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Background Macrophage activation syndrome (MAS) is a potentially fatal complication of systemic inflammation. HMGB1 is a nuclear protein released extracellularly during proinflammatory lytic cell death or secreted by activated macrophages, NK cells, and additional cell types during infection or sterile injury. Extracellular HMGB1 orchestrates central events in inflammation as a prototype alarmin. TLR4 and the receptor for advanced glycation end products operate as key HMGB1 receptors to mediate inflammation. Methods Standard ELISA and cytometric bead array-based methods were used to examine the kinetic pattern for systemic release of HMGB1, ferritin, IL-18, IFN-γ, and MCP-1 before and during treatment of four children with critical MAS. Three of the patients with severe underlying systemic rheumatic diseases were treated with biologics including tocilizumab or anakinra when MAS developed. All patients required intensive care therapy due to life-threatening illness. Add-on etoposide therapy was administered due to insufficient clinical response with standard treatment. Etoposide promotes apoptotic rather than proinflammatory lytic cell death, conceivably ameliorating subsequent systemic inflammation. Results This therapeutic intervention brought disease control coinciding with a decline of the increased systemic HMGB1, IFN-γ, IL-18, and ferritin levels whereas MCP-1 levels evolved independently. Conclusion Systemic HMGB1 levels in MAS have not been reported before. Our results suggest that the molecule is not merely a biomarker of inflammation, but most likely also contributes to the pathogenesis of MAS. These observations encourage further studies of HMGB1 antagonists. They also advocate therapeutic etoposide administration in severe MAS and provide a possible biological explanation for its mode of action.

OBJECTIVE: To evaluate the efficacy and safety of 2 canakinumab monotherapy tapering regimens in order to maintain complete clinical remission in children with systemic juvenile idiopathic arthritis (JIA). METHODS: The study was designed as a 2-part phase IIIb/IV open-label, randomized trial. In the first part, patients received 4 mg/kg of canakinumab subcutaneously every 4 weeks and discontinued glucocorticoids and/or methotrexate as appropriate. Patients in whom clinical remission was achieved (inactive disease for at least 24 weeks) with canakinumab monotherapy were entered into the second part of the trial, in which they were randomized 1:1 into 1 of 2 treatment arms. In arm 1, the dose of canakinumab was reduced from 4 mg/kg to 2 mg/kg and then to 1 mg/kg, followed by discontinuation. In arm 2, the 4 mg/kg dose interval was prolonged from every 4 weeks, to every 8 weeks, and then to every 12 weeks, followed by discontinuation. In both arms, canakinumab exposure could be reduced provided systemic JIA remained in clinical remission for 24 weeks with each step. The primary objective was to assess whether >40% of randomized patients in either arm maintained clinical remission of systemic JIA for 24 weeks in the first part of the study. RESULTS: In part 1 of the study, 182 patients were enrolled, with 75 of those patients randomized before entering part 2 of the trial. Among the 75 randomized patients, clinical remission was maintained for 24 weeks in 27 (71%) of 38 patients in arm 1 (2 mg/kg every 4 weeks) and 31 (84%) of 37 patients in arm 2 (4 mg/kg every 8 weeks) (P ≤ 0.0001 for arm 1 versus arm 2 among those meeting the 40% threshold). Overall, 25 (33%) of 75 patients discontinued canakinumab, and clinical remission was maintained for at least 24 weeks in all 25 of these patients. No new safety signals were identified. CONCLUSION: Reduction of canakinumab exposure may be feasible in patients who have achieved clinical remission of systemic JIA, but consistent interleukin-1 inhibition appears necessary to maintain this response. ispartof: ARTHRITIS & RHEUMATOLOGY vol:73 issue:2 pages:336-346 ispartof: location:United States status: published

Macrophage activation syndrome (MAS) is a potentially fatal complication of systemic inflammation. High mobility group box 1 (HMGB1) is a nuclear protein extensively leaked extracellularly during necrotic cell death or actively secreted by natural killer (NK) cells, macrophages and additional cells during infection or sterile injury. Extracellular HMGB1 orchestrates key events in inflammation as a prototypic alarmin. The redox states of its three cysteines render the molecule mutually exclusive functions: fully reduced “all-thiol HMGB1” exerts chemotactic activity; “disulfide HMGB1” has cytokine-inducing, toll-like receptor 4 (TLR4)-mediated effects—while terminally oxidized “sulfonyl HMGB1” lacks inflammatory activity. This study examines the kinetic pattern of systemic HMGB1 isoform expression during therapy in four children with severe MAS. Three of the four patients with underlying systemic rheumatic diseases were treated with biologics and two suffered from triggering herpes virus infections at the onset of MAS. All patients required intensive care unit therapy due to life-threatening illness. Tandem mass-spectrometric analysis revealed dramatically increased systemic levels of the cytokine-inducing HMGB1 isoform during early MAS. Disease control coincided with supplementary etoposide therapy initiated to boost apoptotic cell death, when systemic HMGB1 levels drastically declined and the molecule emerged mainly in its oxidized, noninflammatory isoform. Systemic interferon (IFN)-γ and ferritin peaked concomitantly with HMGB1, whereas interleukin (IL)-18 and monocyte chemotactic protein (MCP)-1 levels developed differently. In conclusion, this work provides new insights in HMGB1 biology, suggesting that the molecule is not merely a biomarker of inflammation, but most likely also contributes to the pathogenesis of MAS. These observations encourage further studies of disulfide HMGB1 antagonists to improve outcome of MAS.

