The influence of disease duration, clinical course, and immunosuppressive therapy on the synthesis of intrathecal oligoclonal IgG bands in multiple sclerosis

Journal of Neuroimmunology 264 (2013) 100–105

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The influence of disease duration, clinical course, and immunosuppressive therapy on the synthesis of intrathecal oligoclonal IgG bands in multiple sclerosis Markus Axelsson a,⁎, Niklas Mattsson b,c, Clas Malmeström a, Henrik Zetterberg b, Jan Lycke a a b c

Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, Mölndal, Sweden San Francisco VA Medical Center, Center for Imaging of Neurodegenerative Diseases (CIND), University of California San Francisco, San Francisco, CA, USA

a r t i c l e

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Article history: Received 27 February 2013 Received in revised form 4 September 2013 Accepted 9 September 2013 Keywords: Multiple sclerosis Intrathecal IgG Oligoclonal band (OCB) CXCL13 Mitoxantrone B-cell

a b s t r a c t We investigated the impact of disease duration, clinical course and immunosuppressive therapy on intrathecal IgG synthesis in multiple sclerosis (MS). Cerebrospinal fluid (CSF) was obtained twice, 8–10 years apart, from 20 MS patients and 26 healthy controls, and from 22 MS patients before and after two years of mitoxantrone treatment. The oligoclonal IgG band patterns changed in 15 patients at long-term follow-up, but were only influenced in 4 patients by mitoxantrone therapy. The CSF B-cell-regulating chemokine CXCL13 correlated with intrathecal IgG production suggesting a B-cell-dependence of intrathecal IgG synthesis in MS. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Increased intrathecal IgG production (Kabat et al., 1942) and the presence of oligoclonal IgG bands (OCBs) in cerebrospinal fluid (CSF) are characteristic findings of multiple sclerosis (MS) (Link, 1967) and are used for diagnostic purposes (Poser et al., 1983; Polman et al., 2011). Improvements in biochemical methods for detecting OCBs can increase the percentage of OCB-positive MS patients to N 95% (Olsson et al., 1984; Kostulas et al., 1987; Keir et al., 1990; Andersson et al., 1994). The rate of intrathecal IgG synthesis seems, to some extent, to mirror MS disease activity (Calabrese et al., 2011; Lourenco et al., 2013) whereas the OCB pattern is assumed to remain relatively stable throughout life (Correale and de los Milagros Bassani Molinas, 2002; Link and Huang, 2006). However, with few exceptions, the follow-up interval in most investigations has been limited to 2 years or less. OCBs in CSF are mainly produced by clonally expanded plasma cells and plasma blasts (Cepok et al., 2005; Winges et al., 2007), which strongly suggests an involvement of B-cell-dependent immune mechanisms in the pathogenesis of MS. However, the actual pathological relevance of this synthesis and any target antigens remains essentially unknown. The chemokine CXCL13 is a regulator of B-cells and has the

⁎ Corresponding author at: Department of Neurology, Sahlgrenska University Hospital, Blå stråket 7, 413 45 Gothenburg, Sweden. Tel.: +46 31 3426022; fax: +46 31277824. E-mail address: [email protected] (M. Axelsson). 0165-5728/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jneuroim.2013.09.003

potential to indirectly influence IgG synthesis. Elevated CXCL13 levels in CSF are related to increased disease activity and have also been associated with increased intrathecal IgG synthesis and the appearance of OCBs in MS (Sellebjerg et al., 2009; Khademi et al., 2010). Despite the presumed link to B-cell activity, treatment with potent immunomodulating or immunosuppressive treatments seems to have no or little impact on OCB synthesis, as shown by studies of hematopoietic stem cell transplantation (Mancardi et al., 2001; Fassas et al., 2002), B-cell depletion (Piccio et al., 2010) or by the use of fingolimod (Kowarik et al., 2011). However, recently the disappearance of OCBs was reported in 4 of 6 (von Glehn et al., 2011) and 12 of 73 (Harrer et al., 2012) natalizumab-treated MS patients. The aim of this study was to specifically investigate the impact of disease duration, disease course, and immunosuppressive therapy on intrathecal IgG synthesis and formation of OCBs in MS. We also examined the relationship between the B-cell-regulating chemokine CXCL13 and intrathecal IgG synthesis.

