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Synovial membrane protein expression differs between juvenile idiopathic

arthritis subtypes in early disease

Article  in  Arthritis research & therapy · January 2014

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Synovial membrane protein expression differs
between juvenile idiopathic arthritis subtypes in
early disease
Finnegan et al.

Finnegan et al. Arthritis Research & Therapy 2014, 16:R8

http://arthritis-research.com/content/16/1/R8
Finnegan et al. Arthritis Research & Therapy 2014, 16:R8
http://arthritis-research.com/content/16/1/R8

RESEARCH ARTICLE Open Access

Synovial membrane protein expression differs

between juvenile idiopathic arthritis subtypes in
early disease
Sorcha Finnegan1, Joanne Robson2, Caitriona Scaife3, Catherine McAllister4, Stephen R Pennington3,
David S Gibson1,5* and Madeleine E Rooney1,4

Abstract
Introduction: Juvenile idiopathic arthritis (JIA) is the most common rheumatological disease of childhood with a
prevalence of around 1 in 1,000. Without appropriate treatment it can have devastating consequences including
permanent disability from joint destruction and growth deformities. Disease aetiology remains unknown.
Investigation of disease pathology at the level of the synovial membrane is required if we want to begin to
understand the disease at the molecular and biochemical level. The synovial membrane proteome from early
disease-stage, treatment naive JIA patients was compared between polyarticular and oligoarticular subgroups.
Methods: Protein was extracted from 15 newly diagnosed, treatment naive JIA synovial membrane biopsies and
separated by two dimensional fluorescent difference in-gel electrophoresis. Proteins displaying a two-fold or greater
change in expression levels between the two subgroups were identified by matrix assisted laser desorption ionization-
time of flight mass spectrometry with expression further verified by Western blotting and immunohistochemistry.
Results: Analysis of variance analysis (P ≤ 0.05) revealed 25 protein spots with a two-fold or greater difference in
expression levels between polyarticular and oligoarticular patients. Hierarchical cluster analysis with Pearson ranked
correlation revealed two distinctive clusters of proteins. Some of the proteins that were differentially expressed
included: integrin alpha 2b (P = 0.04); fibrinogen D fragment (P = 0.005); collagen type VI (P = 0.03); fibrinogen
gamma chain (P = 0.05) and peroxiredoxin 2 (P = 0.02). The identified proteins are involved in a number of different
processes including platelet activation and the coagulation system.
Conclusions: The data indicate distinct synovial membrane proteome profiles between JIA subgroups at an early
stage in the disease process. The identified proteins also provide insight into differentially perturbed pathways
which could influence pathological events at the joint level.

