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Clinical Neurology and Neurosurgery 133 (2015) 30–33

Clinical Neurology and Neurosurgery 133 (2015) 30–33 Contents lists available at ScienceDirect Clinical Neurology and

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Clinical Neurology and Neurosurgery

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journal homepage: Abnormally high levels of anti-collagen type IV IgG

Abnormally high levels of anti-collagen type IV IgG antibodies in the serum of patients with a clinically isolated syndrome correlate with an increased risk of conversion to MS

Behidhe Sadarzanska-Terzieva a, , Plamen Tzvetanov b , Vishwajit Hegde c , Jasem Y. Al-Hashel d , Rossen T. Rousseff d , Lubomir Haralanov e , Boyko Stamenov a , Milena Atanassova f , Iveta Marinova g , Anna Marinova g , Adelaida Rousseva h

Marinova g , Anna Marinova g , Adelaida Rousseva h a Department of Neurology and Neurosurgery,

a Department of Neurology and Neurosurgery, University Hospital of Pleven, Georgi Kochev st 8A, Pleven 5800, Bulgaria

b Department of Clinical Neurophysiology, Sandringham level 1, Leicester Royal Infirmary, University Hospital of Leicester, Leicester LE1 5WW, UK

c Department of Neurosciences, University Hospital of Coventry and Warwickshire NHS Trust, Clifford Bridge Road, Coventry CV2 2DX, UK

d Department of Neurology, Ibn-Sina Hospital, Kuwait, POB 25427, Safat 13115, Kuwait

e National Hospital in Cardiology, Neurology Clinic, 65 Konjovitsa Str., Sofia 1309, Bulgaria

f Department of Biology, Medical University of Pleven, Kliment Ohridski St. 1, Pleven 5800, Bulgaria

g Department of Psychology, Affiliate “Prof Ivan Mitev” of Vratza, Medical University of Sofia, School Complex 1, Vratza 3000, Bulgaria

h Department of Clinical Laboratory, University Hospital of Pleven, Georgi Kochev St. 8A, Pleven 5800, Bulgaria

a r t i c

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i n f o

Article history:

Received 20 June 2014 Received in revised form 8 February 2015 Accepted 7 March 2015 Available online 17 March 2015


Multiple sclerosis Clinically isolated syndrome Matrix metalloproteinases Metalloproteinases inhibitor Anti-collagen type IV antibodies

a b s t r a c t

Objective: To investigate anti-collagen-type-IV serum antibodies (ACIVAbs) levels in patients with clin- ically isolated syndrome (CIS), and to determine their predictive value for conversion into multiple sclerosis (MS). Material and Methods: Serum levels of IgM and IgG ACIVAbs in 40 untreated patients with CIS (13 male, mean age 34.85 ± 11.4 years, range 16–58 years) were compared to those of 27 gender- and age-matched healthy controls. ACIVAbs were quantified using ELISA. Patients were followed for 5 years by clinical examination and MRI studies. Results: Thirty two patients (80%) converted to MS (converted CIS, C-CIS group) while the rest 8 (20%) did not (non-converted CIS, NC-CIS). The C-CIS patients had significantly higher levels of IgG ACIVAb compared to NC-CIS while the IgM levels did not differ between C-CIS and NC-CIS. Conversion to MS occurred in 66% of patients with IgG ACIVAbs levels exceeding the 95th percentile found in controls. IgG ACIVAbs levels correlated positively with the serum levels of matrix metalloproteinases type 9 (r = 0.37; p = 0.003) and inversely with those of tissue inhibitor of metalloproteinases type 1 (r = 0.43; p = 0.0008). Conclusion: High serum levels of IgG ACIVAbs in patients with CIS correlate strongly with increased risk of conversion to MS.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

Predicting the risk of conversion of clinically isolated syndrome (CIS) into definitive multiple sclerosis (MS) is important in patient management [1–3]. While advances in imaging have contributed to define early treatment strategies, the approach in many cases remains debatable [4–6]. Autoimmune inflammation is cardinal in MS pathogenesis, and therefore, a search for immunological

Corresponding author. Tel.: +359 64 80 22 46; fax: +359 64 80 22 46. E-mail address: (B. Sadarzanska-Terzieva).

