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The improvement of cognitive functions is associated with a decrease of plasma Osteopontin levels in Natalizumab treated relapsing multiple sclerosis
Brain, Behavior, and Immunity 35 (2014) 176–181
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The improvement of cognitive functions is associated with a decrease of plasma Osteopontin levels in Natalizumab treated relapsing multiple sclerosis Pietro Iaffaldano, Maddalena Ruggieri, Rosa Gemma Viterbo, Mariangela Mastrapasqua, Maria Trojano ⇑ Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari Aldo Moro, Bari, Italy
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Article history: Received 29 April 2013 Received in revised form 21 August 2013 Accepted 22 August 2013 Available online 30 August 2013 Keywords: Multiple sclerosis Neuropsychology Cognition Psychoneuroimmunology
a b s t r a c t Objective: To investigate the effect of two-years Natalizumab treatment on plasma Osteopontin levels, cognitive performances and fatigue in relapsing multiple sclerosis (RRMS) patients. Methods: Forty-nine RRMS patients scheduled for Natalizumab treatment as second-line therapy were enrolled. Plasma samples of twenty-four treatment-naïve RRMS and 22 healthy controls (HCs) were used as controls of baseline Osteopontin levels. Plasma Osteopontin levels, using an enzyme-linked immunosorbent assay, cognitive functions using the brief repeatable battery, and fatigue, by the fatigue severity scale (FSS), were assessed at baseline and every 12 months. A global cognitive impairment index (CII) was calculated for each patient. Results: Patients scheduled for Natalizumab treatment had higher baseline Osteopontin levels (mean [SD] 65.42 [22.20] ng/ml) (p = 0.013) than HCs (53.20 [12.68] ng/ml), but not different from those in the treatment-naïve RRMS group (67.70 [24.23] ng/ml); 30.6% of patients showed a cognitive impairment (failure P3 tests) and 47.6% complained fatigue interfering with daily activities(FSS score P4.5). A significant decrease of mean Osteopontin levels (p < 0.005), of mean CII values (p < 0.005) and of mean FSS score (p < 0.05) was found during the treatment. Baseline Osteopontin levels significantly correlated (p = 0.002) with baseline CII values, and the reduction of the CII values during Natalizumab treatment significantly correlated with the decrease of the Osteopontin levels (p < 0.05). No correlations were found between Osteopontin levels and FSS score before and during Natalizumab treatment. Conclusions: Natalizumab treatment reduces plasma Osteopontin levels and improves cognition and fatigue in RRMS patients. The results suggest that the improvement of cognitive functions is associated to a decrease of plasma Osteopontin levels. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Multiple sclerosis (MS) is a chronic inflammatory, demyelinating disease of the central nervous system (CNS). Osteopontin (OPN), a molecule expressed in most tissues, exerts pro-inflammatory properties and has been studied as a potential key player in the pathogenesis of MS (Steinman, 2009). OPN binds to the a4b1 integrin (VLA-4), the main adhesion molecule involved in MS relapses (Steinman, 2009). This interaction stimulates the production of pro-inflammatory cytokines and promotes the survival of autoreactive T cells (Steinman, 2009). OPN transcripts are increased in the CNS of the experimental autoimmune encephalomyelitis (EAE) affected mice (Chabas et al., 2001). OPN / mice ⇑ Corresponding author. Address: Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari Aldo Moro, Piazza Giulio Cesare 11, 70124 Bari, Italy. Tel./fax: +39 080 5478555. E-mail address: [email protected] (M. Trojano). 0889-1591/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbi.2013.08.009
and anti-OPN antibody-treated mice have a milder clinical course of EAE (Chabas et al., 2001). Moreover, OPN appears to be necessary for the induction of recurrent disease activity and a progressive disease course in EAE (Hur et al., 2007; Murugaiyan et al., 2008). In human MS plaques OPN transcripts are highly expressed (Chabas et al., 2001). Plasma OPN levels have been found to be increased in relapsing-remitting (RR) MS in comparison to healthy controls (HCs), especially during relapses (Vogt et al., 2008, 2010; Comabella et al., 2005). Increased cerebrospinal fluid (CSF) OPN levels in RRMS and in primary progressive (PP) MS patients have been also reported by some groups (Braitch et al., 2008; Börnsen et al., 2011). However, until now no correlations have been found between CSF or plasma OPN levels and measures of neurological impairment in MS patients. Only one study reported a significant correlation between CSF OPN levels and expanded disability status scale (EDSS) score in PPMS patients (Börnsen et al., 2011).
P. Iaffaldano et al. / Brain, Behavior, and Immunity 35 (2014) 176–181
Increased plasma OPN levels have been also reported in HIV-infected subjects in comparison to HCs (Burdo et al., 2008). Among HIV-infected patients those with HIV associated dementia had higher plasma OPN levels than those who were cognitively preserved (Burdo et al., 2008). Natalizumab (NTZ) is a monoclonal antibody directed against VLA-4. NTZ has been proven to be efficacious in reducing annualized relapse rate (ARR) and MRI activity (Polman et al., 2006; Rudick et al., 2006) and to have also some beneficial effects on cognitive functions (Iaffaldano et al., 2012) in RRMS patients. Recently, a significant decrease of OPN CSF concentrations in RRMS patients has been reported during NTZ treatment (Khademi et al., 2009). The main objectives of this study were to longitudinally evaluate the effect of 2 year NTZ treatment on plasma OPN levels, cognitive performances and self-reported fatigue scores in RRMS patients, and to assess the relationships between OPN levels and cognition and fatigue measures before and during the NTZ treatment.
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level of fatigue interfering with daily activities, at baseline and every 12 months. If a relapse occurred at the time of scheduled neuropsychological assessments, cognitive testing was delayed until 30 days after the last steroid administration. Patients with a visual function impairment interfering with the performances in the cognitive tests were not included in the study. All the patients who reached 1 and 2 years of NTZ treatment entered into the analyses. Plasma samples from 24 treatment naïve RRMS patients and from 22 age- and sex-matched healthy volunteers (HCs) were also collected, and used as controls for a cross-sectional evaluation of baseline plasma OPN levels. The treatment-naïve RRMS patients group consisted in regularly followed-up, not previously-treated with immunomodulatory drugs, clinically stable RRMS patients. None of these patients had received steroid treatment within 3 months when sampling was performed. The local Ethics Committee of the University of Bari approved the study, and written informed consent was obtained from all patients prior to entering the study.
