Investigation of Subclinical Markers of Multiple Sclerosis

Neurophysiologic measure of RMT (expressed in percentage of maximal stimulator output) will be evaluated with navigated TMS over the primary motor cortex:

• in relapsing-remitting MS receiving corticosteroid treatment patients in 2 phases (before the intake of steroids, 5 weeks after taking the steroids).
• relapsing-remitting MS receiving immunomodulation treatment patients at baseline

The neurophysiologic measure of MEP (amplitude expressed in microvolts and latency expressed in milliseconds) will be evaluated with navigated TMS over the primary motor cortex for upper and lower extremity muscles:

• in relapsing-remitting MS receiving corticosteroid treatment patients in 2 phases (before the intake of steroids, 5 weeks after taking the steroids).
• relapsing-remitting MS receiving immunomodulation treatment patients at baseline

The neurophysiologic measure of CSP duration (expressed in milliseconds) will be evaluated with navigated TMS over the primary motor cortex in relapsing-remitting MS patients in 2 phases (before the intake of steroids, 5 weeks after taking the steroids).

The neurophysiologic measure of SAI phenomena (expressed in milliseconds) will be evaluated with navigated TMS over the primary motor cortex with peripheral electrical stimulation over the median nerve at different interstimulus intervals (20-28 ms) in relapsing-remitting MS patients in 2 phases (before the intake of steroids, 5 weeks after taking the steroids).

• EMNG measures of DML and SNAP latency (expressed in milliseconds) of upper and lower extremities will be evaluated in relapsing-remitting MS patients receiving corticosteroid treatment in 2 phases (before the intake of steroids, 5 weeks after taking the steroids).
• EMNG measures of DML and SNAP latency (expressed in milliseconds) of upper and lower extremities will be evaluated in relapsing-remitting MS receiving immunomodulation treatment patients at baseline.

• EMNG measures of CMAP and SNAP amplitude (expressed in millivolts) of upper and lower extremities will be evaluated in relapsing-remitting MS patients receiving corticosteroid treatment in 2 phases (before the intake of steroids, 5 weeks after taking the steroids).
• EMNG measures of CMAP and SNAP amplitude (expressed in millivolts) of upper and lower extremities will be evaluated in relapsing-remitting MS patients receiving immunomodulation treatment at baseline.

• EMNG measures of CV (expressed in meters per second) of motor and sensory nerves of upper and lower extremities will be evaluated in relapsing-remitting MS patients treated with corticosteroids in 2 phases (before the intake of steroids, 5 weeks after taking the steroids).
• EMNG measures of CV (expressed in meters per second) of motor and sensory nerves of upper and lower extremities will be evaluated in relapsing-remitting MS patients treated with immunomodulation treatment at baseline.

-EMNG measures of F-wave (expressed in milliseconds) for motor nerves of upper and lower extremities will be evaluated in relapsing-remitting MS patients treated with corticosteroids in 2 phases (before the intake of steroids, 5 weeks after taking the steroids).

--EMNG measures of F-wave (expressed in milliseconds) for motor nerves of upper and lower extremities will be evaluated in relapsing-remitting MS patients treated with immunomodulation at baseline.

• CD 14 expression on monocytes will be evaluated in relapsing-remitting MS patients treated with corticosteroids in 2 phases (before the intake of steroids, 5 weeks after taking the steroids). The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive monocytes.
• CD 14 expression on monocytes will be evaluated in relapsing-remitting MS patients treated with immunomodulation at baseline.The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive monocytes.
• CD 14 expression on monocytes will be evaluated in the healthy control group.The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive monocytes.

• CD 16 expression on monocytes will be evaluated in relapsing-remitting MS patients treated with corticosteroids in 2 phases (before the intake of steroids, 5 weeks after taking the steroids). The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive monocytes.
• CD 16 expression on monocytes will be evaluated in relapsing-remitting MS patients treated with immunomodulation at baseline. The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive monocytes.
• CD 16 expression on monocytes will be evaluated in the healthy control group. The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive monocytes.

