Effect of a treat-to-target strategy based on methotrexate and intra-articular betamethasone with or without additional cyclosporin on MRI-assessed synovitis, osteitis, tenosynovitis, bone erosion, and joint space narrowing in early rheumatoid arthritis: results from a 2-year randomized double-blind placebo-controlled trial (CIMESTRA).

OBJECTIVES: To investigate whether a treat-to-target strategy based on methotrexate (MTX) and intra-articular (IA) betamethasone suppresses magnetic resonance imaging (MRI)-determined measures of disease activity and reduces joint destruction in early rheumatoid arthritis (eRA) patients, and to investigate whether concomitant cyclosporin A (CyA) provides an additional effect. METHOD: In the 2-year randomized, double-blind, treat-to-target trial CIMESTRA, 160 patients with eRA (< 6 months) were randomized to MTX, intra-articular betamethasone and CyA, or placebo CyA. A total of 129 patients participated in the MRI substudy, and had contrast-enhanced MR images of the non-dominant hand at months 0, 6, 12, and 24. MR images were evaluated for osteitis, synovitis, tenosynovitis, bone erosion, and joint space narrowing (JSN), using validated scoring methods. RESULTS: Significant reductions were seen at 6 months in all inflammatory parameters [synovitis, mean change -1.6 (p < 0.001, Wilcoxon), tenosynovitis, -3.5 (p < 0.001), and osteitis, -1.3 (p < 0.05)] and at 12/24 months in synovitis and tenosynovitis [-1.6/-2.2 and -3.6/-3.8, respectively; all p < 0.001]. MRI signs of inflammation were not fully eliminated, and increases in erosion and JSN scores were observed at 6 months [0.4 (p < 0.01)/0.1 (p < 0.05)], 12 months [0.8 (p < 0.001)/0.3 (p < 0.01)], and 24 months [1.0 (p < 0.001)/0.4 (p < 0.001)]. Clinical measures decreased significantly (p < 0.001) at all time points. There were no consistent statistically significant differences between treatment groups. CONCLUSIONS: In this eRA treat-to-target trial, MTX and intra-articular glucocorticoids markedly reduced, but did not eliminate, MRI osteitis, synovitis, and tenosynovitis. Accordingly, minimal but statistically significant increases in bone erosion and JSN were observed. No additional effect of CyA was demonstrated.

Differentiation between early rheumatoid arthritis patients and healthy persons by conventional and dynamic contrast-enhanced magnetic resonance imaging.

OBJECTIVES: To identify the magnetic resonance imaging (MRI) parameter that best differentiates healthy persons and patients with early rheumatoid arthritis (RA), and to investigated responsiveness to treatment of various MRI parameters. METHOD: Conventional MRI and dynamic contrast-enhanced (DCE)-MRI of the hand were performed once for 26 healthy persons, and before and after 6 and 12 months of disease-modifying anti-rheumatic drug (DMARD) treatment for 14 early RA patients, using a 1.0-T MRI unit. One-slice DCE-MRI was analysed using Dynamika version 4.2. The number of enhancing voxels (Nvoxel), the initial rate of enhancement (IRE), the maximum enhancement (ME), ME×Nvoxel, and IRE×Nvoxel were calculated for wrist and 2nd-5th metacarpophalangeal (MCP) joints. Conventional MR images were evaluated using the RA MRI scoring system (RAMRIS) synovitis score. RESULTS: Using DCE-MRI, enhancement was demonstrated in 61.5% of healthy persons and in 91.7% of RA patients at baseline, with a median Nvoxel of 3 and 362, respectively. At baseline, all parameters were higher for patients than for healthy persons (all p ≤ 0.003). Only one patient had a baseline RAMRIS synovitis score below the 95th percentile of the healthy persons. The corresponding number of patients was 3 for Nvoxel, ME×Nvoxel and IRE×Nvoxel, and 10 for IRE and ME. The RAMRIS synovitis score and IRE showed the highest responsiveness, with a standardized response mean (SRM) of -1.00 and -0.88, respectively. CONCLUSIONS: The RAMRIS synovitis scoring of conventional MRI, and to a lesser extent the one-slice DCE-MRI parameters of synovial volume, differentiated early RA patients and healthy persons. The decrease in RAMRIS synovitis score, Nvoxel, and IRE showed sensitivity to change during treatment.

