1 Department of Rheumatology, Klinikum Benjamin Franklin, Free University, Berlin,
2 German Rheumatology Research Center, Berlin, Germany,
3 Joint Diseases Laboratory, Shriners Hospitals for Children, Department of Surgery, McGill University, Montreal, Canada and
4 Rheumazentrum Ruhrgebiet, Herne, Germany
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Abstract |
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Methods. Peripheral blood (PB) mononuclear cells (MNC) from 47 AS patients, 22 RA patients and 20 healthy normal controls were exposed in vitro for 6 h to the cartilage-derived autoantigens G1, human cartilage (HC) gp-39 and collagen II. Synovial fluid (SF) MNC from seven AS and four RA patients were similarly analysed. Furthermore, PB MNC of 15 AS and 10 RA patients were examined with overlapping 18-mer peptides covering the whole G1 protein to identify the immunodominant epitopes. T cells were stained by monoclonal antibodies directed against the surface markers CD4, CD69 and against the intracellular cytokines interferon- (IFN
), tumour necrosis factor-
(TNF
), interleukin 4 (IL-4) and IL-10. The percentage of reactive T cells was quantified by flow cytometry.
Results. After antigen-specific stimulation with the G1 protein, the CD4+ T cells of 30 AS patients (61.7%) and of 12 RA patients (54.5%) secreted significant amounts of IFN and TNF
, while, in contrast, only 10% of the normal controls showed a response (P < 0.05). The synovial CD4+ T cells of five AS (71.5%) and of all four RA patients showed antigen-specific responses to the G1. In contrast, stimulation with HC gp-39 and collagen II showed no significant IFN
and TNF
secretion of MNC in all groups. Several G1-derived T-cell epitopes were identified as immunodominant in PB MNC of AS and RA patients and were partly overlapping.
Conclusions. These data show that a cellular immune response to G1 is present in most AS and RA patients. G1-immunodominant epitopes were identified. The relevance of this finding for the pathogenesis of AS and RA remains to be established.
KEY WORDS: Ankylosing spondylitis, Rheumatoid arthritis, G1 immunity, T-cell epitope, Aggrecan.
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Introduction |
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The outcome of 2050% of the patients with reactive arthritis and inflammatory bowel disease carrying HLA-B27 is AS [16, 17]. This association has raised the question of whether the immunopathology in AS is caused or triggered [5, 18] by an antibacterial immune response and/or perpetuated by an immune response directed against an unknown self-antigen. The fact that no bacterial DNA could be detected in biopsies from sacroiliac joints by the polymerase chain reaction technique (PCR) [19], and that in spondylarthropathies such diverse structures as spine, enthesis, eye and aorta are involved, makes bacterial persistence at these sites rather unlikely and argues in favour of an autoimmune response. An increasing amount of studies using magnetic resonance imaging (MRI) have shown that the most severe inflammation in spondylarthropathies is an osteitis occurring at bone/cartilage interphases [7, 20, 21]. This finding has been supplemented by histological investigations indicating that, especially in early phases of spondylarthropathies, mononuclear cells invade and erode the cartilage at different sites [7, 22, 23]. Based on these findings it has been proposed that the cartilage is the primary target of the immune response in spondylarthropathies [18, 2426].
The Montreal group has provided evidence over the last few years that the cartilage proteoglycan aggrecan might be a candidate autoantigen in AS [11, 12, 14, 18, 27, 28], and that it is the G1 globular domain of this molecule where immunity is mainly targeted [27, 28]. These findings are supported by others [15, 29]. While the animal model of HLA-B27-transgenic rats [30] displays a polyarthritis, a spondylitis is less prominent. A mouse model for AS has recently gained more attention. Injection of the G1 domain of aggrecan into BALB/c mice induces not only peripheral arthritis but also spondylitis [27]. It could be shown that T cells play an important role in this model and G1-derived immunodominant T-cell epitopes have been identified [27, 28]. There are limited data in humans about the cellular immune response to the G1 protein to date: these are mainly based on lymphocyte proliferation. With this technique, a T-cell response to the aggrecan has been reported in AS and RA patients, but also in osteoarthritis patients [12, 14, 31]. Furthermore, T lymphocytes have also been reported to respond positively to human cartilage (HC) gp-39 and collagen II, two other cartilage-derived antigens, in RA [3234].