2. Materials and methods 2.1. Patients and healthy controls In total, the study included 42 patients, 16 with relapsing–remitting (RR), 22 with secondary progressive (SP) (Lublin and Reingold, 1996), and 4 with primary progressive (PP) MS according to the McDonald

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criteria (McDonald et al., 2001; Polman et al., 2011) and 26 healthy controls (HCs). The study was approved by the regional ethical board of the University of Gothenburg, Sweden. Informed consent was obtained from all participants. Patients were recruited from two study populations at the Department of Neurology, Sahlgrenska University Hospital, Gothenburg, Sweden. One of these populations has been described previously (Malmestrom et al., 2003; Axelsson et al., 2011). The long-term follow-up (LTF) group consisted of 20 patients who were assessed twice, 8–10 years apart (Table 1). They were recruited from a previously described population consisting of 43 patients (Malmestrom et al., 2003). Of the original cohort, three patients were lost to follow-up, eight chose not to participate, four had died and eight were excluded because of deficiency of sample material. The present study population consisted of nine patients with RRMS, seven with SPMS, and four with RRMS that had converted to SPMS during the longterm follow-up (denoted ‘transitory’). No patient had been treated with disease-modifying therapy (DMT) at the first examination, but four RRMS patients were on treatment with interferon-beta at the final follow-up. Five patients (four RRMS and one transitory) had transiently been on interferon-beta treatment between examinations, but none of these patients were on any DMT 6 months prior to the final examination. No relapse was recorded within 3 months prior to examinations. The mitoxantrone treated (MTX) group consisted of 22 patients: cases were 3 RRMS, 15 SPMS, and 4 PPMS (Table 1) assessed before and after 2 years of mitoxantrone treatment. Patients were consecutively recruited and eligible for mitoxantrone treatment (i.v. 12 mg/m2 at three-month intervals for 2 years; i.e., 8 infusions, total dose 96 mg/m2) according to established regimens (Krapf et al., 2005) and Swedish guidelines (www.mssallskapet.se). At study enrolment, 11 patients (2 RRMS, 6 SPMS, 3 PPMS) were untreated, and 11 (1 RRMS, 9 SPMS, 1 PPMS) were treated with DMT (10 interferon-beta, 1 glatiramer acetate). Patients previously treated with DMT switched to mitoxantrone treatment without a washout period. Healthy control subjects (HCs) were 26 blood donors or students who were assessed twice, 8–10 years apart (Table 1). They were recruited from a previous study population consisting of 50 people (Malmestrom et al., 2003) of the original cohort, 3 were lost to followup, 17 chose not to participate, and 4 were excluded because of deficiency of sample material. None of the controls had a history of neurological disease, and no abnormal signs were found at neurological examination.

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2.2. Clinical assessment and specimen sampling All participants underwent clinical neurological examination, peripheral blood sampling, and lumbar puncture twice. Disease duration was estimated from onset of first demyelinating symptoms. The neurological examinations were performed by trained neurologists, and disability was scored using the Expanded Disability Status Scale (EDSS) (Kurtzke, 1983). Disease progression and severity were measured using the Multiple Sclerosis Severity Score (MSSS) (Roxburgh et al., 2005). Following lumbar puncture, CSF was transported on ice and snap-frozen within 2 h. The lumbar punctures were at both occasions made according to similar procedures that are recommended in the consensus protocol of the BioMS-EU network for CSF biomarker research in MS (Teunissen et al., 2009). The first 12 mL of CSF was carefully mixed; after centrifugation, fractions were frozen in 0.5 mL aliquots at −80 °C until analysis. 2.3. Biochemical analysis of CSF All biochemical analyses were performed at the Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden. The experienced and certified laboratory technicians who performed them were blinded to all clinical data. 2.4. Determination of albumin ratio, IgG index, and OCB Albumin and IgG levels in serum and CSF were measured by immunonephelometry on a Beckman Image Immunochemistry system (Beckman Instruments, Beckman Coulter, Brea, CA, USA). The albumin ratio reflects the integrity of the blood–brain barrier (BBB) (Tibbling et al., 1977) and was calculated as (CSF albumin (mg/L) ⁄ serum albumin (g/L)). The IgG index is a measure of the intrathecal synthesis of IgG adjusted for the degree of disruption of the BBB. It was calculated as (CSF/serum IgG ratio) / (CSF/serum albumin ratio). Two serum and CSF samples from each individual were run in parallel and simultaneously. OCBs were visualized in CSF and serum by isoelectric focusing followed by silver staining and verified by immunoblotting using alkaline phosphatase-conjugated goat anti-human IgG (Sigma-Aldrich Sweden AB, Stockholm, Sweden), as described before (Blennow et al., 1993). Visual assessment of CSF-specific OCBs was done independently by two experienced researchers (NM, HZ). Patients were grouped

