Synovial tissue hypoxia and inflammation in vivo.

INTRODUCTION: Hypoxia is a microenvironmental feature in the inflamed joint, which promotes survival advantage for cells. The aim of this study was to examine the relationship of partial oxygen pressure in the synovial tissue (tPO(2)) in patients with inflammatory arthritis with macroscopic/microscopic inflammation and local levels of proinflammatory mediators. METHODS: Patients with inflammatory arthritis underwent full clinical assessment and video arthroscopy to quantify macroscopic synovitis and measure synovial tPO(2) under direct visualisation. Cell specific markers (CD3 (T cells), CD68 (macrophages), Ki67 (cell proliferation) and terminal deoxynucleotidyl transferase dUTP nick end labelling (cell apoptosis)) were quantified by immunohistology. In vitro migration was assessed in primary and normal synoviocytes (synovial fibroblast cells (SFCs)) using a wound repair scratch assay. Levels of tumour necrosis factor alpha (TNFalpha), interleukin 1beta (IL1beta), interferon gamma (IFNgamma), IL6, macrophage inflammatory protein 3alpha (MIP3alpha) and IL8 were quantified, in matched serum and synovial fluid, by multiplex cytokine assay and ELISA. RESULTS: The tPO(2) was 22.5 (range 3.2-54.1) mm Hg and correlated inversely with macroscopic synovitis (r=-0.421, p=0.02), sublining CD3 cells (-0.611, p<0.01) and sublining CD68 cells (r=-0.615, p<0.001). No relationship with cell proliferation or apoptosis was found. Primary and normal SFCs exposed to 1% and 3% oxygen (reflecting the median tPO(2) in vivo) induced cell migration. This was coupled with significantly higher levels of synovial fluid tumour necrosis factor alpha (TNFalpha), IL1beta, IFNgamma and MIP3alpha in patients with tPO(2) <20 mm Hg (all p values <0.05). CONCLUSIONS: This is the first study to show a direct in vivo correlation between synovial tPO(2), inflammation and cell migration, thus it is proposed that hypoxia is a possible primary driver of inflammatory processes in the arthritic joint.

EULAR evidence-based recommendations for the diagnosis of knee osteoarthritis.

OBJECTIVE: To develop evidence-based recommendations for the diagnosis of knee osteoarthritis (OA). METHODS: The multidisciplinary guideline development group, representing 12 European countries, generated 10 key propositions regarding diagnosis using a Delphi consensus approach. For each recommendation, research evidence was searched systematically. Whenever possible, the sensitivity, specificity and likelihood ratio were calculated for individual diagnostic indicators and a diagnostic ladder was developed using Bayes' method. Secondary analyses were undertaken to test directly the recommendations using multiple predictive models in two populations from the UK and the Netherlands. Strength of recommendation was assessed by the EULAR visual analogue scale. RESULTS: Recommendations covered the definition of knee OA and its risk factors, subsets, typical symptoms and signs, the use of imaging and laboratory tests and differential diagnosis. Three symptoms (persistent knee pain, limited morning stiffness and reduced function) and three signs (crepitus, restricted movement and bony enlargement) appeared to be the most useful. Assuming a 12.5% background prevalence of knee OA in adults aged > or =45 years, the estimated probability of having radiographic knee OA increased with increasing number of positive features, to 99% when all six symptoms and signs were present. The performance of the recommendations in the study populations varied according to the definition of knee OA, background risk and number of tests applied. CONCLUSION: 10 key recommendations for diagnosis of knee OA were developed using both research evidence and expert consensus. Although there is no agreed reference standard, thorough clinical assessment alone can provide a confident rule-in diagnosis.

Comparison of interferon {gamma} release assays and conventional screening tests before tumour necrosis factor {alpha} blockade in patients with inflammatory arthritis.