Introduction heterogeneity of the disease would suggest multiple fac-

Juvenile idiopathic arthritis (JIA) consists of a heteroge- tors are responsible [2]. Adverse outcomes include
neous group of diseases which persist for more than six chronic pain and stiffness, and joint damage and dis-
weeks and commence before the age of 16. It is the most ability as a result of chronic joint inflammation. Despite
common rheumatological disease of childhood, with a improved prognoses for JIA patients in recent years,
prevalence of approximately 1/1,000 [1]. The cause of around half of patients will continue to have active joint
JIA remains unknown; however, environmental and gen- disease into adulthood [3].
etic factors play a role in its pathogenesis and the The International League of Associations for Rheuma-
tology (ILAR) classification system divides JIA into seven
* Correspondence: [email protected]
clinical subgroups [4,5]. The largest subgroup - oligoarti-
1
Arthritis Research Group, Queen’s University Belfast, Centre for Infection and cular JIA - accounts for approximately 65% of all cases
Immunity, Health Sciences Building, 97 Lisburn Road, Belfast BT9 7BL, UK and refers to children with four or fewer joints involved
5
Northern Ireland Centre for Stratified Medicine, C-TRIC Building, Altnagelvin
Hospital campus, Glenshane Road, Londonderry BT47 6SB, UK
within six months of diagnosis. Oligoarticular disease is
Full list of author information is available at the end of the article further defined as persistent when it remains confined to
© 2014 Finnegan et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Finnegan et al. Arthritis Research & Therapy 2014, 16:R8 Page 2 of 12
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four or fewer joints throughout the course of the disease. on ice. Biopsies were snap frozen in liquid nitrogen and
Oligoarticular JIA is more prevalent in females, with a stored at −80°C.
peak age of onset of one to three years [6]. Polyarticular
JIA is defined by the involvement of more than four joints Sample preparation
within six months of diagnosis, it accounts for around 10 Protein was extracted from synovial membrane biopsies
to 15% of all JIA cases [7]. in lysis buffer (8.4 M urea, 2.4 M thiourea, 4% 3-[(3-chola-
Very few studies examine the synovial membrane from midopropyl)dimethylammonio]-1-propanesulfonate, 2 mM
JIA patients and, of those available, most involved pa- dithiothreitol (DTT), 1% ampholytes). Samples were centri-
tients with established disease and on disease modifying fuged at 12,000 rpm for 15 minutes and the supernatant
treatment [8-11]. Our previous immunohistochemical collected. Protein was precipitated in ice cold acetone
study demonstrated significant differences in the inflam- at −20°C overnight, pelleted by centrifugation and the pellet
matory infiltrates and levels of vascularisation in the dif- air dried to remove residual acetone. The protein pellet was
ferent JIA subgroups. It is still unclear, however, why resuspended in difference in-gel electrophoresis (DIGE)
these changes take place in JIA and why they differ lysis buffer (30 mM Tris-Cl, 7 M urea, 2 M thiourea, 4%
between subgroups. Proteomics offers an unsupervised CHAPS, pH 8.5) and protein concentration was determined
means to investigate the disease process within the using a modified Bradford assay.
affected tissue.
This study was designed to investigate how the predom- Difference in-gel electrophoresis (DIGE)
inant protein expression profile within synovial membrane DIGE was performed as previously described [13] with the
varies between persistent oligoarticular and polyarticular exception that isoelectric focusing (IEF) and 2D PAGE
JIA across a group of newly diagnosed, treatment-naïve were performed on 11 cm Immobiline DryStrip pH 4 to 7
patients. We hypothesize that subtype-specific protein linear. Each gel contained one synovium sample from a
expression patterns exist in the early stages of disease. polyarticular patient and one synovium sample from an
Comparison of the synovial proteome between polyarticu- oligoarticular patient (each labelled with either Cy3 or
lar and oligoarticular JIA patients could provide a much Cy5 dyes). Cy3 and Cy5 dyes were swapped randomly bet-
needed insight into the pathology underlying these two ween samples to prevent dye bias. Each gel also contained
distinct subtypes. the internal standard which is a pooled mixture of every
patient sample (labelled with Cy2). Voltage and times
were adjusted accordingly. Two preparative gels loaded
Materials and methods
with 300 μg of protein were Coomassie stained and spots
Patients
excised for identification by mass spectrometry.
Children with newly diagnosed JIA of less than two years
duration were consecutively recruited following informed
Image analysis
consent. Medical Ethics Committee approval was obtained
DIGE gels were scanned using a Typhoon 9410 imager (GE
for this study at Green Park Healthcare Trust and patient
Healthcare, Bucks, UK). Gel images were analysed using
assent and parent informed consent was given (ORECNI
Progenesis Samespots software version 2.0 build 2644.18003
408/03). Recruitment criteria included: involvement of
(Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK) as
one knee joint that required intra-articular steroid injec-
previously described [14]. PermutMatrix [15] was subse-
tion, and the patient being steroid and disease-modifying
quently used to cluster and visualise the spot data as previ-
antirheumatic drug (DMARD) naive. Children were classi-
ously described [14].
fied according to the ILAR criteria at the time of recruit-
ment. Children were then reclassified after two years of
Tryptic digestion of protein spots and MALDI target
follow-up. Detailed clinical examination, biochemical and
spotting
immunological data, and imaging assessments were ob-
Tryptic digestion of protein bands was performed using a
tained. Subgroups were compared with each other as it
ProGest robot (Genomic Solutions, Holliston, MA, USA)
was not ethically possible to obtain synovial membrane
programmed with the long trypsin digestion method. A
biopsies from healthy paediatric controls.
sterile pipette tip, cut to increase the bore width (approxi-
mately 2.0 mm), was used for spot excision. Gel plugs
Tissue collection were then transferred to a 96-well plate. This plate is de-
Synovial membrane samples were obtained by blind signed with microscopic holes at the bottom of the wells
needle biopsy using ultrasound guidance as described pre- which allows for positive displacement of liquids during
viously [12]. Biopsies were obtained prior to the instilla- reagent changes on the robot. Gel pieces were first equili-
tion of steroids. Tissue was wrapped in saline soaked brated in 50 μl of 50 mM ammonium bicarbonate and
gauze, immersed in saline and placed in sterile containers proteins were reductively alkylated with 10 mM DTT and
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100 mM iodacetamide followed by de-staining of the pro- included ensuring the matched peptides were the most
tein bands and desiccation of the gel plugs with aceto- abundant peaks in the mass spectrum, the theoretical
nitrile. Gel plugs were rehydrated with 50 mM isoelectric point and molecular weight of the identified
ammonium bicarbonate containing 5% trypsin and the protein correlated with the spot’s position on the 2D gel,
proteins were digested for 12 hours at 37°C. Resulting and sequence coverage was high.
peptides were extracted from the gel plugs with 2 × 25 μl
washes of 50% acetonitrile, 0.1% TFA. Peptide extracts Immunohistochemistry
were freeze dried and then re-suspended in 10 μl of 0.1% Biopsies were snap frozen in liquid nitrogen for section-
formic acid. A saturated solution of α-cyano-4-hydroxy- ing and stored at −80°C. A total of 7 μm thick frozen
cinnamic acid was prepared in 50% acetonitrile, 0.1%TFA, sections were cut using a Leica CM 1900 cryostat
10 mM ammonium acetate. For each sample, 1 μl of (Leica Microsystems Nussloch GmbH, Germany) and
matrix solution was spotted on the matrix assisted laser allowed to air-dry at room temperature for 30 minutes.
desorption ionisation (MALDI) target immediately Primary antibodies used were monoclonal anti-integrin
followed by 1 μl of sample into the matrix spot and the alpha 2b antibody (ITA2B) (Abcam, Cambridge, UK),
sample/matrix droplet allowed to slowly air dry. monoclonal anti-peroxiredoxin 2 (PRDX2) (Sigma, Dorset,
UK) and polyclonal anti-myosin regulatory light chain 2
MALDI TOF-TOF analyses, database searching and protein (MYL2) (Sigma, Dorset, UK). Immunostaining was per-
identification formed using a standard protocol and staining was visual-
Matrix assisted laser desorption ionisation-tandem time of ized using the diaminobenzidine (DAB) method [16].
flight (MALDI TOF-TOF) analyses was performed on a Sections were counterstained with haematoxylin.
4800 mass spectrometer (AB Sciex UK Limited, Cheshire,
UK) using the following protocol: TOF-MS analyses was Western blotting
first performed on all of the dried target spots using Western immunoblot analysis was performed using a
automated data acquisition and processing under the standard protocol using the above antibodies against
control of Applied Biosystems 4000 series Explorer ITA2B, PRDX2 and MYL2.
software (version 3.5) using reflector mode, a mass
range of 700 to 4,000 m/z, 1,000 total laser shots per Results
spectrum and a laser intensity of 3,300 V. Following ac- Patient demographics and clinical features
quisition the TOF-MS spectra were noise corrected, There were 12 females and 3 males included in the
peak de-isotoped and internally calibrated using the study. The clinical and laboratory data of patients are
trypsin autolysis peaks 842.500 and 2,211.100 m/z. The shown in Table 1.
eight most abundant precursor ions from each spectra
were then selected by the software for fragmentation General laboratory characteristics of JIA patients
and MS-MS analyses using a 1 kV collision ion dissoci- There was no significant difference in the age at disease
ation (CID) fragmentation method collecting 4,000 onset between the two groups. VAS pain scores (parent)
laser shots per spectra with a laser intensity of 3,800 were significantly higher in the polyarticular as were
over the mass range. ESR and CRP levels (Table 1).
Peak lists of ion masses were generated by GPS Ex-
plorer software version 3.6 (Applied Biosystems) from 2D DIGE analysis
the calibrated and de-isotoped MS and MS-MS spectra We compared the synovial membrane proteome from
for each sample. Combined lists of MS and MS-MS data seven persistent oligoarticular JIA patients with eight poly-
were used for database searching with MASCOT ver- articular JIA patients using 2D DIGE. 2D DIGE analysis
sion 2.2 (Matrix Science), against all entries in the Na- revealed an average of 460 spots per pH 4 to 7, 11 cm gel
tional Center for Biotechnology Information (NCBI) which were matched across all gels (Figure 1). ANOVA re-
database. Database search parameters used were the fol- vealed 26 spots that were differentially expressed (two-fold
lowing: digestion enzyme trypsin, single missed cleavage or more change in expression levels) between polyarticu-
allowed, variable modifications of carboxymethyl cyst- lar and oligoarticular patients (P ≤0.05).
eine and oxidised methionine, precursor mass tolerance
of 50 ppm and fragment ion tolerance of 0.2 Da. HTML Hierarchical cluster analysis (HCA)
pages of MASCOT output were generated for each HCA was used to examine the expression patterns of
sample. the 26 differentially expressed proteins across the study
The Mowse scoring algorithm was used to determine cohort with results depicted in heat map form (Figure 2).
the significance of the identity. Additional criteria were Depicted on the horizontal axis is the patient num-
used to ensure correct protein identifications. These ber and on the vertical axis is the spot number/
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Table 1 Demographic and clinical characteristics at the time of biopsy