Table 1 Clinical characteristics and demographics of multiple sclerosis (MS) patients with long-term follow-up (LTF), mitoxantrone treatment (MTX), and healthy controls (HC). Group LTF

N

All MS RRMS SPMS Transitory

20 9 7 4

Group MTX

N

All MS RRMS SPMS PPMS

22 3 15 4

Group controls

N

Agea

Disease-durationa

Time between assessmentsa

EDSS baselineb

MSSS baselineb

EDSS follow-upb

MSSS follow-upb

15:5 9:0 3:4 3:1

41.5 (21–59) 33.7 (21–48) 46.7 (41–52) 50 (44–59)

10.5 (1–26) 9.6 (1–19) 12.6 (3–26) 9.0 (3.0–15.0)

8.9 (8.0–9.5) 8.8 (8.0–9.0) 8.8 (8.5–9.5) 8.8 (8.5–9.0)

3.0 (1.0–8.0) 3.0 (0–8.0) 7.5 (2.0–8.0) 3.25 (2.0–4.0)

4.24 (0.94–9.43) 2.34 (0.94–7.93) 8.21 (3.17–9.43) 4.47 (2.91–7.54)

3.5 (0–9.5) 3.5 (0–3.5) 8.0 (3.5–9.5) 5.5 (2.0–6.5)

3.70 (0.05–4.43) 1.99 (0.05–5.36) 8.84 (3.70–9.96) 5.03 (1.98–6.74)

Sex

Agea

Disease-durationa

Time between assessmentsa

EDSS baselineb

MSSS baselineb

EDSS follow-upb

MSSS follow-upb

11:11 3:0 7:8 1:3

45.2 (22–60) 42.6 (37–50) 46.1 (22–60) 43.5 (34–52)

10.0 (2–28) 4.0 (3–5) 10.4 (2–28) 13.0 (5–28)

1.9 (0.5–2.0) 1.5 (0.5–2.0) 2.0 (1–2) 2.0 (2.0)

7.54 (2.99–9.79) 9.08 (7.75–9.35) 7.33 (2.99–9.79) 8.77 (5.99–9.24)

6.0 (3.0–8.5) 6.5 (6.0–6.5) 5.5 (3.0–8.0) 6.5 (6.0–8.5)

7.25 (2.75–9.47) 9.08 (8.91–9.32) 6.73 (2.75–9.47) 8.27 (5.61–9.03)

Sex

Agea

Disease-duration

Time between assessmentsa

EDSS baselineb

MSSS baselineb

EDSS follow-upb

MSSS follow-upb

33.1 (18–60)

NA

8.6

NA

NA

NA

NA

Sex F:M

F:M 6.0 (3.0–8.0) 6.0 (4.5–6.5) 6.0 (3.0–8.0) 6.5 (6.0–7.5)

F:M HC

26

8:18

Long-term follow up, LTF; mitoxantrone treated, MTX; healthy controls, HC; EDSS, Expanded Disability Status Scale; MSSS, Multiple Sclerosis Severity Score; PPMS, primary progressive MS; RRMS, relapsing–remitting MS; SPMS, secondary progressive MS. a Data are mean (range), years. b Data are median (range).