2. Material and methods 2.1. Osteopontin assay Forty-nine RRMS patients, scheduled to start treatment with 300 mg NTZ intravenously once monthly and previously treated with immunomodulants, were consecutively included. All patients were longitudinally followed-up during NTZ treatment. The relapses occurred in the last year pre-NTZ treatment and every new relapse occurring during the NTZ treatment were recorded. The annualized relapse rate (ARR) in the last year and at the end of the first and second year of NTZ treatment were calculated for each patient. The number of gadolinium (Gd) enhancing lesions at the magnetic resonance imaging (MRI) performed just before the start of the NTZ treatment, and every 6 months during the NTZ treatment, were collected. A complete neurological examination including the EDSS score assessment was performed at baseline and every 3 months throughout the treatment. Cognitive functioning, using the Rao’s brief repeatable battery (BRB) (Rao et al., 1991; Amato et al., 2006) and the Stroop test (ST), was assessed before the first infusion and every 12 months by a trained psychologist. BRB included tests of verbal memory acquisition and delayed recall (selective reminding test – SRT), visual memory acquisition and delayed recall (10/36 spatial recall test – SPART), attention, concentration and speed of information processing (PASAT 3; PASAT 2; symbol digit modalities test – SDMT), and verbal fluency on semantic stimulus (word list generation – WLG). Frontal lobe executive functions were assessed by the Stroop color-word task (ST). Versions A and B of the BRB were used alternatively at each examination. Cognitive impairment (CI) was defined as the failure in at least 3 tests on BRB and ST using a 5th percentile cut-off for each test, which corresponded to a z-score 2 standard deviation (SD) below the mean Italian normative values (Amato et al., 2006). A global score, defined cognitive impairment index (CII), allowing the evaluation of changes in cognitive performances independently by the number of cognitive tests failed at the BRB and the ST, was obtained using the mean and SD from the normative sample of Rao’s battery and the ST (Amato et al., 2006; Camp et al., 1999; Iaffaldano et al., 2012; Patti et al., 2010; Viterbo et al., 2013). The CII was obtained applying a grading system to each cognitive tests of the BRB and the ST. For each patient, the grading system was applied to individual cognitive tests, based on the number of SDs below the control mean (i.e., grade 0 was given if the patient scored at or above the control mean, 1 if he/she scored below the control mean, but at or above 1 SD below the control mean, and so on until all patient scores were accommodated). Finally, all the patient’s scores were summed to give one overall measure of cognitive function. Fatigue was assessed by the fatigue severity scale (FSS) (Krupp et al., 1989), using a cut-off value of 4.5 to identify patients with a
Plasma samples were obtained by venous puncture, at baseline and every 6 months during the NTZ treatment. Plasma was isolated by centrifugation and stored at 80 °C until use.OPN concentrations in plasma samples were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Quantikine ELISA kit, R&D Systems Europe, Abingdon, UK) according to the manufacturer’s instructions. The lower limit of detection was 0.313 ng/ml. For the analysis we diluted plasma samples 1:25 in assay diluent. Baseline and follow-up plasma samples from a patient were analyzed on the same plate. Inter-assay precision (precision between assays): four samples of known concentration were tested in all separate assays to assess inter-assay precision. Moreover, we had coefficient of variation (CV) values less than 10% across the standard curve for both intra- and inter-assay precision. These low CV values allowed us to perform repeated assays and be confident that the results are consistent throughout the study. The samples were stored, coded, and made anonymous in accordance with the MRC (Medical Research Council) guidelines on the ethical use of biological specimen collections in clinical research.
2.2. Statistical analyses Descriptive analyses were performed at the baseline. No imputation of missing data was considered. Differences in OPN levels between patients and HCs were analyzed by the Mann–Whitney test. The association of OPN levels and cognitive measures with other parameters was evaluated by Spearman correlation analysis. To test differences across different time points in related samples non parametric tests were used. The Wilcoxon signed – rank test for paired samples and the Friedman two-way test for repeated measures with post hoc correction were used to compare mean values in paired examinations or when three or more evaluations were available for continuous variables. Cochran Q test for repeated measures and McNemar test for pairwise comparisons were used to assess changes over time in categorical variables. A value of p < 0.05 was considered significant for the Wilcoxon signed – rank test, the Friedman two-way test and the Cochran Q test. For post hoc multiple comparisons, we elected a pre-assigned p/number of comparisons (Bonferroni’s correction) as the threshold for significance, and therefore, a p < 0.017 (0.05/3) and a p < 0.005 (0.05/10) were used as a definition of statistical significance for n = 3 and for n = 10 comparisons, respectively. Analyses were performed by SPSS 17.0.
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3. Results In Table 1 a comparison of the demographic and clinical characteristics between the two RRMS groups and HCs is shown. There was no difference in sex distribution and age in RRMS patients and HCs. The RRMS patients scheduled to NTZ treatment had a longer disease duration (10.54 ± 5.44 vs 5.58 years ± 5.78; p < 0.0001), were more disabled (median [range] EDSS score 3.5 [2.0–7.0] vs 2.0 [1.0 – 4.5]; p < 0.0001) and had a more active course (mean ARR 1.78 ± 0.90 vs 1.21 ± 0.78; p = 0.018) in comparison to treatment-naïve RRMS patients. All RRMS patients scheduled to NTZ treatment received previous first line immunomodulant treatments (Interferon-beta [n = 39] and Glatiramer acetate [n = 10]) with a median exposure time of 60.76 months (Range: 3.87– 170.0). The median wash out time from the last treatment to the start of NTZ treatment was 2.54 months (Range: 0.90–20.97). After 1 year-NTZ treatment, the baseline mean ARR (1.73 ± 0.94) and mean EDSS score (3.69 ± 1.04) significantly decreased (ARR: 0.34 ± 0.66, p < 0.0001; EDSS: 3.52 ± 1.17, p = 0.021). In the group of patients receiving NTZ for two years (n = 21), the mean ARR also significantly decreased during the treatment (Friedman test: p < 0.0001). A Wilcoxon signed-rank test with a Bonferroni correction for multiple tests showed that the ARR significantly improved after 1 (0.87 ± 0.60; p < 0.0001) and 2 years (0.10 ± 0.32; p < 0.0001) in comparison to baseline value (1.76 ± 0.99). There were no significant differences between year 1 and 2 of treatment (p = N.S.). No significant reduction of the baseline EDSS score (3.82 ± 0.97) was observed during the follow-up (Year 1: 3.60 ± 1.03; Year 2: 3.63 ± 1.33; p = N.S.). The number of Gd enhancing lesions significantly decrease during the NTZ treatment (Baseline vs year 1 (n = 42): 1 (0–8) vs 0 (0– 1); p < 0.0001; Baseline vs year 2 (n = 21): 1 (0–8) vs 0; p = 0.003). The percentage of CI patients decreased from 30.6% (13/42 patients) at baseline to 26.2% (11/42) at the end of the first year NTZ treatment. Accordingly, the mean CII values were found to be significantly reduced at year 1 in comparison to baseline values (13.52 ± 6.50 vs 11.67 ± 7.46; p = 0.004) (Fig. 1). In patients receiving NTZ for two years, the percentage of CI decreased from 23.8% (5/21 patients) to 19% (4/21 patients) at 1 year and to 9.5% (2/21 patients) at 2 years, and the mean CII significantly decreased (Friedman test: p < 0.0001) during the NTZ treatment. A Wilcoxon signed-rank test with a Bonferroni correction for multiple tests showed that the mean CII value significantly improved after 1 (10.10 ± 6.47; p = 0.003) and 2 years (7.86 ± 6.59; p < 0.0001) in
Fig. 1. Changes of cognitive performances in MS patients during NTZ treatment. The changes of the cognitive impairment index (CII) after 1 and 2 years of NTZ treatment was evaluate in 42 and 21 patients, respectively.