ILAR subtype JIA patient M/F Disease No. of VAS* VAS* WCC* Hb* ESR* CRP* PC* ANA* RF
study ID duration swollen pain global physician (mg/l) (+/−)
(months) joints (parent)
Oligoarticular (n = 7) 1 F 4 1 5 1.9 7.5 11 8 0 407 0 -
2 M 4 1 0 0.8 12 11 11 0 415 0 -
3 F 6 3 3.6 2.9 12.1 11 10 5.7 453 0 -
4 F 10 2 0.3 1.9 6.9 10.7 4 4 315 0 -
5 F 5 3 7.5 3 12.8 10.6 37 26 407 80 -
6 M 20 1 1.4 1.9 5.6 14.1 5 0 249 0 -
7 F 4 3 4.3 1.8 9.1 8.9 62 66.5 460 0 -
Mean values 7.6 2 3.2 2 9.4 11 19.6 14.6 386.6 11.4
Polyarticular (n = 8) 8 F 2 24 8.7 7.5 12.9 8.3 38 27.5 570 160 -
9 F 2 9 8.3 3.3 13.9 10.4 22 36.1 232 0 -
10 F 2 24 4.7 5.7 12.2 10.5 30 67.3 601 0 -
11 F 2 6 9 4.8 9.9 11.1 26 32.1 446 80 -
12 F 1 2 7.1 1.5 11.2 10.4 96 47.4 522 ND -
13 F 4 15 9.8 3.6 11.8 7.2 110 72 578 0 -
14 M 10 4 3.3 5.3 10.27 11 20 5.4 314 40 +
15 F 5 25 6.5 7.4 9.3 8.4 138 181 401 80 +
Mean values 3.5 13.6 7.2 4.9 11.4 9.7 60 58.6 458 51.4
t-test p value ns ns 0.004 0.002 0.056 0.049 0.029 0.03 0.12 - -
ANA, anti-nuclear antibodies; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; Hb, haemoglobin; ns, not statistically significant at P ≥0.05; PC, platelet
count;; RF, rheumatoid factor; *VAS, visual analogue scale; WCC, white cell count.

Figure 1 A representative 2D DIGE gel image of JIA synovial membrane proteins. Proteins labeled using fluorescent dyes Cy3 (green) and
Cy5 (red). Protein samples were separated on an 11 cm pH4 to 7 IPG strip and on a 12.5% homogenous SDS-PAGE gel. Protein spots analysed by
mass spectrometry are highlighted by spot outlines. Spot numbers correspond with those listed in Table 2, Additional file 1: Table S1 and Figure 2.
Proteins overexpressed in oligoarticular patients are indicated by red spot outlines; proteins overexpressed in polyarticular are indicated by blue
spot outlines. DIGE, difference in-gel electrophoresis; JIA, juvenile idiopathic arthritis.
Finnegan et al. Arthritis Research & Therapy 2014, 16:R8 Page 5 of 12
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Figure 2 Hierarchical cluster analysis of proteins expressed with statistically significant differences between patient subgroups. The
inter-individual and inter-group variation in 19 selected proteins is represented in the form of a heatmap. The protein expression data were reordered by
hierarchical cluster analysis (HCA) using Pearson centred correlation (unweighted pair group method with arithmetic mean (UPGMA)), which revealed
specific expression patterns. Two main clusters of proteins are highlighted by the coloured bars (blue, red). Each patient sample is represented by a single
numbered column (oligarticular 1 to 7 and polyarticular 9 to 15). Spot numbers and protein identifications (UniProt code) are labelled on the vertical axis;
unidentified protein spots are indicated by an asterix (*). The relative amount of a protein is denoted by colour intensity within a spectrum of black to
red, red being the highest amount.

