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Serum and CSF measles antibody levels increase over time in patients with multiple sclerosis or clinically isolated syndrome
Journal of Neuroimmunology 247 (2012) 70–74
Contents lists available at SciVerse ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Serum and CSF measles antibody levels increase over time in patients with multiple sclerosis or clinically isolated syndrome Cecilia Ahlgren a,⁎, Anders Odén b, Tomas Bergström c, Jan Lycke a a b c
Institute of Neuroscience and Physiology, Department of Neurology, Sahlgrenska University Hospital, 413 45 Gothenburg, Sweden Institute of Mathematical Statistics, Department of Mathematical Sciences, Chalmers University of Technology, 412 96 Gothenburg, Sweden Institute of Biomedicine, Department of Virology, Sahlgrenska University Hospital, 413 45 Gothenburg, Sweden
a r t i c l e
i n f o
Article history: Received 2 January 2012 Received in revised form 13 March 2012 Accepted 16 March 2012 Keywords: Multiple sclerosis Epidemiology Measles virus antibodies CSF Polyspecific immune response Duration of disease
a b s t r a c t In general, measles virus (MV) immunoglobulin G (IgG) antibody titres decline over time. However, we found that serum and CSF MV antibody titres increased with age (slope=0.038, p b 0.001 and slope=0.022, p=0.008), respectively, and disease duration (slope=0.031, p=0.002 and slope =0.032, p=0.005), respectively, in patients with multiple sclerosis (MS) or clinically isolated syndrome (CIS). The age dependency of serum antibody levels differed between patients and controls (slope=0.038 versus −0.004, p b 0.001). The increasing MV antibody titres over time in MS/CIS patients support a general nonspecific stimulation of B cells and plasma cells that is not confined only to the CNS/CSF compartment. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Patients with multiple sclerosis (MS) typically demonstrate intrathecally synthesised immunoglobulins visualised as oligoclonal immunoglobulin G (IgG) bands (Link and Kostulas, 1983). In the majority of MS patients, a small fraction of the intrathecally synthesised antibodies is directed against various neurotropic viruses. This phenomenon is considered to be the result of a polyspecific immune response (Jacobi et al., 2007). Measles virus (MV) antibodies have been detected in the CSF of 74–78% of MS patients (Reiber et al., 1998; Ahlgren et al., 2011). The proportion, however, depends on the exposure to wild and vaccine MV in the study population (Ahlgren et al., 2011). Whereas the presence of MV antibodies in serum does not differ between MS patients and healthy individuals (Compston et al., 1986; Ahlgren et al., 2011), the levels of serum MV antibody titres are higher in MS patients (Adams et al., 1970; Panelius et al., 1973; Ahlgren et al., 2011). Normally, MV antibody titres in serum decline over time after natural measles infection and measles vaccination (Krugman, 1983; Davidkin et al., 2008). However, in a recent study we found no indication of a time-related decline in serum and CSF MV antibody titres in patients with MS or clinically isolated syndrome (CIS) (Ahlgren et al., 2011). The aim of the present
⁎ Corresponding author. Tel.: + 46 31 3424785; fax: + 46 31 826227. E-mail address: [email protected] (C. Ahlgren). 0165-5728/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2012.03.014
study was to investigate whether serum and CSF MV antibody titres change during the course of MS. 2. Materials and methods 2.1. Patients and sampling The present study was based on a previously investigated MS/CIS patient cohort, aged 10–39 years at disease onset (n= 161) (Ahlgren et al., 2011). The date of disease onset was defined as the time of the first neurological symptom suggestive of MS. Information concerning measles infection was obtained from health records and questionnaires, whereas information about measles vaccination was obtained from health records only, as described previously (Ahlgren et al., 2011). MS/CIS patients were divided into two subgroups with respect to immunisation status 1) patients with a history of natural measles infection (n= 74), and 2) patients who had received measles vaccination only (n= 65). Eleven patients without reliable information on their history of natural measles infection and/or measles vaccination were excluded from these subgroups. Demographic data, clinical characteristics, and the numbers of subjects and samples from MS/CIS patients and controls are shown in Table 1. MS and CIS patients exhibited similar levels of MV antibody titres in serum and CSF and the proportions of MS and CIS patients in the subgroups did not differ significantly (Ahlgren et al., 2011). Therefore, MS and CIS patients were analysed together throughout the study.
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Table 1 Demographic data, clinical characteristics, and number of subjects and samples. No. of subjects
MS/CIS patients with: Detectable serum and/or CSF titresb Natural measles infection Measles vaccination only Unreliable infection/vaccination data Non-detectable serum and CSF titresb Total Healthy controls with: Detectable serum and/or CSF titresb Non-detectable serum and CSF titresb Total a b
Year of birth
Agea
Durationa
No. of samples
Total
Men
Women
Range
Mean (range)
Mean (range)
Serum
CSF
150 74 65 11 11 161
42 17 17 8 4
108 57 48 3 7
1959–1986 1959–1979 1960–1986 1960–1977 1959–1984
30 (15–49) 33 (19–49) 26 (15–44) 32 (23–42) 29 (16–38)
3.3 (0–21) 3.9 (0–21) 2.4 (0–13) 4.5 (0–17) 1.4 (0–4)
146 73 64 9 10
113 62 42 9 11
46 4 50
31 4
15 0
1940–1978 1940–1978
33 (19–57) 31 (18–56)
NA NA
46 4
17 4
At the time of specimen sampling. Cut-off values for detectable antibodies: serum ≥ 100, CSF ≥ 10.
