Corresponding author:

Petra Nytrova, MD

Dpt. of Neurology and Center for Clinical Neuroscience, First Medical Faculty, Charles

University in Prague,

Katerinska 30, 120 00 Praha 2, Czech Republic

Tel. + 420 2 2496 6422

Fax: + 420 2 2491 7907

e-mail:petranytrova@yahoo.com

Complement activation in patients with Neuromyelitis optica.

Petra Nytrova1,2, Eliska Potlukova3, David Kemlink1, Mark Woodhall2, Dana Horakova1,

Patrick Waters2, Eva Havrdova1, Dana Zivorova4, Angela Vincent2, Marten Trendelenburg5

1Department of Neurology and Center of Clinical Neuroscience First Faculty of Medicine,

General University Hospital and First Faculty of Medicine, Charles University in Prague,

Czech Republic

2Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK

3Third Department of Medicine, General University Hospital and First Faculty of Medicine,

Charles University in Prague, Czech Republic

4Laboratory of Clinical Immunology, Institute of Clinical Biochemistry and Laboratory

Diagnostics, General University in Prague, Czech Republic

5Laboratory of Clinical Immunology, Department of Biomedicine, University Hospital

Basel, Switzerland

Abstract

The role of complement has been demonstrated in experimental models of neuromyeltis

optica (NMO), however, only few studies have analysed complement components

longitudinally in NMO patients. We measured serum or plasma concentrations of anti-C1q

antibodies and complement split products C3a, C4a and soluble C5b-9 in patients with NMO,

multiple sclerosis and healthy controls. NMO patients had higher levels of C3a and anti-C1q

antibodies than healthy controls. C3a levels correlated with disease activity, neurological

disability and aquaporin-4 IgG in NMO patients suggesting a role of the alternative pathway

of complement in the pathogenesis of NMO and supporting the strategy of therapeutic

complement inhibition.

Keywords: neuromyelitis optica, complement, aquaporin-4 IgG, C1q antibodies

1. Introduction

Neuromyelitis optica (NMO), also referred to as Devic´s disease, is a rare, severely disabling

inflammatory disorder of the central nervous system (CNS), usually with a relapsing-

remitting course (Wingerchuk et al., 1999). This autoimmune disease predominantly affects

the optic nerves and spinal cord. NMO is associated with serum antibodies against aquaporin-

4 (also known as NMO-IgG or AQP4-IgG) in up to 80% of cases (Lennon et al., 2004;

Waters et al., 2008).

The pathogenesis of NMO seems to be closely linked to the activation of the complement

system. In experimental models, AQP4-IgG binding to AQP4 causes cytotoxicity only in the

presence of complement (Hinson et al., 2007; Saadoun et al., 2010) suggesting that this is

crucial for pathogenicity in NMO. In addition, recent studies reported that exacerbations of

NMO were reflected by changes in concentrations of complement activation products in sera

(C4d) as well as in the cerebrospinal fluid (C5a) (Kuroda et al., 2012; Tuzun et al., 2011).

Furthermore, although Veszeli et al. did not find substantial systemic complement activation

in NMO patients without disease activity, the complement system was found to be

abnormally affected even during remission (Veszeli et al., 2014). Eculizumab, a therapeutic

monoclonal IgG that neutralises the complement protein C5, has effectively reduced the

relapse frequency of NMO in open label trial (Pittock et al., 2013).

AQP4 is a water channel that is most abundantly expressed in the processes of astrocytes. Its

monomers assemble as tetramers. These AQP4 tetramers aggregate in cell plasma membranes

to form supramolecular clusters which are targeted by AQP4-IgG. These clustered AQP4-IgG

are crucial for complement-dependent cytotoxicity through multivalent C1q binding

(Papadopoulos et al., 2013). AQP4-IgG are mainly of the IgG1 subclass that usually

activates the classical pathway of complement (Waters et al., 2008). Formation of

AQP4:AQP4-IgG immune complexes, with subsequent complement-mediated destruction of

astrocytes, is paralleled by loss of AQP4 and GFAP staining in perivascular lesions in

histological sections. This loss is localised in the same areas that exhibit accumulation of

eosinophils and plasma cells as well as vasculocentric deposits of immunoglobulins and

products of complement activation (Jarius et al., 2008; Lucchinetti et al., 2002; Roemer et al.,

2007). These characteristics also resemble the histopathological features of murine brain

lesions induced by injection of human AQP4-IgG with human complement into the brain

(Saadoun et al. 2010).