In this study we applied the more sensitive and more specific technique of antigen-specific cytometry to investigate the T-cell response in patients with AS, RA and healthy controls to these cartilage-derived autoantigens [35]. Using induction of interferon- (IFN
) by CD4+ T cells as the primary outcome parameter, we examined the antigen-specific T-cell response in peripheral blood (PB) and synovial fluid (SF) to determine whether T cells specific for the G1 domain of aggrecan and to single G1-derived peptides are detectable in AS patients and controls and compared this to the response after stimulation with two other cartilage-derived proteins, the human cartilage glycoprotein (HC gp)-39 and collagen II protein. We used patients with RA as a reference group to compare responses to the proteoglycan aggrecan.
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Patients and methods |
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The cartilage-derived antigen G1 domain of aggrecan
Human aggrecan G1 domain (AG1) proteins were expressed and purified in an adenovirus expression system. Briefly, a cDNA fragment encoding the N-terminal 431 amino acids of human aggrecan with a His-tag at its C-terminus was generated from a human chondrocyte RNA preparation by reverse transcriptase (RT)-PCR (5'-GCAGATCTACTATGGCCACTTTACTCTGGGTTTTCG-3' and 5'-CAGATCTCAATGGTGATGGTGAT GATGCTCAGCGAAGGCAGTGGC-3'). This PCR fragment was cloned into a pCR2.1 vector using a TA cloning kit (Invitrogen Inc., Carlsbad, CA). The construct, including the human aggrecan G1 globular domain and a partial interglobular domain (IGD) plus six histidine residues at its C-terminal, was sub-cloned into the pQBI-AdCMV5-IRES-GFP transfer vector from the ADENO-QUEST KIT (Quantum Biotechnologies Inc., Montreal, QC) at the BglII site. After being linearized by FseI restriction enzyme digestion, 1 mg of the recombinant transfer vector plasmid was co-transfected into 293 cells with 1 mg of QBI-viral DNA from the same kit using Lipofectamine plus reagent (Lifetech Co., Burlington, ON). Screening and purification of recombinant adenovirus were performed according to the Adeno-Quest application manual included in the kit. For recombinant AG1 production, 293 cells were split on to 150-mm dishes in 1 to 10 dilution in Dulbecco's Modified Eagle Medium (DMEM) plus 5% fetal calf serum (FCS), grown for 2 days until cells were 90% confluent, then the media were changed into 293 serum-free media (Lifetech Co., Burlington, ON); meanwhile recombinant virus were added at 50 MOI. On day 3 after infection, the supernatant containing recombinant AG1 protein was collected. The supernatant was applied to a Sephadex G-25 column equilibrated with phoshate-buffered saline (PBS), pH 7.4; the protein-containing fractions were collected and applied to a Ni-NTA agarose column (Qiagen Inc., Mississanga, ON). The column was washed with 40 mM imidazole in PBS containing 0.3 M NaCl, pH 7.4. Recombinant versican G1 (VG1) and AG1 were eluted with 100 mM imidazole in PBS, pH 7.4, containing 0.3 M NaCl.
HC gp-39 and collagen II
HC gp-39 is present in cartilage, but also in other structures [39]. There have been several reports showing that HC gp-39 might be a possible autoantigen both in animal models of RA and in RA patients [32, 33]. HC gp-39, recombinantly produced, was kindly provided by A.M.M. Boots, at Organon, Akzonobel, The Netherlands. Collagen II has also been implicated in the pathogenesis of RA [34] and collagen-induced arthritis is regarded as an animal model of RA [40]. Collagen II, also recombinantly produced (derived from human sequence), was kindly provided by Fibro Gen, San Francisco, USA. It was expressed in yeast and lacks hydroxylysine and the glycosylated forms of hydroxylysine.