2.2. Enzyme-linked immunosorbent assay Serum and CSF samples were analysed by enzyme-linked immunosorbent assay (ELISA) for IgG antibodies to MV, together with positive and negative control sera at the Sahlgrenska University Hospital. Briefly, antigen of MV was prepared by infecting primary cell cultures of human lung fibroblasts with Edmonston strain E 139 as described previously (Forghani and Schmidt, 1979). Infection was continued until a complete cytopathogenic effect was observed. Cells were scraped down, freeze-thawed, ultrasonicated, and centrifuged, and the supernatant was saved as antigen, which was passively coated on to 96 well microtitre plates (Nunc Immuno Maxisorp, Nunc, Gothenburg). Patient samples were incubated in twofold dilutions starting from 1/10 for CSF or 1/100 for serum. For determination of IgG antibodies, alkaline phosphatase-conjugated affinity-purified goat anti-human IgG antibodies (Jackson Immunoresearch, Fisher Scientific, Gothenburg) were used and the colour reaction after addition of p-nitro phenyl phosphate disodium was determined by a spectrophotometer (Molecular Devices VMax kinetic microplate reader). Titres were given as the highest dilution giving positive reaction as compared to the negative controls. The coefficient of variation (CV) was 4% for intra-assay and 13% for inter-assay comparisons. As quality assessment and external control, 2nd International standard antimeasles serum from National Institute for Biological Standards and Controls, UK, was used. Furthermore, the laboratory participated in the quality assessment program of Equalis AB, Uppsala, Sweden for measles serology.
titres (the regression functions of variables for disease duration) in patients versus controls were compared using a t-test. Models including age, duration and duration squared were used to illustrate the relationships between disease duration and antibody titres. Four multivariable regression functions were determined. Because the MV titre may depend on age at disease onset, this age was set at 25 and 35 years in the models, i.e. one above and one below the median age at onset (27 years).
3. Results 3.1. MV IgG antibody titres in MS/CIS patients and healthy controls MS/CIS patients had significantly higher serum and CSF MV antibody titres than healthy controls (serum, median 3200, interquartile range (IQR) 1600–6400 versus 1600, IQR 400–3200, p b 0.001 and
2.3. Detectable MV IgG antibodies in serum and CSF We defined MV antibody titres of ≥100 in serum and ≥10 in CSF as positive cut-off values for detectable antibodies (Table 1). Serum and CSF samples with detectable MV antibodies were either paired (n = 109) or unpaired (serum, n = 37, CSF, n = 4). Fifty healthy blood donors with paired serum and CSF samples served as controls. A total of 150 MS/CIS patients and 46 healthy controls with detectable MV IgG antibody titres in serum and/or CSF were included in analyses of relationships with time (Table 1). 2.4. Statistical analysis The antibody index (AI) for MV was calculated according to the formula AI= (CSF/serum titre) / (albumin ratio · IgG index) (Reiber, 1995). AI ≥ 1.5 indicated intrathecal antibody synthesis (Reiber and Lange, 1991). The median AI and range was calculated for patients and controls. Correlations between the logarithm of MV antibody titres (Y) and age or disease duration on the date of specimen sampling were analysed by linear regression, where Y = constant+ slope· X, and X = age or disease duration (years). The shapes of the curves for MV antibody
Fig. 1. MV IgG antibody titres over time in MS/CIS patients. The figures show the individual serum and/or CSF titre values. The serum and CSF titres refer to (A) age and (B) disease duration on the date of specimen sampling. The values are logarithmic. The estimated increase in MV antibody titres in serum and CSF was 92% and 96%, respectively.
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3.3. Intrathecal MV IgG antibody synthesis
Table 2 MV IgG antibody titres over time in MS/CIS patients and healthy controls. Constant MS/CIS patients, n = 150 Age Duration Natural measles infection, n = 74
Age Duration
Measles vaccination only, n = 65
Age Duration
Slope
p-Valuea
Serum CSF Serum CSF Serum CSF Serum CSF Serum CSF Serum CSF
2.425 1.014 3.465 1.603 2.906 1.576 3.663 1.737 2.462 0.894 3.267 1.471
0.038 0.022 0.031 0.032 0.026 0.009 0.027 0.033 0.033 0.023 0.027 0.025
b0.001 0.008 0.002 0.005 0.009 0.500 0.020 0.009 b0.001 0.129 0.157 0.478
Serum
3.258
− 0.004
0.639
Healthy controls, n = 46 Age a
The p-values for the slopes are given.
CSF, median 20, IQR b10–80 versus b10, IQR b10–10, p b 0.001), as reported previously (Ahlgren et al., 2011).
The median MV AI for MS/CIS patients (n =100) was 2.12 (range 0.24–50.36, 64% ≥1.5) and 1.25 (range 0.56–2.60, 24% ≥1.5) for controls (n =17). The CSF/serum MV antibody titre ratio correlated with increasing IgG index (constant=0.001, slope= 0.020, p=0.011, n =100), but not with age (constant =0.034, slope b 0.001, p=0.469, n =109) or disease duration (constant=0.021, slope b0.001, p= 0.821, n= 109). The degree of disruption to the blood–brain barrier, as determined by the albumin ratio (Tibbling et al., 1977), did not correlate with the CSF/serum MV antibody titre ratio (constant =0.019, slope b 0.001, p=0.861, n =101). 3.4. MV IgG antibody titres over time in MS/CIS patient subgroups In the subgroup restricted to patients with a history of natural measles infection, serum MV antibody titres, but not CSF titres, significantly increased with age. The increase in MV antibody titres with disease duration was significant in both serum and CSF (Table 2). In the subgroup restricted to patients who had received measles vaccination only, the regression coefficients (slopes) for age and disease duration were positive, but significant only for the increase in serum titre with age (Table 2).