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according to the number of IgG bands (0, 1, 2–4, 5–10, and N10). Two or more IgG bands were considered as OCBs (Link and Huang, 2006). 2.5. CXCL13 enzyme-linked immunosorbent assay CXCL13 was measured by ELISA (human CXCL13/BLC/BCA-1 immunoassay, R&D Systems Inc., Abingdon, UK), according to instructions from the manufacturer. Based on measurements of duplicates of the standard samples (concentrations 7.8–500 mg/L), the average intraassay coefficient of variance (CV) was ≤10%. The level of detection was 7.8 pg/mL, and CSF samples below that level were assigned a value of 3.9 pg/mL. 2.6. Statistical analyses Parametric tests were used for evaluating albumin ratio and IgG index. Student's (unpaired) t-test was used for comparing differences between groups, in-group differences over time were calculated with paired t-tests, and correlations between variables were calculated with Pearson's correlation test. Differences in values for CXCL13, EDSS, MSSS, and OCB number and group comparisons were made using the Mann–Whitney U test, and the Wilcoxon signed-rank test was used for evaluating in-group differences over time. Correlations between variables were calculated with Spearman's rank correlation test. For calculating the relationship between change in the OCB pattern and other parameters, patients were grouped based on their pattern of change and compared groupwise. 3. Results 3.1. Influence of disease duration and clinical course on albumin ratio, IgG index, and OCB formation All patients except for two in the LTF group (n = 18) had detectable CSF-specific OCBs (those two patients had only one IgG band). We found no significant differences between albumin ratios, IgG indices, and OCB number among the different clinical courses (Table 2). In most patients (n = 15), the OCB pattern changed between examinations. At the second examination, four had increased numbers of bands, six had decreased numbers of bands, three had both new

bands and lost bands, and two had unchanged OCB numbers but had a changed intensity (Fig. 1). The altered pattern was not related to disease duration or clinical course. The number of OCBs and IgG index correlated at both examinations (r = 0.60, p b 0.01, and r = 0.47, p b 0.01, respectively). Neurological disability (EDSS) or severity of disease (MSSS) did not correlate with OCB number and were not related to the change in band number. However, in RRMS patients (n = 9), EDSS correlated with both OCB number (r = 0.70, p b 0.05) and change in band number between examinations (r = 0.68, p b 0.05). These results were not associated with DMT. 3.2. The influence from mitoxantrone treatment on albumin ratio, IgG index, and formation of OCBs Nineteen patients in the MTX group (n = 22) had CSF-specific OCBs (Table 3). At the post-treatment examination, only four patients (all SPMS) had an altered OCB number or pattern. Two of these four had an increased OCB number, and two had both new and lost bands. We found no significant changes in albumin ratios or IgG indices (Table 2). No correlations were found between OCB counts, clinical course, EDSS, and MSSS. However, again, the number of bands correlated with IgG index at both examinations (r = 0.58, p b 0.01, and r = 0.45, p b 0.01, respectively). 3.3. The relationship between CXCL13 levels in CSF and disease duration, clinical course, albumin ratio, IgG index, and OCB formation CXCL13 levels were determined in CSF obtained at the final assessment of patients in the LTF group and in CSF obtained before and after 2 years of mitoxantrone treatment. In the LTF group, CXCL13 levels correlated with OCB number (r = 0.49, p b 0.05). This relationship was not statistically significant in pre- or post-treatment CSF obtained from mitoxantrone treated patients. CXCL13 levels correlated with IgG indices in both groups (r = 0.51, p b 0.05, and r = 0.61, p b 0.01, respectively). The pre-treatment level of CXCL13 in was significantly higher than in HCs (13.8 ± 1.22 SD vs. 4.6 ± 2.6 SD, p = 0.006), but after 2 years of mitoxantrone treatment, the mean CXCL13 level was significantly reduced (4.3 ± 1.9 SD, p = 0.008).