comparison to the baseline value (13.33 ± 6.44). The CII further decreased in the second year of treatment in comparison to the year 1 value (p = 0.007) (Fig. 1). At baseline the mean FSS score was 3.99 (±1.48), and 20/42 patients (47.6%) reported levels of fatigue which interfered with daily activities (FSS score P4.5). After 1 year of NTZ treatment, the mean FSS score significantly decreased to 3.54 (±1.43) (p = 0.025) and the proportion of patients with FSS score P4.5 decreased to 26.2% (11/42, p = 0.049). In patients with a 2 yearfollow-up, the mean FSS score significantly decreased during the treatment (Friedman test, p = 0.002). A Wilcoxon signed-rank test with a Bonferroni correction for multiple tests showed that the mean FSS score significantly improved at 2 years (3.18 ± 1.78; p = 0.002) in comparison to baseline values (4.31 ± 1.47). There were no significant differences between baseline and year 1 (3.52 ± 1.47), and between year 1 and 2 of treatment (p = N.S.). In this subgroup the proportion of patients with FSS score P4.5 significantly (Cochran Q test, p = 0.021) decreased during the 2 years NTZ treatment from 57.1% (12/21 patients) to 28.6% (6/21 patients) at year 1 and to 33.3% (7/21 patients) at year 2. A McNemar test with a Bonferroni correction for multiple tests showed no significant differences of the proportion of patients with FSS score P4.5 between the different timepoint. Plasma OPN levels of RRMS patients scheduled for NTZ treatment (65.42 ng/ml ± 22.20) did not differ from those of the
Table 1 Demographic and clinical characteristics at baseline.
Sex (F/M) Age (years) Disease duration (years) EDSS ARR Time from the last relapse (months) Last DMDs IFN-b GA Washout (months) No. of Gd enhancing lesions at the MRI performed just before the start of NTZ treatment
RRMS patients scheduled for NTZ treatment (n = 49)
RRMS patients treatment naïve (n = 24)
HCs (n = 22)
p Value
37/12 34.23 (10.12) 10.54 (5.44) 3.5 (2.0–7.0) 1.78 (0.90) 6.13 (5.91)
17/7 35.8 (10.83) 5.58 (5.78) 2.0 (1.0–4.5) 1.21 (0.78) 13.79 (22.79)
12/10 39.18 (10.12)
N.S.* N.S.** <0.0001 <0.0001 0.018
39 (79.6%) 10 (20.4%) 3.57 (4.08) 1 (0–8)
Data are reported as mean (SD) with the exception of: EDSS and No. of Gd enhancing lesions (reported as median (range)) and sex and last DMDs reported as count. Abbreviations: NTZ, Natalizumab; HCs, healthy controls; EDSS, expanded disability status scale; ARR, annualized relapse rate; DMDs, disease modifying drugs; IFN-b, interferon – beta; GA, Glatiramer acetate; Gd, gadolinium. * No significant differences in sex distribution (Pearson v2 test) were found between the 2 patients groups (p = 0.6) and between the patients and HCs (p = 0.08). ** No significant differences of age at time of the blood sampling (Mann–Whitney U test) were found between the 2 patients groups (p = 0.5) and between the patients and HCs (p = 0.06).
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Fig. 2. Baseline plasma OPN levels in MS patients and HCs. Y-Axis represents plasma Osteopontin levels expressed in ng/ml. Plasma OPN levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. Abbreviations: OPN, Osteopontin; RRMS, relapsing-remitting multiple sclerosis; tn, treatment naïve; HCs, healthy controls.
treatment-naïve RRMS group (67.70 ng/ml ± 24.23), but they were significantly higher (p = 0 0.013) than in HCs (53.20 ng/ml ± 12.68). Plasma OPN levels were also significantly (p = 0.039) higher in treatment-naïve RRMS patients in comparison to HCs (Fig. 2). After1 year-NTZ treatment baseline OPN levels significantly (p = 0.003) decreased from 66.98 ng/ml (±22.38) to 55.23 ng/ml (±19.88). No significant differences were found between baseline and 6 months OPN levels (60.79 ng/ml ± 20.23) and between 6 months and year 1 OPN levels (Fig. 3A). In patients receiving NTZ for two years (n = 21), OPN levels also significantly decreased during the treatment (Friedman test: p < 0.0001).A Wilcoxon signed-rank test with a Bonferroni correction for multiple tests showed that the OPN levels significantly decreased after 18 months (32.81 ng/ml ± 22.82; p < 0.0001) and 2 years (45.96 ng/ml ± 17.01; p = 0.001) in comparison to baseline value (69.52 ng/ml ± 26.07). There were also a significant difference between year 1 (59.00 ng/ml ± 19.65; p = 0.004) and 2 of treatment (Fig. 3B).
No correlations were found between baseline OPN levels and age, EDSS score, ARR, FSS score, previous DMD treatment duration and washout time from previous DMDs. A significant positive correlation was found (r = 0.43, p = 0.002) between the baseline CII and the baseline OPN levels (Fig. 4). A significant correlation was found between baseline SRT-long term storage (LTS) (r = 0.378, p = 0.007), SRT-consistent long term retrieval (CTLR) (r = 0.404, p = 0.004), SRT-delayed (D) (r = 0.472, p = 0.001), SDMT (r = 0.307, p = 0.032), PASAT3 (r = 0.298, p = 0.037), ST scores (r = 0.318, p = 0.026) and baseline OPN levels. After 1 and 2 years of NTZ treatment the reduction of the CII was found to be significantly correlated to the reduction of the OPN levels (r = 0.305, p = 0.05 and r = 0.667, p = 0.001, respectively) (Fig. 5A and B). 4. Discussion The results of this study confirm the efficacy of NTZ treatment in reducing disease activity and disability progression in RRMS
Fig. 3. Changes of plasma OPN at 1 (A) and 2 (B) year-NTZ-treatment. Y-Axis represents plasma Osteopontin levels expressed in ng/ml for both the graphs. Plasma OPN levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. (A) Shows the significant reduction of the plasma OPN levels during the first year of NTZ treatment (Study population: baseline n = 49; 6 months n = 43; 1 year n = 42). (B) Shows the significant reduction of the plasma OPN levels during the 2 years of NTZ treatment (Study population: baseline n = 49; 6 months n = 43; 1 year n = 42; 18 months n = 24; 2 years n = 21).