3.2. MV IgG antibody titres over time 4. Discussion In MS/CIS patients, the serum and CSF MV antibody titres significantly increased with age and disease duration (Fig. 1A and B, Table 2), whereas serum MV antibody titres slightly decreased with increasing age in healthy controls (Fig. 2 and Table 2). In male patients (serum, n = 40, CSF, n = 35), MV antibody titres significantly increased with age in both serum (constant = 2.054, slope= 0.048, p b 0.001) and CSF (constant = 0.334, slope= 0.042, p = 0.001), but not with disease duration (serum, constant = 3.382, slope= 0.030, p = 0.066 and CSF, constant= 1.517, slope= 0.028, p = 0.091). In female patients (serum, n = 106, CSF, n = 78), MV antibody titres significantly increased with age in serum (constant= 2.578, slope= 0.034, p b 0.001), but not in CSF (constant = 1.436, slope= 0.010, p = 0.371), whereas the increase with disease duration was significant in both serum (constant= 3.484, slope= 0.036, p = 0.006) and CSF (constant= 1.626, slope= 0.040, p = 0.013). When considering both genders, the changes in serum titres with age significantly differed between patients and controls (slope = 0.038 versus −0.004, p b 0.001) (Figs. 1A and 2.). The serum and CSF MV antibody titres approximately doubled over the 21-year disease course (92% in serum and 96% in CSF) in MS/CIS patients. The annual mean increase was approximately 4– 5%. The shape of each estimated curve for serum and CSF MV antibody titres based on disease duration reached a minimum before (Fig. 3B) or shortly after disease onset (Fig. 3A, C, D), after which the curves sloped upward (increasing and convex) for at least the 21 years of observation. No maximum or level off was observed in any of the curves (Fig. 3A–D).
Fig. 2. MV IgG antibody titres over time in healthy controls. The serum titres refer to age on the date of specimen sampling.
We found that the MV IgG antibody titres in both serum and CSF of MS/CIS patients increased with disease duration, whereas, as expected, MV antibody titres in serum of healthy controls slightly declined over time (Krugman, 1983; Davidkin et al., 2008). The difference between patients and controls with respect to these changes was significant and persistent. The lowest MV antibody titres occurred shortly before or after disease onset and the levels increased thereafter for at least 20 years of disease duration, with no indication of a peak or level off. After 21 years of disease duration, the serum and CSF MV antibody titres were approximately doubled, with an annual MV titre increase of 4–5%. According to the models, the evolution of serum/CSF MV antibody titres over time did not depend on age at disease onset. In the present study two categories of MS/CIS patients were included: those with a history of natural measles infection and those with measles vaccination only. Natural measles infection is known to induce higher MV antibody titres than measles vaccination (Krugman, 1983; Davidkin et al., 2008) which was also shown in the present cohort (Ahlgren et al., 2011). This difference could possibly introduce a confounding factor in the present study, because patients with longer MS duration were more likely to have a history of natural measles infection than those with shorter disease duration. However, serum and CSF MV antibody titres in the patient group restricted to those with a history of natural measles infection significantly increased with disease duration and in the subgroup of patients with measles vaccination only, the MV titres tended to increase over time, significantly, however, only in serum. Furthermore, the slopes for age and disease duration were positive for MS/CIS patients, whereas the slope for age was negative for healthy controls. The observed increase in CSF MV antibodies over time was not related to a damaged blood–brain barrier. The pathologically increased mean MV AI for MS/CIS patients compared to the mean MV AI for healthy controls suggests intrathecally synthesised MV IgG (Reiber and Lange, 1991). The positive correlation between the MV CSF/ serum titre ratio and IgG index in the MS/CIS patients provides further support for intrathecal synthesis of CSF MV IgG. Increased antibody levels against several neurotropic infections were observed previously (Reiber et al., 1998), but only a few studies have reported a change in virus antibody levels during the course of MS. It has been demonstrated that serum MV antibody titres are
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Fig. 3. Models of relationships between disease duration and MV IgG antibody titres. The figures show antibody titres in (A) serum from men, (B) serum from women, (C) CSF from men and (D) CSF from women. The curves for onset at 25 and 35 years coincided in (D).
