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1.  Good correlation between changes in objective and subjective signs of inflammation in patients with short- but not long duration of axial spondyloarthritis treated with tumor necrosis factor-blockers 
The aim of this study was to investigate the influence of symptom duration on treatment response and on the correlation between improvements in patient reported outcomes (PRO) and objective inflammation in patients with axial spondylarthritis (SpA) treated with etanercept (ETA) or adalimumab (ADA).
Data from 112 patients with axial SpA originally enrolled in two randomized controlled clinical trials were pooled and analyzed after one year of treatment with ETA (n = 66) or ADA (n = 46). Patients with <4 years and ≥4 years of disease were compared for improvement in Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Ankylosing Spondylitis Disease Activity Score (ASDAS), C-reactive protein (CRP) and magnetic resonance imaging (MRI) score for sacroiliac joints (SIJ).
Patients with <4 years of disease showed a significantly better improvement than longer diseased patients in BASDAI (3.2 (95% confidence interval (CI): 2.7 to 3.7) vs. 1.7 (1.1 to 2.2)), BASFI, BASMI and ASDAS (1.6 (1.4 to 1.8) vs. 0.9 (0.7 to 1.1)). The change in BASDAI showed a significant correlation with the change in SIJ score (Spearman’s rank correlation coefficient (rho) = 0.37, P = 0.01) and the change in CRP (rho = 0.45, P = 0.001) in patients with <4 years of disease. For long diseased patients this correlation was poor and did not achieve statistical significance (rho = 0.13, P = 0.46; rho = 0.22, P = 0.13 respectively).
The low correlation between change of PROs and change of objective signs of inflammation seen in axial SpA patients with longer symptom duration treated with tumor necrosis factor-blocker seems to indicate that inflammation is not the only cause of the patients’ symptoms, while inflammation seems to be the major cause in short diseased patients.
Trial registration
Clinical NCT00844142 (Trial 1); NCT00235105 (Trial 2)
PMCID: PMC3978619  PMID: 24476416
2.  Long-term outcome of patients with active ankylosing spondylitis with etanercept-sustained efficacy and safety after seven years 
Data from clinical studies on the long-term efficacy and safety of anti-tumor necrosis factor (TNF)-α therapy in patients with ankylosing spondylitis (AS) are scarce. This is the first report on continuous treatment with the TNFα fusion protein etanercept over seven years (y).
Overall, 26 patients with active AS were initially treated with etanercept 2 × 25 mg s.c./week with no concomitant disease modifying anti-rheumatic drugs (DMARDs) or steroids. The clinical response was assessed by standardized parameters. The primary outcome was the proportion of patients in the Spondyloarthritis International Society (ASAS) partial remission at seven years. AS disease activity scores (ASDAS) for status and improvement were compared to conventional outcome measures.
Overall, 21/26 patients (81%) completed two years of treatment and 16/26 patients (62%) completed seven years. In the completer analysis, 31% patients were in ASAS partial remission at seven years, while 44% patients showed an ASDAS inactive disease status. Mean Bath AS activity index (BASDAI) scores, which were elevated at baseline (6.3 ± 0.9), showed constant improvement and remained low: 3.1 ± 2.5 at two years and 2.5 ± 2.2 at seven years, while ASDAS also improved (3.9 ± 0.7 at baseline, 1.8 ± 0.9 at two years, 1.6 ± 0.8 at seven years), all P <0.001. From the 10 dropouts, only 5 patients discontinued treatment due to adverse events. Patients who completed the study had lower baseline Bath AS function index (BASFI) scores vs. patients who discontinued. No other clinical parameter at baseline could predict any long-term outcome.
This study confirms the clinical efficacy and safety of etanercept in patients with active AS over seven years of continuous treatment. After seven years, more than half of the initially treated patients remained on anti-TNF therapy, and one-third were in partial remission.
Trial Registration NCT01289743
PMCID: PMC4060369  PMID: 23786760
ankylosing spondylitis; TNFa; etanercept; ASDAS, BASDAI
3.  TNF signaling drives myeloid-derived suppressor cell accumulation 
The Journal of Clinical Investigation  2012;122(11):4094-4104.
TNF, an inflammatory cytokine that is enriched in the tumor microenvironment, promotes tumor growth and subverts innate immune responses to cancer cells. We previously reported that tumors implanted in TNF receptor–deficient (Tnfr–/–) mice are spontaneously rejected; however, the molecular mechanisms underlying this rejection are unclear. Here we report that TNF signaling drives the peripheral accumulation of myeloid-derived suppressor cells (MDSCs). MDSCs expand extensively during inflammation and tumor progression in mice and humans and can enhance tumor growth by repressing T cell–mediated antitumor responses. Peripheral accumulation of MDSCs was drastically impaired in Tnfr–/– mice. Signaling of TNFR-2, but not TNFR-1, promoted MDSC survival through upregulation of cellular FLICE-inhibitory protein (c-FLIP) and inhibition of caspase-8 activity. Loss of TNFRs impaired the induction of MDSCs from bone marrow cells, but this could be reversed by treatment with caspase inhibitors. These results demonstrate that TNFR-2 signaling promotes MDSC survival and accumulation and helps tumor cells evade the immune system.
PMCID: PMC3484453  PMID: 23064360
4.  IL-17A Expression Is Localised to Both Mononuclear and Polymorphonuclear Synovial Cell Infiltrates 
PLoS ONE  2011;6(8):e24048.
This study examines the expression of IL-17A-secreting cells within the inflamed synovium and the relationship to in vivo joint hypoxia measurements.