Autoantibodies against complement C1q (anti-C1q) have been found in a number of

autoimmune diseases (Potlukova et al., 2008; Siegert et al., 1999; Trendelenburg, 2005;

Wisnieski et al., 1992). Most interestingly, anti-C1q were found in more than 97% of patients

with biopsy-proven active lupus nephritis supporting the hypothesis of a pathogenic role of

anti-C1q in systemic lupus erythematosus (Trendelenburg et al., 2006). In other autoimmune

diseases, the role of anti-C1q remains unclear. Interestingly, C1q-targeting monoclonal

antibodies prevented complement-mediated damage in animal models of NMO (Phuan et al.,

2013), may have a possible role in regulation of the immune response, and providing a proof-

of-concept for C1q-targeted monoclonal antibody therapies in NMO.

In contrast to NMO, studies on complement activation in multiple sclerosis (MS) led to more

conflicting results. As a consequence, complement activation is generally not considered to

be of crucial pathogenic relevance in MS (Brink et al., 2005; Sellebjerg et al., 1998; Urich et

al., 2006). The role of anti-C1q in MS remains to be determined.

The aims of our study were to elucidate the role of complement activation and anti-C1q in

patients with NMO by means of serological assessments during the relapse and remission of

disease and to evaluate the potential use of plasma complement parameters as biomarkers of

disease activity NMO. To support our observations we assessed complement binding to

AQP4-IgG on frozen primate optic nerve.

2. Materials and Methods

2.1. Patients

The study was designed as a prospective cohort study in patients with demyelinating

disorders at the Multiple Sclerosis Centre of the Department of Neurology, General

University Hospital in Prague in 2010-2011. NMO and MS patients were included to the

study. At inclusion, time of relapse and at 6-months follow-up, clinical data were recorded

and serum as well as plasma samples were collected.

The diagnosis of NMO was based on the Wingerchuk’s diagnostic criteria (Wingerchuk,

2007). Plasma (n=43) and serum samples (n=43) were analysed from 19 AQP4-IgG positive

patients. In eight patients with NMO, we obtained three or more samples at different time

points (range 2 weeks to 6 months). For the comparison of all groups, only first sample value

of individual patient was included for statistical analysis. All forty-three AQP4-IgG positive

samples were used for intragroup NMO analysis and correlations. The plasma samples were

used for complement breakdown product assessment and the serum samples for C1q and

AQP4 antibody measurement.

Thirty five MS patients were included in the study for measurement of complement

activation products. Furthermore, for the analysis of anti-C1q, we used additional 41 serum

samples of different MS patients (in total, 76 serum samples were frozen in 2010). All MS

patients fulfilled the McDonald's criteria for MS relapse remitting (n=64), secondary (n=9) or

primary progressive form (n=3) (Polman et al., 2005).

Patient-reported symptoms or objectively observed signs typical of an acute inflammatory

demyelinating event in the CNS with duration of at least 24 hours, in the absence of fever or

infection, were considered as a relapse (Polman et al., 2005). Samples were taken during both

remission and relapse. Samples of the NMO or MS patients in relapse were obtained prior to

initiation of treatment by high-dose methylprednisolone, or plasma exchange. Samples of

patients treated with intravenous immunoglobulins (IVIG), cyclophosphamide, natalizumab

and rituximab were drawn before regular medicament infusion.

All patients were of Caucasian origin except two NMO female patients from Asia. Patients

were observed by a specialist in demyelinating disorders of the CNS. Conventional brain and

spinal cord MRI were used for establishment of diagnosis and evaluation of spinal cord

involvement. Neurological disability was evaluated by Kurtzke expanded disability status

scale (EDSS) (Kurtzke, 1983). As controls, 40 volunteers without signs of an autoimmune

disorder or infection at the time of blood sampling were included; most of them worked as

health care professionals.