G1-derived peptides
Peptides were synthesized by a robotic multiple peptide synthesizer (SYRO, MultiSynTech, Bochum, Germany) using a Fmoc/tBu solid-phase synthesis strategy [41]. Wang resin (p-benzyloxybenzylalcohol-polystyrene) (Novabiochem, Bad Soden, Germany) was used as solid support. Side-chain-protected Fmoc-amino acids were obtained from Senn Chemicals (Dielsdorf, Switzerland) and Novabiochem (Bad Soden, Germany). Peptides were characterized by reversed-phase high-pressure liquid chromatography (HPLC) (M480 pump, UVD-320 S diode-array UV-detector, GINA 160 autosampler, Gynkotek, Germering/Munich, Germany) on Nucleosil C18, 100A, 5 µm (Macherey-Nagel, Düren, Germany) and electrospray mass-spectrometry (ESI-Quattro II, Micromass Ltd, Altrincham, UK). Forty-six overlapping 18-mer peptides overlapping by 10 amino acid, which cover all 394 amino acid residues of G1 protein, were synthesized.
In vitro stimulation of CD4+ T cells by protein antigen or peptides
T cells were stimulated in vitro by protein antigens as described previously [42]. Briefly, 1 ml of whole heparinized peripheral blood or whole synovial fluid (1 ml containing 5x106 cells) was stimulated in the presence of anti-CD28 (Immunotech, Marseille, France; 1 µg/ml) for 6 h with 20 mg/ml G1 protein, or 20 mg/ml HC gp-39, or 20 mg/ml collagen II, or Staphylococcus enterotoxin B (SEB, Sigma, 1 mg/ml; used as positive control). These concentrations were found to be optimal in preliminary experiments (data not shown). HC gp-39 was investigated in AS but not in RA. Brefeldin A (10 mg/ml, Sigma) was added for the last 4 h of the stimulation. The culture tubes were left at a 5° slant at 37°C in a CO2 incubator. Afterwards cells were incubated in EDTA (2 mM final concentration) for 15 min at room temperature. Nine millilitres of FACS lysing solution (1x) (Becton Dickinson, San Jose, CA) was added to 1 ml of blood for 10 min at room temperature to lyse erythrocytes and fix monocytes. Then the cells were washed again with PBS/BSA. For SF, cells were washed with PBS after EDTA incubation and 1 ml of 2% formaldehyde was added to the pellet for 20 min at room temperature, then the cells were washed again with PBS/BSA.
For stimulation with peptides, the 46 G1 peptides were put into five pools of peptides, with 810 in each pool. Pool 1 contained peptides 110, pool 2 peptides 1119, pool 3 peptides 2028, pool 4 peptides 2938, and pool 5 peptides 3946. For stimulation with peptides, mononuclear cells (MC) instead of whole blood were used. Immediatedly after the blood was drawn in heparinized syringes, MC of 15 AS and 10 RA patients, whose blood was available again, were obtained by Ficoll-paque density gradient centrifugation (Pharmacia, Uppsala, Sweden). The cells were then suspended in RPMI 1640 medium (Gibco BRL, Life Technologies, Paisley, Scotland) with 100 unit/ml penicillin, 100 µg/ml streptomycin (Biochrom KG, Berlin, Germany), 2 mM L-glutamine (Biochrom KG), and 10% heat-inactivated fetal calf serum (Gibco BRL). At least 1x106 cells/ml were stimulated with G1 pools of peptides (each peptide 5 mg/ml) in the presence of anti-CD28, in the presence of anti-CD28 alone (negative control), or with SEB as a positive control. Finally, staining with monoclonal antibodies directed against the surface markers CD4 and CD69 and against intracellular cytokines IFN and tumour necrosis factor-
(TNF
) was performed (see below). For fine epitope mapping, fresh blood was taken again from patients who responded to pools of peptides, and MC were stimulated with single peptides.