IL-17A expression was quantified in synovial tissue (ST), serum and synovial fluid (SF) by immunohistochemistry and MSD-plex assays. IL-6 SF and serum levels were measured by MSD-plex assays. Dual immunofluorescence for IL-17A was quantified in ST CD15+ cells (neutrophils), Tryptase+ (mast cells) and CD4+ (T cells). Synovial tissue oxygen (tpO2) levels were measured under direct visualisation at arthroscopy. Synovial infiltration was assessed using immunohistochemistry for cell specific markers. Peripheral blood mononuclear and polymorphonuclear cells were isolated and exposed to normoxic or 3% hypoxic conditions. IL-17A and IL-6 were quantified as above in culture supernatants.
IL-17A expression was localised to mononuclear and polymorphonuclear (PMN) cells in inflamed ST. Dual immunoflourescent staining co-localised IL-17A expression with CD15+ neutrophils Tryptase+ mast cells and CD4+T cells. % IL-17A positivity was highest on CD15+ neutrophils, followed by mast cells and then CD4+T-cells. The number of IL-17A-secreting PMN cells significantly correlated with sublining CD68 expression (r = 0.618, p<0.01). IL-17A SF levels correlated with IL-6 SF levels (r = 0.675, p<0.01). Patients categorized according to tp02< or >20mmHg, showed those with low tp02<20mmHg had significantly higher IL-17A+ mononuclear cells with no difference observed for PMNs. Exposure of mononuclear and polymorphonuclear cells to 3% hypoxia, significantly induced IL-6 in mononuclear cells, but had no effect on IL-17A expression in mononuclear and polymorphonuclear cells.
This study demonstrates IL-17A expression is localised to several immune cell subtypes within the inflamed synovial tissue, further supporting the concept that IL-17A is a key mediator in inflammatory arthritis. The association of hypoxia with Il-17A expression appears to be indirect, probably through hypoxia-induced pro-inflammatory pathways and leukocyte influx within the joint microenvironment.
PMCID: PMC3161104  PMID: 21887369
5.  Analysis of IL-17+ cells in facet joints of patients with spondyloarthritis suggests that the innate immune pathway might be of greater relevance than the Th17-mediated adaptive immune response 
In this study, we analysed the number of IL-17+ cells in facet joints, in the peripheral blood (PB) and synovial fluid (SF) of spondyloarthritis (SpA) patients and compared these results with those of patients with other rheumatic diseases and controls.
Immunohistochemical analysis of IL-17+ cells was performed in facet joints of 33 ankylosing spondylitis (AS) patients and compared with data from 20 osteoarthritis (OA) patients. The frequency of IL-17+CD4+ T cells in PB and SF of SpA patients (PB n = 30, SF n = 11), rheumatoid arthritis (RA) patients (PB n = 14, SF n = 7), OA patients (PB n = 10) and healthy controls (PB n = 12) was analysed after stimulation with Staphylococcus aureus Enterotoxin B and phorbol 12-myristate 13-acetate/ionomycin and quantified by flow cytometry.
In AS facet joints, the frequency of IL-17-secreting cells was significantly higher than in samples obtained from OA patients (P < 0.001), with a slight predominance of IL-17+ cells among the mononuclear cells (61.5% ± 14.9%) compared to cells with polysegmental nuclei. Immunofluorescence microscopy revealed that the majority of IL-17+ cells were myeloperoxidase-positive (35.84 ± 13.06/high-power field (HPF) and CD15+ neutrophils (24.25 ± 10.36/HPF), while CD3+ T cells (0.51 ± 0.49/HPF) and AA-1+ mast cells (2.28 ± 1.96/HPF) were less often IL-17-positive. The frequency of IL-17+CD4+ T cells in the PB and SF of SpA patients did not differ significantly compared to RA patients, OA patients or healthy controls.
Our data suggest an important role for IL-17 in the inflammatory processes in AS. However, the innate immune pathway might be of greater relevance than the Th17-mediated adaptive immune response.
PMCID: PMC3218910  PMID: 21689402
6.  Predicting the outcome of ankylosing spondylitis therapy 
Annals of the Rheumatic Diseases  2011;70(6):973-981.
To create a model that provides a potential basis for candidate selection for anti-tumour necrosis factor (TNF) treatment by predicting future outcomes relative to the current disease profile of individual patients with ankylosing spondylitis (AS).
ASSERT and GO–RAISE trial data (n=635) were analysed to identify baseline predictors for various disease-state and disease-activity outcome instruments in AS. Univariate, multivariate, receiver operator characteristic and correlation analyses were performed to select final predictors. Their associations with outcomes were explored. Matrix and algorithm-based prediction models were created using logistic and linear regression, and their accuracies were compared. Numbers needed to treat were calculated to compare the effect size of anti-TNF therapy between the AS matrix subpopulations. Data from registry populations were applied to study how a daily practice AS population is distributed over the prediction model.
Age, Bath ankylosing spondylitis functional index (BASFI) score, enthesitis, therapy, C-reactive protein (CRP) and HLA-B27 genotype were identified as predictors. Their associations with each outcome instrument varied. However, the combination of these factors enabled adequate prediction of each outcome studied. The matrix model predicted outcomes as well as algorithm-based models and enabled direct comparison of the effect size of anti-TNF treatment outcome in various subpopulations. The trial populations reflected the daily practice AS population.
Age, BASFI, enthesitis, therapy, CRP and HLA-B27 were associated with outcomes in AS. Their combined use enables adequate prediction of outcome resulting from anti-TNF and conventional therapy in various AS subpopulations. This may help guide clinicians in making treatment decisions in daily practice.
PMCID: PMC3086037  PMID: 21402563
7.  Performance of referral recommendations in patients with chronic back pain and suspected axial spondyloarthritis 
Annals of the Rheumatic Diseases  2007;66(11):1479-1484.