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Fig. 4. Relationships between plasma OPN levels and CII at baseline. The figure shows the significant correlation between the baseline CII and the baseline OPN levels (r = 0.43, p = 0.002). Plasma OPN levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. Abbreviation: CII, cognitive impairment index.
patients, as already demonstrated in NTZ pivotal trials (Polman et al., 2006; Rudick et al., 2006), and in improving cognitive functions and self-reported fatigue as suggested in previous observational studies (Mattioli et al., 2011), even on larger MS populations (Iaffaldano et al., 2012). Moreover, according to other groups (Comabella et al., 2005; Vogt et al., 2008) our findings show that plasma OPN levels in RRMS patients are higher than in HCs, and that the immunomodulant therapy do not have any significant effect on these levels. In fact plasma OPN levels of RRMS patients scheduled for NTZ treatment and with a previous treatment with immunomodulants did not differ from those of the treatment-naïve RRMS group. However, we demonstrate that, unlike immunomodulators, NTZ treatment significantly decreases plasma OPN levels and that this decrease is already evident after 1 year NTZ treatment and it is even more prominent in the second year of treatment. NTZ treatment in RRMS have been already found to be associated with a decrease of inflammatory and neurodegenerative markers. Mellergard et al. (2010) reported a global decline of pro-inflammatory
cytokines and chemokines both in the CSF and in the plasma of 31 RRMS patients during 1 year of NTZ treatment. NTZ therapy induced a 3-fold reduction of the CSF neurofilament light levels, a marker of axonal injury, in 92 patients with relapsing forms of MS in a recent Swedish study (Gunnarsson et al., 2010). Finally, Khademi et al. (2009) found that 1 year NTZ treatment reduced CSF OPN and matrix metalloproteinase (MMP)-9 levels in 22 RRMS patients. In our study plasma OPN levels do not show any correlation with age, EDSS score, ARR, FSS score, previous DMD treatment duration and washout time from previous DMDs. This is in line with most of the previous studies (Comabella et al., 2005; Vogt et al., 2008) reporting any significant correlation among plasma and CSF OPN levels and measures of neurological impairment in MS patients. Only one study reported a correlation between CSF OPN levels and the EDSS score in PPMS patients (Börnsen et al., 2011). In our study population the reduction of the inflammatory activity, as revealed by the decrease of the number of Gd enhancing lesions and the reduction of the ARR, goes in parallel with the reduction of plasma levels of a pro-inflammatory cytokine like OPN. It is noteworthy that in the current study we show an association between plasma OPN levels and cognitive deficits in RRMS patients. In particular we find a significant correlation between baseline plasma OPN levels and performances in tests which explore the cognitive domains of verbal memory, attention, information processing speed and executive functions. In addition we demonstrate a significant correlation between the improvement of the cognitive functions and the reduction of the plasma OPN levels during the NTZ treatment. These findings are in accordance with other reports (Burdo et al., 2008; Brown et al., 2011) showing an association between plasma OPN levels and cognitive functions in other neurological diseases. Burdo and colleagues demonstrated that HIV-infected subjects have increased plasma OPN levels in comparison to HCs (Burdo et al., 2008) and that, among HIV-positive patients, those with minor cognitive/motor disorder and those with HIV-associated dementia have higher plasma OPN levels than the cognitive preserved HIV-infected patients (Burdo et al., 2008). More recently OPN has been found to be increased also in CSF of HIV infected patients with dementia (Brown et al., 2011).
Fig. 5. Correlation between the changes (D) of OPN levels and CII at 1 (A) and 2 (B) year-NTZ-treatment. (A) Shows the relationship between the reduction of the CII and the reduction of the OPN levels after 1 year of NTZ treatment (r = 0.305, p = 0.05). (B) Shows the same relationship after 2 years of of NTZ treatment (r = 0.667, p = 0.001).
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OPN is a pro-inflammatory cytokine, which is secreted by activated macrophages, leukocytes and T lymphocytes. It is present in extracellular fluids and is up-regulated at sites of inflammation and it has been shown to be involved in inflammation and autoimmune disorders, including MS (Brown, 2012). In addition, it has been demonstrated that classical mediators of acute inflammation such as TNF-a and IL-1 h strongly induce OPN expression in different cell types (Denhardt and Guo, 1993; Yu et al., 1999). OPN, protecting monocytes from apoptosis and decreasing the number of macrophages returning to the circulation from the brain (Burdo et al., 2007), amplifies the brain inflammatory damage which is considered the pathological substrate of HIV-associated dementia (Glass et al., 1995) and is one of the main mechanisms that may underlie the cognitive deficits in MS (Heesen et al., 2010). In EAE affected mice, it was recently showed that the activated microglia and the inflammatory cytokines released from infiltrating lymphocytes are able to alter the synaptic transmission (Centonze et al., 2009; Mandolesi et al., 2010). This induced synaptopathy is related to cognitive dysfunction in this experimental model of MS (Centonze et al., 2009; Mandolesi et al., 2010).The significant reduction of plasma OPN, we found in this study, together with the already demonstrated reduction of CSF OPN (Khademi et al., 2009), CSF and plasma levels of pro-inflammatory cytokines and CSF levels of light-chain neurofilament during the NTZ treatment (Gunnarsson et al., 2010; Khademi et al., 2009; Mellergard et al., 2010) in RRMS, may be responsible of the concurrent improvement of the cognitive dysfunctions observed in these patients. In conclusion our results suggest that the beneficial effect of NTZ on cognitive performances may be partially related to a reduction of plasma OPN levels and that OPN may act as a bridge protein between inflammation and neurodegeneration in RRMS. Plasma OPN might represent a suitable candidate biomarker for the evaluation of the effect of a treatment on cognition in future clinical trials in RRMS.
Competing interest Maria Trojano received honoraria for consultancy or speaking from Biogen, Sanofi-Aventis, Merck Serono and Bayer-Schering and research grants from Merck Serono, Biogen and Novartis; Rosa Gemma Viterbo serves on scientific advisory boards for BiogenIdec and received honoraria for speaking from Novartis and Biogen; Pietro Iaffaldano, Maddalena Ruggieri and Mariangela Mastrapasqua declare no conflict of interest.
Funding This work was supported by annual research grants from the Italian University and Research Ministry (MIUR) (COFIN 20092011 M.T.) and Strategic Project Apulian Region Neurobiotech PS 124 (M.T.). References Amato, M.P., Portaccio, E., Goretti, B., Zipoli, V., Ricchiuti, L., et al., 2006. The Rao’s brief repeatable battery and Stroop test: normative values with age, education and gender corrections in an Italian population. MultScler 12, 787–793. Börnsen, L., Khademi, M., Olsson, T., Sørensen, P.S., Sellebjerg, F., 2011. Osteopontin concentrations are increased in cerebrospinal fluid during attacks of multiple sclerosis. MultScler 17 (1), 32–42. Braitch, M., Nunan, R., Niepel, G., Edwards, L.J., Constantinescu, C.S., 2008. Increased Osteopontin levels in the cerebrospinal fluid of patients with multiple sclerosis. Arch. Neurol. 65 (5), 633–635.