Blood samples were collected by venepuncture in the collection tubes with EDTA. The serum

and plasma samples were centrifuged at 3000 rpm for 10 minutes and frozen within forty five

minutes after centrifugation and aliquoting. The samples were stored at -80oC and not thawed

until assessment for complement split products and antibodies against AQP4 and C1q.

All subjects gave an informed consent to the procedures, which were approved by the Ethical

Committee of the General University Hospital and the First Faculty of Medicine, Charles

University in Prague.

2.2. Serological analysis

2.2.1. Serum antibodies against aquaporin-4 and complement C1q

All samples were tested in a blinded manner by a commercially available

immunofluorescence cell-based assay (CBA) using recombinant human M1-AQP4

(Euroimmun, Lübeck, Germany) as antigen following the manufacturer’s instructions. Sera

of 19 NMO AQP4-IgG positive patients were retested by an in-house CBA using human

M23-AQP4 expressed on live cells, and serial dilutions to determine the end-point titres. Sera

of all MS and healthy controls were AQP4-IgG negative. Anti-C1q were measured in serum

using a commercially available ELISA kit (Bühlmann Laboratories, Schönenbuch,

Switzerland). In this assay, undigested purified human C1q served as the antigen, and sera

were diluted and incubated in a high-salt buffer (1M NaCl). The optical densities were

measured at 450 nm and converted into international units per ml (IU/ml) using the standards

provided by the manufacturer. The manufacturer’s cut-off of 15 IU/ml was used to

determined positivity.

2.2.2. Assays for complement split products

The soluble terminal complement complex (sC5b-9) as well as C3a and C4a fragments of

complement proteins were measured by commercially available enzyme immunoassays

(MicroVue™SC5b-9 Plus EIA Kit, MicroVue™ C3a Plus EIA Kit, MicroVue™ C4a Plus

EIA Kit; Quidel, San Diego, USA) following the manufacturer’s instructions. Briefly, test

specimens were added to microassay wells precoated with specific monoclonal antibodies to

each of the complement split products used for their capturing. Horseradish peroxidase

conjugated antibodies to different epitopes of the same antigen were added to each test well.

Following addition of a chromogen, the plates were measured spectrophotometrically. The

reference ranges for the complement split products for healthy individuals stated by the

manufacturer were 33.8 – 268.1 ng/ml (mean 129.6 ng/ml) for C3a and 383.5 – 8168.2 ng/ml

(mean 1694.7 ng/ml) for C4a. The reference values for sC5b-9 assay were not provided by

the manufacturer.

Total C3 and C4 serum levels in NMO patients were measured by routine nephelometry

(Dade Behring, Vienna Austria).

2.3. Immunohistochemistry - indirect immunofluorescence

Fourteen serum samples from NMO patients and 20 healthy blood donors were tested on

multiplex biochip based slides (Euroimmun). Each field, that is screened with an individual

serum, consisted of four individual chips with fixed frozen tissue (rat hippocampus, primate

optic nerve, primate and rat cerebellum) and four individual chips with HEK cells transfected

with human M1-AQP4, LGI1, CASRP2 and GlyR-alpha1, giving a total of 8 biochips per

field. Phosphate-buffered saline (PBS) containing 5% Tween-20 was used for samples

dilution. For each serum (diluted 1:10), indirect immunofluorescence was carried out on

individual chips: A) diluted sample / FITC-conjugated anti-human IgG (Euroimmun), B)

diluted sample / serum of blood donors as complement source (Euroimmun)/FITC-

conjugated anti-complement antibodies [polyclonal rabbit anti-human C1q (Dako, Glostrup,

Denmark), polyclonal rabbit anti-human C3c (Euroimmun), polyclonal rabbit anti-human

C4c (Dako)]. Since C3b and C4b were progressively degraded, we used anti-C3c and

anti-C4c antibodies for immunohistochemistry analysis. The lyophilized complement source

(Euroimmun) from 10 healthy donors was resuspended in sterile water prior using. The

patterns were visually interpreted by fluorescence microscopy (Zeiss LSM 700 Confocal,

Carl Zeiss Microscopy GmbH, Jena, Germany).