Ankylosing spondylitis (AS) and its early form account for up to 5% of all patients with chronic back pain. Interest has recently focused on shortening the delay of 5–10 years between the appearance of first symptoms and the diagnosis of AS, particularly because effective treatments have now become available. Referral parameters that are easy for doctors in primary care to apply to patients presenting with possible AS could contribute to earlier diagnosis.
Orthopaedists and primary‐care doctors were requested to refer patients with (1) chronic low back pain (duration >3 months) and (2) onset of back pain before <45 years of age to a specialist rheumatology outpatient clinic for further diagnostic investigation if at least one of the following screening parameters was present: (1) inflammatory back pain, (2) positive human leucocyte antigen B27, and (3) sacroiliitis detected by imaging. The final diagnosis was made according to expert opinion.
In total, 350 referred cases were analysed. A diagnosis of definite axial spondyloarthritis (axial SpA), comprising established AS and pre‐radiographic axial SpA, could be made in 45.4% of all referred patients (of which 50.3% were classified as AS and 49.7% as preradiographic axial SpA), whereas 45.4% were classified as non‐SpA and 9.1% as possible SpA. A diagnosis of definite axial SpA could be made in 34.2% if only one referral parameter was positive, and in 62.6% if there was >1 positive referral parameter.
The proposed referral parameters have proven useful when applied in primary care in identifying patients with AS/pre‐radiographic axial SpA among young to middle‐aged patients with chronic low back pain.
PMCID: PMC2111623  PMID: 17456526
ankylosing spondylitis; spondyloarthritis; pre‐radiographic; screening; early diagnosis
8.  Physical function, disease activity, and health-related quality-of-life outcomes after 3 years of adalimumab treatment in patients with ankylosing spondylitis 
Arthritis Research & Therapy  2009;11(4):R124.
We evaluated the three-year impact of adalimumab on patient-reported physical function and health-related quality-of-life (HRQOL) outcomes in patients with active ankylosing spondylitis (AS).
The Adalimumab Trial Evaluating Long-Term Efficacy and Safety in AS (ATLAS) is an ongoing five-year study that included an initial 24-week, randomized, placebo-controlled, double-blind period, followed by open-label extension treatment with adalimumab. Clinical and HRQOL data collected for up to three years from ATLAS were used for these analyses. Patients were randomized to receive adalimumab 40 mg or placebo by subcutaneous injection every other week. Physical function was assessed by the Bath AS Functional Index (BASFI), as well as by the Short Form 36 (SF-36) Health Survey Physical Component Summary (PCS) and Physical Function subscale scores. HRQOL was assessed using the AS Quality of Life (ASQOL) questionnaire. Disease activity was assessed by the Bath AS Disease Activity Index (BASDAI).
Of 315 patients enrolled in ATLAS, 288 (91%) participated in an open-label adalimumab treatment extension and 82% provided three-year outcome data. During the 24-week double-blind phase, adalimumab-treated patients experienced significant improvement compared with placebo-treated patients in the BASDAI (P < 0.001), BASFI (P < 0.001), ASQOL (P < 0.001), and both the SF-36 PCS (P < 0.001) and Physical Function subscale (P < 0.001) scores, but not the SF-36 Mental Component Summary score (P = 0.181) and Mental Health subscale scores (P = 0.551). Mean changes from baseline through three years of adalimumab treatment were statistically significant for the BASDAI (change score: -3.9, P < 0.001), BASFI (change score: -29.6, P < 0.001), SF-36 PCS (change score: 11.6, P < 0.001), and Physical Function (change score: 23.3, P < 0.001). Comparable results were observed for the other SF-36 scores and for the ASQOL (all P < 0.001).
Adalimumab significantly improved disease activity, patient-reported physical function, and HRQOL. These benefits were maintained over three years of treatment in patients with AS.
Trial registration NCT00085644.
PMCID: PMC2745808  PMID: 19686597
9.  Developments in the scientific and clinical understanding of the spondyloarthritides 
Major advances have been achieved over the last 10 years both in the clinical and scientific understanding of the spondyloarthritides (SpA), which can be separated in predominantly axial and predominantly peripheral SpA. The clinical progress includes the development of classification criteria, strategies for early diagnosis, definition of outcome criteria for clinical studies, and the conduction of a series of clinical studies with a focus on tumor necrosis factor (TNF) blockers. The proven high efficacy of TNF blocker treatment has meant a breakthrough for SpA patients, who until recently had only quite limited treatment options. More and more data have accumulated over recent years in regard to long-term efficacy and safety, prediction of response, and the relevance of extrarheumatic manifestations such as uveitis, psoriasis, and inflammatory bowel disease for treatment decisions with TNF blockers. A better understanding of the interaction of the immune system and inflammation with bone degradation/new bone formation is crucial for the development of optimal treatment strategies to prevent structural damage. Recent results from genetic studies could show that, besides HLA-B27, the interleukin-23 receptor and the ARTS1 enzyme are associated with ankylosing spondylitis. Only when the exact pathogenesis is clarified will a curative treatment be possible.
PMCID: PMC2688219  PMID: 19232062
10.  Serum levels of biomarkers of bone and cartilage destruction and new bone formation in different cohorts of patients with axial spondyloarthritis with and without tumor necrosis factor-alpha blocker treatment 
Arthritis Research & Therapy  2008;10(5):R125.
Recent data about radiographic progression during treatment with tumor necrosis factor-alpha (TNF-α) blocker agents in patients with ankylosing spondylitis (AS) have prompted an intensive discussion about the link between inflammation/bone destruction and new bone formation and the order of events. Therefore, we analysed parameters of cartilage degradation, neoangiogenesis, and new bone formation in different cohorts of patients with axial spondyloarthritis with and without treatment with TNF-α blocker agents.