• CD 40 expression on monocytes will be evaluated in relapsing-remitting MS patients treated with corticosteroids in 2 phases (before the intake of steroids, and 5 weeks after taking the steroids). The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive monocytes.
• CD 40 expression on monocytes will be evaluated in relapsing-remitting MS patients treated with immunomodulation at baseline. The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive monocytes.
• CD 40 expression on monocytes will be evaluated in the healthy control group. The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive monocytes.

• CD 192 expression on monocytes will be evaluated in relapsing-remitting MS patients treated with corticosteroids in 2 phases (before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids). The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive monocytes.
• CD 192 expression on monocytes will be evaluated in relapsing-remitting MS patients treated with immunomodulation at baseline. The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive monocytes.
• CD 192 expression on monocytes will be evaluated in a healthy control group. The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive monocytes

CD 163 levels in serum will be evaluated in relapsing-remitting MS patients in 2 phases (before the intake of steroids, 5 weeks after taking the steroids). CD 163 levels will be measured with ELISA assay (expressed in micro mols per litre).

• CD 4 expression on lymphocytes will be evaluated in relapsing-remitting MS patients treated with immunomodulation at baseline. The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive lymphocytes.
• CD 4 expression on lymphocytes will be evaluated in the healthy control group. The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive lymphocytes.

• CD 25 expression on lymphocytes will be evaluated in relapsing-remitting MS patients treated with immunomodulation at baseline. The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive lymphocytes.
• CD 25 expression on lymphocytes will be evaluated in the healthy control group. The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive lymphocytes.

• CTLA-4 expression on lymphocytes will be evaluated in relapsing-remitting MS patients treated with immunomodulation at baseline. The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive lymphocytes.
• CTLA-4 expression on lymphocytes will be evaluated in the healthy control group. The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive lymphocytes.

• FOXP3 expression on lymphocytes will be evaluated in relapsing-remitting MS patients treated with immunomodulation at baseline. The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive lymphocytes.
• FOXP3 expression on lymphocytes will be evaluated in the healthy control group. The expression will be measured as the median of fluorescence distribution acquired with flow cytometry. The expression will also be quantified as the percentage of positive lymphocytes.

The Edinburgh Handedness Inventory is a well known short questionnaire for determining objectively whether one is left , right handed, or ambidekster. The subjects determines whether he/she is using right or left hand for specific activities (ten activities). The total checks are given for right and left hand, than cumulative sume is given (total checks from right and left hand), difference between total checks for right minus left hand. Final result is the difference result subtracted from cumulative result x 100.

• Relapsing-remitting MS patients treated with corticosteroids will be tested with LDST before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. LDST is interpreted as the number of correct responses in 60 seconds.
• Relapsing-remitting MS patients treated with immunomodulation therapy will be tested with LDST at baseline.

Relapsing-remitting MS patients will be tested with MoCA before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. MoCA is interpreted as the number of points (30 is maximal).

• Relapsing-remitting MS patients treated with corticosteroids will be tested with MSIS before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. The total score is formed as the sum of the rounded answers to the two subscales (physical and psychological scale).
• Relapsing-remitting MS patients treated with immunomodulation will be tested with MSIS at baseline. The total score is formed as the sum of the rounded answers to the two subscales (physical and psychological scale).

• Relapsing-remitting MS patients treated with corticosteroids will be tested with DASS-21 before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids.
• Relapsing-remitting MS patients treated with immunomodulation will be tested with DASS-21 at baseline.

DASS-21 contains three scales for measuring emotional states of depression, anxiety and stress. Each scale contains 7 statements.

The result on the depression, anxiety and stress scales is the sum of the rounded answers to specific statements.

-Relapsing-remitting MS patients treated with corticosteroids will be tested with FSS before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids.

--Relapsing-remitting MS patients treated with immunomodulation will be tested with FSS at baseline.

The subject is asked to read each statement and circle a number from 1 to 7, depending on how appropriate they felt the statement applied to them over the preceding week. A low value indicates that the statement is not very appropriate whereas a high value indicates agreement.The scoring is done by calculating the average response to the questions (adding up all the answers and dividing by nine).