2.4. Statistical analysis

Statistical intergroup analyses were conducted by Kruskal Wallis non-parametric ANOVA

and its post hoc multiple comparison z-test. For correlation statistics we used the Spearman’s

test. For NMO group comparisons we used Mann-Whitney non-parametric tests. Tests were

performed with the help of StatSoft, Inc. (2011) STATISTICA (Statsoft, Tulsa, OK, USA),

version 10 (www.statsoft.com) and GraphPad PRISM 5. For the construction of receiver

operating characteristics (ROC) and its statistics we used the program JROCFIT1.0.2

(www.jrocfit.org). Differences were considered as being statistically significant in case of p <

0.05.

3. Results

3.1. Clinical features and treatment

Nineteen patients with NMO were included in our study. Ten NMO patients had active

disease (defined as ≥1 relapse during the last 6 months prior to sample collecting) and nine

were inactive (relapse-free during the period of 6 months). In eight patients with NMO, we

obtained three or more plasma samples at different time points ranging from 2 weeks to 6

months from the first.

The majority of patients had long histories and had been treated with variable regimens. Six

NMO patients were treated by a combination of drugs, i.e. low dose of prednisone with

azathioprine. Monotherapy was used in 10 patients (low dose of prednisone, mycophenolate

mofetil, cyclophosphamide, rituximab). One female patient underwent autologous stem cell

transplantation three months before samples could be collected. Two patients were without

treatment.

MS patients were treated by a monotherapy (natalizumab, interferon beta, prednisone, IVIG,

mycophenolate mofetil, cyclophosphamide) or received a combination of drugs (interferon

beta and prednisone or azathioprine or methotrexate). Eleven MS patients were without

treatment.

Demographic data of NMO patients, MS patients, and healthy controls are summarised in

Table 1.

3.2. Complement activation products analysis

Plasma levels of complement activation products differed substantially between groups (Fig.

1A-C). Both NMO and MS patient had significantly higher levels of C3a and sC5b-9 and

lower levels of C4a compared with controls. However, patients with NMO had lower levels

of C4a than the MS patients (p < 0.05). Serum levels of anti-C1q were higher in the NMO

group compared to both MS and controls (Fig. 1D). Surprisingly, the MS patients had lower

anti-C1q levels than controls.

3.3. Correlation of complement activation products with disease activity

In NMO patients (Table 2) there was a significant correlation between EDSS score and C3a

or the ratio C3a:C3 (Fig. 2A). This correlation was as strong as the correlation between

EDSS score and titres of AQP4-IgG (Fig. 2B). In addition, plasma levels of C3a in NMO

patients with clinically active disease were significantly higher than in NMO patients with

inactive disease (Fig. 3A), and C3a levels were higher during relapses than during remission

periods (Fig. 3B). Using ROC curves, the best cut-off for the distinction between patients

with active disease compared to those who were inactive was 500 ng/ml with a sensitivity of

94.7% and a specificity of 90.3% (Fig. 4). However, we did not find any correlation between

C3a levels and the time to the next relapse after plasma sampling.

In contrast to C3a, the other complement split products and anti-C1q did not correlate with

disease activity. We also did not find any correlation between complement split products or

C1q antibodies and the clinical activity or severity of disease including EDSS score in MS

patients.

3.4. Immunohistochemical studies

Using diluted patients´ sera and FITC-conjugated anti-human IgG, all patients with NMO

were found to be AQP4-IgG positive, showing weak reactivity with optic nerve and

cerebellum, but strong membrane reactions to the AQP4-expressing recombinant cells which

have high AQP4 expression (Fig.5 row I-III: B). All control sera remained negative (Fig.5

row I-III: A).