TNF-α blocker-naïve AS patients were investigated for serum levels of metalloproteinase-3 (MMP-3) (n = 71), vasoendothelial growth factor (VEGF) (n = 50), and bone-specific alkaline phosphatase (BALP) (n = 71) at baseline and after 1 and 2 years. This was compared with 34 adalimumab-treated patients with axial spondyloarthritis (22 AS and 12 non-radiographic axial spondyloarthritis patients) before and after 36 to 52 weeks of treatment.
There were no significant changes in serum levels of MMP-3 (P > 0.05), VEGF (P > 0.05), and BALP (P > 0.05) in a large cohort of TNF-α blocker-naïve AS patients followed for 2 years. In contrast, adalimumab-treated spondyloarthritis (AS and non-radiographic axial spondyloarthritis) patients had a significant decrease of VEGF (P < 0.001) and MMP-3 (P = 0.022) after 36 to 52 weeks of therapy. Most interestingly, the level of BALP increased significantly after 36 to 52 weeks of therapy (P < 0.001). A decrease in MMP-3 serum levels correlated significantly to an increase of BALP (r = -0.398, P = 0.02). In the case of VEGF, there was a negative correlation without significance (r = -0.214, P > 0.05).
Rising levels of BALP and the negative correlation between MMP-3 and BALP in spondyloarthritis patients with TNF-α blocker treatment indicate that new bone formation in AS occurs if inflammation is successfully treated and might be part of a healing process.
PMCID: PMC2592815  PMID: 18945353
11.  The relationship between inflammation and new bone formation in patients with ankylosing spondylitis 
Arthritis Research & Therapy  2008;10(5):R104.
Spinal inflammation as detected by magnetic resonance imaging and new bone formation as identified by conventional radiographs are characteristic of ankylosing spondylitis. Whether and how spondylitis and syndesmophyte formation are linked are unclear. Our objective was to investigate whether and how spinal inflammation are associated with new bone formation in ankylosing spondylitis.
Spinal magnetic resonance images and conventional radiographs from 39 ankylosing spondylitis patients treated with anti-tumour necrosis factor (anti-TNF) agents at baseline and after 2 years were analysed for syndesmophyte formation at vertebral edges with or without inflammatory lesions at baseline.
Overall, 922 vertebral edges at the cervical and lumbar spine were analysed. At baseline, the proportion of vertebral edges with and without inflammation (magnetic resonance imaging) that showed structural changes (conventional radiographs) was similar (in total, 16.6% of all vertebral edges in 71.4% of patients). From the perspective of syndesmophyte formation (n = 26, 2.9%) after 2 years, there were more vertebral edges without (62%) than with (38%) inflammation at baseline (P = 0.03). From the perspective of spinal inflammation at baseline (n = 153 vertebral edges), more syndesmophytes developed at vertebral edges with (6.5%) than without (2.1%) inflammation (P = 0.002, odds ratio 3.3, 95% confidence interval 1.5 to 7.4). Inflammation persisted in 31% of the initially inflamed vertebral edges (n = 132), and new lesions developed in 8% of the vertebral edges without inflammation at baseline (n = 410). From the perspective of spinal inflammation after 2 years (n = 72 vertebral edges), 5.6% of the vertebral edges showed syndesmophyte development in contrast to 1.9% of the vertebral edges with new syndesmophytes without inflammation (P = 0.06).
These findings obtained in patients treated with anti-TNF agents suggest linkage and some dissociation of inflammation and new bone formation in ankylosing spondylitis. Although syndesmophytes were also found to develop at sites where no inflammation had been seen by magnetic resonance imaging at baseline, it was more likely that syndesmophytes developed in inflamed vertebral edges. More effective suppression of spinal inflammation may be required to inhibit structural damage in ankylosing spondylitis.
PMCID: PMC2592781  PMID: 18761747
12.  The human anti-IL-1β monoclonal antibody ACZ885 is effective in joint inflammation models in mice and in a proof-of-concept study in patients with rheumatoid arthritis 
IL-1β is a proinflammatory cytokine driving joint inflammation as well as systemic signs of inflammation, such as fever and acute phase protein production.
ACZ885, a fully human monoclonal antibody that neutralizes the bioactivity of human IL-1β, was generated to study the potent and long-lasting neutralization of IL-1β in mechanistic animal models as well as in a proof-of-concept study in patients with rheumatoid arthritis (RA).
The mouse IL-1 receptor cross-reacts with human IL-1β, and it was demonstrated that ACZ885 can completely suppress IL-1β-mediated joint inflammation and cartilage destruction in mice. This observation prompted us to study the safety, tolerability and pharmacodynamic activity of ACZ885 in RA patients in a small proof-of-concept study – the first to be conducted in humans. Patients with active RA despite treatment with stable doses of methotrexate were enrolled in this dose escalation study. The first 32 patients were split into four cohorts of eight patients each (six were randomly assigned to active treatment and two to placebo). ACZ885 doses were 0.3, 1, 3 and 10 mg/kg, administered intravenously on days 1 and 15. To explore efficacy within 6 weeks of treatment, an additional 21 patients were randomly assigned to the 10 mg/kg cohort, resulting in a total of 20 patients dosed with 10 mg/kg and 15 patients treated with placebo. There was clinical improvement (American College of Rheumatology 20% improvement criteria) at week 6 in the 10 mg/kg treatment group; however, this did not reach statistical significance (P = 0.085). A statistically significant reduction in disease activity score was observed after 4 weeks in the 10 mg/kg group. Onset of action was rapid, because most responders exhibited improvement in their symptoms within the first 3 weeks. C-reactive protein levels decreased in patients treated with ACZ885 within 1 week. ACZ885 was well tolerated. Three patients receiving ACZ885 developed infectious episodes that required treatment. No anti-ACZ885 antibodies were detected during the study.