Extracellular high mobility group box-1 protein (HMGB1) plays important roles in the pathogenesis of nerve injury- and cancer-induced pain. However, the involvement of spinal HMGB1 in arthritis-induced pain has not been examined previously and is the focus of this study. Immunohistochemistry showed that HMGB1 is expressed in neurons and glial cells in the spinal cord. Subsequent to induction of collagen antibody-induced arthritis (CAIA), Hmgb1 mRNA and extranuclear protein levels were significantly increased in the lumbar spinal cord. Intrathecal (i.t.) injection of a neutralizing anti-HMGB1 monoclonal antibody or recombinant HMGB1 box A peptide (Abox), which each prevent extracellular HMGB1 activities, reversed CAIA-induced mechanical hypersensitivity. This occurred during ongoing joint inflammation as well as during the postinflammatory phase, indicating that spinal HMGB1 has an important function in nociception persisting beyond episodes of joint inflammation. Importantly, only HMGB1 in its partially oxidized isoform (disulfide HMGB1), which activates toll-like receptor 4 (TLR4), but not in its fully reduced or fully oxidized isoforms, evoked mechanical hypersensitivity upon i.t. injection. Interestingly, although both male and female mice developed mechanical hypersensitivity in response to i.t. HMGB1, female mice recovered faster. Furthermore, the pro-nociceptive effect of i.t. injection of HMGB1 persisted in Tlr2- and Rage-, but was absent in Tlr4-deficient mice. The same pattern was observed for HMGB1-induced spinal microglia and astrocyte activation and cytokine induction. These results demonstrate that spinal HMGB1 contributes to nociceptive signal transmission via activation of TLR4 and point to disulfide HMGB1 inhibition as a potential therapeutic strategy in treatment of chronic inflammatory pain.

Summary Objective To explore the possibility of cartilage protection in osteoarthritis (OA) by intraarticular injection of a chemically modified hyaluronan (HA) gel and investigate whether the chemical modifications provide intrinsic anti-inflammatory activity. Method OA was induced in C57BL/6 mice by anterior cruciate ligament transection (ACLT) and HA gel or carbazate-modified component was injected intra-articularly. Assessment of cartilage rescue was performed by histology, immunohistochemistry and TUNEL analysis. Serum levels of proinflammatory cytokines were evaluated with cytometric bead array, measuring IL-1β, TNF, IFN-γ, KC/CXCL1 and MCP-1. Results Intraarticular injection of the HA gel showed significantly reduced cartilage destruction and decreased osteophyte formation. Besides the biological and biomechanical effects of HA, we investigated lipid peroxidation products as an alternative inflammatory and potential mechanism contributing to OA. To address this, injection of the carbazate-modified component alone was performed, which also demonstrated a cartilage-saving effect. Besides the cartilage amelioration effects, decreased apoptosis, 4-hydroxynonenal (4-HNE) and MHC class II staining was recorded. No changes in serum levels of proinflammatory cytokines were detected. Conclusion We have shown that the HA gel has an anti-destructive effect on articular cartilage (AC). Our results demonstrated that the carbazate-modified component could suppress apoptotic events, potentially by quenching of ROS/LPO products such as 4-HNE in OA joints. Modification of the HA molecule offers opportunities to introduce (covalent) coupling of additional molecules to the gel, with controlled retention and subsequent release in the joint.

Objectives Polymyositis and dermatomyositis are characterised by muscle weakness and fatigue even in patients with normal muscle histology via unresolved pathogenic mechanisms. In this study, we investigated the mechanisms by which high mobility group box protein 1 (HMGB1) acts to accelerate muscle fatigue development. Methods Intact single fibres were dissociated from flexor digitorum brevis (FDB) of wild type, receptor for advanced glycation endproduct (RAGE) knockout and toll like receptor 4 (TLR4) knockout mice and cultured in the absence or presence of recombinant HMGB1. A decrease in sarcoplasmic reticulum Ca 2+ release during a series of 300 tetanic contractions, which reflects the development of muscle fatigue, was determined by measuring myoplasmic free tetanic Ca 2+ . TLR4 and major histocompatibility complex (MHC)-class I expression in mouse FDB fibres were investigated by immunofluorescence and confocal microscopy. Immunohistochemistry was used to investigate TLR4, MHC-class I and myosin heavy chain expression in muscle fibres of patients. Results Our results demonstrate that TLR4 is expressed in human and mouse skeletal muscle fibres, and coexpressed with MHC-class I in muscle fibres of patients with myositis. Furthermore, we show that HMGB1 acts via TLR4 but not RAGE to accelerate muscle fatigue and to induce MHC-class I expression in vitro. In order to bind and signal via TLR4, HMGB1 must have a reduced cysteine 106 and a disulphide linkage between cysteine 23 and 45. Conclusions The HMGB1-TLR4 pathway may play an important role in causing muscle fatigue in patients with polymyositis or dermatomyositis and thus is a potential novel target for future therapy.

High mobility group box chromosomal protein 1 (HMGB1) is a DNA-binding nuclear protein that can be released from dying cells and activated myeloid cells. Extracellularly, HMGB1 promotes inflammation. Experimental studies demonstrate HMGB1 to be a pathogenic factor in many inflammatory conditions including arthritis. HMGB1-blocking therapies in arthritis models alleviate disease and confer significant protection against cartilage and bone destruction. So far, the most successful HMGB1-targeted therapies have been demonstrated with HMGB1-specific polyclonal antibodies and with recombinant A box protein, a fragment of HMGB1. The present study is the first to evaluate the potential of a monoclonal anti-HMGB1 antibody (2G7, mouse IgG2b) to ameliorate arthritis. Effects of repeated injections of this antibody have now been studied in two conceptually different models of arthritis: collagen type II–induced arthritis (CIA) in DBA/1 mice and in a spontaneous arthritis disease in mice with combined deficiencies for genes encoding for the enzyme DNase type II and interferon type I receptors. These mice are unable to degrade phagocytozed DNA in macrophages and develop chronic, destructive polyarthritis. Therapeutic intervention in CIA and prophylactic administration of anti-HMGB1 monoclonal antibody (mAb) in the spontaneous arthritis model significantly ameliorated the clinical courses. Anti-HMGB1 mAb therapy also partially prevented joint destruction, as demonstrated by histological examination. The beneficial antiarthritic effects by the anti-HMGB1 mAb in two diverse models of arthritis represent additional proof-of-concept, indicating that HMGB1 may be a valid target molecule to consider for development of future clinical therapy.