OBJECTIVE: To compare the performance of two interferon gamma release assays (IGRAs) and conventional screening tests in patients with inflammatory arthritis undergoing screening for latent tuberculosis infection (LTBI) before treatment with anti-tumour necrosis factor alpha (anti-TNFalpha) compounds. METHODS: Successive patients were subjected to conventional LTBI screening, including a tuberculin skin test (TST). The T-SPOT.TB test was performed on all patients and the QuantiFERON-TB Gold test was performed on a large subset. The results of the IGRAs were compared with the results of conventional screening tests. RESULTS: A total 150 patients were evaluated. The majority (57.9%) had rheumatoid arthritis. Previous vaccination with Bacille Calmette-Guerin was confirmed in 82% of patients. No patient had received prior anti-TB treatment. A total of 57 patients (38.0%) had at least one positive conventional risk factor. In contrast, an unequivocally positive T-SPOT.TB test was seen in only 14/143 (9.8%). There was 98.2% agreement between the two IGRAs. Statistically significant associations were found between each of the IGRAs and both TST and risk history, but not chest x-ray (CXR). A positive IGRA result was significantly associated with increased age. TB was not reactivated in any patient during the follow-up period. INTERPRETATION: This study suggests that IGRAs may be useful when screening for LTBI before anti-TNFalpha therapy in patients with immune-mediated inflammatory diseases. The observations reported here also highlight the inadequate performance of CXR as a marker of LTBI.

Disparities in rheumatoid arthritis disease activity according to gross domestic product in 25 countries in the QUEST-RA database.

OBJECTIVE: To analyse associations between the clinical status of patients with rheumatoid arthritis (RA) and the gross domestic product (GDP) of their resident country. METHODS: The Quantitative Standard Monitoring of Patients with Rheumatoid Arthritis (QUEST-RA) cohort includes clinical and questionnaire data from 6004 patients who were seen in usual care at 70 rheumatology clinics in 25 countries as of April 2008, including 18 European countries. Demographic variables, clinical characteristics, RA disease activity measures, including the disease activity score in 28 joints (DAS28), and treatment-related variables were analysed according to GDP per capita, including 14 "high GDP" countries with GDP per capita greater than US$24,000 and 11 "low GDP" countries with GDP per capita less than US$11,000. RESULTS: Disease activity DAS28 ranged between 3.1 and 6.0 among the 25 countries and was significantly associated with GDP (r = -0.78, 95% CI -0.56 to -0.90, r(2) = 61%). Disease activity levels differed substantially between "high GDP" and "low GDP" countries at much greater levels than according to whether patients were currently taking or not taking methotrexate, prednisone and/or biological agents. CONCLUSIONS: The clinical status of patients with RA was correlated significantly with GDP among 25 mostly European countries according to all disease measures, associated only modestly with the current use of antirheumatic medications. The burden of arthritis appears substantially greater in "low GDP" than in "high GDP" countries. These findings may alert healthcare professionals and designers of health policy towards improving the clinical status of patients with RA in all countries.

An audit of hospital based outpatient infusions and a pilot program of community-based monoclonal antibody infusions.

INTRODUCTION: Infliximab, a chimeric monoclonal antibody to tumour necrosis factor alpha, is administered as an intravenous infusion requiring a costly hospital day case or inpatient admission. METHODS: An audit of all current therapies given by intravenous infusions in an outpatient setting in St Vincent's University Hospital (SVUH) was undertaken. Furthermore, in conjunction with TCP homecare, we established in a general practise health clinic, the first Irish community infusion centre for the administration of infliximab in August 2006. RESULTS: All outpatient departments indicated that they would favour a centralized hospital infusion unit. There were no adverse events and the mean global satisfaction improved in the community infliximab infusion pilot programme of seven patients. CONCLUSION: This study suggests efficiencies in providing centralized infusion facilities, while the community based infusion of infliximab is feasible and safe in this small cohort and identifies the community infusion unit as a viable and cost efficient alternative for administration of infliximab.

Melanoma inhibitory activity, a biomarker related to chondrocyte anabolism, is reversibly suppressed by proinflammatory cytokines in rheumatoid arthritis.

OBJECTIVE: In mice, melanoma inhibitory activity (MIA) is a chondrocyte-specific molecule with similar regulation to collagen type II. As MIA is a small secreted protein, its value as cartilage biomarker in human inflammatory arthritis was assessed. METHODS: MIA tissue distribution was studied by quantitative PCR and immunohistochemistry. The regulation of MIA production was studied in vivo in rheumatoid arthritis (RA) (n = 37) and spondyloarthritis (SpA) (n = 30) synovial fluid (SF), and in vitro in alginate embedded human chondrocytes. Therapeutic modulation of serum MIA was evaluated during tumour necrosis factor (TNF)alpha and interleukin (IL)1 blockade in RA. RESULTS: MIA was primarily expressed by chondrocytes in the human joint. SF MIA levels were lower in RA than in SpA despite similar levels of overall synovial inflammation. Further analysis indicated that these levels were inversely correlated with the degree of joint inflammation in RA, but not in SpA, and that the levels of TNFalpha and IL1beta were significantly increased in RA versus SpA. Accordingly, these proinflammatory cytokines suppressed MIA mRNA and protein in cultured chondrocytes. This suppression was paralleled by suppression of cartilage anabolism as assessed by collagen type 2 and aggrecan mRNA. Treatment of patients with RA with TNF blockade or IL1 blockade induced an increase of serum MIA levels. CONCLUSION: The decreased levels of MIA in the inflamed RA joint and the coregulation of MIA and cartilage matrix molecules by proinflammatory cytokines indicate that joint inflammation in RA not only drives accelerated cartilage degradation but also suppresses cartilage anabolism. This inflammation-driven suppression is reversible in vivo.