protein ID. Pearson ranked correlation revealed two distinct quantities of proteins generated using Samespots software
clusters of proteins. Cluster 1 contains proteins that were (Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK).
overexpressed in the polyarticular patients, whereas pro- The levels of proteins 27 to 31 (listed in Table 2) did not
teins in cluster 2 are overexpressed in the oligoarticular differ between oligoarticular and polyarticular patient
subgroup. It seemed conceivable that these two distinct groups. There were a number of proteins that could not
clusters could be used in combination to differentiate these be identified due to their low abundance in the gel.
two disease subgroups.
Confirmation of protein localisation and expression
MALDI-TOF MS protein identification Figure 4 shows representative images of MYL2, PRDX2
Differentially expressed proteins were identified by and ITA2B immunostaining in polyarticular and oligoar-
MALDI MS (Table 2). Peptide ion validation data is avail- ticular synovial membrane: (A) MYL2 staining is limited
able as an additional file (Additional file 1: Table S1). Pro- to a small number of discrete cells within the sublining
teins that were overexpressed in the polyarticular group layer in the oligoarticular tissue (arrows), its expression in
included: chain A of profilin-beta-actin (P = 0.04); alpha-1 the polyarticular tissue (B) is more intense. PRDX2 stain-
subunit of F-actin capping protein (P = 0.03); beta actin, ing is sparse in the oligoarticular tissue (arrows) (C) with
(P = 0.04); beta actin (P = 0.02); myosin regulatory light more intense staining in the polyarticular tissue (D).
chain 2 (MYL2) isoform B (P = 0.02); myosin regulatory ITA2B staining is localised to blood vessels in oligoarticu-
light chain 9 (MYL9), isoform a (P = 0.04); beta actin lar tissue (E), some ITA2B staining was also observed in
variant (P = 0.005); peroxiredoxin 2 (PRDX2), isoform a the polyarticular tissue (F) around blood vessels (arrows).
(P = 0.02); mixture of protein kinase C inhibitor protein 1 Figure 5 depicts the synovial membrane expression levels
(KCIP-1) and annexin A5 (P = 0.003); fibrinogen gamma for the aforementioned proteins across a representative
chain (P = 0.004 and P = 0.05). subset of the study patients.
Proteins overexpressed in the oligoarticular group
included: collagen type VI, alpha 2 (P = 0.03); integrin alpha Discussion
2b (ITA2B) (P = 0.04); chain C of fibrinogen fragment D This is the first study to identify synovial membrane pro-
(P = 0.005); Chain C, crystal structure of fibrinogen fragment tein expression patterns that discriminate clinical sub-
D (P = 0.0002). Figure 3 shows representative differentially groups in early treatment of naive JIA. By understanding
expressed proteins along with a 3D image of the relative the early pathological changes taking place at the disease
 http://arthritis-research.com/content/16/1/R8
Finnegan et al. Arthritis Research & Therapy 2014, 16:R8
Table 2 Mass spectrometry and DIGE quantification data of synovial membrane proteins
Spot Protein Gene Accession # Mowse % Sequence CalculatedpI Nominal Number matched Normalised Normalised ANOVA
number symbol score coverage value mass (Mr) peptides volume (Oligo) volume (Poly) P-value
1 No ID - - - - - - - 0.73 3.02 0.001
2 Protein kinase C inhibitor protein 1 KCIP-1 gi|114629705 88 40 4.78 24,166 14 1.07 2.1 0.03
Annexin A5 ANXA5 gi|149698420 79 33 4.98 35,977 10 - - -
3 Myosin regulatory l ight chain 9 MYL9 gi|29568111 52 29 4.8 19,814 8 0.92 2.02 0.04
4 No ID - - - - - - - 0.79 2.35 0.006
5 Beta Actin ACTB gi|15277503 76 21 5.55 40,194 9 1.14 2.28 0.04
6 Beta Actin ACTB gi|15277503 176 37 5.55 40,194 17 0.35 1.24 0.02
7 Fibrinogen gamma chain FGG gi|930064 94 50 6.49 24,109 11 0.88 2.23 0.004
8 Beta actin variant ACTB gi|62897625 451 53 5.37 41,738 23 0.56 1.94 0.005
9 No ID - - - - - - - 0.64 2.04 0.02
10 Chain A, profi l in-beta-actin PFN1 gi|157111829 106/130 44 5.29 41,616 21 0.39 1.76 0.04
11 F-actin capping protein alpha-1 CAPZA1 gi|5453597 196 55 5.45 32,902 17 0.3 1.38 0.03
12 Myosin regulatory l ight chain 2 MYL2 gi|222144328 86 55 4.26 17,745 10 0.66 2.03 0.02
13 No ID - - - - - - - 0.62 1.72 0.05
14 Peroxiredoxin 2 PRDX2 gi|4758638 348 57 6 25,019 18 0.3 1.73 0.02
15 Chain C, fibrinogen fragment D FFD gi|2781209 89 42 5.87 36,157 12 2.08 0.58 0.0002
16 Fibrinogen gamma chain FGG gi|930064 71 44 6.49 24,109 7 0.9 2.14 0.05
17 Collagen, type VI, alpha 2 COL6A2 gi|41350923 81 18 5.85 182,860 18 2.77 1.09 0.03
18 Chain C, fibrinogen fragment D FFD gi|24987625 102 44 5.86 35,155 14 2.1 0.96 0.005
19 No ID - - - - - - - 1.6 0.9 0.008
20 No ID - - - - - - - 1.6 0.8 0.02
21 No ID - - - - - - - 1.4 0.7 0.006
22 No ID - - - - - - -
23 Integrin, alpha 2b ITGA2B gi|119571979 95 15 5.41 103,127 16 1.87 0.64 0.04
24 Alpha 3 type VI collagen COL6A3 gi|62088852 119 17 8.73 182,860 27 1.78 0.78 0.05
25 No ID - - - - - - - 1.72 0.74 0.04
26 No ID - - - - - - - 1.56 0.72 0.02
27 Peroxiredoxin 6 PRDX6 gi|4758638 348 57 6 25,019 18 - - -

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Finnegan et al. Arthritis Research & Therapy 2014, 16:R8
Table 2 Mass spectrometry and DIGE quantification data of synovial membrane proteins (Continued)
28 Triosephosphate isomerase 1 TPI1 gi|17389815 111 53 6.45 26,625 15 - - -
29 Albumin, isoform CRA_j ALB gi|119626073 59 23 6.4 26,233 7 - - -
30 Calreticul in precursor variant CRT gi|62897681 236 29 4.3 46,890 19 - - -
31 Fibrinogen, beta chain FGB gi|70906435 93 34 8.54 55,892 21 - - -
Spot trypsin digests were identified using matrix assisted laser desorption ionization (MALDI-TOF/TOF), correlated to compiled peptide data (Matrix Science). P-values highlight inter-subgroup comparisons which
reached statistical significance (P <0.05) by ANOVA. Full mass spectra peak lists for each identified protein are provided in Additional file 1: Table S1.