higher in MS patients than controls (Adams et al., 1970; Panelius et al., 1973; Ahlgren et al., 2011), and that the majority of MS patients have detectable MV antibodies also in their CSF (Reiber et al., 1998; Ahlgren et al., 2011). High serum titres against MV, Epstein Barr virus (EBV), and human herpes virus 6 (HHV-6) were shown to significantly increase the risk of developing MS (Sundstrom et al., 2004). Epstein-Barr nuclear antigen (EBNA) complex antibody titres in serum, showed a sharp and significant increase in early adulthood, followed by a plateau at around 30 years of age, prior to MS onset (Levin et al., 2005). The first follow-up study of virus antibody levels in CSF after MS onset revealed that the proportion of MS patients with intrathecal antibodies to MV, but not to rubella or varicella zoster virus (VZV), increased over time. Individual determinations of virus antibody response in CSF from repeated lumbar punctures showed that the antibody reactivity may expand from one to more viruses. In this relatively short follow-up study of 2–5 years, intrathecal measles–rubella–varicella zoster virus (MRZ) antibody titres persisted at a stable level (Petereit and Reske, 2005). In a 5-year follow-up study, 10 paediatric MS patients showed stable or possibly increasing MRZ antibody titre levels (Reiber et al., 2009). An antibody reaction against two or more MRZ viruses was shown in less than half of CIS patients (Brettschneider et al., 2009) compared to almost all MS patients (Reiber et al., 1998). Taken together, these observations suggest that MV antibody titres, and perhaps other neurotropic virus antibody titres, increase with disease duration. However, compared to previous studies we present compelling data on this relationship, probably a consequence of a considerably broader range of disease duration, from 0 to 21 years in our present population. Evidence has accumulated in recent years about the importance of B cells in the pathogenesis of MS. Most striking is the rapid reduction in MS disease activity achieved by anti-CD 20 monoclonal antibody therapy (Bar-Or et al., 2008; Hauser et al., 2008; Kappos et al., 2011), which results in B cell depletion in the peripheral blood. However, the mechanism seems to not be associated with decreased autoreactive antibody synthesis as the IgG levels are only slightly affected (Bar-Or et al., 2008; Hauser et al., 2008; Kappos et al., 2011). Another important finding is the detection of ectopic B cell follicles in the meninges of MS patients (Prineas, 1979; Serafini et al., 2004; Magliozzi et al., 2007). Associations between these tertiary germinal centres
and a gradient of subpial cortical demyelination, neuronal loss, and cortical atrophy were recently reported (Magliozzi et al., 2010). Although B cells and plasma cells aggregate in these follicles and may explain the persistence of oligoclonal IgG bands in MS patients (Frischer et al., 2009), it is not known if they are responsible for the intrathecal production of autoreactive antibodies against myelin proteins or the frequently found neurotropic virus antibodies. The production of CSF IgG has been proposed to be maintained mainly by long-lived plasma cells (Prineas and Wright, 1978; Serafini et al., 2004) and mature plasma cells also occurred more often in the CSF of MS patients with long disease duration (Zeman et al., 2001), which is in line with our results. However, because we found increasing MV antibody titres also outside the CNS/CSF compartment, our results point to a more general activation of B cells and plasma cells in MS. Although we cannot exclude a specific immune stimulation by measles-related proteins, this phenomenon is most likely part of a polyspecific immune response, probably with no pathogenic role. This is the first study to report increasing MV IgG antibody levels with MS disease duration. Further studies remain to prove if our observation of increasing MV IgG synthesis is part of a general phenomenon in MS that also involves antibodies against other infectious agents and even autoreactive antibodies against CNS-derived proteins. Previous studies have shown that MV IgG antibodies in CSF may have diagnostic implications (Reiber et al., 2009). The pathogenic role of anti-virus antibodies and their usefulness as biomarkers for disease activity remains to be explored. References Adams, J.M., Brooks, M.B., Fisher, E.D., Tyler, C.S., 1970. Measles antibodies in patients with multiple sclerosis and with other neurological and nonneurological diseases. Neurology 20 (10), 1039–1042. Ahlgren, C., Oden, A., Haghighi, S., Andersen, O., Bergstrom, T., Lycke, J., 2011. The effect of live, attenuated measles vaccine and measles infection on measles antibody levels in serum and CSF of patients with multiple sclerosis or clinically isolated syndrome. J. Neuroimmunol. 235 (1–2), 98–103. Bar-Or, A., Calabresi, P.A., Arnold, D., Markowitz, C., Shafer, S., Kasper, L.H., Waubant, E., Gazda, S., Fox, R.J., Panzara, M., Sarkar, N., Agarwal, S., Smith, C.H., 2008. Rituximab in relapsing-remitting multiple sclerosis: a 72-week, open-label, phase I trial. Ann. Neurol. 63 (3), 395–400. Brettschneider, J., Tumani, H., Kiechle, U., Muche, R., Richards, G., Lehmensiek, V., Ludolph, A.C., Otto, M., 2009. IgG antibodies against measles, rubella, and varicella
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Magliozzi, R., Howell, O.W., Reeves, C., Roncaroli, F., Nicholas, R., Serafini, B., Aloisi, F., Reynolds, R., 2010. A Gradient of neuronal loss and meningeal inflammation in multiple sclerosis. Ann. Neurol. 68 (4), 477–493. Panelius, M., Salmi, A., Halonen, P.E., Kivalo, E., Rinne, U.K., Penttinen, K., 1973. Virus antibodies in serum specimens from patients with multiple sclerosis, from siblings, and matched controls. A final report. Acta Neurol. Scand. 49 (1), 85–107. Petereit, H.F., Reske, D., 2005. Expansion of antibody reactivity in the cerebrospinal fluid of multiple sclerosis patients — follow-up and clinical implications. Cerebrospinal Fluid Res. 2, 3. Prineas, J.W., 1979. Multiple sclerosis: presence of lymphatic capillaries and lymphoid tissue in the brain and spinal cord. Science 203 (4385), 1123–1125. Prineas, J.W., Wright, R.G., 1978. Macrophages, lymphocytes, and plasma cells in the perivascular compartment in chronic multiple sclerosis. Lab. Invest. 