To assess complement activation by the antibodies, patients’ sera together with a complement

source and FITC-conjugated anti-human complement (anti-human C1q, anti-human C3c,

anti-human C4c, in separate assays) were applied to the cells and the tissue sections. First, as

a positive control, the recombinant AQP4 expressing cells were tested in this three-step

staining procedure (Fig.5, row I: C-E). All NMO sera but none of the controls produced

fluorescent signals on the AQP4-expressing cells indicating complement activation by the

bound human antibodies. The primate optic nerve transverse sections fluoresced in a

mesh-like staining pattern (Fig. 5, row II: C-E) corresponding to the glial supporting

meshwork that separates the optic nerve fibers. The cerebellum exhibited mainly membrane

staining, particularly in the granule cell layers of cerebellum (Fig. 5, row III: C-E).

4. Discussion

Previous studies have implicated complement-activating IgG1 AQP4 antibodies in the

pathogenesis of NMO, but these have concentrated on the classical pathway. Here we provide

clinical, serological and immunohistochemical data suggesting that activation of C3 is

implicated in the pathogenesis of NMO, and might provide a biomarker of disease activity.

Moreover, by immunofluorescence staining we show that patients´ AQP4-IgG form immune

complexes, including C1q, C3c and C4c, not only on AQP4 transfected cells but also on

native neuronal tissues when normal human serum is added as a source of complement. The

staining pattern was similar to that described by Waters et al. on mouse brain tissue sections

(Waters et al., 2008). Based on the similar staining patterns observed for AQP4 and

AQP4:AQP4-IgG complexes and complement deposits, particularly C3c, we conclude that

AQP4-IgG binds to brain tissue AQP4 with subsequent complement activation of the

alternative pathway as well as the better classical pathway.

We did not find any correlation between C3a and sC5b-9 levels, which might suggest a more

complex mechanism of astrocyte damage including the possibility of C3a-mediated

recruitment of eosinophils and neutrophils into NMO lesions. C3a is proinflammatory

mediator and anaphylotoxin with immune and non-immune biological functions. For

instance, C3a is involved in excitotoxicity-mediated neuronal death through astrocytes

stimulation (van Beek et al., 2001). Receptor for C3aR is expresses by

monocytes/macrophages, microglia, astrocytes, neurons and endothelial cell too (Davoust et

al., 1999; Ischenko et al., 1998; Klos et al., 1992). Interestingly, C3a has been shown to

induce an increase in interleukin 6 (IL-6) mRNA expression by astrocyte cell lines (Jauneau

et al., 2003; Sayah et al., 1999), which is in line with the observation that cerebrospinal fluid

concentrations of IL-6 are increased during the initial attack of NMO (Uzawa et al., 2013).

Low C4a levels as observed in our NMO patients might be the result of complement

dysregulation and/or the more complex interplay between complement-activating AQP4-IgG

and anti-C1q. The biological function of C4a in NMO remains more speculative but might be

linked to regulatory properties of the fragment. Recombinant C4a has been demonstrated to

inhibit C3a and C5a-stimulated degranulation of mast cells (Xie et al., 2012). In addition, an

anti-inflammatory role of C4a in glomerulonephritis has been described (Welch et al., 2001).

Considering that the lack of early components of the classical pathway of complement (C1q

and C4) are associated with systemic autoimmunity (Pickering et al., 2000), these

components might also have a protective effects in NMO.

To date, only a few analyses of complement split products in plasma/serum or cerebrospinal

fluid have been reported and led to conflicting results. Tuzun et al. described an increased

activity of the classical pathway during relapses of NMO by measuring the levels of

breakdown products for the classical (C4d), alternative (fragment Bb) and terminal

complement (sC5b-9) pathways (Tuzun et al., 2011). Moreover, they described a negative

correlation between EDSS and levels of C4d, fragment Bb and sC5b-9. The differing results

of our study could partially be explained by the use of different assays, by different sampling

time points, differences in the accompanying treatment of the patients and the assessment in

sera of AQP4-IgG negative NMO patients. More in line with our findings is the report

demonstrating significantly elevated C5a levels in the cerebrospinal fluid of NMO patients, in

particular in patients with multiple enhanced lesions on MRI (Kuroda et al., 2013).

Independent of the complement activation products, we found that anti-C1q Abs levels were

significantly higher in NMO patients as compared to the MS patients and healthy controls,

respectively. This might be of particular interest with regard to the recent study by Phuan et

al. who demonstrated that monoclonal neutralising antibodies targeting C1q significantly

improved the course of NMO in an experimental model of NMO (Phuan et al.,2013).