Table 2 Albumin ratio, IgG index, and CXCL13 levels with long-term follow-up (LTF), mitoxantrone treatment (MTX), and healthy controls (HC). Group LTF

N

All MS RRMS SPMS Transit

20 9 7 4

Group MTX

N

All MS RRMS SPMS PPMS

22 3 15 4

Group controls

N

HC

26

Albumin ratio

Albumin ratio

IgG index

Baseline

Follow-up

Baseline

6.0 5.3 6.6 6.7

± ± ± ±

2.1 1.8 2.1 2.8

6.7 5.2 8.0 7.9

± ± ± ±

2.7 1.5 2.7 3.4

0.75 0.74 0.70 0.87

± ± ± ±

0.35a 0.36a 0.19a 0.58a

IgG index Follow-up 0.88 0.89 0.57 1.38

± ± ± ±

0.81a 0.59a 0.91a 1.64a

CXCL13

CXCL13

Baseline

Follow-up

ND ND ND ND

6.1 5.1 5.1 10.1

± ± ± ±

6.1 3.6 3.3 12.4

Albumin ratio baseline

Albumin ratio follow-up

IgG index baseline

IgG index follow-up

CXCL13 baseline

CXCL13 follow-up

Baseline

Follow-up

Baseline

Follow-up

Baseline

Follow-up

5.9 6.2 5.9 11.4

1.1 1.35 1.08 0.90

6.7 7.5 6.3 7.6

± ± ± ±

3.0b 2.1 3.3 2.3

± ± ± ±

3.3 3.4 3.3 4.6

± ± ± ±

0.65a 1.11a 0.64a 0.65a

0.94 1.80 0.82 0.74

± ± ± ±

0.78a 1.94a 0.41a 0.20a

13.8 24.5 13.9 5.7

± ± ± ±

22.1a,c 30.9a,d 23.4b 3.6

4.3 3.9 4.5 3.9

± ± ± ±

1.9 0.0 2.3 0.0

Albumin ratio baseline

Albumin ratio follow-up

IgG index baseline

IgG index follow-up

CXCL13 baseline

CXCL13 follow-up

Baseline

Follow-up

Baseline

Follow-up

Baseline

Follow-up

4.5 ± 1.5

5.6 ± 3.8

0.45 ± 0.04c

0.48 ± 0.03

ND

4.6 ± 2.6

Long-term follow up, LTF; mitoxantrone treated, MTX; healthy controls, HC; PPMS, primary progressive MS; RRMS, relapsing–remitting MS; SPMS, secondary progressive MS; Transit, transitory, converted to SPMS during follow-up. All data are mean ± 1 standard deviation. a p b 0.01 vs. controls. b p b 0.05 vs. controls. c p b 0.01 vs. examination 2. d p b 0.05 vs. examination 2.

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4. Discussion

Fig. 1. Photographs of IgG oligoclonal bands detected by isoelectric focusing and silver staining. A: No change in OCBs; B: two new bands and one with increased intensity; C: fading of 3 OCBs; and D: increased intensity of 2 OCBs.

No correlations were found between CXCL13 levels and sex, age, disease duration, or albumin ratio. In CSF obtained from patients in the LTF group (at follow-up) and the MTX group (pre-treatment), a weak correlation was found between CXCL13 levels and EDSS (r = 0.32, p b 0.05), as well as for MSSS (r = 0.32, p b 0.05). Table 3 The distribution of cerebrospinal fluid (CSF) IgG bands in patients before long-term follow-up (LTF), mitoxantrone treatment (MTX), and in healthy controls (controls). Participants are grouped according to the number of CSF IgG bands.

Group LTF (n = 20) RRMS (n = 9) SPMS (n = 7) Transit (n = 4) Group MTX (n = 22) RRMS (n = 3) SPMS (n = 15) PPMS (n = 4) Controls (n = 26)

0

1

2–4

5–10

N10

0 0 0 0 3 0 1 2 23

2 1 1 0 0 0 0 0 0

5 3 1 1 1 0 1 0 2

6 0 4 2 12 2 8 2 1

7 5 1 1 6 1 5 0 0

Long-term follow up; LTF, Mitoxantrone treated; MTX, Healthy controls; HC, PPMS, primary progressive MS; RRMS, relapsing–remitting MS; SPMS, secondary progressive MS; Transit, transitory, converted to SPMS during follow-up.