Analysis of HLA class II restriction
Monoclonal antibodies (mAbs) specific for the three main human class II MHC products, HLA-DR, HLA-DQ and HLA-DP were purchased from Becton Dickinson (murine Ig G1, San Jose, CA). These antibodies have been observed to block the corresponding class II mediated responses in vitro. The mAbs were thoroughly dialysed against PBS and were used at a predetermined optimal blocking concentration of 2.5 mg/ml in the culture. To determine whether the responding T cells were stimulated by peptide presented by antigen-presenting cells (APC) in the context of MHC class II molecules, blocking antibodies specific for HLA-DR, -DQ or -DP were added to cultures for 30 min at 37°C and samples without blocking antibodies were included as controls. Then the cells were washed with PBS/BSA and resuspended in RPMI 1640 medium with anti-CD28 and stimulated with the peptides of interest for 6 h as described above.
Staining for T-cell surface markers, intracellular cytokines and analysis by flow cytometry
T cells were stained after in vitro stimulation as described previously [42]. Briefly, cells from whole PB or SF or MC were washed with PBS/BSA, centrifuged (300 g, 10 min, 4°C), and cells were quadruple stained for CD4-, CD69-surface markers and two intracellular cytokines, either IFN/TNF
or interleukin 4 (IL-4)/IL-10. All stainings were performed in FACSTM Permeabilizing Solution (Becton Dickinson, Heidelberg, Gemany). To avoid non-specific binding of antibodies to Fc-receptors, all the staining was done in the presence of Beriglobin (3 mg/ml, Centeon pharma, Berlin, Germany). The following antibodies were used: anti-human CD4 PerCP (clone Leu-3a), anti-CD69 FITC and anti-CD69 phycoerythrin (PE) (Leu-23) obtained from Becton Dickinson. The antibodies against TNF
(Hölzel Diagnostika, Köln, Germany) were coupled to FITC (Sigma), antibodies to IFN
were coupled to Cy5 (Amersham Pharmacia Biotech, Freiburg, Germany), antibodies to IL-4 (4D9) were coupled to PE (Becton Dickinson), antibodies to IL-10 were labelled to APC (Pharmingen, San Jose, CA). Positive cells were subsequently quantified by flow cytometry using a FACSCalibur from Becton Dickinson (San Jose, CA) with Cellquest-software. After gating on CD4+ T cells, only cytokine-positive T cells which were also positive for the early activation surface antigen CD69 were counted. To analyse whether the two cytokines, which were stained simultaneously, were produced by the same or different cells, CD4+ T cells positive for two cytokines were also counted at the same time.
CD4+ T cells were regarded as positive after antigen-specific stimulation as judged by the percentage of CD69/cytokine double-positive cells if at least 30 cells and 0.02% of the gated CD4+ T cells were positive without background staining (stimulation with anti-CD28 without antigen only) [42, 43]. If the background staining was above 10 cells the percentage of T cells positive for intracellular cytokine staining had to be at least three times higher than the background staining to be accepted as positive. CD69 is an early T-cell activation marker and is up-regulated shortly after stimulation with specific antigens [44]. Thus, specificity of intracellular cytokine staining is increased by excluding cytokine+/CD69- T cells (non-specific staining of the intracellular cytokines) from analysis.
HLA typing
HLA-DR typing was done by PCR-based methods using sequence-specific primers.
Statistics
2-test was used to compare frequency in different groups; the unpaired version of the Wilcoxon test was used to analyse between-group differences of percentages of G1-specific positive T cells; the paired version of the Wilcoxon test was used to analyse differences of percentages of G1-specific positive T cells between PB and SF. Differences were considered to be significant if there was a two-tailed P value of less than 0.05.
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Results |
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In SF, 71.5% (5/7) of the AS patients responded to in vitro stimulation with G1 by IFN production and 57.2% (4/7) by TNF
production (Fig. 4
). In SF from RA patients, a response to G1 was detectable in all four patients (100%) as judged by IFN
production and in 50% of the patients by TNF
production (Fig. 4
). Interestingly, in SF the percentage of patients responding by IFN
production was higher than the percentage responding by TNF
synthesis, while it was the other way around in PB (Figs 1 and 4
). An example of IFN
and TNF
production of synovial and PB CD4+ T cells in response to these antigens is shown for one AS patient in Fig. 5
. The G1-specific T-cell response in SF [0.12% (0.070.23%) for IFN
; 0.14% (0.010.23%) for TNF
] was significantly (P=0.005 for IFN
, P=0.008 for TNF
) higher than that in PB [0.04% (0.020.12%) for IFN
; 0.04% (0.010.06%) for TNF
] (Fig. 4
).