However, the effects of anti-C1q on C1q could be different from neutralizing monoclonal

antibodies as mentioned before. Again, data derived from studies in systemic autoimmunity

point towards a disease exacerbating effect of autoantibodies against C1q (Trendelenburg et

al., 2006), e.g. by the inhibition of protective effects of the C1q molecule.

Our study has several limitations: the descriptive character of our data does not allow final

statements on the role of complement in NMO. In addition, the relatively small and locally

recruited cohort of NMO patients, due to the fact that NMO is a rare disease in Caucasians,

does not allow definite conclusions on the correlation between plasma/serum parameters and

disease activity. The limited relapse samples and the time frame of the study may preclude

the finding of associations between these products and disease activity. Finally, different

treatment profiles in MS and NMO patients might have strongly affected our comparative

analyses. Thus, further studies on the role of complement in NMO, in particular with a focus

on outcome measures such as disability, will be necessary.

In conclusion, our data strongly support the hypothesis that complement activation is

involved in the pathogenesis of NMO in vivo. Independently, complement C3a as a

biomarker might play an important role not only for the diagnosis of NMO but also for the

evaluation of disease activity at follow-up. Moreover, our data support strategies of

therapeutic complement inhibition in NMO patients in particular during relapses of disease.

Abbreviations

AQP4 aquaporin-4; AQP4-IgG antibodies against aquaporin-4; Anti-C1q antibodies against

C1q; AUC Area Under Curve; CASPR2 contactin-associated protein 2; CNS central nervous

system; EDSS Expanded Disability Status Scale; FPF False Positive Fraction; GlyR glycine

receptor; HSD Honestly Significant Difference; IVIG intravenous immunoglobulins; LGI1

leucine-rich, glioma inactivated 1; MS multiple sclerosis; NMO neuromyelitis optica; ROC

Receiver Operating Characteristic; sC5b-9 soluble terminal complement complex; TPF True

Positive Fraction

Competing interests

Nytrova Petra, her research stay at Dpt. of Clinical Neurosciences at University of Oxford

was supported by Euroimmun and a John Newsom-Davis Fellowship from the Guarantors of

Brain.

Potlukova Eliska has nothing to disclose.

Kemlink David has nothing to disclose.

Woodhall Mark has nothing to disclose.

Waters Patrick is a named inventor on patents for antibody assays and has received

royalties, and he has received a speaker honorarium from Biogen-Idec Japan.

Horakova Dana received speaker honoraria and consultant fees from Biogen Idec, Novartis,

Merck Serono, Teva and Bayer Healthcare and financial support for research activities from

Biogen Idec.

Havrdova Eva received speaker and consulting honoraria from Biogen Idec, Novartis,

Sanofi Genzyme, Roche, Merck Serono, Teva and Bayer Healthcare.

Zivorova Dana has nothing to disclose.

Vincent Angela has received funding from Euroimmun AG and is a consultant for Athena

Diagnostics. The University of Oxford holds patents and receives royalties and payments for

antibody tests.

Trendelenburg Marten receives financial support for research activities from Roche Pharma

and is member of the Swiss advisory board of GSK.

Acknowledgements

Supported by Grant Agency of the Charles University (grant GAUK 132010), the Czech

Ministries of Education and Health (PRVOUK-P26/LF1/4, NT13237-4/2012) and by the

National Health Service National Specialised Commissioning Group for Neuromyelitis

Optica and the National Institute for Health Research Oxford Biomedical Research Centre for

funding. The research stay of main author PN at Dpt.of Clinical Neuroscience at University

of Oxford was supported by a John Newsom-Davis Fellowship from the Guarantors of Brain,

Fond Mobility of the Charles University and Euroimmun. MT is a recipient of a grant from

the Swiss National Foundation (310030_134900/1).

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Figure legends

Figure 1. Comparison of plasma levels of complement activation products and serum levels

of anti-C1q antibodies in patients with demyelinating disorders versus normal controls. A:

C3a; B: C4a; C: sC5b-9; D: anti-C1q. For this analysis, only the first samples of each patient

(before escalation of treatment) were used. Horizontal lines depict the medians of the

measurements, the dotted horizontal lines represent the mean of normal controls (C3a and

C4a, not available for sC5b-9) or the upper limit of normal values (anti-C1q) as stated by the

manufacturer. (n.s.: not significant).