Page 7 of 12
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Figure 3 DIGE image analysis of differentially expressed synovial membrane proteins. Spots 22, 23 (integrin alpha 2b) and 24 (collagen)
were overexpressed in the oligoarticular group. Spots 14 (peroxiredoxin 2) and 16 (fibrinogen gamma chain) were overexpressed in the
polyarticular group. JIA, juvenile idiopathic arthritis.

Figure 4 Immunohistochemical staining of differentially expressed synovial membrane proteins. A and B Distribution of myosin
regulatory light chain 2 (MYL2), C and D peroxiredoxin 2 (PRDX2), E and F integrin alpha 2b (ITA2B) in oligoarticular and polyarticular synovial
membrane. Sections were counterstained with haematoxylin. Arrows indicate areas of positive staining.
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Figure 5 Validation of protein expression changes by Western immunoblot probing for MYL2, PRDX2 and ITA2B. Each lane represents
an individual patient synovial membrane protein sample. β-actin was used as a loading control. ITA2B, integrin alpha 2b; MYL2, myosin regulatory
light chain 2; PRDX2, peroxiredoxin 2.

site we can begin to elucidate the disease process. There suggesting a process of serial post translational modifica-
have been a number of proteomic investigations of syn- tions. ITA2B contains five N-linked glycosylation points
ovial tissue in adult inflammatory disease [17-19] but not [24]. Furthermore, intense ITA2B immunostaining was
in children. To date, proteomic studies of JIA have observed around blood vessels in the oligoarticular
focused on synovial fluid and plasma [13,14,20-22], due to group, relative to polyarticular tissue. ITA2B is a major
the fact that synovial membrane is extremely difficult to integrin found on platelet plasma membranes, assisting
acquire. The synovial membrane has an advantage over platelet activation and adhesion and thrombus formation
synovial fluid in that it is the actual site of pathology in at sites of vascular injury [25]. A previous study reported
JIA. It is also important to note that all synovial mem- similar ITA2B expression patterns in rheumatoid arth-
branes used in this study were from treatment-naive early ritis (RA) synovial membrane, restricted to vascular
disease-stage patients (average disease duration at time of endothelial cells [26]. ITA2B could act as a key target in
biopsy was 5.4 months). For the purpose of this study the controlling inflammation, as platelets can elicit cytokine
differences in two JIA subgroups were noted. Although responses from synovial fibroblasts via interleukin-1 in
control tissue from healthy subjects would have been use- RA patients. The apparent increase in ITA2B observed
ful, such tissue is not obtainable for ethical reasons. in oligoarticular patients may result from leaky and tor-
A number of proteins identified in this study were part of tuous vessels observed in the development of synovitis.
a ‘charge train’ on the 2D gels, indicating post translational Fibrinogen fragment D was also expressed at higher
modification (PTM). A recently published study by our levels in the oligoarticular group as part of a ‘charge
group indicates that protein modification acts as a surrogate train’. The D-dimer of fibrin is a degradation product
marker of inflammation spread in JIA [23]. Glycosylation of (FDP) found in the blood following fibrinolysis of a
VDBP- Vitamin D-binding protein, which confers macro- blood clot [27]. Under various inflammatory conditions,
phage activation activity, was decreased in patients at risk including RA, fibrin degradation product levels are
of disease spread. Subtype-specific protein isoforms uncov- increased [28,29], suggesting an imbalance in the coa-
ered within the current study should also be carefully inves- gulation/fibrinolysis system. D-dimers have also been
tigated for post translational modifications. In early disease shown to trigger release of IL-1β, IL-6 in peripheral
discrete protein isoforms may play a role in the different blood monocytes [30]. Fibrinogen is one of several pro-
pathologies observed between oligoarticular and polyarticu- tein targets for citrullination in RA [31] and citrul-
lar synovium. In an earlier study we reported histological linated fibrinogen D fragments have been detected in
features which distinguish these JIA subgroups, including synovial exosomes from patients with RA and osteo-
levels of vascularisation and white blood cell infiltration arthritis (OA).
[16]. The likely pathological impact of synovial protein iso- In contrast, fibrinogen beta and gamma chains were
forms identified in the current study are subsequently dis- overexpressed in the polyarticular group. Fibrinogen
cussed, along with their potential as therapeutic targets. gamma chains are involved in fibrin polymerization and
ITA2B was overexpressed in the oligoarticular group cross-linking, interacting with platelets and mediating
as part of a ‘charge train’ of spots (Figure 3), strongly thrombin binding to fibrin [32]. Rosenkranz et al. also
Finnegan et al. Arthritis Research & Therapy 2014, 16:R8 Page 10 of 12
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observed that fibrinogen gamma chain levels were in- Peroxiredoxins constitute a ubiquitous family of antioxi-
creased in systemic JIA synovial fluid relative to oligoarti- dant enzymes that are involved in the control of cytokine-
cular patients [21]. Fibrinogen beta chain is also increased induced peroxide levels [38]. Lymphocytes from RA pa-
in patients with recurrent joint inflammation [22]. A re- tients have been shown to have increased levels of intra-
cent study also demonstrated strong reactivity of JIA sera cellular PRDX2 when compared to healthy controls [39].
to anti-citrullinated fibrinogen antibodies [33]. Fibrinogen Autoantibodies against peroxiredoxin have also been iden-
has been shown to be the target of citrullination not only tified in RA [40]. Increased levels of PRDX2 in the pol-
in JIA but also in adult RA [34], suggesting it may have an yarticular patients may indicate a dysregulated redox
important role to play in autoimmunity. response system similar to that observed in adult RA.
During a normal inflammatory response, activation of
coagulation and deposition of fibrin are crucial events Conclusions
that help regulate and contain inflammatory activity to There are still substantial gaps in our understanding of
the site of injury or infection. It is, therefore, possible JIA disease pathogenesis. As a result, JIA specific treat-
that altered levels of coagulation system proteins re- ments have lagged behind the management of other