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Brain, Behavior, and Immunity journal homepage: www.elsevier.com/locate/ybrbi
The improvement of cognitive functions is associated with a decrease of plasma Osteopontin levels in Natalizumab treated relapsing multiple sclerosis Pietro Iaffaldano, Maddalena Ruggieri, Rosa Gemma Viterbo, Mariangela Mastrapasqua, Maria Trojano ⇑ Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari Aldo Moro, Bari, Italy
a r t i c l e
i n f o
Article history: Received 29 April 2013 Received in revised form 21 August 2013 Accepted 22 August 2013 Available online 30 August 2013 Keywords: Multiple sclerosis Neuropsychology Cognition Psychoneuroimmunology
a b s t r a c t Objective: To investigate the effect of two-years Natalizumab treatment on plasma Osteopontin levels, cognitive performances and fatigue in relapsing multiple sclerosis (RRMS) patients. Methods: Forty-nine RRMS patients scheduled for Natalizumab treatment as second-line therapy were enrolled. Plasma samples of twenty-four treatment-naïve RRMS and 22 healthy controls (HCs) were used as controls of baseline Osteopontin levels. Plasma Osteopontin levels, using an enzyme-linked immunosorbent assay, cognitive functions using the brief repeatable battery, and fatigue, by the fatigue severity scale (FSS), were assessed at baseline and every 12 months. A global cognitive impairment index (CII) was calculated for each patient. Results: Patients scheduled for Natalizumab treatment had higher baseline Osteopontin levels (mean [SD] 65.42 [22.20] ng/ml) (p = 0.013) than HCs (53.20 [12.68] ng/ml), but not different from those in the treatment-naïve RRMS group (67.70 [24.23] ng/ml); 30.6% of patients showed a cognitive impairment (failure P3 tests) and 47.6% complained fatigue interfering with daily activities(FSS score P4.5). A significant decrease of mean Osteopontin levels (p < 0.005), of mean CII values (p < 0.005) and of mean FSS score (p < 0.05) was found during the treatment. Baseline Osteopontin levels significantly correlated (p = 0.002) with baseline CII values, and the reduction of the CII values during Natalizumab treatment significantly correlated with the decrease of the Osteopontin levels (p < 0.05). No correlations were found between Osteopontin levels and FSS score before and during Natalizumab treatment. Conclusions: Natalizumab treatment reduces plasma Osteopontin levels and improves cognition and fatigue in RRMS patients. The results suggest that the improvement of cognitive functions is associated to a decrease of plasma Osteopontin levels. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Multiple sclerosis (MS) is a chronic inflammatory, demyelinating disease of the central nervous system (CNS). Osteopontin (OPN), a molecule expressed in most tissues, exerts pro-inflammatory properties and has been studied as a potential key player in the pathogenesis of MS (Steinman, 2009). OPN binds to the a4b1 integrin (VLA-4), the main adhesion molecule involved in MS relapses (Steinman, 2009). This interaction stimulates the production of pro-inflammatory cytokines and promotes the survival of autoreactive T cells (Steinman, 2009). OPN transcripts are increased in the CNS of the experimental autoimmune encephalomyelitis (EAE) affected mice (Chabas et al., 2001). OPN / mice ⇑ Corresponding author. Address: Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari Aldo Moro, Piazza Giulio Cesare 11, 70124 Bari, Italy. Tel./fax: +39 080 5478555. E-mail address: [email protected] (M. Trojano). 0889-1591/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbi.2013.08.009
and anti-OPN antibody-treated mice have a milder clinical course of EAE (Chabas et al., 2001). Moreover, OPN appears to be necessary for the induction of recurrent disease activity and a progressive disease course in EAE (Hur et al., 2007; Murugaiyan et al., 2008). In human MS plaques OPN transcripts are highly expressed (Chabas et al., 2001). Plasma OPN levels have been found to be increased in relapsing-remitting (RR) MS in comparison to healthy controls (HCs), especially during relapses (Vogt et al., 2008, 2010; Comabella et al., 2005). Increased cerebrospinal fluid (CSF) OPN levels in RRMS and in primary progressive (PP) MS patients have been also reported by some groups (Braitch et al., 2008; Börnsen et al., 2011). However, until now no correlations have been found between CSF or plasma OPN levels and measures of neurological impairment in MS patients. Only one study reported a significant correlation between CSF OPN levels and expanded disability status scale (EDSS) score in PPMS patients (Börnsen et al., 2011).
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Increased plasma OPN levels have been also reported in HIV-infected subjects in comparison to HCs (Burdo et al., 2008). Among HIV-infected patients those with HIV associated dementia had higher plasma OPN levels than those who were cognitively preserved (Burdo et al., 2008). Natalizumab (NTZ) is a monoclonal antibody directed against VLA-4. NTZ has been proven to be efficacious in reducing annualized relapse rate (ARR) and MRI activity (Polman et al., 2006; Rudick et al., 2006) and to have also some beneficial effects on cognitive functions (Iaffaldano et al., 2012) in RRMS patients. Recently, a significant decrease of OPN CSF concentrations in RRMS patients has been reported during NTZ treatment (Khademi et al., 2009). The main objectives of this study were to longitudinally evaluate the effect of 2 year NTZ treatment on plasma OPN levels, cognitive performances and self-reported fatigue scores in RRMS patients, and to assess the relationships between OPN levels and cognition and fatigue measures before and during the NTZ treatment.
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level of fatigue interfering with daily activities, at baseline and every 12 months. If a relapse occurred at the time of scheduled neuropsychological assessments, cognitive testing was delayed until 30 days after the last steroid administration. Patients with a visual function impairment interfering with the performances in the cognitive tests were not included in the study. All the patients who reached 1 and 2 years of NTZ treatment entered into the analyses. Plasma samples from 24 treatment naïve RRMS patients and from 22 age- and sex-matched healthy volunteers (HCs) were also collected, and used as controls for a cross-sectional evaluation of baseline plasma OPN levels. The treatment-naïve RRMS patients group consisted in regularly followed-up, not previously-treated with immunomodulatory drugs, clinically stable RRMS patients. None of these patients had received steroid treatment within 3 months when sampling was performed. The local Ethics Committee of the University of Bari approved the study, and written informed consent was obtained from all patients prior to entering the study.