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Characterization of immunodominant G1 epitopes
A positive response of CD4+ T cells derived from PB to pools of G1 peptides was observed in 15 AS and in 10 RA patients, who also showed a positive response to the whole protein (Tables 2 and 3
). Some of the AS patients recognized only one peptide while others showed a T-cell response to two or more peptides. In all of the RA patients a T-cell response to two or more peptides was detectable. Restimulation of PB T cells from the responding patients with single peptides (all out of the positive pools) indicated that peptides 13, 17 and 35 were stimulatory both in AS and RA patients, while peptide 30 was stimulatory only in some AS but not in RA patients, and peptide 9 was stimulatory only in some RA but not in AS patients (Tables 2
and 3
). The amino acid sequence and its position in the G1 protein is shown for the single peptides in Tables 2
and 3
.
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HLA typing
In the majority of the AS patients a full HLA class II typing was performed. The results for the HLA-DR typing are shown in Table 2. None of the peptides was confined to a single HLA-DR type indicating that they can be presented by different -DR types. However, not all of the -DR types seem to be associated with these peptides. There is a negative association of peptides 13 and 17 with HLA-DR3 and -DR5, of peptide 30 with HLA-DR4 and -DR7 and of peptide 35 with HLA-DR7. This association was statistically not significant, possibly because of a small number. The T-cell response to peptide 13 and 35 could be blocked by anti-class HLA-DR antibodies by about 50% in five patients investigated: a reduction of the percentage of IFN
-positive CD4+ T cells from a median of 0.020 (range 0.0150.035) to 0.01 (00.02) (P=0.038 for this difference) was found for peptide 13 and a reduction from 0.046 (0.0250.06) to 0.024 (0.010.035) (P=0.041 for the difference) for peptide 35. No inhibition of the T-cell response was observed after adding anti-DQ or anti-DP antibodies (data not shown). Thus, these data indicate that the immune response to G1-derived peptides is HLA class II restricted, although several G1-derived peptides can be presented by several HLA class II types.
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Discussion |
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The results presented here do not prove that the G1-specific T-cell response plays a primary role in causing immunopathology in AS and/or RA. It may be a secondary event after cartilage destruction caused by other mechanisms. The fact that the T-cell response to this autoantigen does not seem to be specific for one rheumatic disease is compatible with: (i) aggrecan being a major component of human cartilage which is affected by various rheumatic diseases such as AS, RA and also osteoarthritis; (ii) the physiological role, cleavage and breakdown of aggrecan; and (iii) previous results describing immune reactivity to the G1 domain at both the cellular and the humoral level in different rheumatic diseases [14, 31, 51]. None the less, the demonstration of such a cellular response in both PB and SF of AS and RA patients is encouraging enough to pursue this question in future experiments, particularly since experimental immunity to G1 causes the induction of an inflammatory erosive polyarthritis (as in RA) and a spondylitis (as in AS) [27, 28].
Furthermore, the T-cell response to the aggrecan G1 domain is so far the clearest and strongest autoimmune reaction to cartilage-derived autoantigens both in AS and RA. The T-cell response to both the whole G1 protein and to G1-derived pools of peptides and to single peptides, as shown in our study, confirms the presence of a G1-specific immune response and, importantly, it also excludes a false-positive response due to contamination in the protein/peptide preparations. In comparison, it has been difficult to show T-cell responses to other potential autoantigens such as collagen II and gp-39 in RA [33, 34] and no such investigations have been performed in AS. Thus, independently of its potential role as a causative autoantigen, the aggrecan G1 domain could be a candidate for antigen-specific tolerance induction through bystander suppression, both for AS and RA, similarly as it has been tried with oral collagen II treatment in RA [52].