High-mobility group box 1 (HMGB1) is a nuclear and cytosolic protein that is increasingly recognized as an important proinflammatory mediator actively secreted from monocytes and macrophages and passively released from necrotic cells. In antineutrophilic cytoplasmatic antibody (ANCA)-associated vasculitis (AAV), the kidneys are commonly affected vital organs, characterized by focal necrotizing and/or crescentic pauci-immune glomerulonephritis. The aim of the study was to determine whether HMGB1 serum levels are elevated in AAV with renal manifestations. A total of 30 AAV patients (16 female and 14 male; median age 59 years, range 17–82) with Wegener granulomatosis, microscopic polyangiitis and Churg-Strauss syndrome with available renal biopsies and serum samples were included. In seven cases, serum was also obtained at rebiopsy in remission. HMGB1 was analyzed with Western blot. Birmingham Vasculitis Activity Score (BVAS, version 2003), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), urinanalysis, creatinine, estimated glomerular filtration rate, sex and age were included in the analysis. Twenty-five episodes of biopsy-proven active disease with BVAS 17.9 ± 4.6 and 13 cases with inactive biopsies and BVAS 2.3 ± 3.7 (P = 0.0001) were identified. CRP, ESR, hematuria and proteinuria were significantly higher in active cases. HMGB1 was significantly elevated (P = 0.01) comparing active with inactive cases (120 ± 48 versus 78 ± 46 ng/mL) and significantly lower in the seven control patients (P = 0.03) at rebiopsy in remission. HMGB1 remained higher in inactive cases compared with historic healthy controls (10.9 ± 10.5 ng/mL). HMGB1 levels did not differ significantly between AAV subgroups. CRP and ESR did not correlate with HMGB1. HMGB1 is significantly increased in AAV with renal involvement. Residual HMGB1 elevation in remission could possibly reflect low-grade inflammatory activity or tissue damage. Future studies may further reveal whether HMGB1 is useful as a marker of disease activity and a predictor of outcome in AAV.

Objective. High mobility group box chromosomal protein 1 (HMGB-1) is a DNA binding nuclear protein that can be released from dying cells and activated myeloid cells. Extracellularly, HMGB-1 promotes inflammation. Clinical and experimental studies demonstrate that HMGB-1 is a pathogenic factor in chronic arthritis. Mice with combined gene deficiency for DNase II and IFNRI spontaneously develop chronic, destructive polyarthritis with many features shared with rheumatoid arthritis. DNase II is needed for macrophage degradation of engulfed DNA. The aim of this study was to evaluate a potential pathogenic role of HMGB-1 in this novel murine model. Methods. The course of arthritis, assessed by clinical scoring and histology, was studied in DNase II / IFNRI / mice, in comparison with heterozygous and wild-type mice. Synovial HMGB-1 expression was analyzed by immunohistochemistry. Serum levels of HMGB-1 were determined by Western immunoblotting and enzyme-linked immunosorbent assay (ELISA), and anti–HMGB-1 autoantibodies were detected by ELISA. Macrophage activation was studied by immunostaining for intracellular interleukin-1 and HMGB-1. HMGB-1 was targeted with truncated HMGB-1–derived BoxA protein, acting as a competitive antagonist, with intraperitoneal injections every second day for 5 weeks. Results. DNase II / IFNRI / mice developed symmetric polyarthritis with strong aberrant cytosolic and extracellular HMGB-1 expression in synovial tissue, in contrast to that observed in control animals. Increased serum levels of HMGB-1 and HMGB-1 autoantibodies were recorded in DNase II / IFNRI / mice, both prior to and during the establishment of disease. Systemic HMGB-1–specific blockade significantly ameliorated the clinical disease course, and a protective effect on joint destruction was demonstrated by histologic evaluation. Conclusion. HMGB-1 is involved in the pathogenesis of this spontaneous polyarthritis, and intervention with an HMGB-1 antagonist can mediate beneficial effects.

Objective Extracellular high mobility group box chromosomal protein 1 (HMGB-1) is a recently identified, endogenous, potent tumor necrosis factor– and interleukin-1 (IL-1)–inducing protein detectable in inflamed synovia in both human and experimental disease. In the present study, we examined clinical effects in collagen-induced arthritis (CIA) using therapeutic administration of neutralizing HMGB-1 antibodies or truncated HMGB-1–derived A-box protein, a specific, competitive antagonist of HMGB-1. Methods CIA was induced in DBA/1j mice or dark agouti rats, and animals were examined daily for signs of arthritis. Treatment with polyclonal anti–HMGB-1 antibodies or the A-box protein was initiated at the onset of disease and was administered intraperitoneally twice daily for 7 days. Animals were killed 8 days after initiation of therapy, and immunohistochemical analysis of synovial tissue specimens was performed. Results Systemic administration of anti–HMGB-1 antibodies or A-box protein significantly reduced the mean arthritis score, the disease-induced weight loss, and the histologic severity of arthritis. Beneficial effects were observed in both mice and rats. Immunohistochemical analysis revealed pronounced synovial IL-1β expression and articular cartilage destruction in vehicle-treated mice. Both these features were significantly less manifested in animals treated with anti–HMGB-1 antibodies or A-box protein. Conclusion Counteracting extracellular HMGB-1 with either neutralizing antibodies or a specific HMGB-1 antagonist may offer a new method for the successful treatment of arthritis. Inflammation and tissue destruction were suppressed in CIA after HMGB-1 blockade.