ACZ885 administration to methotrexate-refractory patients resulted in clinical improvement in a subset of patients. Additional studies to characterize efficacy in RA and to determine the optimal dose regimen appear warranted.
Trial Registration identifier NCT00619905.
PMCID: PMC2483458  PMID: 18534016
13.  Homologous high-throughput expression and purification of highly conserved E coli proteins 
Genetic factors and a dysregulated immune response towards commensal bacteria contribute to the pathogenesis of Inflammatory Bowel Disease (IBD). Animal models demonstrated that the normal intestinal flora is crucial for the development of intestinal inflammation. However, due to the complexity of the intestinal flora, it has been difficult to design experiments for detection of proinflammatory bacterial antigen(s) involved in the pathogenesis of the disease. Several studies indicated a potential association of E. coli with IBD. In addition, T cell clones of IBD patients were shown to cross react towards antigens from different enteric bacterial species and thus likely responded to conserved bacterial antigens. We therefore chose highly conserved E. coli proteins as candidate antigens for abnormal T cell responses in IBD and used high-throughput techniques for cloning, expression and purification under native conditions of a set of 271 conserved E. coli proteins for downstream immunologic studies.
As a standardized procedure, genes were PCR amplified and cloned into the expression vector pQTEV2 in order to express proteins N-terminally fused to a seven-histidine-tag. Initial small-scale expression and purification under native conditions by metal chelate affinity chromatography indicated that the vast majority of target proteins were purified in high yields. Targets that revealed low yields after purification probably due to weak solubility were shuttled into Gateway (Invitrogen) destination vectors in order to enhance solubility by N-terminal fusion of maltose binding protein (MBP), N-utilizing substance A (NusA), or glutathione S-transferase (GST) to the target protein. In addition, recombinant proteins were treated with polymyxin B coated magnetic beads in order to remove lipopolysaccharide (LPS). Thus, 73% of the targeted proteins could be expressed and purified in large-scale to give soluble proteins in the range of 500 μg.
Here, we report a cost-efficient procedure to produce around 200 soluble recombinant E. coli proteins in large-scale, including removal of LPS by polymyxin B coated beads for subsequent use of the proteins in downstream immunological studies.
PMCID: PMC1914363  PMID: 17553160
14.  Correlation of histopathological findings and magnetic resonance imaging in the spine of patients with ankylosing spondylitis 
Ankylosing spondylitis (AS) is a chronic inflammatory disease which affects primarily the sacroiliac joints and the spine. In patients with active disease, magnetic resonance imaging (MRI) of the spine shows areas of bone marrow edema, the histopathological equivalent of which is unknown. In this study we correlate inflammation in the spine of patients with AS as revealed by histological examination with bone marrow edema as detected by MRI. We have compared the histopathological findings of zygapophyseal joints from 8 patients with AS (age: 30 to 64, disease duration 7 to 33 years) undergoing spinal surgery with findings in MRI. For histopathological analysis, we quantified infiltrates of CD3+, CD4+ and CD8+ T cells as well as CD20+ B cells immunohistochemically. Bone marrow edema was evaluated in hematoxylin and eosin stained sections and quantified as the percentage of the bone marrow area involved. All patients with AS showed interstitial mononuclear cell infiltrates and various degrees of bone marrow edema (range from 10% to 60%) in histopathological analysis. However, in only three of eight patients histopathological inflammation and edema in the zygapophyseal joints correlated with bone marrow edema in zygapophyseal joints of the lumbar spine as detected by MRI. Interestingly, two of these patients showed the highest histological score for bone marrow edema (60%). This first study correlating histopathological changes in the spine of patients with AS with findings in MRI scans suggests that a substantial degree of bone marrow inflammation and edema is necessary to be detected by MRI.
PMCID: PMC1779434  PMID: 16925803
16.  Clinical response to discontinuation of anti-TNF therapy in patients with ankylosing spondylitis after 3 years of continuous treatment with infliximab 
Arthritis Research & Therapy  2005;7(3):R439-R444.
We analyzed the clinical response and the time to relapse after discontinuation of continuous long-term infliximab therapy in patients with ankylosing spondylitis (AS). After 3 years of infliximab therapy, all AS patients (n = 42) discontinued treatment (time point (TP)1) and were visited regularly for 1 year in order to assess the time to relapse (TP2). Relapse was defined as an increase to a value ≥ 4 on the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and a physician's global assessment ≥ 4 according to the recommendations of the Assessments in Ankylosing Spondylitis (ASAS) working group. After 52 weeks, 41 of the 42 patients (97.6%) had to be reinfused because of relapse. The mean change in the BASDAI between TP1 and TP2 was 3.6 ± 1.7 and that in the physician's global assessment was 4.4 ± 1.8 (both P < 0.001). The mean time to relapse was 17.5 weeks (± 7.9 weeks, range 7 to 45). Ten patients (24%) showed a relapse within 12 weeks and 38 patients (90.5%), within 36 weeks. After 52 weeks, only one patient had remained in ongoing remission without further treatment with anti-tumor-necrosis factor. Patients who were in partial remission according to the ASAS criteria and those with normal C-reactive protein levels at the time point of withdrawal had longer times to relapse after discontinuation of the treatment. Retreatment with infliximab was safe and resulted in clinical improvement in all patients to a state similar to that before the treatment was stopped. Discontinuation of long-term therapy with infliximab eventually led to relapse of disease activity in all patients but one.