In the present study, we showed that OCB patterns changed during long-term follow-up in MS but that this change was not related to disease course or influenced by immunosuppressive treatment. Our results contradict the widespread opinion that OCB patterns are stable over the course of MS and represent fingerprints of individual patients (Norrby, 1979; Olsson and Nilsson, 1979; Bloom, 1980; Keshgegian et al., 1980; Rolak et al., 1996; Correale and de los Milagros Bassani Molinas, 2002; Link and Huang, 2006). It is possible that this assumption to some extent is based on unpublished observations, or that changes in OCB patterns have been considered unreliable or were ignored due to methodological problems (Link and Huang, 2006). In fact, since the introduction of agarose gel electrophoresis over 50 years ago, investigations into the dynamics of OCB patterns in CSF over the course of MS have been scarce and contradictory (Olsson and Link, 1973; Delmotte and Gonsette, 1977; Vandvik, 1977; Mattson et al., 1980; Thompson et al., 1983; Rudick et al., 1999). With one exception (Rudick et al., 1999), study populations have been small, follow-up periods relatively short, and the applied methodologies for CSF OCB detection variable in sensitivity and reliability because of improvements in biochemical assays (Andersson et al., 1994; Link and Huang, 2006). Unchanged OCB patterns in sequential CSF have been reported in four cases of progressive MS (Vandvik, 1977), 22 of relapsing MS (Olsson and Link, 1973), 20 (unspecified) MS patients (Delmotte and Gonsette, 1977), and 137 RRMS patients who participated in a randomized placebo-controlled, double-blind phase III trial of interferon-beta1a (Rudick et al., 1999). All of these studies were limited to 2 years of follow-up, some used either less-sensitive methods (Olsson and Link, 1973; Delmotte and Gonsette, 1977; Vandvik, 1977) or unspecified standard methods (Rudick et al., 1999), and validity could be questioned due to the use of multiple clinical labs (Rudick et al., 1999). In contrast, we tried to optimize the conditions and observed patients up to 10 years, with samples obtained, stored, and analyzed in a standardized way similarly those recommended by the BioMS-EU network for CSF biomarker research in MS (Teunissen et al., 2009). The sequential serum and CSF samples of each patient were run simultaneously and in parallel. The CSF OCBs were detected by isoelectric focusing with silver staining (Blennow et al., 1990), allowing separation of OCBs with high precision (Link and Huang, 2006), and the patterns of OCBs were determined by two experienced researchers unaware of other clinical features. This procedure was complemented with immunoblot to ensure the IgG content of the OCBs (Freedman et al., 2005). The impact of storage of CSF in −80 °C on proteins over a longer period (8–10 years) of time is probably low since the OCB patterns changed in different directions and IgG indices of HCs at long-term follow-up were stable. Furthermore, in a previous study of ours, HCs had increasing CSF levels of glial fibrillary acidic protein (GFAP) during long-term follow-up (Axelsson et al., 2011) supporting its age dependence, and the absence of substantial protein degradation from −80 °C storage. Two previous investigations have demonstrated changed CSF OCB patterns over the course of MS (Mattson et al., 1981; Thompson et al., 1983). Based on results of isoelectric focusing of sequential CSF samples, 7 of 10 (Mattson et al., 1981) and 12 of 25 MS patients (Thompson et al., 1983) showed changed OCB patterns after 3–56 months and 4–17 months of follow-up, respectively. Of interest, different OCB patterns of eluted IgG have been found in different MS plaques within the same MS brain (Mattson et al., 1980). This finding is in line with our result, suggesting a dynamic shift of clonally expanded cells of the B-cell lineage over the course of MS. We found no impact of disease duration or clinical course on the rate of intrathecal IgG synthesis or OCB formation. Remarkably, the clonal synthesis of intrathecal IgG also seems to be unaffected even by very potent immunomodulating therapies such as hematopoietic stem cell transplantation (Mancardi et al., 2001; Fassas et al., 2002) or B-cell depletion by rituximab (Piccio et al., 2010). We showed that mitoxantrone, a cytotoxic