Objective High mobility group box chromosomal protein 1 (HMGB-1) is a ubiquitous chromatin component expressed in nucleated mammalian cells. It has recently and unexpectedly been demonstrated that stimulated live mononuclear phagocytes secrete HMGB-1, which then acts as a potent factor that causes inflammation and protease activation. Macrophages play pivotal roles in the pathogenesis of arthritis. The aim of this study was to determine whether synovial macrophage expression of HMGB-1 is altered in human and experimental synovitis. Methods Intraarticular tissue specimens were obtained from healthy Lewis rats, Lewis rats with Mycobacterium tuberculosis–induced adjuvant arthritis, and from patients with rheumatoid arthritis (RA). Specimens were immunohistochemically stained for cellular HMGB-1. Extracellular HMGB-1 levels were assessed in synovial fluid samples from RA patients by Western blotting. Results Immunostaining of specimens from normal rats showed that HMGB-1 was primarily confined to the nucleus of synoviocytes and chondrocytes, with occasional cytoplasmic staining and no extracellular matrix deposition. In contrast, inflammatory synovial tissue from rats with experimental arthritis as well as from humans with RA showed a distinctly different HMGB-1 staining pattern. Nuclear HMGB-1 expression was accompanied by a cytoplasmic staining in many mononuclear cells, with a macrophage-like appearance and an extracellular matrix deposition. Analysis of synovial fluid samples from RA patients further confirmed the extracellular presence of HMGB-1; 14 of 15 samples had HMGB-1 concentrations of 1.8–10.4 μg/ml. Conclusion The proinflammatory mediator HMGB-1 was abundantly expressed as a nuclear, cytoplasmic, and extracellular component in synovial tissues from RA patients and from rats with experimental arthritis. These findings suggest a pathogenetic role for HMGB-1 in synovitis and indicate a new potential therapeutic target molecule.

Macrophage activation syndrome (MAS) is a potentially fatal complication of systemic inflammation. High mobility group box 1 (HMGB1) is a nuclear protein extensively leaked extracellularly during necrotic cell death or actively secreted by natural killer (NK) cells, macrophages and additional cells during infection or sterile injury. Extracellular HMGB1 orchestrates key events in inflammation as a prototypic alarmin. The redox states of its three cysteines render the molecule mutually exclusive functions: fully reduced “all-thiol HMGB1” exerts chemotactic activity; “disulfide HMGB1” has cytokine-inducing, toll-like receptor 4 (TLR4)-mediated effects—while terminally oxidized “sulfonyl HMGB1” lacks inflammatory activity. This study examines the kinetic pattern of systemic HMGB1 isoform expression during therapy in four children with severe MAS. Three of the four patients with underlying systemic rheumatic diseases were treated with biologics and two suffered from triggering herpes virus infections at the onset of MAS. All patients required intensive care unit therapy due to life-threatening illness. Tandem mass-spectrometric analysis revealed dramatically increased systemic levels of the cytokine-inducing HMGB1 isoform during early MAS. Disease control coincided with supplementary etoposide therapy initiated to boost apoptotic cell death, when systemic HMGB1 levels drastically declined and the molecule emerged mainly in its oxidized, noninflammatory isoform. Systemic interferon (IFN)-γ and ferritin peaked concomitantly with HMGB1, whereas interleukin (IL)-18 and monocyte chemotactic protein (MCP)-1 levels developed differently. In conclusion, this work provides new insights in HMGB1 biology, suggesting that the molecule is not merely a biomarker of inflammation, but most likely also contributes to the pathogenesis of MAS. These observations encourage further studies of disulfide HMGB1 antagonists to improve outcome of MAS.

This study was performed to elucidate pathophysiological events before and during the course of collagen-induced arthritis in Dark Agouti rats, a model for rheumatoid arthritis. Kinetic studies of local cytokine responses were determined using immunohistochemical techniques, quantified by computer-assisted image analysis. We recently reported that the macrophage-pacifying agent CNI-1493 successfully ameliorated collagen-induced arthritis. In the present trial, we investigated the potential of CNI-1493 to down-regulate pro-inflammatory cytokines. Synovial cryosections were analyzed at various time points for the presence of interleukin (IL)-1β, tumor necrosis factor (TNF), and transforming growth factor (TGF)-β. Unexpectedly, an early simultaneous TNF and IL-1β expression was detected in resident cells in the lining layer, preceding disease onset and inflammatory cell infiltration by >1 week. The predominant cytokine synthesis by synovial (ED1 + ) macrophages coincided with clinical disease. TNF production greatly exceeded that of IL-1β. CNI-1493 treatment did not affect the early disease-preceding TNF and IL-1β synthesis in the lining layer. However, after disease onset, CNI-1493 intervention resulted in a pronounced reduced IL-1β and in particular TNF expression. Furthermore, CNI-1493 significantly up-regulated synthesis of the anti-inflammatory cytokine TGF-β and thereby shifted the balance of pro-inflammatory and anti-inflammatory cytokines in the arthritic joint in a beneficial way.

The purpose of this study was to analyse kinematic and kinetic gait changes in rheumatoid arthritis (RA) patients in comparison to healthy controls and to examine whether levels of functional disability (Health Assessment Questionnaire (HAQ)-scores) were associated with gait parameters. Using a three-dimensional motion analysis system, kinematic and kinetic gait parameters were measured in 50 RA patients and 37 healthy controls. There was a significant reduction in joint motions, joint moments and work in the RA cohort compared with healthy controls. The following joint motions were decreased: hip flexion-extension range (Delta6 degrees ), hip abduction (Delta4 degrees ), knee flexion-extension range (Delta8 degrees ) and ankle plantarflexion (Delta10 degrees ). The following joint moments were reduced: hip extensor (Delta0.30Nm/kg) and flexor (Delta0.20Nm/kg), knee extensor (Delta0.11Nm/kg) and flexor (Delta0.13Nm/kg), and ankle plantarflexor (Delta0.44Nm/kg). Work was lower in hip positive work (Delta0.07J/kg), knee negative work (Delta0.08J/kg) and ankle positive work (Delta0.15J/kg). Correlations were fair although significant between HAQ and hip flexion-extension range, hip abduction, knee flexion-extension range, hip abductor moment, stride length, step length and single support (r=-0.30 to -0.38, p0.05). Our findings suggest that RA patients have overall less joint movement and specifically restricted joint moments and work across the large joints of the lower limbs during walking than healthy controls. There were only fair associations between levels of functional disability and gait parameters. The findings of this study help to improve the understanding how RA affects gait changes in the lower limbs.