38 (4), 409–421. Reiber, H., 1995. External quality assessment in clinical neurochemistry: survey of analysis for cerebrospinal fluid (CSF) proteins based on CSF/serum quotients. Clin. Chem. 41 (2), 256–263. Reiber, H., Lange, P., 1991. Quantification of virus-specific antibodies in cerebrospinal fluid and serum: sensitive and specific detection of antibody synthesis in brain. Clin. Chem. 37 (7), 1153–1160. Reiber, H., Ungefehr, S., Jacobi, C., 1998. The intrathecal, polyspecific and oligoclonal immune response in multiple sclerosis. Mult. Scler. 4 (3), 111–117. Reiber, H., Teut, M., Pohl, D., Rostasy, K.M., Hanefeld, F., 2009. Paediatric and adult multiple sclerosis: age-related differences and time course of the neuroimmunological response in cerebrospinal fluid. Mult. Scler. 15 (12), 1466–1480. Serafini, B., Rosicarelli, B., Magliozzi, R., Stigliano, E., Aloisi, F., 2004. Detection of ectopic B-cell follicles with germinal centers in the meninges of patients with secondary progressive multiple sclerosis. Brain Pathol. 14 (2), 164–174. Sundstrom, P., Juto, P., Wadell, G., Hallmans, G., Svenningsson, A., Nystrom, L., Dillner, J., Forsgren, L., 2004. An altered immune response to Epstein-Barr virus in multiple sclerosis: a prospective study. Neurology 62 (12), 2277–2282. Tibbling, G., Link, H., Ohman, S., 1977. Principles of albumin and IgG analyses in neurological disorders. I. Establishment of reference values. Scand. J. Clin. Lab. Invest. 37 (5), 385–390. Zeman, D., Adam, P., Kalistova, H., Sobek, O., Andel, J., Andel, M., 2001. Cerebrospinal fluid cytologic findings in multiple sclerosis. A comparison between patient subgroups. Acta Cytol. 45 (1), 51–59.
Contents lists available at SciVerse ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Serum and CSF measles antibody levels increase over time in patients with multiple sclerosis or clinically isolated syndrome Cecilia Ahlgren a,⁎, Anders Odén b, Tomas Bergström c, Jan Lycke a a b c
Institute of Neuroscience and Physiology, Department of Neurology, Sahlgrenska University Hospital, 413 45 Gothenburg, Sweden Institute of Mathematical Statistics, Department of Mathematical Sciences, Chalmers University of Technology, 412 96 Gothenburg, Sweden Institute of Biomedicine, Department of Virology, Sahlgrenska University Hospital, 413 45 Gothenburg, Sweden
a r t i c l e
i n f o
Article history: Received 2 January 2012 Received in revised form 13 March 2012 Accepted 16 March 2012 Keywords: Multiple sclerosis Epidemiology Measles virus antibodies CSF Polyspecific immune response Duration of disease
a b s t r a c t In general, measles virus (MV) immunoglobulin G (IgG) antibody titres decline over time. However, we found that serum and CSF MV antibody titres increased with age (slope=0.038, p b 0.001 and slope=0.022, p=0.008), respectively, and disease duration (slope=0.031, p=0.002 and slope =0.032, p=0.005), respectively, in patients with multiple sclerosis (MS) or clinically isolated syndrome (CIS). The age dependency of serum antibody levels differed between patients and controls (slope=0.038 versus −0.004, p b 0.001). The increasing MV antibody titres over time in MS/CIS patients support a general nonspecific stimulation of B cells and plasma cells that is not confined only to the CNS/CSF compartment. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Patients with multiple sclerosis (MS) typically demonstrate intrathecally synthesised immunoglobulins visualised as oligoclonal immunoglobulin G (IgG) bands (Link and Kostulas, 1983). In the majority of MS patients, a small fraction of the intrathecally synthesised antibodies is directed against various neurotropic viruses. This phenomenon is considered to be the result of a polyspecific immune response (Jacobi et al., 2007). Measles virus (MV) antibodies have been detected in the CSF of 74–78% of MS patients (Reiber et al., 1998; Ahlgren et al., 2011). The proportion, however, depends on the exposure to wild and vaccine MV in the study population (Ahlgren et al., 2011). Whereas the presence of MV antibodies in serum does not differ between MS patients and healthy individuals (Compston et al., 1986; Ahlgren et al., 2011), the levels of serum MV antibody titres are higher in MS patients (Adams et al., 1970; Panelius et al., 1973; Ahlgren et al., 2011). Normally, MV antibody titres in serum decline over time after natural measles infection and measles vaccination (Krugman, 1983; Davidkin et al., 2008). However, in a recent study we found no indication of a time-related decline in serum and CSF MV antibody titres in patients with MS or clinically isolated syndrome (CIS) (Ahlgren et al., 2011). The aim of the present
⁎ Corresponding author. Tel.: + 46 31 3424785; fax: + 46 31 826227. E-mail address: [email protected] (C. Ahlgren). 0165-5728/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2012.03.014
study was to investigate whether serum and CSF MV antibody titres change during the course of MS. 2. Materials and methods 2.1. Patients and sampling The present study was based on a previously investigated MS/CIS patient cohort, aged 10–39 years at disease onset (n= 161) (Ahlgren et al., 2011). The date of disease onset was defined as the time of the first neurological symptom suggestive of MS. Information concerning measles infection was obtained from health records and questionnaires, whereas information about measles vaccination was obtained from health records only, as described previously (Ahlgren et al., 2011). MS/CIS patients were divided into two subgroups with respect to immunisation status 1) patients with a history of natural measles infection (n= 74), and 2) patients who had received measles vaccination only (n= 65). Eleven patients without reliable information on their history of natural measles infection and/or measles vaccination were excluded from these subgroups. Demographic data, clinical characteristics, and the numbers of subjects and samples from MS/CIS patients and controls are shown in Table 1. MS and CIS patients exhibited similar levels of MV antibody titres in serum and CSF and the proportions of MS and CIS patients in the subgroups did not differ significantly (Ahlgren et al., 2011). Therefore, MS and CIS patients were analysed together throughout the study.