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drug that inhibits both B- and T-cells, did not change OCB patterns during 2 years of treatment. Only natalizumab treatment has been shown to cause disappearance of OCBs in MS (von Glehn et al., 2011; Harrer et al., 2012). Intrathecally synthesized IgG OCBs are a characteristic finding in MS but are also common in neurotropic virus infections and other autoimmune CNS diseases. However, only in MS does this polyspecific antibody response seem to persist, indicating that the CNS of patients with MS provides a B-cell-fostering environment. CXCL13 is a chemokine that can attract and maintain B-cells within the CSF/CNS compartment. We confirmed previous findings that the CSF level of CXCL13 was associated with the rate of intrathecal IgG synthesis (Khademi et al., 2010). Our result supports the concept of a B-cell dependence of intrathecal IgG synthesis. However, here, mitoxantrone treatment reduced IgG indices and CXCL13 levels whereas the OCB patterns remained essentially stable, which suggests a non-specific inhibition of the B-cell lineage. The low CXCL13 levels found in LTF patients were probably due to their low disease activity. Rearranged immunoglobulin genes with extensive mutations have been demonstrated in CSF B-cells (Cameron et al., 2009), as well as in the brains of MS patients (Ligocki et al., 2010), and such mutations might be antigen driven (Meinl et al., 2006). Thus, the exposure of new antigens following inflammation and degeneration might induce mutations in immunoglobulin genes, a process that could explain the changed OCB pattern we found in patients with MS at long-term follow-up. In addition the changed OCB pattern in the course of MS could be due to the recognition of a different set of antigens compared to the initial assessment. Conflict of interest statement Markus Axelsson has travel expenses reimbursed and lecture honoraria from Biogen Idec and Merck Serono. Niklas Mattsson has reported no disclosures. Clas Malmeström has received travel support and/or lecture honoraria from Biogen Idec, Merck Serono, and Novartis and unconditional research grants from Biogen Idec. Henrik Zetterberg has reported no disclosures. Jan Lycke has received travel support and/or lecture honoraria from Bayer Schering Pharma, Biogen Idec, Novartis, and Sanofi-Aventis; has served on scientific advisory boards for Almirall, Teva, Biogen Idec, and Genzyme/Sanofi-Aventis; serves on the editorial board of the Acta Neurologica Scandinavica; and has received unconditional research grants from Biogen Idec and Novartis. Acknowledgments This study was funded by grants from the Swedish Federal Government (LUA/ALF agreement), the Swedish Society of the Neurologically Disabled, the Research Foundation of the Multiple Sclerosis Society of Gothenburg, the Edit Jacobson Foundation, and Biogen Idec (unconditional grants). References Andersson, M., Alvarez-Cermeno, J., Bernardi, G., Cogato, I., Fredman, P., Frederiksen, J., Fredrikson, S., Gallo, P., Grimaldi, L.M., Gronning, M., et al., 1994. Cerebrospinal fluid in the diagnosis of multiple sclerosis: a consensus report. J. Neurol. Neurosurg. Psychiatry 57, 897–902. Axelsson, M., Malmestrom, C., Nilsson, S., Haghighi, S., Rosengren, L., Lycke, J., 2011. Glial fibrillary acidic protein: a potential biomarker for progression in multiple sclerosis. J. Neurol. 258, 882–888. Blennow, K., Wallin, A., Fredman, P., Gottfries, C.G., Karlsson, I., Svennerholm, L., 1990. Intrathecal synthesis of immunoglobulins in patients with Alzheimer's disease. Eur. Neuropsychopharmacol. 1, 79–81. Blennow, K., Fredman, P., Wallin, A., Gottfries, C.G., Skoog, I., Wikkelso, C., Svennerholm, L., 1993. Protein analysis in cerebrospinal fluid. III. Relation to blood–cerebrospinal fluid barrier function for formulas for quantitative determination of intrathecal IgG production. Eur. Neurol. 33, 134–142. Bloom, B.R., 1980. Immunological changes in multiple sclerosis. Nature 287, 275–276. Calabrese, M., Federle, L., Bernardi, V., Rinaldi, F., Favaretto, A., Varagnolo, M.C., Perini, P., Plebani, M., Gallo, P., 2011. The association of intrathecal immunoglobulin synthesis

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