ported in polyarticular tissue may indirectly assist in the adult arthritides. Identifying pathology specific proteins
spread of inflammation to multiple joints, a key charac- with functions in key cellular processes may provide
teristic of these patients. It would be intriguing to target help to prioritize putative therapeutic targets. We hy-
these fragments in an animal model of arthritis to deter- pothesize that an imbalance in clotting factors, which
mine their function in the synovium. primarily control plasma cell activation and thrombus
The collagens are a superfamily of extracellular matrix formation, is associated with the involvement of multiple
proteins that play a crucial role in maintaining tissue in- synovial joints in early stage JIA. In the case of polyarti-
tegrity. Type VI collagen is a prominent constituent of the cular patients, diminished levels of specific clotting fac-
synovial extracellular matrix [35]. A study by Alexopoulos tors may prevent the repair of leaky neovasculature and
et al. suggests that type VI collagen has an important role increase the chances that multiple joints will be affected.
in the regulation of normal synovial joint physiology and Further targeted investigations of the circulating levels
pericellular matrix displayed significantly reduced me- of the aforementioned factors in a suitably powered in-
chanical properties in mice lacking type IV collagen. Thus, dependent study cohort are required to robustly test this
as the mice aged, accelerated development of joint de- theory. Murine knockout models of arthritis which can
generation, as well as a number of other musculoskeletal sequentially test the function of the identified members
abnormalities, was noted [36]. Collagen type VI was found of clotting pathways could also be used to verify this
at higher levels in the oligoarticular subgroup. Disease hypothesis.
pathology is much less severe in this group, and it is pos- Alternately, if the integrin alpha 2b is expressed exclu-
sible that this is due to increased tissue integrity and a sively on pathogenic vasculature, this property could be
more stable extracellular and pericellular matrix in the exploited to specifically target poorly formed vessels with
synovial membrane. integrin recognition (RGD) peptides. Integrin homing-
A number of actin family proteins were overexpressed RGD peptides have been shown to preferentially target in-
in the polyarticular group. Citrullinated F-actin capping flamed synovium vessels without damaging other synovial
protein alpha-1 subunit autoantigens have been identified tissues [41]. These small proteins could be linked to thera-
in RA synovium [19]. It is thought that one of the patho- peutic agents, or clotting agents to clog the vessels which
genic mechanisms taking place in RA is the promotion of fuel the ensuing synovitis.
actin polymerization and rearrangement of the actin cyto- In summary, proteomic analysis of synovial tissue from
skeleton. Indeed, a previous study found a number of early disease-stage JIA patients has revealed novel patho-
deregulated genes in synovial fibroblasts known to be in- logical protein expression patterns. With further work,
volved in actin filament and cytoskeleton organisation molecular pathways which discriminate JIA subtypes
[37]. Reorganisation of the actin cytoskeleton and the as- could be exploited to develop new treatment strategies
sociated deregulation of ECM adhesion are known to be which suppress disease and reduce the risk of long term
an intrinsic property of arthritic synovial fibroblasts. The joint damage.
presence of higher levels of these cytoskeletal proteins in
the polyarticular group could reflect the severe disease
Additional file
pathology in this group and is consistent with much
higher levels of synovial hyperplasia and cytoskeletal Additional file 1: Table S1. Peptide ion validation data from collision
reorganisation. ion dissociation (CID) MS/MS mass spectrometry which correspond with
PRDX2 was found at higher levels in the polyarticular Table 2. Peptide position in the matched protein, observed mass (m/z)
and amino acid sequence are listed for each protein match found.
group. Peroxiredoxin is involved in redox regulation.
Finnegan et al. Arthritis Research & Therapy 2014, 16:R8 Page 11 of 12
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Abbreviations 5. Petty RE, Southwood TR, Baum J, Bhettay E, Glass DN, Manners P,
2D DIGE: Two dimensional fluorescent difference in-gel electrophoresis; 2D Maldonado-Cocco J, Suarez-Almazor M, Orozco-Alcala J, Prieur AM: Revision
PAGE: Two dimensional polyacrylamide gel electrophoresis; ANOVA: Analysis of of the proposed classification criteria for juvenile idiopathic arthritis:
variance analysis; CHAPS: 3-((3-Cholamidopropyl) dimethylammonio)-1- Durban, 1997. J Rheumatol 1998, 25:1991–1994.
propanesulfonate; CID: Collision induced dissociation; CRP: C-reactive protein; 6. Sullivan DB, Cassidy JT, Petty RE: Pathogenic implications of age of onset
Da: Dalton; DAB: 3,3'-Diaminobenzidine; DMARD: Disease modifying anti- in juvenile rheumatoid arthritis. Arthritis Rheum 1975, 18:251–255.
rheumatic drug; DTT: Dithiothreitol; ECM: Extracellular matrix; ESR: Erythrocyte 7. Hui-Yuen JS, Imundo LF: A review guide to oligoarticular and polyarticular
sedimentation rate; FDP: Fibrin degradation product; HCA: Hierarchical cluster juvenile idiopathic arthritis. Pediatr Ann 2012, 41:1–8.
analysis; HTML: Hypertext markup language; IEF: Isoelectric focusing; IL-1β/ 8. Gattorno M, Gregorio A, Ferlito F, Gerloni V, Parafioriti A, Felici E, Sala E,
6: Interleukin 1 beta or 6; ILAR: International league of associations for Gambini C, Picco P, Martini A: Synovial expression of osteopontin
rheumatology; ITA2B: Integrin alpha 2b; JIA: Juvenile Idiopathic Arthritis; correlates with angiogenesis in juvenile idiopathic arthritis. Rheumatology
KCIP-1: Protein kinase C inhibitor protein 1; kV: Kilovolt; m/z: Ion mass over (Oxford) 2004, 43:1091–1096.
charge; MALDI-TOF: Matrix assisted laser desorption ionization-time of flight; 9. Gregorio A, Gambini C, Gerloni V, Parafioriti A, Sormani MP, Gregorio S, De
MS-MS: Tandem mass spectrometry; MYL2/9: Myosin regulatory light chain 2 or Marco G, Rossi F, Martini A, Gattorno M: Lymphoid neogenesis in juvenile
9; NCBI: National center for biotechnology information; OA: Osteoarthritis; idiopathic arthritis correlates with ANA positivity and plasma cells
ppm: Parts per million; PRDX2: Periredoxin 2; PTM: Post-translational infiltration. Rheumatology (Oxford) 2007, 46:308–313.
