Clinical Neurology and Neurosurgery 133 (2015) 3033

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Clinical Neurology and Neurosurgery

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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
a

Department of Neurology and Neurosurgery, University Hospital of Pleven, Georgi Kochev st 8A, Pleven 5800, Bulgaria
Department of Clinical Neurophysiology, Sandringham level 1, Leicester Royal Inrmary, 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., Soa 1309, Bulgaria
f
Department of Biology, Medical University of Pleven, Kliment Ohridski St. 1, Pleven 5800, Bulgaria
g
Department of Psychology, Afliate Prof Ivan Mitev of Vratza, Medical University of Soa, School Complex 1, Vratza 3000, Bulgaria
h
Department of Clinical Laboratory, University Hospital of Pleven, Georgi Kochev St. 8A, Pleven 5800, Bulgaria
b

a r t i c l e

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
Keywords:
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 clinically 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 1658 years) were compared to those of 27 gender- and age-matched
healthy controls. ACIVAbs were quantied 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 signicantly 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 denitive multiple sclerosis (MS) is important in patient
management [13]. While advances in imaging have contributed
to dene early treatment strategies, the approach in many cases
remains debatable [46]. Autoimmune inammation 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: tzvetanovplamen@hotmail.com (B. Sadarzanska-Terzieva).
http://dx.doi.org/10.1016/j.clineuro.2015.03.011
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 autoantibodies studied are not very encouraging at this stage [69]. The
presence of IgG oligoclonal bands in cerebrospinal uid 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 inconclusive.
Collagen type IV is a principal constituent of brainblood barrier
and inammatory disruption of the bloodbrain 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) 3033

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 inammation, namely the proinammatory serum metalloproteinase type 9 and the anti-inammatory
tissue inhibitor of metalloproteinases type 1.
2. Patients and Methods
2.1. Patients
The study was carried out at a University Hospital and a specialized 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 1658). 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 112) 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.

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 rst
episode (at nadir)
EDSS at 5 years
MRI
RI 12 criteria of
Barkhof
RI 34 criteria of
Barkhof
Number of initial
RI lesions > 9
Number of initial
RI lesions < 9
Gadoliniumenhancing RI
lesions

C-CIS (n = 32)

NC-CIS (n = 8)

39.5 8.33
13 (40.6)

32.5 8.4
3 (37.5)

0.04
NS

12 (37.5)
5 (15.63)
3 (9.38)

1 (12.5)
2 (25.0)
2 (25.0)

0.071
NS
NS

2 (6.25)
6 (18.75)
2 (6.25)

0 (0.0)
3 (37.5)
0 (0.0)

NS

2 (6.25)

0 (0.0)

3.1 0.2
(2.54.0)
3.0 1.3
(0.05.5)
24 (75)
8 (25)

2.1 0.18
(2.02.5)
0.25 0.46
(0.01.0)
6 (80)
0 (0.0)

<0.001

26 (81.25)

1 (12.5)

0.0002

6 (8.75)

7 (87.5)

0.0002

5 (63.6)

0 (0.0)

0.001

<0.0001

31

Indirect ELISA for testing anti-collagen type IV antibodies of IgG and Ig class in human serum. Microtiter plates
(Microlon U-bottom, high binding, Greiner Bio One, Frickenhausen 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 PBSTween, were dropped in 100 l per
well and incubated for 1 h at 37 C. That was followed by staining with drops of anti-human IgG or IgM peroxidase conjugate
(Sigma) in dilution 1:16 000, and 1:20 000 respectively and incubation 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 2 O2 . The wells were washed three times
with PBSTween 20, after each stage of testing. The reaction
was stopped by adding 50 l 8 NH2 SO4 and the absorbance was
measured on an automated MicroELISA Reader, at a wavelength
of 490 nm. The following controls were used: (1) a substratecontrol: 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 control to assess the specicity 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 anticollagen 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
(95) 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 manufacturers instructions, in each well of the plates 50 l tested serum was dropped,
diluted 1:100 with calibrator diluent or standard in different concentration 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 antibody 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 reaction was stopped with 50 l stop solution and the extinction was
measured on an automated ELISA reader (Ceres UV900CBioTek 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 concentrations 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 concentrations.

0.069

2.3. Statistical Analysis

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 signicance; NS=non-signicant difference; KruskalWallis test for distribution different from normal.

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 signicant difference (LSD) was made in the
case of statistically signicant differences and parametric distribution of the data. In nonparametric distribution, the KruskalWallis
test was used. The correlations between the studied parameters
were evaluated by the Pearson method. For all tests, a signicance
level of p < 0.05 was accepted. SPSS program for Windowsversion
1.5 was used for the statistical data processing.