The examination, quantification and visualization of cellular immune responses have recently become more sophisticated by using flow cytometry [4244], which allows determination not only of the cellular subtypes under investigation but also, at the same time, the measurement of the intracellular cytokines secreted by the same cells after non-specific or antigen-specific stimulation of T cells in vitro [42]. The technique is capable of detecting antigen-specific T-cell frequencies as low as 1x10-5 [35, 43, 53]. Such an improvement in sensitivity is essential if T-cell responses to autoantigens are sought, which are normally difficult to detect because of low frequencies [43]. In our study, we concentrated on CD4+ T-cell responses because we started by investigating whole recombinant proteins, which are normally processed via the pathway II of antigen presentation. The epitopes created are normally only presented to CD4+ T cells.
In parallel to the IFN response, we also observed a high G1-specific TNF
response. However, it remains to be determined whether T-cell responses to candidate antigens including autoantigens can be assessed by TNF
secretion. Our data indicate that antigen-triggered TNF
secretion by T cells could be more sensitive but less specific than IFN
secretion. The investigation of the TNF
response is of special interest in consideration of: (i) the high amount of TNF
present in inflamed sacroiliac joints of AS patients [22], (ii) the reportedly lower amount of TNF
secreted in peripheral blood of AS patients [54] and (iii) the efficacy of anti-TNF
treatment in AS and other spondylarthropathy patients [55].
An antigen-specific T-cell secretion of IL-4 or IL-10 could not be detected in this study. The production of the TH1 cytokines IFN and TNF
upon antigen contact but not of TH2 (IL-4) or TH regulatory (IL-10) cytokines might indicate that: (i) the G1 response might play a role in the immunopatholgy of AS and possibly in RA and (ii) that this does not appear to be counteracted by suppressive cytokines.
The identification of T-cell epitopes is crucial for the understanding of the host response in autoimmune diseases. MHC molecules on the surface of antigen-presenting cells present peptide fragments derived from proteins to T lymphocytes. Once a target protein is defined for a T-cell response, the antigenic epitope can be mapped with synthetic peptides [56]. In our study, four T-cell epitopes within the G1 protein (amino acid residues 116133, 148165, 252269 and 292309) were identified in AS patients and also in RA patients (amino acid residues 84101, 116133, 148165 and 292309). Previous work on G1 induction of an erosive polyarthritis and spondylitis in BALB/c mice has revealed that the G1 domain of the proteoglycan aggrecan contains an immunodominant arthritogenic region identified by two distinct T-cell epitopes [28]. Adoptive transfer of T cells specific for these peptides also induced arthritis [28]. The identified immunodominant epitopes found in mice and in our study were not identical, a finding which is not surprising if a different MHC background is considered.
We showed in this study that not a single but several peptides out of the G1 domain are recognized by T cells, that the immunodominant peptides identified in AS and RA patients are overlapping, and that the immunodominant peptides might, at least partly, be determined by the HLA-DR type. All this indicates that there does not seem to be a single causative antigen, at least not on the CD4 T-cell level, but rather a broad T-cell response to the G1 domain with the implications discussed above. However, in context of the strong HLA-B27 association in AS, the identification of epitopes presented by HLA-B27 to CD8+ T cells [5] would certainly be of great interest. Thus, while the CD4+ T-cell response might be rather non-specific, this does not exclude a CD8 T-cell response to one or a few arthritogenic peptides derived from the G1 domain. The experiments to address this question are currently in progress.
Final proof for a critical role of the G1 molecule in the pathogenesis of AS will come from the detection of antigen-specific T cells in cartilage [7, 22], possibly through tetramer technology [57], or, both for AS and RA, by induction of G1-specific T-cell tolerance, possibly through mucosal tolerance [52]. The latter approach could be used for both AS and RA even if the G1 molecule is not pathogenetic. Owing to the strong HLA-B27 association in AS, it will also be important to investigate the G1-specific response of CD8+ T cells. Furthermore, it will be very interesting to look for G1-directed immune responses in other spondylarthropathies such as reactive arthritis and psoriatic arthritis in which, clinically, the same anatomical structures are involved.
Taken together, a new piece has been added to the puzzle of the immunopathology of AS: this study clearly suggests that CD4+ T cells of the majority of AS and RA patients recognize the G1 domain of aggrecana molecule that is present in clinically relevant anatomical structures involved in AS and other spondylarthropathies.
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Conflict of interest |
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Notes |
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References |
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