Upregulated baseline plasma CCL19 and CCR7 cell-surface expression on monocytes in early rheumatoid arthritis normalized during treatment and CCL19 correlated with radiographic progression.

OBJECTIVES: The aim of this study was to measure, in early rheumatoid arthritis (RA) patients, the concentration of CC-chemokine ligand 19 (CCL19) in plasma and the cell-surface expression of CC-chemokine receptor 7 (CCR7) on circulating monocytes and CD4+ T lymphocytes and to analyse correlations with disease activity and 5-year radiographic progression. METHOD: In disease-modifying anti-rheumatic drug (DMARD)-naïve RA patients (disease duration < 6 months), we measured plasma CCL19 by enzyme-linked immunosorbent assay (ELISA) (n = 160) and CCR7 cell-surface expression on monocytes and CD4+ T lymphocytes by flow cytometry (n = 40) at baseline and after 1 year of treatment with methotrexate (MTX) or methotrexate+cyclosporin A (MTX/CyA). Radiographic progression was scored by the van der Heijde-modified Total Sharp Score (TSS) from 0 to 5 years. RESULTS: Increased baseline CCL19 (median 85 pg/mL, range 31-1008 pg/mL, p = 0.01) decreased after 1 year (median 31 pg/mL, range 31-1030 pg/mL, p < 0.001) and 5 years (median 31 pg/mL, range 31-247 pg/mL, p < 0.001) to a level below the controls (n = 45) (median 60 pg/mL, range 31-152 pg/mL). Baseline plasma CCL19 levels [p = 0.011, 95% confidence interval (CI) 0.0030-0.0176], anti-cyclic citrullinated peptide (anti-CCP) antibody status (p = 0.002, 95% CI 0.61-2.38), and TSS > 0 at baseline (p < 0.001, 95% CI 1.21-3.16) were independent predictors of 5-year radiographic progression evaluated by multiple logistic regression in contrast to never smoked, C-reactive protein (CRP), gender, age, number of tender (NTJ) and swollen joints (NSJ), and 28-joint Disease Activity Score (DAS28). Increased CCR7 expression on monocytes (p = 0.008) correlated to CRP (p = 0.006, r = 0.52) and normalized (n = 15) after 1 year (p = 0.02). CONCLUSIONS: In DMARD-naïve RA patients, CCL19 plasma level and CCR7 surface expression on monocytes were upregulated and normalized after 1 year of treatment. Increased baseline plasma CCL19 level, anti-CCP antibody status, and TSS > 0 at baseline correlated independently with 5-year radiographic progression.

Periarticular and generalised bone loss in patients with early rheumatoid arthritis: influence of alendronate and intra-articular glucocorticoid treatment. Post hoc analyses from the CIMESTRA trial.

OBJECTIVES: The aims of this study were to investigate the influence of alendronate and intra-articular betamethasone treatment on bone mineral density (BMD) changes in hand, lumbar spine and femoral neck during 1 year of a treat-to-target study (Cyclosporine, Methotrexate, Steroid in RA (CIMESTRA)). PATIENTS AND METHODS: A hundred and sixty patients with early, active rheumatoid arthritis (RA) received methotrexate, intra-articular betamethasone and ciclosporin /placebo-ciclosporin. Patients with Z-score ≤0 also started alendronate 10 mg/day. BMD of the hand (digital x-ray radiogrammetry (DXR-BMDhand)), BMD of lumbar spine and femoral neck (dual x-ray absorptiometry (DXA-BMDlumbar spine and DXA-BMDfemoral neck)) and x-rays of hands, wrists and forefeet (modified Sharp-van der Heijde score) were measured at baseline and 1 year, with complete data available in 107 patients. RESULTS: The change in BMD in hand, lumbar spine and femoral neck was negatively associated with the dose of intra-articular betamethasone (p<0.01 for all), but the bone loss in hand was modest and in the axial skeleton comparable with that of healthy individuals. Alendronate did not influence changes in DXR-BMDhand, which averaged -2.8%, whereas significant changes were observed in DXA-BMDlumbar spine and DXA-BMDfemoral neck in alendronate-treated patients (1.8% and 0.8%) compared with untreated patients (-1.8% and -2.2%) (p<0.01 and 0.02). Alendronate did not affect the radiographic progression (alendronate-treated patients: 0 (range 0-19), non-alendronate: 0 (0-18)). CONCLUSIONS: In early active RA, intra-articular betamethasone injections added to disease-modifying antirheumatic drug (DMARD) treatment led to minimal loss of hip and lumbar BMD, and the loss could be prevented by treatment with alendronate. Alendronate treatment did not affect radiographic progression.