0303-8467/© 2015 Elsevier B.V. All rights reserved.

markers for the presence and activity of disease had begun long ago and still continues. The results concerning the predictive value of individual autoan- tibodies studied are not very encouraging at this stage [6–9]. The presence of IgG oligoclonal bands in cerebrospinal fluid is a well established and important predictive factor for conversion [4,10], but the role of autoantibodies against synthesized N-glycosylated peptides [11], anti-alpha-glucose-based glycan [12], myelin basic protein, and myelin oligodendrocyte protein [13], remains incon- clusive. Collagen type IV is a principal constituent of brain–blood barrier and inflammatory disruption of the blood–brain barrier is a major mechanism in MS [1]. We, therefore, decided to assess the levels

B. Sadarzanska-Terzieva et al. / Clinical Neurology and Neurosurgery 133 (2015) 30–33


of anti-collagen-type-IV serum antibodies (ACIVAbs) in patients with CIS and to determine their predictive value for conversion into MS. We also compared the ACIVAbs levels with some established immunological markers of inflammation, namely the proinflam- matory serum metalloproteinase type 9 and the anti-inflammatory tissue inhibitor of metalloproteinases type 1.

2. Patients and Methods

2.1. Patients

The study was carried out at a University Hospital and a spe- cialized medical practice. The hospital is the tertiary care center for a referral population of over 800,000 inhabitants. Primary and secondary care providers were encouraged to refer patients with CIS for participation in this study. Patients were recruited between January 1 and December 31, 2008. Forty patients, diagnosed with CIS according to McDonalds criteria 2005 gave informed consent to participate in the study. They included 27 females and 13 males, of mean age 34.85 ± 11.4 years old (range 16–58). Twenty-seven age- and sex-matched healthy controls also volunteered for this study. Patients were followed over 5 years by phone interviews, detailed history, and clinical examination, at least one time every 6 months. They were instructed to contact the investigators if any events, suspicious of relapse, occurred. MRI with contrast was performed at baseline and every 6 months during the follow-up period. The clinical and demographic characteristics of the patients are presented in Table 1.

2.2. Immunological Methods

Sera from patients were collected within 12 weeks (median 5 weeks, range 1–12) after the onset of symptoms. At that time they were untreated by steroids or disease-modifying therapies.

Table 1 Clinical and MRI characteristic of patients at baseline.


C-CIS (n = 32)

NC-CIS (n = 8)


Mean age Male - n (%) Initial symptoms Motor symptoms Sensory symptoms Brain-stem symptoms Cerebellar symptoms Visual symptoms Afferent

(sensory + visual) Efferent (motor + cerebellar) EDSS during first episode (at nadir) EDSS at 5 years MRI МRI 1–2 criteria of Barkhof МRI 3–4 criteria of Barkhof Number of initial МRI lesions > 9 Number of initial МRI lesions < 9

39.5 ± 8.33 13 (40.6)

32.5± 8.4


3 (37.5)


12 (37.5)

1 (12.5)


5 (15.63)

2 (25.0)




2 (25.0)


2 (6.25)

0 (0.0)

6 (18.75)

3 (37.5)




0 (0.0)



0 (0.0)


± 0.2

2.1 ± 0.18





± 1.3

0.25 ± 0.46















1 (12.5)




7 (87.5)





0 (0.0)


enhancing МRI


C-CIS=(clinically isolated syndrome converted to MS); NC-CIS=(clinically isolated syndrome not converted to MS); EDSS=Kurtzke scale of disability; n=number; P=level of significance; NS=non-significant difference; Kruskal–Wallis test for dis- tribution different from normal.