Decreased CD20 expression in rheumatoid arthritis synovium following 8 weeks of rituximab therapy.

OBJECTIVE: To characterise the effects of rituximab on synovial tissue of patients with refractory rheumatoid arthritis (RA). METHODS: Arthroscopic biopsy of knee joint synovium was performed on 6 patients with seropositive RA prior to commencing rituximab. Four patients underwent repeat biopsy eight weeks following completion of their rituximab infusion schedule. Cryostat sections of synovium were prepared and stained with mouse monoclonal specific antibodies including CD20, plasma cell antibody and CD68. RESULTS: Eight weeks after treatment mean DAS28 fell from 6.6+/-0.43 to 4.7+/-0.49 (p=0.068). Mean CRP fell from 86.7+/-27 mg/L to 20.5+/-7 mg/L (p<0.05). Subsynovial CD20+ B cells were demonstrated in all six patients at baseline. B cells were completely depleted in two patients at follow-up biopsy. Complete depletion was associated with excellent clinical response. No change in subsynovial B cells was seen in one patient. One patient's follow-up arthroscopy yielded inadequate tissue. A reduction was also seen in subsynovial plasma cells and CD68+ cells after treatment. CONCLUSION: B cells were present in synovial tissue of all patients with refractory RA. Complete depletion of B cells was associated with an excellent clinical response. These preliminary results suggest that early depletion of synovial B cells precedes a decrease in local inflammation leading to clinical improvement.

Differential expression of syndecans and glypicans in chronically inflamed synovium.

BACKGROUND: Membrane-bound heparan sulphate proteoglycans (HSPGs) act as co-receptors and presenters of cytokines and are involved in cell-matrix and cell-cell adhesion. AIM: To investigate which HSPGs are expressed in knee joint synovia from patients with different forms of arthritis and normal individuals. METHODS: Synovial samples were obtained from patients with early rheumatoid arthritis (n = 8), longstanding rheumatoid arthritis (n = 13), psoriatic arthritis (n = 7), osteoarthritis (n = 6) and normal joints (n = 12). Expression of syndecan-1, -2, -3 and -4 and glypican-1, -3 and -4 was analysed by immunohistochemistry and dual label immunofluorescence. RESULTS: The expression of HSPGs in chronically inflamed synovium exhibited a differential distribution. Syndecan-1 was present in the mononuclear infiltrates of synovia from patients with rheumatoid and psoriatic arthritis where it was expressed by plasma cells. Syndecan-2 was present mainly in blood vessels where it occurred on endothelial cells, pericytes and smooth muscle cells. Syndecan-3 stained intensely in endothelial cells but also occurred in sublining macrophages and the lining layer. Glypican-4 occurred in the lining layer and blood vessels. Increased expression of these HSPGs was apparent in rheumatoid and psoriatic compared to osteoarthritic and normal synovia. Little or no staining for syndecan-4, glypican-1 and glypican-3 was seen in all samples. DISCUSSION: Selected HSPGs, such as syndecan-1, -2 and -3 and glypican-4, could play a part in the pathophysiology of arthritis, such as the migration and retention of leukocytes and angiogenesis in the chronically inflamed synovium.

Quality of life and economic impact of switching from established infliximab therapy to adalimumab in patients with rheumatoid arthritis.