modification; RA: Rheumatoid arthritis; RGD: Arginine-glycine-aspartate integrin 10. Bramlage CP, Kaps C, Ungethüm U, Bramlage P, Koziolek M, Wessels J,
recognition sequence; rpm: Revolutions per minute; TFA: Trifluoroacetic acid; Krenn V, Pruss A, Müller GA, Strutz F, Burmester GR, Häupl T: Modulatory
TOF-MS: Time of flight mass spectra; UPGMA: Unweighted pair group method effects of inflammation and therapy on GDF-5 expression in rheumatoid
with arithmetic mean; VAS: Visual analogue scale. arthritis synovium. Scand J Rheumatol 2008, 37:401–409.
11. Walker JG, Ahern MJ, Coleman M, Weedon H, Papangelis V, Beroukas D,
Competing interests Roberts-Thomson PJ, Smith MD: Changes in synovial tissue Jak-STAT
The authors declare they have no competing interests. expression in rheumatoid arthritis in response to successful DMARD
treatment. Ann Rheum Dis 2006, 65:1558–1564.
Authors' contributions 12. Koski JM, Helle M: Ultrasound guided synovial biopsy using portal and
SF and CS carried out the DIGE analysis. JLR performed the mass forceps. Ann Rheum Dis 2005, 64:926–929.
spectrometry. SF conducted the Western blot analyses and 13. Gibson DS, Blelock S, Curry J, Finnegan S, Healy A, Scaife C, McAllister C,
immunohistochemistry. CMcA collated all the patient demographic and Pennington S, Dunn M, Rooney M: Comparative analysis of synovial fluid
clinical data. MR obtained consent from patients and collected tissue and plasma proteomes in juvenile arthritis–proteomic patterns of joint
biopsies. DSG conceived the study and, along with SRP and MR, participated inflammation in early stage disease. J Proteomics 2009, 72:656–676.
in its design and coordination. SF, MR and DSG wrote the manuscript. All 14. Gibson DS, Finnegan S, Jordan G, Scaife C, Brockbank S, Curry J, McAllister C,
authors read and approved the final manuscript. Pennington S, Dunn M, Rooney ME: Stratification and monitoring of juvenile
idiopathic arthritis patients by synovial proteome analysis. J Proteome Res
Acknowledgements 2009, 8:5601–5609.
DSG and MR would like to acknowledge the funding of this research by 15. Caraux G, Pinloche S: PermutMatrix: a graphical environment to arrange
Arthritis Research UK in the form of a Project Grant (No. 18748). The UCD gene expression profiles in optimal linear order. Bioinformatics 2005,
Conway Institute and the Proteome Research Centre are funded by the 21:1280-1281. http://www.atgc-montpellier.fr/permutmatrix/
Programme for Research in Third Level Institutions, as administered by the 16. Finnegan S, Clarke S, Gibson D, McAllister C, Rooney M: Synovial membrane
Higher Education Authority of Ireland. SRP acknowledges support for immunohistology in early untreated juvenile idiopathic arthritis: differences
equipment from Science Foundation Ireland. between clinical subgroups. Ann Rheum Dis 2011, 70:1842–1850.
17. Yan X, Zhao Y, Pan J, Fang K, Wang Y, Li Z, Chang X: Vitamin D-binding
Author details protein (group-specific component) has decreased expression in
1 rheumatoid arthritis. Clin Exp Rheumatol 2012, 30:525–533.
Arthritis Research Group, Queen’s University Belfast, Centre for Infection and
Immunity, Health Sciences Building, 97 Lisburn Road, Belfast BT9 7BL, UK. 18. Sun S, Fang K, Zhao Y, Yan X, Chang X: Increased expression of alpha
2
School of Biological and Biomedical Sciences, Durham University, Durham 1-anti-trypsin in the synovial tissues of patients with ankylosing
DH1 3LE, UK. 3Proteome Research Centre, UCD Conway Institute for spondylitis. Clin Exp Rheumatol 2012, 30:39–44.
Biomolecular and Biomedical Research, University College Dublin, Dublin, 19. Matsuo K, Xiang Y, Nakamura H, Masuko K, Yudoh K, Noyori K, Nishioka K,
Ireland. 4Pediatric Rheumatology, Withers Ward, Musgrave Park Hospital, 20 Saito T, Kato T: Identification of novel citrullinated autoantigens of
Stockman's Lane, Belfast BT9 7JB, UK. 5Northern Ireland Centre for Stratified synovium in rheumatoid arthritis using a proteomic approach.
Medicine, C-TRIC Building, Altnagelvin Hospital campus, Glenshane Road, Arthritis Res Ther 2006, 8:R175.
Londonderry BT47 6SB, UK. 20. Ling XB, Park JL, Carroll T, Nguyen KD, Lau K, Macaubas C, Chen E, Lee T,
Sandborg C, Milojevic D, Kanegaye JT, Gao S, Burns J, Schilling J, Mellins ED:
Received: 11 December 2012 Accepted: 31 December 2013 Plasma profiles in active systemic juvenile idiopathic arthritis: biomarkers
Published: 13 January 2014 and biological implications. Proteomics 2010, 10:4415–4430.
21. Rosenkranz ME, Wilson DC, Marinov AD, Decewicz A, Grof-Tisza P, Kirchner D,
References Giles B, Reynolds PR, Liebman MN, Kolli VS, Thompson SD, Hirsch R: Synovial
1. Symmons DP, Jones M, Osborne J, Sills J, Southwood TR, Woo P: Pediatric fluid proteins differentiate between the subtypes of juvenile idiopathic
rheumatology in the United Kingdom: data from the British Pediatric arthritis. Arthritis Rheum 2010, 62:1813–1823.
Rheumatology Group National Diagnostic Register. J Rheumatol 1996, 22. Gibson DS, Blelock S, Brockbank S, Curry J, Healy A, McAllister C, Rooney ME:
23:1975–1980. Proteomic analysis of recurrent joint inflammation in juvenile idiopathic
2. Prakken BJ, Albani S: Using biology of disease to understand and guide arthritis. J Proteome Res 2006, 5:1988–1995.
therapy of JIA. Best Pract Res Clin Rheumatol 2009, 23:599–608. 23. Gibson DS, Newell K, Evans AN, Finnegan S, Manning G, Scaife C, McAllister C,
3. Minden K, Niewerth M, Listing J, Biedermann T, Bollow M, Schöntube M, Pennington SR, Duncan MW, Moore TL, Rooney ME: Vitamin D binding
Zink A: Long-term outcome in patients with juvenile idiopathic arthritis. protein isoforms as candidate predictors of disease extension in childhood
Arthritis Rheum 2002, 46:2392–2401. arthritis. J Proteomics 2012, 75:5479–5492.
4. Petty RE, Southwood TR, Manners P, Baum J, Glass DN, Goldenberg J, He X, 24. Calvete JJ, Henschen A, Gonzalez-Rodriguez J: Complete localization of the
Maldonado-Cocco J, Orozco-Alcala J, Prieur AM, Suarez-Almazor ME, Woo P, intrachain disulphide bonds and the N-glycosylation points in the
International League of Associations for Rheumatology: International alpha-subunit of human platelet glycoprotein IIb. Biochem J 1989,
League of Associations for Rheumatology classification of juvenile 261:561–568.
idiopathic arthritis: second revision, Edmonton, 2001. J Rheumatol 2004, 25. Calvete JJ: On the structure and function of platelet integrin alpha IIb
31:390–392. beta 3, the fibrinogen receptor. Proc Soc Exp Biol Med 1995, 208:346–360.
 Finnegan et al. Arthritis Research & Therapy 2014, 16:R8 Page 12 of 12
http://arthritis-research.com/content/16/1/R8