PMCID: PMC1174938  PMID: 15899030
17.  Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in Chlamydia-triggered reactive arthritis 
Arthritis Research & Therapy  2004;6(6):R521-R534.
Reports of the use of HLA-B27/peptide tetrameric complexes to study peptide-specific CD8+ T cells in HLA-B27+-related diseases are rare. To establish HLA-B27 tetramers we first compared the function of HLA-B27 tetramers with HLA-A2 tetramers by using viral epitopes. HLA-B27 and HLA-A2 tetramers loaded with immunodominant peptides from Epstein–Barr virus were generated with comparable yields and both molecules detected antigen-specific CD8+ T cells. The application of HLA-B27 tetramers in HLA-B27-related diseases was performed with nine recently described Chlamydia-derived peptides in synovial fluid and peripheral blood, to examine the CD8+ T cell response against Chlamydia trachomatis antigens in nine patients with Chlamydia-triggered reactive arthritis (Ct-ReA). Four of six HLA-B27+ Ct-ReA patients had specific synovial T cell binding to at least one HLA-B27/Chlamydia peptide tetramer. The HLA-B27/Chlamydia peptide 195 tetramer bound to synovial T cells from three of six patients and HLA-B27/Chlamydia peptide 133 tetramer to synovial T cells from two patients. However, the frequency of these cells was low (0.02–0.09%). Moreover, we demonstrate two methods to generate HLA-B27-restricted T cell lines. First, HLA-B27 tetramers and magnetic beads were used to sort antigen-specific CD8+ T cells. Second, Chlamydia-infected dendritic cells were used to stimulate CD8+ T cells ex vivo. Highly pure CD8 T cell lines could be generated ex vivo by magnetic sorting by using HLA-B27 tetramers loaded with an EBV peptide. The frequency of Chlamydia-specific, HLA-B27 tetramer-binding CD8+ T cells could be increased by stimulating CD8+ T cells ex vivo with Chlamydia-infected dendritic cells. We conclude that HLA-B27 tetramers are a useful tool for the detection and expansion of HLA-B27-restricted CD8+ T cells. T cells specific for one or more of three Chlamydia-derived peptides were found at low frequency in synovial fluid from HLA-B27+ patients with Ct-ReA. These cells can be expanded ex vivo, suggesting that they are immunologically functional.
PMCID: PMC1064864  PMID: 15535830
HLA-B27; T cells; tetramers; reactive arthritis
18.  Kaposi's Sarcoma-Associated Herpesvirus Cytotoxic T Lymphocytes Recognize and Target Darwinian Positively Selected Autologous K1 Epitopes 
Journal of Virology  2003;77(7):4306-4314.
Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of Kaposi's sarcoma (KS) and certain lymphoproliferations particularly in the context of human immunodeficiency virus (HIV) type 1-induced immunosuppression. The introduction of effective therapies to treat HIV has led to a decline in the incidence of KS, suggesting that immune responses may play a role in controlling KSHV infection and pathogenesis. Cytotoxic-T-lymphocyte (CTL) activity against KSHV proteins has been demonstrated; however, the identification of KSHV CTL epitopes remains elusive and problematic. Although the herpesvirus genomic layout is generally conserved, KSHV encodes a unique hypervariable protein, K1, with intense biological selection pressure at specific amino acid sites. To investigate whether this variability is partly driven by cellular immunity, we designed K1 peptides that match only the unique viral sequence for every individual studied here (autologous peptides). We identified functional CTL epitopes within K1's most variable areas, and we show that a given individual responds only to autologous peptides and not to peptides from other individuals. Furthermore, these epitopes are highly conserved sequences within KSHV isolates from a specific strain but are not conserved between different strains. We conclude that CTL recognition contributes to K1, and therefore to KSHV, evolution.
PMCID: PMC150628  PMID: 12634388
19.  Therapy of ankylosing spondylitis and other spondyloarthritides: established medical treatment, anti-TNF-α therapy and other novel approaches 
Arthritis Research  2002;4(5):307-321.
Therapeutic options for patients with more severe forms of spondyloarthritis (SpA) have been rather limited in recent decades. There is accumulating evidence that anti-tumor-necrosis-factor (anti-TNF) therapy is highly effective in SpA, especially in ankylosing spondylitis and psoriatic arthritis. The major anti-TNF-α agents currently available, infliximab (Remicade®) and etanercept (Enbrel®), are approved for the treatment of rheumatoid arthritis (RA) in many countries. In ankylosing spondylitis there is an unmet medical need, since there are almost no disease-modifying antirheumatic drugs (DMARDs) available for severely affected patients, especially those with spinal manifestations. Judging from recent data from more than 300 patients with SpA, anti-TNF therapy seems to be even more effective in SpA than in rheumatoid arthritis. However, it remains to be shown whether patients benefit from long-term treatment, whether radiological progression and ankylosis can be stopped and whether long-term biologic therapy is safe.
PMCID: PMC128942  PMID: 12223105
ankylosing spondylitis; anti-TNF-α therapy; conventional and innovative treatment; psoriatic arthritis
20.  Early response to adalimumab predicts long-term remission through 5 years of treatment in patients with ankylosing spondylitis 
Annals of the Rheumatic Diseases  2011;71(5):700-706.
To describe the efficacy and safety through 5 years of adalimumab treatment in patients with ankylosing spondylitis (AS), and to identify predictors of remission.