OBJECTIVE: To evaluate the quality of life and economic impact of switching therapy from infliximab to adalimumab in patients with rheumatoid arthritis (RA). METHODS: In this open-label study, patients demonstrating a clinical response to infliximab were switched to treatment with adalimumab and followed for 16 weeks. Both generic (Health Assessment Questionnaire and Short Form 36 Physical Component Summary and Mental Component Summary) and specific (Rheumatoid Arthritis Quality of Life questionnaire) assessment instruments of physical function and of quality of life were employed. An economic analysis of treatment-related costs was also performed. Disease activity was assessed by the composite 28-joint count Disease Activity Score (DAS28). C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were measured as acute phase markers. RESULTS: Nineteen patients were enrolled and completed the study. No changes in functional and quality-of-life measures were observed. One-year extrapolation data showed potential reductions in costs following switching to adalimumab that could be attributed primarily to reductions in patient- and staff-related costs. Safety and tolerability were similar for both treatments. Although there was a significant reduction in DAS28 (P < 0.005) and CRP (P < 0.001) after switching to adalimumab, there were no significant changes in individual DAS28 components, including swollen and tender joint counts and ESR. CONCLUSIONS: A switch from infliximab to adalimumab in patients with RA who have responded to infliximab is a feasible, well-tolerated treatment option, with the potential for direct and indirect economic advantages.

Downregulation of the inhibitor of apoptosis protein survivin in keratinocytes and endothelial cells in psoriasis skin following infliximab therapy.

BACKGROUND: Survivin, an inhibitor of apoptosis protein (IAP), has been implicated in endothelial cell stability, through inhibition of apoptosis and in cell proliferation. OBJECTIVES: To evaluate the effect of antitumour necrosis factor (TNF)-alpha therapy on survivin expression in psoriasis skin at 0, 2 and 12 weeks after infliximab therapy. METHODS: Skin biopsies were obtained from 16 patients; 11 also had arthritis with active skin/joint disease. Clinical scores [Psoriasis Area and Severity Index (PASI), involved body surface area (BSA), Disease Activity Score (DAS28) and Health Assessment Questionnaire] were recorded. Inflammatory infiltration and survivin protein expression were examined and graded by immunohistochemical staining, and mRNA levels were determined by real-time polymerase chain reaction. RESULTS: Survivin mRNA and protein were demonstrated in all baseline lesional biopsies. Survivin mRNA and protein expression was significantly greater in lesional compared with nonlesional baseline skin (P < 0.05). Differential cellular localization of survivin was demonstrated with cytoplasmic survivin protein expression localized to the perivascular/endothelial regions and strong nuclear staining localized in the basal layer of the epidermis. Infliximab produced a dramatic clinical response in skin and joints (P < 0.05), paralleled by significant reduction in the inflammatory infiltrate and survivin protein expression (P < 0.05) which was reflected at the mRNA level where expression was significantly reduced by week 12 (P < 0.01). Survivin protein levels before and after treatment significantly correlated with PASI (r = 0.478, P < 0.05) and BSA scores (r = 0.528, P < 0.024). PASI strongly correlated with BSA (r = 0.949, P < 0.0001) and DAS28 (r = 0.717, P < 0.002) scores. CONCLUSIONS: Survivin correlates with disease activity in patients with psoriasis and is significantly downregulated following anti-TNF-alpha treatment. Understanding the role of IAPs in cell survival/antiapoptosis and proliferation mechanisms may provide important insights into downstream therapeutic targeting in inflammation.

Standardisation of synovial tissue infiltrate analysis: how far have we come? How much further do we need to go?

Changes in cellular infiltrate and expression of cytokines, chemokines, and cell adhesion molecules as a result of therapeutic interventions in rheumatoid arthritis can be demonstrated in the synovial membrane. However, before synovial tissue analysis can be used as an outcome measure in such studies, standardisation of the site and method of synovial tissue acquisition, methods of tissue processing, and appropriate methods of detection and measurement of cell lineage specific markers and relevant biological proteins is needed.

Synovial tissue inflammation in early and late osteoarthritis.