MRI bone oedema is the strongest predictor of subsequent radiographic progression in early rheumatoid arthritis. Results from a 2-year randomised controlled trial (CIMESTRA).

OBJECTIVE: To identify predictors of radiographic progression in a 2-year randomised, double-blind, clinical study (CIMESTRA) of patients with early rheumatoid arthritis (RA). METHODS: Patients with early RA (n = 130) were treated with methotrexate, intra-articular betamethasone and ciclosporin/placebo-ciclosporin. Baseline magnetic resonance imaging (MRI) of the wrist (wrist-only group, n = 130) or MRI of wrist and metacarpophalangeal (MCP) joints (wrist+MCP group, n = 89) (OMERACT RAMRIS), x-ray examination of hands, wrists and forefeet (Sharp/van der Heijde Score (TSS)), Disease Activity Score (DAS28), anti-cyclic citrullinated peptide antibodies (anti-CCP), HLA-DRB1-shared epitope (SE) and smoking status were assessed. Multiple regression analysis was performed with delta-TSS (0-2 years) as dependent variable and baseline DAS28, TSS, MRI bone oedema score, MRI synovitis score, MRI erosion score, anti-CCP, smoking, SE, age and gender as explanatory variables. RESULTS: Baseline values: median DAS28 5.6 (range 2.4-8.0); anti-CCP positive 61%; radiographic erosions 56%. At 2 years: DAS28 2.0 (0.5-5.7), in DAS remission: 56%, radiographic progression 26% (wrist+MCP group, similar for wrist-only group). MRI bone oedema score was the only independent predictor of delta-TSS (wrist+MCP group: coefficient = 0.75 (95% CI 0.55 to 0.94), p<0.001; wrist-only group: coefficient = 0.59 (95% CI 0.40 to 0.77), p<0.001). Bone oedema score explained 41% of the variation in the progression of TSS (wrist+MCP group), 25% in wrist-only group (Pearson's r = 0.64 and r = 0.50, respectively). Results were confirmed by sensitivity analyses. CONCLUSION: In a randomised controlled trial aiming at remission in patients with early RA, baseline RAMRIS MRI bone oedema score of MCP and wrist joints (and of wrist only) was the strongest independent predictor of radiographic progression in hands, wrists and forefeet after 2 years. MRI synovitis score, MRI erosion score, DAS28, anti-CCP, SE, smoking, age and gender were not independent risk factors. TRIAL REGISTRATION NUMBER: NCT00209859.

Circulating levels of osteopontin, osteoprotegerin, total soluble receptor activator of nuclear factor-kappa B ligand, and high-sensitivity C-reactive protein in patients with active rheumatoid arthritis randomized to etanercept alone or in combination with methotrexate.

OBJECTIVE: To determine whether circulating levels of osteopontin (OPN), osteoprotegerin (OPG), total soluble receptor activator of nuclear factor-kappa B ligand (total sRANKL), and high-sensitivity C-reactive protein (hsCRP) change in patients with rheumatoid arthritis (RA) during immunosuppressive therapy. METHODS: Twenty-five active RA patients were randomized to treatment with either etanercept alone or in combination with methotrexate (MTX). The treatment response after 16 weeks was assessed using the European League Against Rheumatism (EULAR) response criteria. Blood samples were taken before the start of and every fourth week during the study. OPN, OPG, and total sRANKL were measured by enzyme-linked immunosorbent assays (ELISAs) and hsCRP by highly sensitive turbidometry. RESULTS: At baseline, OPN and hsCRP were significantly (p<0.001) elevated compared to healthy persons. Compared to baseline only hsCRP levels decreased significantly (p<0.05 to p<0.001) in the EULAR responders through the study. OPN remained significantly (p<0.05) elevated at 16 weeks in patients with a low disease activity score (DAS< or =3.2). Total sRANKL increased significantly (p<0.05) from baseline to week 12. No statistically significant changes were observed in the non-responders. CONCLUSION: Active RA patients showed increased circulating levels of hsCRP and OPN, but only hsCRP decreased during etanercept therapy. Our findings suggest that OPN, OPG, total sRANKL, and hsCRP reflect different aspects of the inflammatory process in RA.