Patients with active AS were followed up to 5 years during a 24-week randomised, controlled period, followed by an open-label extension. Disease activity and clinical improvement were evaluated by Assessment in Spondyloarthritis International Society (ASAS) responses, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Ankylosing Spondylitis Disease Activity Score (ASDAS). Kaplan–Meier was used to identify patients with sustained ASAS partial remission (PR) or ASDAS inactive disease (ID) for three or more consecutive visits spanning ≥6 months. Logistic regression was used to identify factors associated with remission. Explanatory variables included baseline demographic and disease characteristics and week 12 responses.
Of the 311 patients who received at least one dose of adalimumab, 202 (65%) completed the 5-year study. Among 125 patients who received 5 years of adalimumab, 70%, 77%, 51% and 61% achieved ASAS40, BASDAI 50, ASAS PR and ASDAS ID, respectively. Of 311 adalimumab-treated patients, 45% and 55% achieved sustained ASAS PR and ASDAS ID at any time during study participation. The strongest predictor of remission at years 1 and 5 and of sustained remission was achieving remission at 12 weeks of treatment; baseline characteristics showed weaker associations. Adverse events were comparable with previous reports on adalimumab safety.
In patients with active AS, the efficacy and safety of adalimumab were maintained through 5 years with about half of the patients experiencing sustained remission at any time during the study. Early achievement of remission was the best predictor of long-term and sustained remission.
PMCID: PMC3329233  PMID: 22128084
21.  Efficacy and safety of adalimumab in patients with non-radiographic axial spondyloarthritis: results of a randomised placebo-controlled trial (ABILITY-1) 
Annals of the Rheumatic Diseases  2012;72(6):815-822.
To evaluate the efficacy and safety of adalimumab in patients with non-radiographic axial spondyloarthritis (nr-axSpA).
Patients fulfilled Assessment of Spondyloarthritis international Society (ASAS) criteria for axial spondyloarthritis, had a Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score of ≥ 4, total back pain score of ≥ 4 (10 cm visual analogue scale) and inadequate response, intolerance or contraindication to non-steroidal anti-inflammatory drugs (NSAIDs); patients fulfilling modified New York criteria for ankylosing spondylitis were excluded. Patients were randomised to adalimumab (N=91) or placebo (N=94). The primary endpoint was the percentage of patients achieving ASAS40 at week 12. Efficacy assessments included BASDAI and Ankylosing Spondylitis Disease Activity Score (ASDAS). MRI was performed at baseline and week 12 and scored using the Spondyloarthritis Research Consortium of Canada (SPARCC) index.
Significantly more patients in the adalimumab group achieved ASAS40 at week 12 compared with patients in the placebo group (36% vs 15%, p<0.001). Significant clinical improvements based on other ASAS responses, ASDAS and BASDAI were also detected at week 12 with adalimumab treatment, as were improvements in quality of life measures. Inflammation in the spine and sacroiliac joints on MRI significantly decreased after 12 weeks of adalimumab treatment. Shorter disease duration, younger age, elevated baseline C-reactive protein or higher SPARCC MRI sacroiliac joint scores were associated with better week 12 responses to adalimumab. The safety profile was consistent with what is known for adalimumab in ankylosing spondylitis and other diseases.
In patients with nr-axSpA, adalimumab treatment resulted in effective control of disease activity, decreased inflammation and improved quality of life compared with placebo. Results from ABILITY-1 suggest that adalimumab has a positive benefit–risk profile in active nr-axSpA patients with inadequate response to NSAIDs.
PMCID: PMC3664374  PMID: 22772328
22.  Assessment of short-term symptomatic efficacy of tocilizumab in ankylosing spondylitis: results of randomised, placebo-controlled trials 
Annals of the Rheumatic Diseases  2013;73(1):95-100.
BUILDER-1 and BUILDER-2 aimed to assess the efficacy and safety of tocilizumab (TCZ) in patients with ankylosing spondylitis (AS).
BUILDER-1 was a two part, phase II–III parallel-group trial in patients with AS naive to antitumour necrosis factor (aTNF) treatment. Patients in part 1 received TCZ 8 mg/kg or placebo for 12 weeks. In part 2 (beginning after part 1 enrolment ended), newly enrolled patients received TCZ 4 or 8 mg/kg or placebo for 24 weeks. The same treatment arms were used in BUILDER-2, a phase III study in aTNF-inadequate responders. The primary endpoint for both studies was the proportion of patients achieving 20% improvement in the Assessments in Axial SpondyloArthritis international Society (ASAS). Secondary and exploratory endpoints included ASAS40 response rates, Bath Ankylosing Spondylitis Disease Activity Index improvement, changes in joint counts, enthesitis score and C reactive protein (CRP).
102 patients were randomised in BUILDER-1 part 1; 99 (48 TCZ, 51 placebo) completed 12 weeks. Week 12 ASAS20 response rates were 37.3% and 27.5% in the TCZ and placebo arms, respectively (p=0.2823). Secondary and exploratory endpoints did not differ between treatment arms. CRP levels declined with TCZ treatment, suggesting adequate IL-6 receptor blockade. As a result, BUILDER-1 part 2 and BUILDER-2 were terminated. TCZ safety results were consistent with previous observations in rheumatoid arthritis, except for a cluster of anaphylactic and hypersensitivity events at Bulgarian study sites. No apparent explanation for this clustering could be found.
BUILDER-1 failed to demonstrate TCZ efficacy in treating aTNF-naive patients with AS.
Clinical trial registration numbers:
NCT01209702 and NCT01209689 (
PMCID: PMC3888605  PMID: 23765873
Ankylosing Spondylitis; Treatment; Inflammation
23.  Treating spondyloarthritis, including ankylosing spondylitis and psoriatic arthritis, to target: recommendations of an international task force 
Therapeutic targets have been defined for diseases like diabetes, hypertension or rheumatoid arthritis and adhering to them has improved outcomes. Such targets are just emerging for spondyloarthritis (SpA).