Bone loss represents a major unsolved problem in rheumatoid arthritis (RA). The receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for the development and activation of osteoclasts, which are key mediators of bone erosions. This study was performed to determine temporal and spatial expression of RANKL compared with the potentially destructive cytokine interleukin-1beta (IL-1beta), related to progression of synovitis and joint destruction in collagen-induced arthritis (CIA), a model of RA. CIA was induced in dark agouti (DA) rats, and tissue specimens were obtained for immunohistochemical analyses at various time points before and after disease onset. Arthritis was monitored visually, and joint pathology was examined histologically. No disease-preceding expression of RANKL was detected. However, a marked increase of both RANKL- and IL-1beta-expressing cells correlated with the progression of synovial inflammation and clinical disease severity. Abundant and concomitant expression of these cytokines was detected at sites of bone erosion, where a colocalization by osteoclast-like multinuclear tartrate-resistant acid phosphatase (TRAP)+ cells was noted. In contrast to the paucity of RANKL expression in cartilage, an abundant expression of IL-1beta was demonstrated, particularly in superficial cartilage layers. These data support the hypothesis that RANKL and IL-1beta are central contributors to joint destruction in CIA.

To analyse changes in the rates of hospital admission and use of orthopaedic surgery to the lower limbs in Swedish patients with rheumatoid arthritis between 1987 and 2001.Data for all rheumatoid patients admitted to hospital between 1987 and 2001 were abstracted from the Swedish National Hospital Discharge Register (SNHDR). The data in the register are collected prospectively, recording all inpatient admissions throughout Sweden. The SNHDR uses the codes for diagnoses at discharge and surgical procedures according to the Swedish version of the International Classification of Diseases (ICD).In all, 49,802 individual patients with rheumatoid arthritis were identified, accounting for 159,888 inpatient visits. Hospital admissions for rheumatoid arthritis decreased by 42% (p0.001) during the period 1987 to 2001. Twelve per cent of all admissions were for a rheumatoid arthritis related surgical procedure to the lower limbs; those admissions decreased markedly (by 16%) between 1987 and 1996, and by 12% between 1997 and 2001, as did the overall number of rheumatoid arthritis related surgical procedures to the lower limbs during both time periods. Between 1997 and 2001, 47% of all rheumatoid arthritis related surgical procedures were total joint arthroplasties. There was an overall trend towards reduced length of hospital stay after orthopaedic surgery to the lower limbs during the study period.Rates of hospital admission and rheumatoid arthritis related surgical procedures to the lower limbs in Swedish patients with rheumatoid arthritis decreased between 1987 and 2001. This may reflect trends in disease severity, management, and health outcomes of this disease in Sweden.

Objective: To investigate if administration of CNI-1493, an inhibitor of the synthesis of proinflammatory cytokines and NO, protects against development of joint destruction in collagen induced arthritis (CIA) in rats. Methods: In a placebo controlled experiment, CNI-1493 was given once daily intraperitoneally after onset of clinical arthritis in DA rats. Disease progression was studied by clinical scoring of arthritis, serial measurement of serum levels of COMP, and histological examination of joints. Results: Clinical signs of arthritis were significantly reduced in the CNI-1493 treated group of rats in comparison with the placebo treated group. Histological examinations of paws demonstrated a significant reduction of cartilage destruction in the CNI-1493 treated group, but marked destruction of cartilage in the placebo group. Serum levels of COMP increased in the placebo group, whereas in the CNI-1493 treated group levels were low and decreased significantly during the observation time. Conclusions: Treatment with CNI-1493 provides efficient protection against synovial inflammation and cartilage destruction when used therapeutically in CIA. The protective effect against cartilage destruction can be monitored by measuring serum COMP. These observations make CNI-1493 an attractive candidate for therapeutic studies in human arthritis, and COMP an attractive serum marker for monitoring joint protective effects.

Background and objectives The nuclear protein HMGB1 (high mobility group chromosomal protein 1) is secreted by activated macrophages/monocytes and promotes inflammation. Extracellular HMGB1 is present in the synovitis of rheumatoid arthritis (RA) patients and intra-articular injections of recombinant HMGB1 induce arthritis in mice. Thus, the authors hypothesise that HMGB1 represents an endogenous mediator of arthritis as well as a target molecule for successful therapeutic intervention in RA. Polyclonal anti-HMGB1 therapy or recombinant boxA protein works in experimental arthritis models. However, no monoclonal anti-HMGB1 therapy has proven efficient in arthritis models. The aim was to evaluate the antiarthritic potential of a monoclonal anti-HMGB1 antibody (2G7) in two different models of arthritis; collagen-induced arthritis (CIA) in DBA/1 mice and DNase II −/− × IFN-IR −/− mice which spontaneously develop chronic polyarthritis resembling RA. DNase II is specifically needed for macrophage degradation of engulfed DNA. This model is associated with abundant extracellular HMGB1 expression including serum levels of several 100 ng/ml before and during onset of disease. Materials and methods Female and male DNase II −/− × IFN-IR −/− mice, initially 5 weeks old, were treated in 5 weeks every second day with intraperitoneally injections with 100 μg of a mouse monoclonal anti-HMGB1 antibody 2G7 (IgG2b, non-commercial antibody, available upon request) or buffer control. Nine animals were included in each group with an equal sex distribution. Treatment of CIA in female DBA/one mice with anti-HMGB1 antibody 2G7 was initiated when the animal reached an arthritis score of ≤2. The animals were treated for 7 days with daily intraperitoneal injections of 70 μg 2G7 (12 animals) or 70 μg of irrelevant antibody (MOPC-195, mouse IgG2b, 7 animals). The outcome was evaluated by daily clinical scoring. Statistical evaluation was performed using Student9s t test calculated on area under the curve values. Serial joint sections were stained with H&E to evaluate inflammatory cell infiltration or using staining with Safranin O to visualise cartilage damage and bone erosions. Results Systemic HMGB1-specific blockade significantly ameliorated the clinical course in CIA as well as in DNase II −/− × IFN-IR −/− mice, and a protective effect on joint destruction was observed by histological evaluation. Conclusion This study identifies beneficial effect of the HMGB1-specific mAb 2G7 in two different experimental arthritis models. The conserved structure of HMGB1 and the strong cross-reactivity between species of the investigated mAb, has thus enabled them to define a potential mAb candidate for further genetic engineering and clinical testing.