Relapsing-remitting MS patients will be tested with numerical Pain Rating Scale before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. The result is interpreted as the total sum of points. The greater the results the more pain is detected.

Relapsing-remitting MS patients will be tested with SAEHAN Squeeze Dynamometer before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. Six measurement results are recorded for each subject - three for the grip with the dominant hand and three for the grip with the non-dominant hand. The final result is the mean of the three measurements for each arm expressed in kilograms.

Relapsing-remitting MS patients will be tested with Lafayette Manual Muscle Tester Dynamometer before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. Muscle strength measured by Lafayette Manual Muscle Tester is recorded in kilograms and normalized to body-mass index (BMI). Handheld dynamometry will be used to measure ankle dorsiflexion; knee, flexion and extension; hip flexion, extension, abduction, and adduction; and trunk lateral flexion.

Relapsing-remitting MS patients will be tested with O'Conner Finger Dexterity Test before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. The O'Connor Finger Dexterity Test requires hand placement of 3 pins per hole. Result (seconds) = time required to complete the first part of the test + (1.1 x time required to complete the second part of the test) / 2

Relapsing-remitting MS patients will be tested with Barthel Index for Activities of Daily Living (ADL) before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. The examiner is rank the patient's independence in the following areas:feeding, bathing, grooming, dressing, bowel control, bladder control, toilet use, transfers (bed to chair and back), mobility on level surfaces, and stairs.

• 0 - 20 = complete dependence; 21 - 60 = severe addiction; 61 - 90 = moderate dependence

Correlation and regression methods will be used to examine the following data:

• neurophysiological data acquired with TMS (RMT; MEP; CSP; SAI);
• neurophysiological data acquired with EMNG (DML and SNAP latency; CMAP and SNAP amplitude; motor and sensory CV; F-wave);
• immunological data (expression profile of CD40, CD192, CD14, CD16 on monocytes and CD25, CTLA-4 and FOXP3 on lymphocytes; expression of CD163 in serum);
• neuropsychological data; Edinburgh Handedness Inventory test; Pain rating scale; Barthel Index for Activities of Daily Living (ADL); FSS; LDST; MoCA; MSIS-29; DASS-21; O'Conner Finger Dexterity Test; muscle strength - hand grip and upper and lower extremity muscles with a dynamometer
• clinical: EDSS,T25-FW; MRI; biochemical (complete blood count, differential blood count, AST, ALT, GGT, blood sugar test, creatinine, urine, B12, Na, K, Cl, folic acid, neurotrophic virus in serum and liquor, oligoclonal band)

• EDSS applied in relapsing-remiting MS patients before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids.
• EDSS applied in relapsing-remiting MS patients treated with immunomodulation at baseline.

The EDSS scale ranges from 0 to 10 in 0.5 unit increments that represent higher levels of disability. Scoring is based on an examination by a neurologist.

T25-FW applied in relapsing-remiting MS patients before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids.The T25-FW is a quantitative mobility and leg function performance test based on a timed 25-walk. The score for the T25-FW is the average of the two completed trials.

Clinical biochemical assessment of differential blood count will be provided before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. Following components of the blood are measured: leukocytes (x10^9/L) (ref. interval. 3.4-9.7), erythrocytes (x10^12/L) (ref. interval 3.86-5.08), haemoglobin (g/L) (ref. interval 119-157), hematocrit (L/L) (ref. interval 0.356-0.470), MCV (fL) (ref. interval 83.0-97.2), MCH (pg) (ref. interval 27.4-33.9), MCHC (g/L) (ref. interval 320-345), index of scattering of erythrocytes (%) (ref. interval 9.0-15.0), thrombocytes (x10^9/L) (ref. interval 158-424), MPV (fL) (ref. interval 6.8-10.4), PDW (%) (ref. interval 35-65), PCT (L/L) (ref. interval 0.001-0.004)

