Immunoadsorption Therapy in Patients With Multiple Sclerosis With Steroid-Refractory Optical Neuritis

Michael J Koziolek; Desiree Tampe; Matthias Bühr; Hassan Dihazi; Klaus Jung; Dirk Fitzner; Reinhard Klinge; Gerhard A Müller; Bernd Kitze

Disclosures

J Neuroinflammation. 2012;9(80) 

In This Article

Methods

Patients

We prospectively included 11 consecutive patients with MS who had functionally disabling acute optical neuritis. Patients fulfilled the indications for apheresis treatment due to this steroid-unresponsive MS relapse according to German guidelines (http://www.dgn.org).[7]

The study protocol had been approved by the local ethics committee prior to study initiation (no. 2/4/07) and registered at the local government (no. DE/CA25/00007080-00). All patients gave their written informed consent before enrolment.

Immunoadsorption Treatment

IA was performed using the tryptophan-linked polyvinyl alcohol adsorber TR-350, after membrane plasma separation with the polyethylene plasma separator OP-05 W (Asahi Kasei Kuraray, Tokyo, Japan) in combination with the Octo Nova extracorporeal circuit technology (SW 4.30.2, front 4.30.0) (Diamed Medizintechnik, Cologne, Germany). The adsorber, plasma separator and tubing system were for single use only. Combined anticoagulation, with citrate and unfractionated heparin, was used for all treatments. The treated plasma volume was 2,500 mL plasma for all treatments of all patients. In total, five sessions were performed in each patient on alternate days. In case of complications or decrease of fibrinogen below 100 mg/dL, treatment-free intervals were extended individually. Internal jugular veins were used for central vascular access with double lumen catheters in all patients.

Baseline and Follow-up Visits

All patients were followed up by a neurologist. Visits were performed at baseline (visit 0), after each IA treatment (visits 2 to 5) and after 30 ± 5 days (visit 6), 60 ± 10 days (visit 7) and 180 ± 10 days (visit 8). The neurological findings were assessed using the Expanded Disability Status Scale (EDSS) and the Incapacity Status Scale (ISS).[11–13] Changes of visual acuity were monitored using standardized near vision types after correction of refractive error at each visit and confirmed by an ophthalmologist before and after IA, as well as at 180 ± 10 days. Visual evoked potentials were determined at baseline, after the last IA and 60 ± 10 days post-intervention with the Neuropack M1 (Nihon Koden, Surbiton, UK).

Classification of Side Effects

Side effects were defined as any unexpected or symptomatic event that had a possible, probable or definite causal relationship with IA treatment.[14] They were classified as mild, moderate or severe as described previously[15] with small modifications. Briefly, mild side effects included those of transient nature with little or no clinical significance and without any temporary break of the procedure. Side effects that required medical intervention but were not life-threatening were classified as moderate. Unstable and life-threatening events requiring termination of the procedure were classified as severe.

Clinical Chemistry

All laboratory parameters were measured by standard methods. The complement components C3c and C4 as well as the immunoglobulins G (IgG), A (IgA) and M (IgM) were measured by nephelometry (Behring Nephelometer II Analyzer, Germany). Soluble interleukin-2 receptor (sIL-2R) was detected on an immulite system (Siemens, Germany).

Identification of Immunoadsorption Column-binding Proteins by Elution and Proteomics

Immusorba TR-350 column-binding proteins were eluted after the first IA treatment in five of eleven patients. Prior to elution, the column was washed with PBS buffer. The protein elution was carried out as following: PBS-washing step was followed by a three-step elution protocol using solution A (100 mM sodium acetate, 1 M NaCl, pH 5), solution B (20 mM Tris–HCl, 1 M NaCl, pH 8.5) and solution C (20% acetonitril in double diluted H2O). Proteins eluted from all three steps were pooled together and aliquots of 10 mL were used for protein precipitation, protein estimation and two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis, protein visualization and image analysis, in-gel digestion, mass spectrometry analysis of the digestion products and protein identification using a database search were performed as described in detail in our previous publication.[16] For protein identification, qualitative criteria encompassed optimized mass accuracy (<50 ppm), minimal mass deviation (in the millidalton range) and highest possible probability score, which were assigned to each identified protein. Proteins identified by mass spectrometry are described qualitatively.

Western Blot Analysis

Western blot analyses were performed according to previously published data[17] with 20 μg of plasma proteins. Equal loading was ascertained by Coomassie staining. For immunodetection of proteins, the following antibodies were used: rabbit polyclonal to human CD5 ligand (CD5L) and mouse monoclonal to human myelin basic protein (both Abcam, Cambridge, UK); and horseradish peroxidase-linked donkey anti-rabbit antibody (Amersham Biosciences, Freiburg, Germany). Results are expressed as mean ± SD.

Statistics

Visit and response effects were studied by two-way repeated measures analysis of variance using the mixed procedure for the software SAS (version 9.1, SAS Institute). In this global analysis, P < 0.05 indicated a significant effect. In the case of a significant effect at a particular visit, subsequent pairwise comparisons to baseline values were performed using one-way repeated measures analysis of variance to detect the point in time when the effect occurred. These comparisons were performed at Bonferroni-adjusted significance levels (related P-values are labeled with either Bonf. sig. (significant) or with Bonf. n.s. (non-significant)).

Smoothing splines with three degrees of freedom were fitted to mean values of each visit to illustrate the trends and the potential interactions between visits and response effects. Fitting was performed using the free software R (version 2.8, http://www.r-project.org).

Comparative statistical analyses of changes within-subject and within-treatment were performed using t-tests for paired samples in case of normal data and Mann–Whitney U-tests for non-normal data. Normality was checked by quantile-quantile plots. Again, test results with P < 0.05 were considered significant.

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