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Table 1 Demographic data, clinical characteristics, and number of subjects and samples. No. of subjects
MS/CIS patients with: Detectable serum and/or CSF titresb Natural measles infection Measles vaccination only Unreliable infection/vaccination data Non-detectable serum and CSF titresb Total Healthy controls with: Detectable serum and/or CSF titresb Non-detectable serum and CSF titresb Total a b
Year of birth
Agea
Durationa
No. of samples
Total
Men
Women
Range
Mean (range)
Mean (range)
Serum
CSF
150 74 65 11 11 161
42 17 17 8 4
108 57 48 3 7
1959–1986 1959–1979 1960–1986 1960–1977 1959–1984
30 (15–49) 33 (19–49) 26 (15–44) 32 (23–42) 29 (16–38)
3.3 (0–21) 3.9 (0–21) 2.4 (0–13) 4.5 (0–17) 1.4 (0–4)
146 73 64 9 10
113 62 42 9 11
46 4 50
31 4
15 0
1940–1978 1940–1978
33 (19–57) 31 (18–56)
NA NA
46 4
17 4
At the time of specimen sampling. Cut-off values for detectable antibodies: serum ≥ 100, CSF ≥ 10.
2.2. Enzyme-linked immunosorbent assay Serum and CSF samples were analysed by enzyme-linked immunosorbent assay (ELISA) for IgG antibodies to MV, together with positive and negative control sera at the Sahlgrenska University Hospital. Briefly, antigen of MV was prepared by infecting primary cell cultures of human lung fibroblasts with Edmonston strain E 139 as described previously (Forghani and Schmidt, 1979). Infection was continued until a complete cytopathogenic effect was observed. Cells were scraped down, freeze-thawed, ultrasonicated, and centrifuged, and the supernatant was saved as antigen, which was passively coated on to 96 well microtitre plates (Nunc Immuno Maxisorp, Nunc, Gothenburg). Patient samples were incubated in twofold dilutions starting from 1/10 for CSF or 1/100 for serum. For determination of IgG antibodies, alkaline phosphatase-conjugated affinity-purified goat anti-human IgG antibodies (Jackson Immunoresearch, Fisher Scientific, Gothenburg) were used and the colour reaction after addition of p-nitro phenyl phosphate disodium was determined by a spectrophotometer (Molecular Devices VMax kinetic microplate reader). Titres were given as the highest dilution giving positive reaction as compared to the negative controls. The coefficient of variation (CV) was 4% for intra-assay and 13% for inter-assay comparisons. As quality assessment and external control, 2nd International standard antimeasles serum from National Institute for Biological Standards and Controls, UK, was used. Furthermore, the laboratory participated in the quality assessment program of Equalis AB, Uppsala, Sweden for measles serology.
titres (the regression functions of variables for disease duration) in patients versus controls were compared using a t-test. Models including age, duration and duration squared were used to illustrate the relationships between disease duration and antibody titres. Four multivariable regression functions were determined. Because the MV titre may depend on age at disease onset, this age was set at 25 and 35 years in the models, i.e. one above and one below the median age at onset (27 years).
3. Results 3.1. MV IgG antibody titres in MS/CIS patients and healthy controls MS/CIS patients had significantly higher serum and CSF MV antibody titres than healthy controls (serum, median 3200, interquartile range (IQR) 1600–6400 versus 1600, IQR 400–3200, p b 0.001 and
2.3. Detectable MV IgG antibodies in serum and CSF We defined MV antibody titres of ≥100 in serum and ≥10 in CSF as positive cut-off values for detectable antibodies (Table 1). Serum and CSF samples with detectable MV antibodies were either paired (n = 109) or unpaired (serum, n = 37, CSF, n = 4). Fifty healthy blood donors with paired serum and CSF samples served as controls. A total of 150 MS/CIS patients and 46 healthy controls with detectable MV IgG antibody titres in serum and/or CSF were included in analyses of relationships with time (Table 1). 2.4. Statistical analysis The antibody index (AI) for MV was calculated according to the formula AI= (CSF/serum titre) / (albumin ratio · IgG index) (Reiber, 1995). AI ≥ 1.5 indicated intrathecal antibody synthesis (Reiber and Lange, 1991). The median AI and range was calculated for patients and controls. Correlations between the logarithm of MV antibody titres (Y) and age or disease duration on the date of specimen sampling were analysed by linear regression, where Y = constant+ slope· X, and X = age or disease duration (years). The shapes of the curves for MV antibody
Fig. 1. MV IgG antibody titres over time in MS/CIS patients. The figures show the individual serum and/or CSF titre values. The serum and CSF titres refer to (A) age and (B) disease duration on the date of specimen sampling. The values are logarithmic. The estimated increase in MV antibody titres in serum and CSF was 92% and 96%, respectively.