32

B. Sadarzanska-Terzieva et al. / Clinical Neurology and Neurosurgery 133 (2015) 3033

Mean Plot of Var1 grouped by Var2

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

0,50
0,45

Y - ACIVAb IgG ng/ml

0,40
0,35
0,30
0,25
0,20
0,15
0,10
0,05
Median
1%-99%
Raw Data
Outliers
Extremes

0,00
1

- Groups: 1. C - CIS 2. NC - CIS 3 - Controls

Fig. 1. Comparison of ACIVAb IgG serum levels in CCIS (clinically isolated syndrome converted to MS), NCCIS (clinically isolated syndrome non-converted to MS) and
controls.

3. Results
During the observation period, 32 patients (80%, condence
interval 67.692.4% at p = 0.95) converted to MS. They represent
the C-CIS cohort. The rest 8 patients (20%, condence interval
7.632.4%) 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). CCIS patients had
higher IgG ACIVAbs levels compared to NC-CIS (p = 0.041), while
IgM levels did not differ signicantly between C-CIS and NCCIS
cohorts (p = 0.44). The controls had signicantly lower levels of both
classes of antibodies, at a very high statistical level of signicance
(p < 0.0005).
The prospective follow-up for 5 years (Table 2) showed that
about 66% of patients with IgG ACIVAbs exceeding the 95th percentile established in controls converted to MS.
IgG ACIVAbs levels correlated positively to the activity of metalloproteinases 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, multifocal 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 rst 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
ACIVAb IgM
<95th
Percentile
>95th
Percentile
ACIVAb IgG
<95th
percentile
>95th
percentile

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); signicant 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 signicantly more
frequent in C-CIS patients. To the best of our knowledge, this is the
rst 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 brillary protein may be defragmented due to various 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 anticollagen autoantibodies directed against antigenic determinants of

B. Sadarzanska-Terzieva et al. / Clinical Neurology and Neurosurgery 133 (2015) 3033

the intact collagen [1719], as observed in certain autoimmune diseases [2022]. 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 positively 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 identied [26].
Autoimmune process in MS is considered to start in the peripheral lymph tissues, and then the pathological process is transferred
in the central nervous system [4,27,28]. The immune cells penetrate
through bloodbrain 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 antibodies [30], which in turn may further destroy collagen bers and
deteriorate bloodbrain barrier function.
Our results demonstrate the presence of elevated IgM and IgG
anti-collagen antibodies in patients with CIS. They most likely
reect the mechanism described above (as an epiphenomenon following the release of CIVDP after destruction of the bloodbrain
barrier). This conjecture is conrmed 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 inammation
and further reactivation.
One disadvantage of this study is the relatively small number 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 modications [8]. Finally,
the statistical levels of signicance show quite strong trends, implying clinical signicance to the statistical as well.
Aside from the possible implications regarding MS pathogenesis, our ndings, if replicated and conrmed in larger patient
cohorts, might have therapeutic signicance, providing a marker
of continuing CNS inammation in patients with CIS and an additional criterion for starting disease-modifying treatment. However,
to this end we should provide for specicity of the marker, either
by looking for ACIVAbs in the CSF or by combining it with other
serum markers of bloodbrain 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.