Indirect ELISA for testing anti-collagen type IV antibod- ies of IgG and IgМ class in human serum. Microtiter plates (Microlon U-bottom, high binding, Greiner Bio One, Frick- enhausen Germany) were sensitized respectively by human placental collagen type IV (Sigma) at a concentration of 10 g/ml in carbonate buffer pH 9.6, of 100 l per well. After incubation for 2 h at 37 C and overnight at 4 C, the wells were blocked with 0.1% bovine serum albumin and incubated for 1 h at 37 C. The tested serums diluted 1:40 in PBS–Tween, were dropped in 100 l per well and incubated for 1 h at 37 C. That was followed by stain- ing with drops of anti-human IgG or IgM peroxidase conjugate (Sigma) in dilution 1:16 000, and 1:20 000 respectively and incu- bation for 1 h at 37 C. As a colorimetric substrate it was 0.8 g/ml O-phenylenediamine (Sigma), dissolved in 0.05 M citrate buffer (pH 5.0) with 0.01% of Н 2 O 2 . The wells were washed three times with PBS–Tween 20, after each stage of testing. The reaction was stopped by adding 50 l 8 NH 2 SO 4 and the absorbance was measured on an automated MicroELISA Reader, at a wavelength of 490 nm. The following controls were used: (1) a substrate- control: in sensitized wells with collagen it was added only as buffer for sample dissolution, and colorimetric substrate; (2) conjugate control: peroxidase-conjugated antibody was added directly to the wells sensitized with an antigen; (3) negative con- trol to assess the specificity of the reaction: the antigen was replaced with solution of human serum albumin; (4) positive control: the tested sera were replaced with anti- -elastin or anti- collagen type IV polyclonal antibodies (Elastin Product Company, USA), diluted 1:2000 in buffer sera. All sera were tested three times and the mean value ± SE was calculated. In cases other than the normal distribution, the median values and 95 percentile (Р95) were used. For recording MMP-9 and TIMP-1 concentration it used a kit based on the principle of the sandwich ELISA (R&D systems, cat. No. DMP900, DTM100). According to the manufacturer’s instruc- tions, in each well of the plates 50 l tested serum was dropped, diluted 1:100 with calibrator diluent or standard in different con- centration doses for the construction of a standard straight line. After 2 h at room temperature and agitation on a shaker, the plates were washed three times with 300 l washing buffer per well. After the last wash, 200 l anti-MMP-9 or anti-TIMP-1 anti- body conjugated with peroxidase are added, and incubated for 1 h at room temperature on a shaker. They were washed again and 200 l of substrate solution was added to each well. That was incubated for 30 min at room temperature in the dark. The reac- tion was stopped with 50 l stop solution and the extinction was measured on an automated ELISA reader (“Ceres UV900C”—Bio- Tek Instruments Inc.) at 450 nm. From each tested serum or standard for all testing it was dropped in three wells and the arithmetic mean of extinction was determined. The concentra- tions of MMP-9 and TIMP-1 were calculated for each tested serum by a formula derived from the standard line that was constructed, based on the extinctions of the standards at different concentra- tions.

2.3. Statistical Analysis

The data from the study were presented as mean values (X) ± standard error of the mean (SE). The differences between the test groups were analyzed with ANOVA, as post hoc analysis by the method of the least significant difference (LSD) was made in the case of statistically significant differences and parametric distribu- tion of the data. In nonparametric distribution, the Kruskal–Wallis test was used. The correlations between the studied parameters were evaluated by the Pearson method. For all tests, a significance level of p < 0.05 was accepted. SPSS program for Windows–version 1.5 was used for the statistical data processing.


B. Sadarzanska-Terzieva et al. / Clinical Neurology and Neurosurgery 133 (2015) 30–33

Mean Plot of Var1 grouped by Var2

Х - Groups:





3- Controls

Y - ACIVAb IgG ng/ml

0,55 Var1: F(2;64) = 9,845; p = 0,0002; 0,50 KW-H(2;67) = 21,1451; p = 0,00003
F(2;64) = 9,845; p = 0,0002;
KW-H(2;67) = 21,1451; p = 0,00003
Raw Data
Х - Groups:
1. C - CIS
2. NC - CIS
3 - Controls
Y - ACIVAb IgG ng/ml

Fig. 1. Comparison of ACIVAb IgG serum levels in C–CIS (clinically isolated syndrome converted to MS), NC–CIS (clinically isolated syndrome non-converted to MS) and controls.

3. Results

During the observation period, 32 patients (80%, confidence interval 67.6–92.4% at p = 0.95) converted to MS. They represent the C-CIS cohort. The rest 8 patients (20%, confidence interval 7.6–32.4%) remained clinically stable, without any new symptoms or signs, or MRI activity. They form the NC-CIS cohort. The levels of IgM and IgG ACIVAbs in the serum in C-CIS, NC-CIS, and the control group were compared (Fig. 1). C–CIS patients had higher IgG ACIVAbs levels compared to NC-CIS (p = 0.041), while IgM levels did not differ significantly between C-CIS and NC–CIS cohorts (p = 0.44). The controls had significantly lower levels of both classes of antibodies, at a very high statistical level of significance (p < 0.0005). The prospective follow-up for 5 years (Table 2) showed that about 66% of patients with IgG ACIVAbs exceeding the 95th per- centile established in controls converted to MS. IgG ACIVAbs levels correlated positively to the activity of metal- loproteinases type 9 (r = 0.37; p = 0.003) and negatively with tissue inhibitor of metalloproteinases type 1 (r = 0.43; p = 0.0008). The antibody changes were independent of gender, age, mul- tifocal neurological symptoms, EDSS, number of MRI lesions, and baseline MMP type 2 (p > 0.05).

4. Discussion

This pilot study established that abnormally high levels of serum IgM and IgG ACIVAbs appear during a first demyelinating episode in more than half of the patients. However, the acute phase IgM is increased in both C-CIS and NC-CIS groups and has no prognostic

Table 2 Conversion to MS according to ACIVAb baseline serum levels.