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3.3. Intrathecal MV IgG antibody synthesis
Table 2 MV IgG antibody titres over time in MS/CIS patients and healthy controls. Constant MS/CIS patients, n = 150 Age Duration Natural measles infection, n = 74
Age Duration
Measles vaccination only, n = 65
Age Duration
Slope
p-Valuea
Serum CSF Serum CSF Serum CSF Serum CSF Serum CSF Serum CSF
2.425 1.014 3.465 1.603 2.906 1.576 3.663 1.737 2.462 0.894 3.267 1.471
0.038 0.022 0.031 0.032 0.026 0.009 0.027 0.033 0.033 0.023 0.027 0.025
b0.001 0.008 0.002 0.005 0.009 0.500 0.020 0.009 b0.001 0.129 0.157 0.478
Serum
3.258
− 0.004
0.639
Healthy controls, n = 46 Age a
The p-values for the slopes are given.
CSF, median 20, IQR b10–80 versus b10, IQR b10–10, p b 0.001), as reported previously (Ahlgren et al., 2011).
The median MV AI for MS/CIS patients (n =100) was 2.12 (range 0.24–50.36, 64% ≥1.5) and 1.25 (range 0.56–2.60, 24% ≥1.5) for controls (n =17). The CSF/serum MV antibody titre ratio correlated with increasing IgG index (constant=0.001, slope= 0.020, p=0.011, n =100), but not with age (constant =0.034, slope b 0.001, p=0.469, n =109) or disease duration (constant=0.021, slope b0.001, p= 0.821, n= 109). The degree of disruption to the blood–brain barrier, as determined by the albumin ratio (Tibbling et al., 1977), did not correlate with the CSF/serum MV antibody titre ratio (constant =0.019, slope b 0.001, p=0.861, n =101). 3.4. MV IgG antibody titres over time in MS/CIS patient subgroups In the subgroup restricted to patients with a history of natural measles infection, serum MV antibody titres, but not CSF titres, significantly increased with age. The increase in MV antibody titres with disease duration was significant in both serum and CSF (Table 2). In the subgroup restricted to patients who had received measles vaccination only, the regression coefficients (slopes) for age and disease duration were positive, but significant only for the increase in serum titre with age (Table 2).
3.2. MV IgG antibody titres over time 4. Discussion In MS/CIS patients, the serum and CSF MV antibody titres significantly increased with age and disease duration (Fig. 1A and B, Table 2), whereas serum MV antibody titres slightly decreased with increasing age in healthy controls (Fig. 2 and Table 2). In male patients (serum, n = 40, CSF, n = 35), MV antibody titres significantly increased with age in both serum (constant = 2.054, slope= 0.048, p b 0.001) and CSF (constant = 0.334, slope= 0.042, p = 0.001), but not with disease duration (serum, constant = 3.382, slope= 0.030, p = 0.066 and CSF, constant= 1.517, slope= 0.028, p = 0.091). In female patients (serum, n = 106, CSF, n = 78), MV antibody titres significantly increased with age in serum (constant= 2.578, slope= 0.034, p b 0.001), but not in CSF (constant = 1.436, slope= 0.010, p = 0.371), whereas the increase with disease duration was significant in both serum (constant= 3.484, slope= 0.036, p = 0.006) and CSF (constant= 1.626, slope= 0.040, p = 0.013). When considering both genders, the changes in serum titres with age significantly differed between patients and controls (slope = 0.038 versus −0.004, p b 0.001) (Figs. 1A and 2.). The serum and CSF MV antibody titres approximately doubled over the 21-year disease course (92% in serum and 96% in CSF) in MS/CIS patients. The annual mean increase was approximately 4– 5%. The shape of each estimated curve for serum and CSF MV antibody titres based on disease duration reached a minimum before (Fig. 3B) or shortly after disease onset (Fig. 3A, C, D), after which the curves sloped upward (increasing and convex) for at least the 21 years of observation. No maximum or level off was observed in any of the curves (Fig. 3A–D).
Fig. 2. MV IgG antibody titres over time in healthy controls. The serum titres refer to age on the date of specimen sampling.
We found that the MV IgG antibody titres in both serum and CSF of MS/CIS patients increased with disease duration, whereas, as expected, MV antibody titres in serum of healthy controls slightly declined over time (Krugman, 1983; Davidkin et al., 2008). The difference between patients and controls with respect to these changes was significant and persistent. The lowest MV antibody titres occurred shortly before or after disease onset and the levels increased thereafter for at least 20 years of disease duration, with no indication of a peak or level off. After 21 years of disease duration, the serum and CSF MV antibody titres were approximately doubled, with an annual MV titre increase of 4–5%. According to the models, the evolution of serum/CSF MV antibody titres over time did not depend on age at disease onset. In the present study two categories of MS/CIS patients were included: those with a history of natural measles infection and those with measles vaccination only. Natural measles infection is known to induce higher MV antibody titres than measles vaccination (Krugman, 1983; Davidkin et al., 2008) which was also shown in the present cohort (Ahlgren et al., 2011). This difference could possibly introduce a confounding factor in the present study, because patients with longer MS duration were more likely to have a history of natural measles infection than those with shorter disease duration. However, serum and CSF MV antibody titres in the patient group restricted to those with a history of natural measles infection significantly increased with disease duration and in the subgroup of patients with measles vaccination only, the MV titres tended to increase over time, significantly, however, only in serum. Furthermore, the slopes for age and disease duration were positive for MS/CIS patients, whereas the slope for age was negative for healthy controls. The observed increase in CSF MV antibodies over time was not related to a damaged blood–brain barrier. The pathologically increased mean MV AI for MS/CIS patients compared to the mean MV AI for healthy controls suggests intrathecally synthesised MV IgG (Reiber and Lange, 1991). The positive correlation between the MV CSF/ serum titre ratio and IgG index in the MS/CIS patients provides further support for intrathecal synthesis of CSF MV IgG. Increased antibody levels against several neurotropic infections were observed previously (Reiber et al., 1998), but only a few studies have reported a change in virus antibody levels during the course of MS. It has been demonstrated that serum MV antibody titres are
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Fig. 3. Models of relationships between disease duration and MV IgG antibody titres. The figures show antibody titres in (A) serum from men, (B) serum from women, (C) CSF from men and (D) CSF from women. The curves for onset at 25 and 35 years coincided in (D).