2. Material and methods 2.1. Osteopontin assay Forty-nine RRMS patients, scheduled to start treatment with 300 mg NTZ intravenously once monthly and previously treated with immunomodulants, were consecutively included. All patients were longitudinally followed-up during NTZ treatment. The relapses occurred in the last year pre-NTZ treatment and every new relapse occurring during the NTZ treatment were recorded. The annualized relapse rate (ARR) in the last year and at the end of the first and second year of NTZ treatment were calculated for each patient. The number of gadolinium (Gd) enhancing lesions at the magnetic resonance imaging (MRI) performed just before the start of the NTZ treatment, and every 6 months during the NTZ treatment, were collected. A complete neurological examination including the EDSS score assessment was performed at baseline and every 3 months throughout the treatment. Cognitive functioning, using the Rao’s brief repeatable battery (BRB) (Rao et al., 1991; Amato et al., 2006) and the Stroop test (ST), was assessed before the first infusion and every 12 months by a trained psychologist. BRB included tests of verbal memory acquisition and delayed recall (selective reminding test – SRT), visual memory acquisition and delayed recall (10/36 spatial recall test – SPART), attention, concentration and speed of information processing (PASAT 3; PASAT 2; symbol digit modalities test – SDMT), and verbal fluency on semantic stimulus (word list generation – WLG). Frontal lobe executive functions were assessed by the Stroop color-word task (ST). Versions A and B of the BRB were used alternatively at each examination. Cognitive impairment (CI) was defined as the failure in at least 3 tests on BRB and ST using a 5th percentile cut-off for each test, which corresponded to a z-score 2 standard deviation (SD) below the mean Italian normative values (Amato et al., 2006). A global score, defined cognitive impairment index (CII), allowing the evaluation of changes in cognitive performances independently by the number of cognitive tests failed at the BRB and the ST, was obtained using the mean and SD from the normative sample of Rao’s battery and the ST (Amato et al., 2006; Camp et al., 1999; Iaffaldano et al., 2012; Patti et al., 2010; Viterbo et al., 2013). The CII was obtained applying a grading system to each cognitive tests of the BRB and the ST. For each patient, the grading system was applied to individual cognitive tests, based on the number of SDs below the control mean (i.e., grade 0 was given if the patient scored at or above the control mean, 1 if he/she scored below the control mean, but at or above 1 SD below the control mean, and so on until all patient scores were accommodated). Finally, all the patient’s scores were summed to give one overall measure of cognitive function. Fatigue was assessed by the fatigue severity scale (FSS) (Krupp et al., 1989), using a cut-off value of 4.5 to identify patients with a
Plasma samples were obtained by venous puncture, at baseline and every 6 months during the NTZ treatment. Plasma was isolated by centrifugation and stored at 80 °C until use.OPN concentrations in plasma samples were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Quantikine ELISA kit, R&D Systems Europe, Abingdon, UK) according to the manufacturer’s instructions. The lower limit of detection was 0.313 ng/ml. For the analysis we diluted plasma samples 1:25 in assay diluent. Baseline and follow-up plasma samples from a patient were analyzed on the same plate. Inter-assay precision (precision between assays): four samples of known concentration were tested in all separate assays to assess inter-assay precision. Moreover, we had coefficient of variation (CV) values less than 10% across the standard curve for both intra- and inter-assay precision. These low CV values allowed us to perform repeated assays and be confident that the results are consistent throughout the study. The samples were stored, coded, and made anonymous in accordance with the MRC (Medical Research Council) guidelines on the ethical use of biological specimen collections in clinical research.
2.2. Statistical analyses Descriptive analyses were performed at the baseline. No imputation of missing data was considered. Differences in OPN levels between patients and HCs were analyzed by the Mann–Whitney test. The association of OPN levels and cognitive measures with other parameters was evaluated by Spearman correlation analysis. To test differences across different time points in related samples non parametric tests were used. The Wilcoxon signed – rank test for paired samples and the Friedman two-way test for repeated measures with post hoc correction were used to compare mean values in paired examinations or when three or more evaluations were available for continuous variables. Cochran Q test for repeated measures and McNemar test for pairwise comparisons were used to assess changes over time in categorical variables. A value of p < 0.05 was considered significant for the Wilcoxon signed – rank test, the Friedman two-way test and the Cochran Q test. For post hoc multiple comparisons, we elected a pre-assigned p/number of comparisons (Bonferroni’s correction) as the threshold for significance, and therefore, a p < 0.017 (0.05/3) and a p < 0.005 (0.05/10) were used as a definition of statistical significance for n = 3 and for n = 10 comparisons, respectively. Analyses were performed by SPSS 17.0.
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3. Results In Table 1 a comparison of the demographic and clinical characteristics between the two RRMS groups and HCs is shown. There was no difference in sex distribution and age in RRMS patients and HCs. The RRMS patients scheduled to NTZ treatment had a longer disease duration (10.54 ± 5.44 vs 5.58 years ± 5.78; p < 0.0001), were more disabled (median [range] EDSS score 3.5 [2.0–7.0] vs 2.0 [1.0 – 4.5]; p < 0.0001) and had a more active course (mean ARR 1.78 ± 0.90 vs 1.21 ± 0.78; p = 0.018) in comparison to treatment-naïve RRMS patients. All RRMS patients scheduled to NTZ treatment received previous first line immunomodulant treatments (Interferon-beta [n = 39] and Glatiramer acetate [n = 10]) with a median exposure time of 60.76 months (Range: 3.87– 170.0). The median wash out time from the last treatment to the start of NTZ treatment was 2.54 months (Range: 0.90–20.97). After 1 year-NTZ treatment, the baseline mean ARR (1.73 ± 0.94) and mean EDSS score (3.69 ± 1.04) significantly decreased (ARR: 0.34 ± 0.66, p < 0.0001; EDSS: 3.52 ± 1.17, p = 0.021). In the group of patients receiving NTZ for two years (n = 21), the mean ARR also significantly decreased during the treatment (Friedman test: p < 0.0001). A Wilcoxon signed-rank test with a Bonferroni correction for multiple tests showed that the ARR significantly improved after 1 (0.87 ± 0.60; p < 0.0001) and 2 years (0.10 ± 0.32; p < 0.0001) in comparison to baseline value (1.76 ± 0.99). There were no significant differences between year 1 and 2 of treatment (p = N.S.). No significant reduction of the baseline EDSS score (3.82 ± 0.97) was observed during the follow-up (Year 1: 3.60 ± 1.03; Year 2: 3.63 ± 1.33; p = N.S.). The number of Gd enhancing lesions significantly decrease during the NTZ treatment (Baseline vs year 1 (n = 42): 1 (0–8) vs 0 (0– 1); p < 0.0001; Baseline vs year 2 (n = 21): 1 (0–8) vs 0; p = 0.003). The percentage of CI patients decreased from 30.6% (13/42 patients) at baseline to 26.2% (11/42) at the end of the first year NTZ treatment. Accordingly, the mean CII values were found to be significantly reduced at year 1 in comparison to baseline values (13.52 ± 6.50 vs 11.67 ± 7.46; p = 0.004) (Fig. 1). In patients receiving NTZ for two years, the percentage of CI decreased from 23.8% (5/21 patients) to 19% (4/21 patients) at 1 year and to 9.5% (2/21 patients) at 2 years, and the mean CII significantly decreased (Friedman test: p < 0.0001) during the NTZ treatment. A Wilcoxon signed-rank test with a Bonferroni correction for multiple tests showed that the mean CII value significantly improved after 1 (10.10 ± 6.47; p = 0.003) and 2 years (7.86 ± 6.59; p < 0.0001) in
Fig. 1. Changes of cognitive performances in MS patients during NTZ treatment. The changes of the cognitive impairment index (CII) after 1 and 2 years of NTZ treatment was evaluate in 42 and 21 patients, respectively.
