NEUROLOGY MS ID#: NEUROLOGY/2006/139451

Appendix (E) methods

Neutralizing antibodies hamper IFN-beta bioactivityand treatment effect on MRI in MS patients

Per Soelberg Sorensen MD1, Thomas Tscherning MD1, Henrik Kahr Mathiesen MD2, Annika R. Langkilde MD2, Christian Ross MD3,,Mads Ravnborg MD1, Klaus Bendtzen MD3

Neutralizing antibodies

A major problem with IFN-beta therapy of multiple sclerosis is the induction of neutralizing anti-IFN-beta antibodies (NABs) that causes a reduction or abolition of the therapeutic benefits (1-5).

Neutralizing anti-IFN-beta antibodies (NABs) occur in patients treated with IFN-beta with frequencies and titers that vary considerably depending on the IFN-beta preparation, the frequency and route of administration, and the type of assay being used (6). The mechanism by which antibodies are induced is mainly unknown (7). However, NABs that bind with high affinity are likely to weaken or abrogate the cellular response to IFN-beta and to attenuate subsequent downstream effects on viral resistance, cellular proliferation, and immunological responses (8). NABs are associated with reduced gene induction after an IFN-beta injection reflected either as a decrease in response of MxA at the mRNA or protein level and of neopterin or beta-2-microglobulin (9-12).

Although the clinical importance of NABs has been debated, it is now widely accepted that the occurrence of NABs is associated with a weakened or abolished therapeutic response on relapses and MRI activity (13). The main cause of the dispute has been that the effect of NABs on the clinical response does not show in short term studies of 2 years or less (12, 14-18), simply because the clinical effects of NABs are not apparent before 12-18 months after treatment start (19), whereas the negative consequences of NABs are readily shown in all longer trials of 3 years or more where the consequences of NAB have time to show (3, 20, 21).

Measurement of neutralizing antibodies

We used a cytopathic effect assay (CPE) to measure NABs against IFN-beta(8). Briefly, a subclone of A549 human lung carcinoma cells (CCL 185, American Type Culture Collection, Bethesda, MD, USA), were seeded in micro-trays at a concentration of 10,000 cells per well and incubated for 24 h at 37o C in a humidified 5% CO2 atmosphere. IFN-beta (using recombinant IFN-beta homologous to the treatment received by the individual patient) at a concentration of 10 LU per mL was preincubated for 1 h with diluted serum at 5% concentration in a volume of 100 l and then transferred to MC-5 cells challenged with EMC virus at a the lowest concentration which by itself caused 100% cell death. After another 24 h, the antiviral effect of IFN-beta was measured by use of a 3-(4,5 dimethyl thiazol-2-yl)-2,5-difenyl tetrazolium bromide assay. To avoid false-negative and false-positive results, controls for endogenous antiviral activity and serum toxicity were included in each assay.The neutralizing capacity of each serum sample was measured; i.e. the percentage of the added IFN-beta which was neutralized by the NABs. We defined a NAB-positive blood sample as a sample with a serum neutralization capacity of 20% or above. This cut-off value was chosen as the lowest concentration of NABs that had significant clinical importance as shown in a previous study (3).

MRI

Brain MRI was performed at 1.0 Tesla (Siemens Impact Magnetom). The patients were scanned 4 times with intervals of 1 month using the same scanning protocol: 1) Axial, coronal and sagittal scouts were performed for reposition. For all following sequences, 25 axial slices with 5 mm slice thickness and no gaps (continuous) was planed from the midsagittal image using the hyfaline (the line connecting the inferior border of the pineal gland and the fastigium of the fourth ventricle) which was moved to the inferior border of the splenium of the corpus callosum. 2) A T1-weighted spin-echo sequence was performed (TR 700 ms, TE 15 ms, 2 acquisitions, matrix 168x256, FOV 230 mm, Rect. FOV 7/8). 3) 0.2 mmol/kg (double-dose) gadolinium-DTPA (Magnevist) was injected intravenously without moving the patient in the scanner using long catheters. 4) A double spin-echo proton density (PD) and T2-weighted sequence was obtained (TR 2400 ms, TE 20/80 ms, 1 acquisition, matrix 224 x 256, FOV 230 mm, Rect. FOV 7/8). 5) Approximately 9 minutes after contrast injection the T1-weighted sequence was run again.

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