Macrophage activation syndrome (MAS) is a life-threatening complication of childhood systemic inflammatory disorders. HMGB1 is a nuclear protein that extracellularly orchestrates key events in inflammation. Recent data revealed that different redox states of three cysteines within HMGB1 render it with mutually exclusive activities: reduced all-thiol-HMGB1 exerts chemotactic activity, disulfide-HMGB1 cytokine-inducing effects, and terminally oxidized sulfonyl-HMGB1 without inflammatory activity.

This study was performed to elucidate pathophysiological events prior and during the course of collagen-induced arthritis (CIA) in DA rats. Kinetic studies of local cytokine responses were determined using immunohistochemisrty and computer-aided image analysis. We also investigated the effect of the macrophage-pacifying compound CNI-1493 on proinflammatory cytokine expressions. Synovial cryosections were analysed at various time points for the presence of IL-1β, TNF and TGF-β. Unexpectedly, an early simultaneous TNF and IL-1β expression was detected in resident cells in the lining layer, preceding disease onset by more than one week. The predominant cytokine synthesis by synovial (ED-1+) macrophages coincided with clinical disease. TNF-production greatly exceeded that of IL-1β. CNI-1493 treatment did not affect the early TNF and IL-1β synthesis, while disease-associated TNF and IL-1β production was greatly reduced. Furthermore, CNI-1493 significantly up-regulated synthesis of the anti-inflammatory cytokine TGF-β and thereby shifted the balance of pro-inflammatory and anti-inflammatory cytokines in the arthritic joint in a beneficial way.

Backgroundand objectives The endogenous danger signal High Mobility Group Box protein 1 (HMGB1) promotes inflammation. HMGB1 has been implicated as a mediator of RA while there are no reports describing its presence in JIA patient samples. HMGB1 is aberrantly expressed in the synovitis of RA patients, intraarticular injection of HMGB1 induces arthritis in mice and HMGB1 blockade has beneficial effects in several different experimental disease models, including arthritis. In this study, the aim was to determine whether HMGB1 is extracellularly increased during JIA and, if so, to correlate the HMGB1 levels both with other more well-described laboratory parameters and with clinical parameters such as age at onset and disease duration. This type of descriptive study forms the basis for the evaluation of HMGB1 as a biomarker of inflammatory activity during JIA and as a potential therapeutic target. The newly established JIA biobank JABBA coupled to a clinical register gives us a unique opportunity to study JIA pathogenesis. Materials and methods Plasma and synovial fluid (SF) was collected from 23 patients with JIA (median age 12 (2–18)) at Astrid Lindgren Children9s hospital, Stockholm, Sweden and at Tartu University Children9s Hospital, Estonia. Samples were analysed by ELISA and Cytometric bead array (CBA) to measure levels of HMGB1, MMP-3, sRAGE, IL-12p70, TNF, IL-10, IL-6, IL-1β, IL-8, MCP-1, IP-10, RANTES, IFNγ, IFNα and IL-17A. Results Increased HMGB1 levels were recorded in SF as compared to plasma from JIA patients. Highest levels of HMGB1 were recorded in patients with a disease onset in early age (before age 10), while no correlation between the HMGB1 levels and disease duration was evident. In contrast, both S100 and IL-8 levels correlated with disease duration being highest during early stages of disease. MMP-3, a marker of cartilage destruction, was higher in patients with late disease onset which indicates similarities with RA. Conclusion The increased levels of HMGB1 in inflamed joints of JIA patients warrants further studies of HMGB1 as a biomarker for inflammatory activity and as a target for therapy. The correlation of HMGB1 levels with age at disease onset and continuously high levels irrespective of disease duration indicates, together with the decreasing levels of S100 and IL-8 during the disease course, that the inflammatory process in JIA evolves over time. Different mediators might thus be of varying importance during the disease progression. The authors conclude that the role of HMGB1 in the pathogenesis of JIA deserves further investigation.