comparison to the baseline value (13.33 ± 6.44). The CII further decreased in the second year of treatment in comparison to the year 1 value (p = 0.007) (Fig. 1). At baseline the mean FSS score was 3.99 (±1.48), and 20/42 patients (47.6%) reported levels of fatigue which interfered with daily activities (FSS score P4.5). After 1 year of NTZ treatment, the mean FSS score significantly decreased to 3.54 (±1.43) (p = 0.025) and the proportion of patients with FSS score P4.5 decreased to 26.2% (11/42, p = 0.049). In patients with a 2 yearfollow-up, the mean FSS score significantly decreased during the treatment (Friedman test, p = 0.002). A Wilcoxon signed-rank test with a Bonferroni correction for multiple tests showed that the mean FSS score significantly improved at 2 years (3.18 ± 1.78; p = 0.002) in comparison to baseline values (4.31 ± 1.47). There were no significant differences between baseline and year 1 (3.52 ± 1.47), and between year 1 and 2 of treatment (p = N.S.). In this subgroup the proportion of patients with FSS score P4.5 significantly (Cochran Q test, p = 0.021) decreased during the 2 years NTZ treatment from 57.1% (12/21 patients) to 28.6% (6/21 patients) at year 1 and to 33.3% (7/21 patients) at year 2. A McNemar test with a Bonferroni correction for multiple tests showed no significant differences of the proportion of patients with FSS score P4.5 between the different timepoint. Plasma OPN levels of RRMS patients scheduled for NTZ treatment (65.42 ng/ml ± 22.20) did not differ from those of the
Table 1 Demographic and clinical characteristics at baseline.
Sex (F/M) Age (years) Disease duration (years) EDSS ARR Time from the last relapse (months) Last DMDs IFN-b GA Washout (months) No. of Gd enhancing lesions at the MRI performed just before the start of NTZ treatment
RRMS patients scheduled for NTZ treatment (n = 49)
RRMS patients treatment naïve (n = 24)
HCs (n = 22)
p Value
37/12 34.23 (10.12) 10.54 (5.44) 3.5 (2.0–7.0) 1.78 (0.90) 6.13 (5.91)
17/7 35.8 (10.83) 5.58 (5.78) 2.0 (1.0–4.5) 1.21 (0.78) 13.79 (22.79)
12/10 39.18 (10.12)
N.S.* N.S.** <0.0001 <0.0001 0.018
39 (79.6%) 10 (20.4%) 3.57 (4.08) 1 (0–8)
Data are reported as mean (SD) with the exception of: EDSS and No. of Gd enhancing lesions (reported as median (range)) and sex and last DMDs reported as count. Abbreviations: NTZ, Natalizumab; HCs, healthy controls; EDSS, expanded disability status scale; ARR, annualized relapse rate; DMDs, disease modifying drugs; IFN-b, interferon – beta; GA, Glatiramer acetate; Gd, gadolinium. * No significant differences in sex distribution (Pearson v2 test) were found between the 2 patients groups (p = 0.6) and between the patients and HCs (p = 0.08). ** No significant differences of age at time of the blood sampling (Mann–Whitney U test) were found between the 2 patients groups (p = 0.5) and between the patients and HCs (p = 0.06).
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Fig. 2. Baseline plasma OPN levels in MS patients and HCs. Y-Axis represents plasma Osteopontin levels expressed in ng/ml. Plasma OPN levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. Abbreviations: OPN, Osteopontin; RRMS, relapsing-remitting multiple sclerosis; tn, treatment naïve; HCs, healthy controls.
treatment-naïve RRMS group (67.70 ng/ml ± 24.23), but they were significantly higher (p = 0 0.013) than in HCs (53.20 ng/ml ± 12.68). Plasma OPN levels were also significantly (p = 0.039) higher in treatment-naïve RRMS patients in comparison to HCs (Fig. 2). After1 year-NTZ treatment baseline OPN levels significantly (p = 0.003) decreased from 66.98 ng/ml (±22.38) to 55.23 ng/ml (±19.88). No significant differences were found between baseline and 6 months OPN levels (60.79 ng/ml ± 20.23) and between 6 months and year 1 OPN levels (Fig. 3A). In patients receiving NTZ for two years (n = 21), OPN levels also significantly decreased during the treatment (Friedman test: p < 0.0001).A Wilcoxon signed-rank test with a Bonferroni correction for multiple tests showed that the OPN levels significantly decreased after 18 months (32.81 ng/ml ± 22.82; p < 0.0001) and 2 years (45.96 ng/ml ± 17.01; p = 0.001) in comparison to baseline value (69.52 ng/ml ± 26.07). There were also a significant difference between year 1 (59.00 ng/ml ± 19.65; p = 0.004) and 2 of treatment (Fig. 3B).
No correlations were found between baseline OPN levels and age, EDSS score, ARR, FSS score, previous DMD treatment duration and washout time from previous DMDs. A significant positive correlation was found (r = 0.43, p = 0.002) between the baseline CII and the baseline OPN levels (Fig. 4). A significant correlation was found between baseline SRT-long term storage (LTS) (r = 0.378, p = 0.007), SRT-consistent long term retrieval (CTLR) (r = 0.404, p = 0.004), SRT-delayed (D) (r = 0.472, p = 0.001), SDMT (r = 0.307, p = 0.032), PASAT3 (r = 0.298, p = 0.037), ST scores (r = 0.318, p = 0.026) and baseline OPN levels. After 1 and 2 years of NTZ treatment the reduction of the CII was found to be significantly correlated to the reduction of the OPN levels (r = 0.305, p = 0.05 and r = 0.667, p = 0.001, respectively) (Fig. 5A and B). 4. Discussion The results of this study confirm the efficacy of NTZ treatment in reducing disease activity and disability progression in RRMS
Fig. 3. Changes of plasma OPN at 1 (A) and 2 (B) year-NTZ-treatment. Y-Axis represents plasma Osteopontin levels expressed in ng/ml for both the graphs. Plasma OPN levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. (A) Shows the significant reduction of the plasma OPN levels during the first year of NTZ treatment (Study population: baseline n = 49; 6 months n = 43; 1 year n = 42). (B) Shows the significant reduction of the plasma OPN levels during the 2 years of NTZ treatment (Study population: baseline n = 49; 6 months n = 43; 1 year n = 42; 18 months n = 24; 2 years n = 21).