26. Korkusuz P, Dagdeviren A, Eksioglu F, Ors U: Immunohistological analysis

of normal and osteoarthritic human synovial tissue. Bull Hosp Jt Dis 2005,
63:63–69.
27. Adam SS, Key NS, Greenberg CS: D-dimer antigen: current concepts and
future prospects. Blood 2009, 113:2878–2887.
28. Iba T, Kidokoro A, Fukunaga M, Sugiyama K, Sawada T, Kato H: Association
between the severity of sepsis and the changes in hemostatic molecular
markers and vascular endothelial damage markers. Shock 2005,
23:25–29.
29. Ichikawa Y, Yamada C, Horiki T, Hoshina Y, Uchiyama M: Serum matrix
metalloproteinase-3 and fibrin degradation product levels correlate with
clinical disease activity in rheumatoid arthritis. Clin Exp Rheumatol 1998,
16:533–540.
30. Robson SC, Shephard EG, Kirsch RE: Fibrin degradation product D-dimer
induces the synthesis and release of biologically active IL-1 beta, IL-6
and plasminogen activator inhibitors from monocytes in vitro.
Br J Haematol 1994, 86:322–326.
31. van Venrooij WJ, van Beers JJ, Pruijn GJ: Anti-CCP antibodies: the past, the
present and the future. Nat Rev Rheumatol 2011, 7:391–398.
32. Mosesson MW: Fibrinogen gamma chain functions. J Thromb Haemost
2003, 1:231–238.
33. Gilliam BE, Reed MR, Chauhan AK, Dehlendorf AB, Moore TL: Evidence of
fibrinogen as a target of citrullination in IgM rheumatoid factor-positive
polyarticular juvenile idiopathic arthritis. Pediatr Rheumatol Online J 2011, 9:8.
34. Quirke AM, Fisher BA, Kinloch AJ, Venables PJ: Citrullination of
autoantigens: upstream of TNFα in the pathogenesis of rheumatoid
arthritis. FEBS Lett 2011, 585:3681–3688.
35. Bathon JM, Hwang JJ, Shin LH, Precht PA, Towns MC, Horton WE Jr: Type VI
collagen-specific messenger RNA is expressed constitutively by cultured
human synovial fibroblasts and is suppressed by interleukin-1.
Arthritis Rheum 1994, 37:1350–1356.
36. Alexopoulos LG, Youn I, Bonaldo P, Guilak F: Developmental and
osteoarthritic changes in Col6a1-knockout mice: biomechanics of type VI
collagen in the cartilage pericellular matrix. Arthritis Rheum 2009,
60:771–779.
37. Aidinis V, Carninci P, Armaka M, Witke W, Harokopos V, Pavelka N, Koczan D,
Argyropoulos C, Thwin MM, Möller S, Waki K, Gopalakrishnakone P, Ricciardi-
Castagnoli P, Thiesen HJ, Hayashizaki Y, Kollias G: Cytoskeletal rearrangements
in synovial fibroblasts as a novel pathophysiological determinant of
modeled rheumatoid arthritis. PLoS Genet 2005, 1:e48.
38. Rhee SG, Chae HZ, Kim K: Peroxiredoxins: a historical overview and
speculative preview of novel mechanisms and emerging concepts in cell
signaling. Free Radic Biol Med 2005, 38:1543–1552.
39. Szabó-Taylor KÉ, Eggleton P, Turner CA, Faro ML, Tarr JM, Tóth S, Whiteman M,
Haigh RC, Littlechild JA, Winyard PG: Lymphocytes from rheumatoid arthritis
patients have elevated levels of intracellular peroxiredoxin 2, and a greater
frequency of cells with exofacial peroxiredoxin 2, compared with healthy
human lymphocytes. Int J Biochem Cell Biol 2012, 44:1223–1231.
40. Karasawa R, Ozaki S, Nishioka K, Kato T: Autoantibodies to peroxiredoxin I
and IV in patients with systemic autoimmune diseases. Microbiol Immunol
2005, 49:57–65.
41. Gerlag DM, Borges E, Tak PP, Ellerby HM, Bredesen DE, Pasqualini R,
Ruoslahti E, Firestein GS: Suppression of murine collagen-induced arthritis
by targeted apoptosis of synovial neovasculature. Arthritis Res 2001,
3:357–361.

doi:10.1186/ar4434
Cite this article as: Finnegan et al.: Synovial membrane protein
expression differs between juvenile idiopathic arthritis subtypes in early
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