Serum levels

C-CIS (n = 32)

NC-CIS (n = 8)

Controls (n = 27)



56.3% (18 patients)

37.5% (3 patients)

92.6% (25 persons)



43.7% (14 patients)

62.5% (5 patients)

7.4% (2 persons)




34.4% (11 patients)

75.0% (6 patients)

96.3% (26 persons)



65.6% (21 patients)

25.0% (2 patients)

3.7% (1 person)


ACIVAb IgG, ACIVAb IgM=(anti-collagen type IV antibodies of IgG and IgM class); C-CIS=(clinically isolated syndrome converted to MS); NC-CIS=(clinically isolated syndrome non converted to MS); no statistical difference between C-CIS and NC-CIS for IgM (p = 0.44); significant difference between C-CIS and NC-CIS for IgG (p = 0.041; Fisher exact probability test).

value regarding conversion. On the contrary, IgG ACIVAbs correlate with increased risk of conversion to MS and are significantly more frequent in C-CIS patients. To the best of our knowledge, this is the first report on the subject. Collagen type IV together with proteoglycans and glycoproteins is a major component of basal membranes of almost all tissues [14]. This fibrillary protein may be defragmented due to vari- ous pathological processes, and the products of its destruction like collagen type IV degradation peptides (CIVDP) appear in the serum [15,16]. In some cases, the destruction is caused by anti- collagen autoantibodies directed against antigenic determinants of

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the intact collagen [17–19], as observed in certain autoimmune dis- eases [20–22]. In another scenario, the synthesis of antibodies is an epiphenomenon, following initial collagen destruction through other mechanisms and release of immunogenic CIVDP. In recent years, it was found that serum levels of IgG ACIVAbs correlate pos- itively with the development of microvascular complications of diabetes mellitus with diabetic nephropathy, retinopathy [23,24], and polyneuropathy [25]. Some antigenic determinants provoking humoral response to collagen type IV have been identified [26]. Autoimmune process in MS is considered to start in the periph- eral lymph tissues, and then the pathological process is transferred in the central nervous system [4,27,28]. The immune cells penetrate through blood–brain barrier releasing matrix metalloproteinases [29], among which collagenase plays an important role, causing collagen fragmentation and the appearance of CIVDP in the serum. They are a powerful stimulus for synthesis of anti-collagen anti- bodies [30], which in turn may further destroy collagen fibers and deteriorate blood–brain barrier function. Our results demonstrate the presence of elevated IgM and IgG anti-collagen antibodies in patients with CIS. They most likely reflect the mechanism described above (as an epiphenomenon fol- lowing the release of CIVDP after destruction of the blood–brain barrier). This conjecture is confirmed by the positive correlation with metalloproteinases type 9 levels and the inverse one with their tissue inhibitor. The IgM are equally distributed among C-CIS and NC-CIS patients and correspond to the transient acute phase response. The elevated IgG titers in patients prone to convert to MS but not in NC-CIS may indicate a tendency for chronic inflammation and further reactivation. One disadvantage of this study is the relatively small num- ber of patients. Another shortcoming is that the majority of our patients had multifocal CIS with multiple demyelinating lesions; they belong to a group with higher risk for conversion to MS and might be not entirely representative of the general CIS patient population. We do not have an explanation for this clustering of patients; closest to mind is some referral bias, whereby persons with mildest presentations were not referred by the primary or secondary care levels. On the other hand, involving the patients with highest risk of conversion focuses the study on a cohort of particular interest. Indeed, nowadays many of our patients with multifocal CIS would be started on treatment. At the time, in 2008, the National Guidelines in the country of origin did not support such an approach. We see the strengths of our study in its prospective nature, the relatively long observation period, high level of compliance (we did not lose a single patient from follow up). We also followed strictly internationally accepted criteria in their modifications [8]. Finally, the statistical levels of significance show quite strong trends, imply- ing clinical significance to the statistical as well. Aside from the possible implications regarding MS pathogen- esis, our findings, if replicated and confirmed in larger patient cohorts, might have therapeutic significance, providing a marker of continuing CNS inflammation in patients with CIS and an addi- tional criterion for starting disease-modifying treatment. However, to this end we should provide for specificity of the marker, either by looking for ACIVAbs in the CSF or by combining it with other serum markers of blood–brain barrier disruption. In conclusion, patients with CIS who later convert to MS have abnormally high levels of IgG ACIVAbs. These antibodies deserve further study as possible biomarkers predicting disease progression in CIS, in patients with radiologically isolated syndrome (RIS) and in MS with different disease course.



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