Figure 2. Linear regression between neurological disability as expressed by EDSS score and

plasma levels of C3a (A) and AQP4-IgG (B). The analysis was performed using only the

first samples available.

Figure 3. Plasma C3a levels in NMO patients with 1 ≥ relapse within the last 6 months prior

to sample collecting (Group I) versus patients > 6 months relapse-free (Group II) (A); and

during the relapse as compared to remission periods (B). Analysis in Fig. 3A was performed

using only the first samples available whereas in Fig. 3B all available samples from 19

patients were used (i.e. including those at follow-up).

Figure 4. ROC curve of complement C3a levels for the distinction between patients with ≥ 1

relapse during the last 6 months prior to sampling and patients that remained free of relapse

during the last 6 months. AUC: Area Under Curve, TPF: True Positive Fraction, FPF: False

Positive Fraction

Figure 5.

Row I: Cell-based assay for AQP4-IgG. AQP4 transfected HEK cells incubated with serum

of a healthy control and a patient’s serum and FITC-conjugated anti-hIgG (A,B); AQP4

transfected HEK cells incubated with patient’s serum, complement source and

FITC-conjugated anti-hC1q (C), anti-hC3c (D) and anti-hC4c (E).

Row II: Primate optic nerve tissue staining. Primate optic nerve transversal section was

incubated with serum of a healthy control and a patient’s serum and FITC-conjugated

anti-hIgG (A,B); with patient’s serum, complement source and FITC-conjugated anti-hC1q

(C), anti-hC3c FITC (D) and anti-hC4c (E).

Row III: Rat cerebellum tissue staining. Rat cerebellum section was incubated with serum of

a healthy control and a patient’s serum and FITC-conjugated anti-hIgG (A,B); with patient’s

serum, complement source and FITC-conjugated anti-hC1q (C), anti-hC3c (D) and anti-hC4c

(E).

Table 1. Baseline characteristics of patients with NMO and MS.

CharacteristicsNMO

(n=19)

MS analysed for

complement split

products (n=35)

MS analysed

only for anti-C1q

(n=76)

Controls

(n=40)

P–value

(ANOVA)

Post hoc

HSD

Females 15 (79%) 30 (75%) 57 (75%) 30 (75%) n.s.

Age (years) 44.5 (28-68) 40.9 (25-65) 39.3 (18-65) 40.0 (18-74) n.s.

Disease

duration

(years)

8.9 (0.6-30) 13.5 (3-41) 11.9 (0.4-41) 0 n.s.

Annual

relapse rate1.58 (0-5) 0.63 (0-3) 0.65 (0-3) 0 P=0.043

EDSS score 4.0 (1.5-8.0) 3.5 (1.0-7.5) 3.0 (1.0-7.5) 0 n.s.

AQP4-IgG+

19/19 (100) 0/35 (0) 0/76 (0) 0/40 (0) P<0.0001

Data are presented as median (range) or n (%) of patients. EDSS: Kurzke Expanded Disability Status

Scale; HSD: Honestly Significant Difference; n.s.: not significant

Table 2. Spearman rank order coefficients of correlations between complement split

products, AQP4-IgG, anti-C1q and EDSS in NMO.

EDSS

scoreAnti-C1q C3a C4a sC5b-9

AQP4-IgG

titresC3a/C3 C4a/C4

EDSS

score- n.s.

0.683

p<0.05n.s. n.s.

0.590

p<0.05

0.705

p<0.05n.s.

AQP4-IgG

titres

0.590

p<0.05n.s.

0.447

p<0.05n.s. n.s. -

0.482

p<0.05n.s.

Anti-C1q n.s. - n.s. n.s. n.s. n.s. n.s. n.s.

For this analysis, all available samples were used, including those from follow-up (n=43). EDSS:

Kurzke expanded disability status scale; n.s. not significant

Figures

Figure 5.