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Fig. 4. Relationships between plasma OPN levels and CII at baseline. The figure shows the significant correlation between the baseline CII and the baseline OPN levels (r = 0.43, p = 0.002). Plasma OPN levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. Abbreviation: CII, cognitive impairment index.
patients, as already demonstrated in NTZ pivotal trials (Polman et al., 2006; Rudick et al., 2006), and in improving cognitive functions and self-reported fatigue as suggested in previous observational studies (Mattioli et al., 2011), even on larger MS populations (Iaffaldano et al., 2012). Moreover, according to other groups (Comabella et al., 2005; Vogt et al., 2008) our findings show that plasma OPN levels in RRMS patients are higher than in HCs, and that the immunomodulant therapy do not have any significant effect on these levels. In fact plasma OPN levels of RRMS patients scheduled for NTZ treatment and with a previous treatment with immunomodulants did not differ from those of the treatment-naïve RRMS group. However, we demonstrate that, unlike immunomodulators, NTZ treatment significantly decreases plasma OPN levels and that this decrease is already evident after 1 year NTZ treatment and it is even more prominent in the second year of treatment. NTZ treatment in RRMS have been already found to be associated with a decrease of inflammatory and neurodegenerative markers. Mellergard et al. (2010) reported a global decline of pro-inflammatory
cytokines and chemokines both in the CSF and in the plasma of 31 RRMS patients during 1 year of NTZ treatment. NTZ therapy induced a 3-fold reduction of the CSF neurofilament light levels, a marker of axonal injury, in 92 patients with relapsing forms of MS in a recent Swedish study (Gunnarsson et al., 2010). Finally, Khademi et al. (2009) found that 1 year NTZ treatment reduced CSF OPN and matrix metalloproteinase (MMP)-9 levels in 22 RRMS patients. In our study plasma OPN levels do not show any correlation with age, EDSS score, ARR, FSS score, previous DMD treatment duration and washout time from previous DMDs. This is in line with most of the previous studies (Comabella et al., 2005; Vogt et al., 2008) reporting any significant correlation among plasma and CSF OPN levels and measures of neurological impairment in MS patients. Only one study reported a correlation between CSF OPN levels and the EDSS score in PPMS patients (Börnsen et al., 2011). In our study population the reduction of the inflammatory activity, as revealed by the decrease of the number of Gd enhancing lesions and the reduction of the ARR, goes in parallel with the reduction of plasma levels of a pro-inflammatory cytokine like OPN. It is noteworthy that in the current study we show an association between plasma OPN levels and cognitive deficits in RRMS patients. In particular we find a significant correlation between baseline plasma OPN levels and performances in tests which explore the cognitive domains of verbal memory, attention, information processing speed and executive functions. In addition we demonstrate a significant correlation between the improvement of the cognitive functions and the reduction of the plasma OPN levels during the NTZ treatment. These findings are in accordance with other reports (Burdo et al., 2008; Brown et al., 2011) showing an association between plasma OPN levels and cognitive functions in other neurological diseases. Burdo and colleagues demonstrated that HIV-infected subjects have increased plasma OPN levels in comparison to HCs (Burdo et al., 2008) and that, among HIV-positive patients, those with minor cognitive/motor disorder and those with HIV-associated dementia have higher plasma OPN levels than the cognitive preserved HIV-infected patients (Burdo et al., 2008). More recently OPN has been found to be increased also in CSF of HIV infected patients with dementia (Brown et al., 2011).
Fig. 5. Correlation between the changes (D) of OPN levels and CII at 1 (A) and 2 (B) year-NTZ-treatment. (A) Shows the relationship between the reduction of the CII and the reduction of the OPN levels after 1 year of NTZ treatment (r = 0.305, p = 0.05). (B) Shows the same relationship after 2 years of of NTZ treatment (r = 0.667, p = 0.001).
P. Iaffaldano et al. / Brain, Behavior, and Immunity 35 (2014) 176–181
OPN is a pro-inflammatory cytokine, which is secreted by activated macrophages, leukocytes and T lymphocytes. It is present in extracellular fluids and is up-regulated at sites of inflammation and it has been shown to be involved in inflammation and autoimmune disorders, including MS (Brown, 2012). In addition, it has been demonstrated that classical mediators of acute inflammation such as TNF-a and IL-1 h strongly induce OPN expression in different cell types (Denhardt and Guo, 1993; Yu et al., 1999). OPN, protecting monocytes from apoptosis and decreasing the number of macrophages returning to the circulation from the brain (Burdo et al., 2007), amplifies the brain inflammatory damage which is considered the pathological substrate of HIV-associated dementia (Glass et al., 1995) and is one of the main mechanisms that may underlie the cognitive deficits in MS (Heesen et al., 2010). In EAE affected mice, it was recently showed that the activated microglia and the inflammatory cytokines released from infiltrating lymphocytes are able to alter the synaptic transmission (Centonze et al., 2009; Mandolesi et al., 2010). This induced synaptopathy is related to cognitive dysfunction in this experimental model of MS (Centonze et al., 2009; Mandolesi et al., 2010).The significant reduction of plasma OPN, we found in this study, together with the already demonstrated reduction of CSF OPN (Khademi et al., 2009), CSF and plasma levels of pro-inflammatory cytokines and CSF levels of light-chain neurofilament during the NTZ treatment (Gunnarsson et al., 2010; Khademi et al., 2009; Mellergard et al., 2010) in RRMS, may be responsible of the concurrent improvement of the cognitive dysfunctions observed in these patients. In conclusion our results suggest that the beneficial effect of NTZ on cognitive performances may be partially related to a reduction of plasma OPN levels and that OPN may act as a bridge protein between inflammation and neurodegeneration in RRMS. Plasma OPN might represent a suitable candidate biomarker for the evaluation of the effect of a treatment on cognition in future clinical trials in RRMS.
Competing interest Maria Trojano received honoraria for consultancy or speaking from Biogen, Sanofi-Aventis, Merck Serono and Bayer-Schering and research grants from Merck Serono, Biogen and Novartis; Rosa Gemma Viterbo serves on scientific advisory boards for BiogenIdec and received honoraria for speaking from Novartis and Biogen; Pietro Iaffaldano, Maddalena Ruggieri and Mariangela Mastrapasqua declare no conflict of interest.
Funding This work was supported by annual research grants from the Italian University and Research Ministry (MIUR) (COFIN 20092011 M.T.) and Strategic Project Apulian Region Neurobiotech PS 124 (M.T.). References Amato, M.P., Portaccio, E., Goretti, B., Zipoli, V., Ricchiuti, L., et al., 2006. The Rao’s brief repeatable battery and Stroop test: normative values with age, education and gender corrections in an Italian population. MultScler 12, 787–793. Börnsen, L., Khademi, M., Olsson, T., Sørensen, P.S., Sellebjerg, F., 2011. Osteopontin concentrations are increased in cerebrospinal fluid during attacks of multiple sclerosis. MultScler 17 (1), 32–42. Braitch, M., Nunan, R., Niepel, G., Edwards, L.J., Constantinescu, C.S., 2008. Increased Osteopontin levels in the cerebrospinal fluid of patients with multiple sclerosis. Arch. Neurol. 65 (5), 633–635.
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