Background and objectives High mobility group box protein 1 (HMGB1) is a nuclear protein present in all cells. HMGB1 is passively released extracellularly during cell death and, similarly, HMGB1 can be actively secreted from activated cells. Extracellular HMGB1 mediates multiple inflammatory processes. High levels of extracellular HMGB1 have been demonstrated in synovial joints of arthritis patients, intraarticular injection of HMGB1 causes synovitis in mice and neutralisation of HMGB1 by peptide inhibitors or polyclonal antibodies ameliorates experimental arthritis. In this study, we investigated and compared the effects of an HMGB1 peptide inhibitor and a mouse monoclonal antibody (mAb) targeting HMGB1 in an experimental model of arthritis and in acute autoimmune hepatitis. Defining a mAb or peptide inhibitor with ameliorative effects will forward the development of HMGB1 blockade suitable for clinical trials. Methods Collagen induced arthritis (CIA) was induced in DBA/1 mice. Arthritis severity was evaluated by clinical grading of inflammation. Mice with established arthritis were treated every other day with mAb 2G7 or irrIgG and monitored for 7 days. Autoimmune hepatitis was induced by ConcavalinA injection in C57/bl6 mice pretreated with Abox, mAb 2G7 or PBS. After 8 h, sera and liver biopsies were collected. Serum ALT levels, along with HTX staining of liver biopsies were assessed. Liver myeloid peroxidase (MPO) were measured as an indication of neutrophil infiltration. Pro-inflammatory mediator levels were quantified by ELISA. HMGB1 levels were evaluated by ELISA and immunohistochemistry. Results mAb 2G7 treatment significantly ameliorated inflammation in CIA mice and also reduced destruction, as assessed by histological analysis. In mice with autoimmune hepatitis, we recorded an upregulated HMGB1 expression damaged liver lobules. Abox treatment minimised liver damage and mAb 2G7 reduced neutrophil infiltration. Reduced interferon γ and CXCL1 levels were demonstrated in both Abox and mAb 2G7 treated mice. Conclusion By these studies, we have for the first time defined a mAb targeting HMGB1 that effectively reduces inflammation and destruction during CIA. Beneficial effects of this mAb were also demonstrated in a model of autoimmune hepatitis. Furthermore, we could confirm the inhibitory effects of the peptide inhibitor A box, previously demonstrated in CIA, in experimental autoimmune hepatitis. Hence, our current work with humanisation of mAb 2G7 and assessements of its therapeutic effects can be performed in the autoimmune hepatitis model. This allows us to rapidly screen multiple humanised mAb clones and also avoids xenoreactivity effects. Hence, we have facilitated the development of HMGB1 blockade for clinical use.

Introduction: High mobility group box 1 protein (HMGB1) is a nuclear DNA binding protein acting as a proinflammatory mediator following extracellular release. HMGB1 has been increasingly recognized as a pathogenic mediator in several inflammatory diseases. Elevated serum levels of HMGB1 have been detected in autoimmune diseases including Systemic lupus erythematosus (SLE). However, the local expression of HMGB1 in active lupus nephritis (LN) is not known. Here we aimed to study both tissue expression and serum levels of HMGB1 in LN patients with active disease and after induction therapy. Methods: Thirty-five patients with active LN were included. Renal biopsies were performed at baseline and after standard induction therapy; corticosteroids combined with immunosuppressive drugs. The biopsies were evaluated according to the World Health Organization (WHO) classification and renal disease activity was estimated using the British Isles lupus assessment group (BILAG) index. Serum levels of HMGB1 were analysed by western blot. HMGB1 expression in renal tissue was assessed by immunohistochemistry at baseline and follow-up biopsies in 25 patients. Results: Baseline biopsies showed WHO class III, IV or V and all patients had high renal disease activity (BILAG A/B). Follow-up biopsies showed WHO I to II (n = 14), III (n = 6), IV (n =3 ) or V( n = 12), and 15/35 patients were regarded as renal responders (BILAG C/D). At baseline HMGB1 was significantly elevated in serum compared to healthy controls (P < 0.0001). In all patients, serum levels decreased only slightly; however, in patients with baseline WHO class IV a significant decrease was observed (P = 0.03). Immunostaining revealed a pronounced extranuclear HMGB1 expression predominantly outlining the glomerular endothelium and in the mesangium. There was no clear difference in HMGB1 expression comparing baseline and follow-up biopsies or any apparent association to histopathological classification or clinical outcome. Conclusions: Renal tissue expression and serum levels of HMGB1 were increased in LN. The lack of decrease of HMGB1 in serum and tissue after immunosuppressive therapy in the current study may reflect persistent inflammatory activity. This study clearly indicates a role for HMGB1 in LN.

High-mobility group chromosomal box protein 1 (HMGB1) is a structural nuclear protein that promotes inflammation when present extracellularly. Aberrant, extracellular HMGB1 expression has been demonstrated in human and experimental synovitis. The aim of the present study was to elucidate the temporal and spatial expression of HMGB1 compared to that of the central mediators tumor necrosis factor (TNF) and interleukin-1-beta (IL-1beta) during the course of collagen-induced arthritis. Thus, Dark Agouti rats were immunized with homologous type II collagen and synovial tissue specimens were obtained at various time points prior to and during the course of clinical arthritis. Local cytokine responses were assessed by immunohistochemistry and by in situ hybridization. We demonstrate a distinct nuclear expression of HMGB1 at early disease-preceding time points. Preceding clinical onset by a few days, cytoplasmic HMGB1 expression was evident in synoviocytes within the non-proliferative lining layer. Pronounced cytoplasmic and additional extracellular HMGB1 expression coincided with the progression of clinical disease. In advanced arthritis, the number of cells with cytoplasmic HMGB1 expression was quantitatively comparable to that of cells expressing TNF and IL-1beta. Interestingly, although HMGB1 was abundantly expressed throughout the inflamed synovium at a protein level, upregulation of HMGB1 mRNA was restricted mainly to areas of cartilage and bone destruction. In conclusion, these new findings implicate a role for HMGB1 in both inducing and perpetuating inflammatory events of significant importance in the destructive processes in chronic arthritis.

Autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in patients with rheumatoid arthritis and have been suggested to be involved in the disease pathogenesis. The targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deiminase (PAD), which converts positively charged arginine to polar but uncharged citrulline. The aim of this study was to explore the effects of citrullination on the immunogenicity of autoantigens as well as on potential arthritogenicity. Thus, immune responses to citrullinated rat serum albumin (Cit-RSA) and to unmodified rat serum albumin (RSA) were examined as well as arthritis development induced by immunisation with citrullinated rat collagen type II (Cit-CII) or unmodified CII. In addition, to correlate the presence of citrullinated proteins and the enzyme PAD4 with different stages of arthritis, synovial tissues obtained at different time points from rats with collagen-induced arthritis were examined immunohistochemically. Our results demonstrate that citrullination of the endogenous antigen RSA broke immunological tolerance, as was evident by the generation of antibodies directed against the modified protein and cross-reacting with the native protein. Furthermore we could demonstrate that Cit-CII induced arthritis with higher incidence and earlier onset than did the native counterpart. Finally, this study reveals that clinical signs of arthritis precede the presence of citrullinated proteins and the enzyme PAD4. As disease progressed into a more severe and chronic state, products of citrullination appeared specifically in the joints. Citrullinated proteins were detected mainly in extracellular deposits but could also be found in infiltrating cells and on the cartilage surface. PAD4 was detected in the cytoplasm of infiltrating mononuclear cells, from day 21 after immunisation and onwards. In conclusion, our data reveal the potency of citrullination to break tolerance against the self antigen RSA and to increase the arthritogenic properties of the cartilage antigen CII. We also show that citrullinated proteins and the enzyme PAD4 are not detectable in healthy joints, and that the appearance and amounts in arthritic joints of experimental animals are correlated with the severity of inflammation.

Introduction In addition to its direct proinflammatory activity, extracellular high mobility group box protein 1 (HMGB1) can strongly enhance the cytokine response evoked by other proinflammatory molecules, such as lipopolysaccharide (LPS), CpG-DNA and IL-1β, through the formation of complexes. Extracellular HMGB1 is abundant in arthritic joint tissue where it is suggested to promote inflammation as intra-articular injections of HMGB1 induce synovitis in mice and HMGB1 neutralizing therapy suppresses development of experimental arthritis. The aim of this study was to determine whether HMGB1 in complex with LPS, interleukin (IL)-1α or IL-1β has enhancing effects on the production of proinflammatory mediators by rheumatoid arthritis synovial fibroblasts (RASF) and osteoarthritis synovial fibroblasts (OASF). Furthermore, we examined the toll-like receptor (TLR) 4 and IL-1RI requirement for the cytokine-enhancing effects of the investigated HMGB1-ligand complexes. Methods Synovial fibroblasts obtained from rheumatoid arthritis (RA) and osteoarthritis (OA) patients were stimulated with HMGB1 alone or in complex with LPS, IL-1α or IL-1β. Tumour necrosis factor (TNF) production was determined by enzyme-linked immunospot assay (ELISPOT) assessment. Levels of IL-10, IL-1-β, IL-6 and IL-8 were measured using Cytokine Bead Array and matrix metalloproteinase (MMP) 3 production was determined by ELISA. Results Stimulation with HMGB1 in complex with LPS, IL-1α or IL-1β enhanced production of TNF, IL-6 and IL-8. HMGB1 in complex with IL-1β increased MMP production from both RASF and OASF. The cytokine production was inhibited by specific receptor blockade using detoxified LPS or IL-1 receptor antagonist, indicating that the synergistic effects were mediated through the partner ligand-reciprocal receptors TLR4 and IL-1RI, respectively. Conclusions HMGB1 in complex with LPS, IL-1α or IL-1β boosted proinflammatory cytokine- and MMP production in synovial fibroblasts from RA and OA patients. A mechanism for the pathogenic role of HMGB1 in arthritis could thus be through enhancement of inflammatory and destructive mechanisms induced by other proinflammatory mediators present in the arthritic joint.

Introduction High mobility group box chromosomal protein 1 (HMGB1) is a nuclear protein that acts as a pro-inflammatory mediator following extracellular release. The protein is aberrantly expressed extracellularly in the settings of clinical and experimental synovitis. Therapy based on HMGB1 antagonists has shown encouraging results in experimental arthritis and warrants further scientific exploration using independent methods. In the present study we asked whether nuclear sequestration of HMGB1 preventing HMGB1 release would be beneficial for synovitis treatment. Methods Oxaliplatin-based therapy was evaluated in collagen type II-induced arthritis in DBA/1 mice by clinical scoring and immunostaining of articular tissue. Oxaliplatin is an antineoplastic platinum-based compound that generates DNA adducts which tightly bind HMGB1. Secretion and intracellular location of HMGB1 were assessed by a novel HMGB1-specific ELISPOT assay and immunofluorescent staining. Results Intraperitoneal injections of oxaliplatin in early collagen type II-induced arthritis trapped HMGB1 with a distinct biphasic response pattern. Oxaliplatin therapy showed beneficial results for approximately 1 week. Microscopic evaluation of synovitis during this period showed strong nuclear HMGB1 staining in the oxaliplatin treated animals with much lower quantities of extracellular HMGB1 when compared to control treated animals. Furthermore, cellular infiltration, as well as cartilage and bone damage, were all reduced in the oxaliplatin treated group. A dramatic and as yet unexplained clinical relapse occurred later in the oxaliplatin exposed animals, which coincided with a massive synovial tissue expression of extracellular HMGB1 in all treated animals. This rebound-like reaction was also accompanied by a significantly increased incidence of arthritis in the oxaliplatin treated group. These results indicate a distinct temporal and spatial relationship between the clinical course of disease and the cellular localization of HMGB1. Beneficial effects were noted when extracellular HMGB1 expression was low, while severe inflammation coincided with substantial extracellular synovial HMGB1 expression. Conclusion Therapeutic compounds like oxaliplatin and gold salts share a capacity to inhibit nuclear HMGB1 release and to ameliorate the course of synovial inflammation. These observations support the hypothesis that HMGB1 plays an important functional role in the pathogenesis of arthritis and may represent a novel target molecule for therapy.