Clinical biochemical assessment of differential blood count will be provided before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. The blood collection is taken early in the morning, before breakfast. Following components of the blood are measured: neutrophile granulocytes (%) ( ref. interval 44-72), lymphocyte (%) (ref. interval 20-46), monocyte (%) (ref.interval 2-12), eosinophile (%) (ref. interval 0-7), basophile (%) (ref. interval 0-1), neutrophile # (x10^9/L) (ref. interval 2.06-6.49), lymphocyte # (x10^9/L) (ref. interval 1.19-3.35), monocyte # (x10^9/L) (ref. interval 0.12-0.84), eosinophile # (x10^9/L) (ref. interval 0.00-0.43), basophile # (x10^9/L) (0.00-0.06)

Clinical biochemical assessment of glucose will be provided before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. The glucose is measured in mmol/L, and ref. interval is 4.4-6.4

Clinical biochemical assessment of urea will be provided before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. The urea is measured in mmol/L, and ref. interval is 2.8-8.3

Clinical biochemical assessment of creatinine will be provided before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. The creatinine is measured in micro mol/L, and ref. interval is 49-90

Clinical biochemical assessment of AST test will be provided before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. A high AST level is a sign of liver damage, but it can also mean the patient have damage to another organ like heart or kidneys. The normal range of values for AST is about 8 to 30 units per liter of serum (the liquid part of the blood).

Clinical biochemical assessment of ALT test will be provided before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. A high ALT level is a sign of liver damage, but it can also mean the patient have damage to another organ like heart or kidneys. The normal range of values for ALT is about 10 to 36 units per liter of serum (the liquid part of the blood).

Clinical biochemical assessment of GGT test will be provided before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. GGT is elevated in the blood in most diseases that cause damage to the liver or bile ducts. The normal range for GGT levels is 9-35 units per liter (U/L). Normal values can vary due to age and sex.

Clinical biochemical assessment of urine will be provided before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. Urine examination will include colour (expressed as a qualitative unit), acidity (pH, ref. interval 5-9), specific gravity (kg/L, 1.002-1.030), glucose (pos or neg), bilirubin (pos or neg), ketones (pos or neg), haemoglobin (pos or neg), proteins (pos or neg), urobilinogen (pos or neg).

Clinical biochemical assessment of B12 will be provided before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. The normal range for vitamin B12 can vary slightly depending on the lab. But a normal level of vitamin B12 in blood is generally between 145 and 569 picomols per litre (pmol/L).

Clinical biochemical assessment of Na will be provided before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. The normal range for blood sodium levels is 137 to 146 millimoles per litre (mmol/L).

Clinical biochemical assessment of K will be provided before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. The normal range for blood potassium levels is 3.9 to 5.1 millimoles per litre (mmol/L).

Clinical biochemical assessment of Cl will be provided before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. The normal range for Cl levels is 97 to 108 millimoles per litre (mmol/L).

Clinical biochemical assessment of FA will be provided before the intake of steroids, 5 weeks after taking the steroids and 6 months after taking the steroids. The normal range for FA levels is 8.83 to 60.8 nanomoles per litre (nmol/L).

Serologic test for neurotropic viruses and bacteria in blood serum and cerebrospinal liquor will be provided before the intake of steroids. Seroconversion for Epstein Barr virus, Cytomegalovirus, Herpes simplex 1/2 virus, Borrelia burgdoferi, German measles virus, Measles virus. Seroconversion is expressed as positive or negative.

Microscopic and biochemical assessment of cerebrospinal liquor includes: colour (qualitative units), transparency (qualitative units), leukocytes # (10^6/L), erythrocytes # (10^6/L), glucose (mmol/L, ref interval 2.49 to 4.44), lactate (mmol/L, ref interval 1.1-2.2), total protein (mg/L, ref interval 170-370) , chlorides (mmol/L, ref interval 115-129)

Oligoclonal bands in cerebrospinal liquor will be assessed at baseline, measured as present or absent.

• Each MS patient treated with corticosteroid will have MRI scanning before intake of steroids and 6 months after the intake. Number and location of different radiological lesions will be noted.
• Each MS patient treated with immunomodulation will have MRI scanning at baseline (also verifying pre-baseline MRI if available).