higher in MS patients than controls (Adams et al., 1970; Panelius et al., 1973; Ahlgren et al., 2011), and that the majority of MS patients have detectable MV antibodies also in their CSF (Reiber et al., 1998; Ahlgren et al., 2011). High serum titres against MV, Epstein Barr virus (EBV), and human herpes virus 6 (HHV-6) were shown to significantly increase the risk of developing MS (Sundstrom et al., 2004). Epstein-Barr nuclear antigen (EBNA) complex antibody titres in serum, showed a sharp and significant increase in early adulthood, followed by a plateau at around 30 years of age, prior to MS onset (Levin et al., 2005). The first follow-up study of virus antibody levels in CSF after MS onset revealed that the proportion of MS patients with intrathecal antibodies to MV, but not to rubella or varicella zoster virus (VZV), increased over time. Individual determinations of virus antibody response in CSF from repeated lumbar punctures showed that the antibody reactivity may expand from one to more viruses. In this relatively short follow-up study of 2–5 years, intrathecal measles–rubella–varicella zoster virus (MRZ) antibody titres persisted at a stable level (Petereit and Reske, 2005). In a 5-year follow-up study, 10 paediatric MS patients showed stable or possibly increasing MRZ antibody titre levels (Reiber et al., 2009). An antibody reaction against two or more MRZ viruses was shown in less than half of CIS patients (Brettschneider et al., 2009) compared to almost all MS patients (Reiber et al., 1998). Taken together, these observations suggest that MV antibody titres, and perhaps other neurotropic virus antibody titres, increase with disease duration. However, compared to previous studies we present compelling data on this relationship, probably a consequence of a considerably broader range of disease duration, from 0 to 21 years in our present population. Evidence has accumulated in recent years about the importance of B cells in the pathogenesis of MS. Most striking is the rapid reduction in MS disease activity achieved by anti-CD 20 monoclonal antibody therapy (Bar-Or et al., 2008; Hauser et al., 2008; Kappos et al., 2011), which results in B cell depletion in the peripheral blood. However, the mechanism seems to not be associated with decreased autoreactive antibody synthesis as the IgG levels are only slightly affected (Bar-Or et al., 2008; Hauser et al., 2008; Kappos et al., 2011). Another important finding is the detection of ectopic B cell follicles in the meninges of MS patients (Prineas, 1979; Serafini et al., 2004; Magliozzi et al., 2007). Associations between these tertiary germinal centres
and a gradient of subpial cortical demyelination, neuronal loss, and cortical atrophy were recently reported (Magliozzi et al., 2010). Although B cells and plasma cells aggregate in these follicles and may explain the persistence of oligoclonal IgG bands in MS patients (Frischer et al., 2009), it is not known if they are responsible for the intrathecal production of autoreactive antibodies against myelin proteins or the frequently found neurotropic virus antibodies. The production of CSF IgG has been proposed to be maintained mainly by long-lived plasma cells (Prineas and Wright, 1978; Serafini et al., 2004) and mature plasma cells also occurred more often in the CSF of MS patients with long disease duration (Zeman et al., 2001), which is in line with our results. However, because we found increasing MV antibody titres also outside the CNS/CSF compartment, our results point to a more general activation of B cells and plasma cells in MS. Although we cannot exclude a specific immune stimulation by measles-related proteins, this phenomenon is most likely part of a polyspecific immune response, probably with no pathogenic role. This is the first study to report increasing MV IgG antibody levels with MS disease duration. Further studies remain to prove if our observation of increasing MV IgG synthesis is part of a general phenomenon in MS that also involves antibodies against other infectious agents and even autoreactive antibodies against CNS-derived proteins. Previous studies have shown that MV IgG antibodies in CSF may have diagnostic implications (Reiber et al., 2009). The pathogenic role of anti-virus antibodies and their usefulness as biomarkers for disease activity remains to be explored. References Adams, J.M., Brooks, M.B., Fisher, E.D., Tyler, C.S., 1970. Measles antibodies in patients with multiple sclerosis and with other neurological and nonneurological diseases. Neurology 20 (10), 1039–1042. Ahlgren, C., Oden, A., Haghighi, S., Andersen, O., Bergstrom, T., Lycke, J., 2011. The effect of live, attenuated measles vaccine and measles infection on measles antibody levels in serum and CSF of patients with multiple sclerosis or clinically isolated syndrome. J. Neuroimmunol. 235 (1–2), 98–103. Bar-Or, A., Calabresi, P.A., Arnold, D., Markowitz, C., Shafer, S., Kasper, L.H., Waubant, E., Gazda, S., Fox, R.J., Panzara, M., Sarkar, N., Agarwal, S., Smith, C.H., 2008. Rituximab in relapsing-remitting multiple sclerosis: a 72-week, open-label, phase I trial. Ann. Neurol. 63 (3), 395–400. Brettschneider, J., Tumani, H., Kiechle, U., Muche, R., Richards, G., Lehmensiek, V., Ludolph, A.C., Otto, M., 2009. IgG antibodies against measles, rubella, and varicella
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