33

References
[1] Miller DH, Barkhof F, Montalban X, Thompson A. Clinically isolated syndromes
suggestive of MS: part 1 natural history, pathogenesis, diagnosis and treatment.
Lancet Neurol 2005;4:2818.
[2] Garcia-Merino A, Blasco M. Conrming the MS diagnosis. Int MS J
2007;14:5863.
[3] Hutchinson M. Predicting and preventing the future: active managing multiple
sclerosis. Pract Neurol 2009;9:13343.
[4] Miller DH, Chard DT, Cicarelli O. Clinically isolated syndromes review. Lancet
Neurol 2012;11:15769.
[5] Bunyan RF, Pittock JS. Do not treat from CIS onset: evaluate disease course and
prognosis rst-yes. Mult Scler J 2012;18:3913.
[6] Freedman M. Do not treat from CIS onset: evaluate disease course and prognosis
rst-No (treat!). Mult Scler J 2012;18:3945.
[7] Bielekova B, Martin R. Development of biomarkers in multiple sclerosis. Brain
2004;127:146378.
[8] Polman CH, Reingold SC, Banwell B, Clanet M, Cohen JA, Filippi M, et al. Diagnostic criteria for multiple sclerosis: 2010 revision to the McDonald criteria.
Ann Neurol 2011;69:292302.
[9] Kuhle J, Pohl C, Mehling M, Edan G, Freedman M, Hartung H, et al. Lack of association between antimyelin antibodies and progression to multiple sclerosis. N
Engl J Med 2007;356:3718.
[10] Bennett JL, Haubold K, Ritchie AM, Edwards SJ, Burgoon M, Shearer AJ, et al. CSF
IgG heavy-chain bias in patients at the time of clinically isolated syndrome. J
Neuroimmunol 2008;199:12632.
[11] Lolli F, Lolli B, Carotenuto A. An N-glucosylated peptide detecting diseasespecic autoantibodies, biomarkers of multiple sclerosis. Proc Natl Acad Sci
U S A 2005;102:102738.
[12] Freedman M, Laks J, Dotan N, Altstock R, Dukler A. Anti-alphaglucose-based
glycan IgM antibodies predict relapse activity in multiple sclerosis after the
rst neurological event. Mult Scler 2009;15:42230.
[13] Berger T, Rubner P, Schautzer F. Antimyelin antibodies as a predictor of clinically denite multiple sclerosis after a rst demyelinating event. N Engl J Med
2003;349:13945.
[14] Fauri G. Function structural relationship of elastic arteries in evolution: from
microbrils to elastin and elastic bers. Pathol Biol 2001;49(4):31025.
[15] Nicoloff G, Baydanoff S, Stanimirova N. Serum collagen type IV in healthy
children and children with insulin-dependent diabetes mellitus. Clin Appl
Immunol Invest 2001;1:1658.
[16] Risteli L, Risteli J. Analysis of extracellular matrix proteins in biological uids.
Methods Enzymol 1987;145:391411.
[17] Susumu T, Uchugu T, Shoichi T. Measurement of serum anti-type IV collagen
antibody in diabetic patients. Annals of Gunma University School of Health
Sciences 2003;24:8791.
[18] Menge T, Lalive PH, von Budingen HC, Cree B, Hauser SL, Genain CP. Antibody
responses against galactocerebroside is potential stage-specic biomarkers in
multiple sclerosis. J Allergy Clin Immunol 2005;116:4539.
[19] Yang CL, Brinckmann J, Rui F, Vehring K. Autoantibodies to cartilage collagens
in relapsing polychondritis. Arch Dermatol Res 1993;285(5):2459.
[20] Moreland L, Gay R, Gay S. Collagen autoantibodies in patients with vasculitis and systemic lupus erythematosus. Clin Immunnopathol 1991;60(3):
4128.
[21] Gyorgy B, Tothfalusi L, Nagy G. Natural auto antibodies reactive with glycosaminoglycans in rheumatoid arthritis. Arthritis Res Ther 2008;10:R10.
[22] Colburn K, Langa-Sharif E, Kely G, Malto M, Sandberg L, Baydanoff S, et al.
Abnormalities of serum anti-elastin antibodies in connective tissue disease. J
Investig Med 2003;51(2):1049.
[23] Nicoloff G, Baydanoff S, Petrova Ch, Christova P. Serum antibodies to collagen type IV and development of diabetic vascular complications in children
with type 1 (insulin-dependent) diabetes mellitus. A longitudinal study. Vascul
Pharmacol 2002;38(3):1437.
[24] Nicoloff G, Baydanoff S, Stanimirova N, Petrova C, Christova P. Detection of
serum collagen type IV in children with type 1 (insulin-dependent) diabetes
mellitusa longitudinal study. Pediatr Diabetes 2001;2(4):18490.
[25] Nicoloff G, Valcova D, Baydanoff S. Specicity and age-related changes
of antibodies to collagen type I in normal human sera. Autoimmunity
1994;17(3):2578.
[26] Mahieu PM, Lambert PH, Maghuin-Rogister GR. Primary structure of a small glycopeptide isolated from human glomerular basement membrane and carrying
a major antigenic site. Eur J Biochem 1973;40:599606.
[27] Steinman L. Multiple sclerosis: a two-stage disease. Nat Immunol
2001;2:7624.
[28] Chitnis T. The role of CD4T cells in the pathogenesis of multiple sclerosis. Int
Rev Neurobiol 2007;79:4372.
[29] Rozenberg G. Matrix metalloproteinases and neuroinamation in multiple sclerosis. Neuroscience 2002;8:58693.
[30] Van Horssen J, B L, Dijkstra CD, de Vries HE. Extensive extracellular matrix
depositions in active multiple sclerosis lesions. Neurobiol Dis 2006;24:
48491.