To define the treatment target for SpA including ankylosing spondylitis and psoriatic arthritis (PsA) and develop recommendations for achieving the target, including a treat-to-target management strategy.
Based on results of a systematic literature review and expert opinion, a task force of expert physicians and patients developed recommendations which were broadly discussed and voted upon in a Delphi-like process. Level of evidence, grade and strength of the recommendations were derived by respective means. The commonalities between axial SpA, peripheral SpA and PsA were discussed in detail.
Although the literature review did not reveal trials comparing a treat-to-target approach with another or no strategy, it provided indirect evidence regarding an optimised approach to therapy that facilitated the development of recommendations. The group agreed on 5 overarching principles and 11 recommendations; 9 of these recommendations related commonly to the whole spectrum of SpA and PsA, and only 2 were designed separately for axial SpA, peripheral SpA and PsA. The main treatment target, which should be based on a shared decision with the patient, was defined as remission, with the alternative target of low disease activity. Follow-up examinations at regular intervals that depend on the patient's status should safeguard the evolution of disease activity towards the targeted goal. Additional recommendations relate to extra-articular and extramusculoskeletal aspects and other important factors, such as comorbidity. While the level of evidence was generally quite low, the mean strength of recommendation was 9–10 (10: maximum agreement) for all recommendations. A research agenda was formulated.
The task force defined the treatment target as remission or, alternatively, low disease activity, being aware that the evidence base is not strong and needs to be expanded by future research. These recommendations can inform the various stakeholders about expert opinion that aims for reaching optimal outcomes of SpA.
PMCID: PMC3888616  PMID: 23749611
Treat-to-target; spondyloarthritis; ankylosing spondylitis; psoriatic arthritis; therapy
24.  Spinal Inflammation in the Absence of Sacroiliac Joint Inflammation on Magnetic Resonance Imaging in Patients With Active Nonradiographic Axial Spondyloarthritis 
To evaluate the presence of spinal inflammation with and without sacroiliac (SI) joint inflammation on magnetic resonance imaging (MRI) in patients with active nonradiographic axial spondyloarthritis (SpA), and to compare the disease characteristics of these subgroups.
ABILITY-1 is a multicenter, randomized, controlled trial of adalimumab versus placebo in patients with nonradiographic axial SpA classified using the Assessment of SpondyloArthritis international Society axial SpA criteria. Baseline MRIs were centrally scored independently by 2 readers using the Spondyloarthritis Research Consortium of Canada (SPARCC) method for the SI joints and the SPARCC 6–discovertebral unit method for the spine. Positive evidence of inflammation on MRI was defined as a SPARCC score of ≥2 for either the SI joints or the spine.
Among patients with baseline SPARCC scores, 40% had an SI joint score of ≥2 and 52% had a spine score of ≥2. Forty-nine percent of patients with baseline SI joint scores of <2, and 58% of those with baseline SI joint scores of ≥2, had a spine score of ≥2. Comparison of baseline disease characteristics by baseline SI joint and spine scores showed that a greater proportion of patients in the subgroup with a baseline SPARCC score of ≥2 for both SI joints and spine were male, and patients with spine and SI joint scores of <2 were younger and had shorter symptom duration. SPARCC spine scores correlated with baseline symptom duration, and SI joint scores correlated negatively with the baseline Bath Ankylosing Spondylitis Disease Activity Index, but neither correlated with the baseline Ankylosing Spondylitis Disease Activity Score, total back pain, the patient's global assessment of disease activity, the Bath Ankylosing Spondylitis Functional Index, morning stiffness, nocturnal pain, or C-reactive protein level.
Assessment by experienced readers showed that spinal inflammation on MRI might be observed in half of patients with nonradiographic axial SpA without SI joint inflammation.
PMCID: PMC4033572  PMID: 24574227
25.  Referral Patterns, Diagnosis, and Disease Management of Patients With Axial Spondyloarthritis 
Journal of Clinical Rheumatology  2014;20(8):411-417.
Supplemental digital content is available in the text.
Recognition, diagnosis, and management of axial spondyloarthritis (axial SpA) continue to advance.
The objectives of this study were to compare referrals, diagnosis, and management of axial SpA in Western Europe (WE), North America (US and Canada), and the rest of world (RoW) in academic and community rheumatology practices and to identify areas for further education.
Rheumatologists responded online to the MAXIMA (Management of Axial SpA International and Multicentric Approaches) survey. Questions pertained to referral, diagnosis, and management of axial SpA.
Rheumatologists (N = 809) from 56 countries completed the survey about patients with chronic back pain (≥3 months) starting before age 45 years. Responses from academic and community practice rheumatologists were generally similar. Most referrals were from primary care providers. Symptom duration of 3 years or more at referral was reported more frequently by WE and RoW than US respondents. More WE and RoW than US rheumatologists referred to the Assessment of SpondyloArthritis International Society criteria for axial SpA in clinical practice. Rheumatologists reported prescribing disease-modifying antirheumatic drugs for the management of axial SpA. Sulfasalazine was frequently prescribed across regions; methotrexate was more commonly prescribed by US rheumatologists compared with other regions.
Referral patterns, diagnosis, and disease management for axial SpA were similar among WE, North America, and RoW rheumatologists and in academic/community practices, although more WE and RoW rheumatologists referred to Assessment of SpondyloArthritis International Society criteria in clinical practice. Disease-modifying antirheumatic drugs were commonly prescribed for axial SpA patients, although it was unclear whether these were prescribed for axial or peripheral symptoms.
PMCID: PMC4243795  PMID: 25417676
axial spondyloarthritis; referral patterns; diagnosis; disease management; survey

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