14 patients) was 93%, 86% and 29% respectively. The sensitivity for detecting Clinically Isolated Syndrome (eight patients) was 75%, 25% and 13% respectively.

The number of OligoClonal Bands in IEF was higher in inflammatory than in Non-Inflammatory Neurological Diseases or symptoms, but similar in MS and Other Inflammatory Diseases.

Conclusion
IEF is the method of choice in diagnosing Intrathecal IgG synthesis.

Introduction
Intrathecal ImmunoGlobulins (Ig) synthesis, reflected by OligoClonal Bands (OCBs) in CerebroSpinal Fluid (CSF) is observed in up to 90 percent of patients with clinically definite Multiple Sclerosis (MS).

The gold standard laboratory test to establish the presence of OCBs in CSF of MS patients is IsoElectric Focusing (IEF).

However, a quicker and less expensive method has been developed: ImmunoFixation (IF).

Methods
The aim of this study was to compare these two methods carried out 74 CSF/Serum pairs of MS, 103 CSF/Serum pairs of subject controls and to determine their sensitivity and specificity.

Results
The agreement between results from IEF and IF was excellent (Kappa = 0.84).

IEF sensitivity (78 percent) was not significantly different from that of IF (74 percent) (p = 0.3).

Similarly, the specificity of IEF (93 percent) was not significantly different from that of IF (95 percent) (p = 0.2).

Conclusion
IF is a semi automated method which is easier to perform than IEF and which appears to be as efficient as IEF.

Background
The detection of Intrathecal synthesis of ImmunoGlobulins is used in the diagnosis of Multiple Sclerosis (MS).

We tested the semiautomated immunofixation-peroxidase (IFPOD) technique, which uses High-Resolution Agarose Gel Electrophoresis (HRAGE) directly followed by ImmunoFixation with a peroxidase-labeled anti-IgG AntiSerum to detect OligoClonal ImmunoGlobulins in CerebroSpinal Fluid (CSF).

Methods
We analyzed 230 consecutive matched Serum/CSF pairs that arrived in the laboratory over a 6-month period with both IFPOD and our routine techniques, ImmunoFixation Electrophoresis (IFE) and HRAGE.

For IFPOD, CSF samples were not concentrated before testing.

Results
Among the 230 samples were 12 clinically Definite MS, 33 clinically Probable, and 20 clinically Possible MS samples.

IFPOD and HRAGE + IFE each detected OligoClonal IgG in CSF in 10, 16, and 7 cases of these respective groups.

For clinically definite MS, sensitivity and specificity (95% confidence intervals) were, respectively, 83% (51-97%) and 79% (73-84%).

Conclusions
The IFPOD technique performs comparably to other analytical methods, without the requirement for sample concentration, and may represent an attractive alternative in testing for Intrathecal ImmunoGlobulin synthesis.

Two or more OligoClonal IgG Bands (OB) detected by separation of CerebroSpinal Fluid (CSF) proteins while not demonstrable in corresponding Serum reflect a local B-Cell response accompanying Central Nervous System (CNS) inflammation.

Using optimized, standardized methodology, preferentially protein separation by IsoelEctric Focusing followed by ImmunoBlotting, more than 95% of patients with Multiple Sclerosis (MS) have CSF OB of IgG class.

Not detectable in Serum, thereby providing powerful evidence for the diagnosis of MS. Once present, CSF OB persists in the individual patient irrespective of MS course or therapy.

Because of the high sensitivity of CSF OB in MS as well as its high specificity in the appropriate clinical setting, examination of CSF for OB of IgG class can be strongly recommended,

To obtain support for the diagnosis of MS and identify patients with Clinically Isolated Syndrome (CIS) at increased risk of developing MS.

The IgG index equal to CSF/Serum IgG:CSF/Serum Albumin is elevated in about 70% of MS patients, but rarely in CSF OB-negative MS.

Because of lower diagnostic sensitivity, IgG index cannot be recommended as replacement of CSF OB in the diagnosis of MS.

But, when elevated, as additional evidence for an augmented B-Cell response within the CNS that is compatible with MS.

Although the clinical picture as well as findings from Magnetic Resonance Imaging of the Brain and Spinal Cord are essential for an MS diagnosis, this should be re-evaluated in CSF OB-negative patients, keeping in mind the many disease entities imitating MS.

Recommended diagnostic criteria for MS must include definitions of the role of Lumbar Puncture and of clearly specified, optimized and standardized routine CSF investigations including for the presence of CSF IgG OB.

There is a need for concerted long-term follow-up studies of the subgroup of MS patients without CSF OB regarding e.g. prognostic and Immunologic features.

For inclusion in trials of disease-modulating drugs, it is recommended that patients with MS or CIS are selected regarding presence vs. absence of CSF OB.

Development and evaluation of new technologies to define local vs. systemic B-Cell responses in patients with MS or CIS vs. patients with Other Inflammatory Neurological Diseases should shed new light on the role of CSF OB, which remains enigmatic.

Although the diagnosis of Multiple Sclerosis (MS) may be clinically suspect and the Magnetic Resonance Imaging findings compatible, CerebroSpinal Fluid (CSF) analysis remains mandatory in order to support the diagnosis.

This is especially important since our understanding of the defining disease pathogenesis remains incomplete.

However, there is no specifically diagnostic CSF test. And until recently, laboratory techniques for CSF analysis had not been rigorously standardized.

Unconcentrated CSF without fixative should be used for the determinations of cell count and differential, protein and Glucose, Lactate, Myelin Basic Protein, and the CSF/Serum Albumin Ratio which is an indicator of Blood-CSF Barrier disruption.

Additionally, CSF ImmunoGlobulin-gamma (IgG) determinations are of major importance and are now included in the MS diagnostic criteria.

Testing for OligoClonal IgG Bands utilizing isoelectric focusing with IgG ImmunoBlotting, the IgG synthesis rate, and the IgG index should be included.

CSF analysis for kappa light chains and IGM may be diagnostically helpful.

The search for BioMarkers including those possibly present in the CSF which could predict and assess the course as well as response to treatment in a particular MS patient has not yet been successful.

CSF ImmunoGlobulin and T-Cell/B-Cell patterns, soluble HLA Class I and II Antigens, Nitric Oxide metabolites, NeuroFilament and MicroTubule components and AntiBodies, tau Protein, 14-3-3-Protein, Neuronal Cell and InterCellular Adhesion Molecule 1 Molecules, and Chemokines are actively being investigated as MS BioMarkers.