OBJECTIVE: To compare selected immunohistological features of inflammation in synovial tissue from patients with early and late osteoarthritis (OA). METHODS: Synovial tissue samples were obtained from 10 patients with knee pain, normal radiographs, and arthroscopic manifestations of OA (early OA), and from 15 patients with OA undergoing knee joint arthroplasty (late OA). Conventional immunohistochemical techniques were used to measure microscopic manifestations of inflammation. The inflammatory cell infiltrate, blood vessel formation, and angiogenic factors, NF-kappaB activation, expression of tumour necrosis factor alpha (TNFalpha) and interleukin 1beta (IL1beta), and the presence of cyclo-oxygenase (COX)-1 and COX-2 were quantified. Fibroblast-like synoviocytes (FLS) were isolated from early and late OA tissue samples to compare in vitro production of prostaglandin E2 (PGE2) RESULTS: Synovial tissue from patients with early OA demonstrated significantly greater CD4+ (p = 0.017) and CD68+ (p<0.001) cell infiltration, blood vessel formation (p = 0.01), vascular endothelial growth factor (p = 0.001), and intercellular adhesion molecule-1 expression (p<0.001). Numbers of cells producing TNFalpha and IL1beta were also significantly greater in early OA (p<0.001). Manifestations of inflammation in early OA were associated with increased expression of the NF-kappaB1 (p<0.001) and RelA (p = 0.015) subunits, and with increased COX-2 expression (p = 0.04). Cytokine-induced PGE2 production by cultured FLS was similar in both groups. CONCLUSION: Increased mononuclear cell infiltration and overexpression of mediators of inflammation were seen in early OA, compared with late OA. Isolated FLS were functionally similar in both groups, consistent with microenvironmental differences in the synovial tissue during different phases of OA. These observations may have important therapeutic implications for some patients during the early evolution of OA.

Synovial tissue macrophages: a sensitive biomarker for response to treatment in patients with rheumatoid arthritis.

BACKGROUND: Previous work identified synovial sublining macrophage numbers as a potential biomarker for clinical efficacy in rheumatoid arthritis. OBJECTIVE: To investigate the association between changes in infiltration of synovial macrophages and clinical improvement after antirheumatic treatment. METHODS: 88 patients who participated in various clinical trials were studied. All patients underwent serial arthroscopy before initiation of treatment and after different time intervals. Immunohistochemical and digital image analysis were performed according to standardised procedures to detect changes in CD68+ synovial sublining macrophages in relationship to changes in the 28 joint count Disease Activity Score (DAS28). Statistical analysis was performed using one way analysis of variance, the independent samples t test, linear regression, and the standardised response mean (SRM). RESULTS: For good, moderate, and non-responders, according to the DAS28 response criteria, there was a significant difference in the change in sublining macrophages (mean (SEM) cells/mm(2) -643 (124), -270 (64), and -95 (60), respectively; p<0.0003). There was a significant correlation between the change in the number of macrophages and the change in DAS28 (Pearson correlation 0.874, p<0.01). The change in sublining macrophages explained 76% of the variation in the change in DAS28 (p<0.02). The sensitivity to change of the biomarker was high in patients treated actively (SRM >0.8), whereas the ability to detect changes in placebo treated patients was weak (SRM <0.3). CONCLUSION: The results suggest that changes in synovial sublining macrophages can be used to predict possible efficacy of antirheumatic treatment.

Synovial tissue interleukin-18 expression and the response to treatment in patients with inflammatory arthritis.

OBJECTIVE: To measure synovial tissue interleukin-18 (IL-18) expression in patients with inflammatory arthritis, and to identify associations with serum levels, disease activity, and response to treatment. METHODS: Synovial tissue biopsies and serum samples were obtained from patients with early, active, rheumatoid arthritis (RA) (n = 12), undifferentiated seronegative arthritis (SnA) (n = 9), psoriatic arthritis (PsA) (n = 5), and reactive arthritis (ReA) (n = 2) before and one year after introduction of disease modifying antirheumatic drug (DMARD) treatment. Osteoarthritis (OA) tissues were compared. Tissue IL-18 expression was determined after immunohistochemical staining using a semiquantitative scale. Serum IL-18 was measured by enzyme linked immunosorbent assay. RESULTS: Before treatment was started, tissue IL-18 expression was increased in each diagnostic group compared with OA (p<0.05). Tissue IL-18 expression was correlated with serum C reactive protein levels (r = 0.53, p = 0.003) but not with serum IL-18. After DMARD treatment, 12 patients (five RA, four SnA, three PsA) were re-evaluated. Decreases in tissue IL-18 expression were observed in eight, although the trend did not reach significance (p = 0.068). Changes in tissue IL-18 expression were correlated with changes in serum IL-18 (r = 0.62, p = 0.041) and C reactive protein (r = 0.72, p = 0.009). CONCLUSIONS: Synovial tissue IL-18 expression was correlated with disease activity in inflammatory arthritis. After treatment, tissue levels changed in parallel with changes in serum IL-18 and with changes in the acute phase response. These observations support a role for IL-18 in the pathophysiology of inflammatory arthritis.