Aggressive combination therapy with intra-articular glucocorticoid injections and conventional disease-modifying anti-rheumatic drugs in early rheumatoid arthritis: second-year clinical and radiographic results from the CIMESTRA study.

OBJECTIVE: To investigate whether clinical and radiographic disease control can be achieved and maintained in patients with early, active rheumatoid arthritis (RA) during the second year of aggressive treatment with conventional disease-modifying antirheumatic drugs (DMARDs) and intra-articular corticosteroid. This paper presents the results of the second year of the randomised, controlled double-blind CIMESTRA (Ciclosporine, Methotrexate, Steroid in RA) study. METHODS: 160 patients with early RA (duration <6 months) were randomised to receive intra-articular betamethasone in any swollen joint in combination with step-up treatment with either methotrexate and placebo-ciclosporine (monotherapy) or methotrexate plus ciclosporine (combination therapy) during the first 76 weeks. At week 68 hydroxychlorochine 200 mg daily was added. From week 76-104 ciclosporine/placebo-ciclosporine was tapered to zero. RESULTS: American College of Rheumatology 20% improvement (ACR20), ACR50 and ACR70 levels were achieved in 88%, 79% and 59% of patients in the combination vs 72%, 62% and 54% in the monotherapy group (p = 0.03, 0.02 and 0.6 between groups). The patients globally declined from 50 to 12 vs 52 to 9, with 51% and 50% in Disease Activity Score (DAS) remission, respectively. Mean (SD) progressions in total Sharp-van der Heijde scores were 1.42 (3.52) and 2.03 (5.86) in combination and monotherapy groups, respectively (not significant). Serum creatinine levels increased by 7% in the combination group (4% in monotherapy), but hypertension was not more prevalent. CONCLUSION: Continuous methotrexate and intra-articular corticosteroid treatment resulted in excellent clinical response and disease control at 2 years, and the radiographic erosive progression was minimal. Addition of ciclosporine during the first 76 weeks resulted in significantly better ACR20 and ACR50 responses, but did not have any additional effect on remission rate and radiographic outcome.

Bone loss in unclassified polyarthritis and early rheumatoid arthritis is better detected by digital x ray radiogrammetry than dual x ray absorptiometry: relationship with disease activity and radiographic outcome.

OBJECTIVE: To compare changes in regional bone mineral density (BMD) of the metacarpal joints measured by dual x ray absorptiometry (DXA) and digital x ray radiogrammetry (DXR) in relation to disease activity and radiographic outcome in a two year follow up study of patients with early RA and unclassified polyarthritis. PATIENTS AND METHODS: 72 patients with symmetrically swollen and tender second and third metacarpophalangeal or proximal interphalangeal joints for at least four weeks and less than two years were included. 51 patients fulfilled the ACR criteria for RA. 21 patients had unclassified polyarthritis. The patients with RA were divided into groups according to mean disease activity, average glucocorticoid dose, and MRI and x ray detected bone erosions in the hands. Clinical and biochemical measurements were made every month and an x ray examination of the hands and BMD of the metacarpal joints every six months. RESULTS: DXR BMD decreased significantly only in patients with RA from month 6 and was associated with the mean disease activity. Patients with RA and erosive as well as non-erosive disease showed a significant decrease in the rate of bone loss, greatest in those with erosive disease. No changes in BMD measured by DXA were seen in any patient group. CONCLUSION: DXR is a useful measure of the destructive disease activity in patients with RA and unclassified polyarthritis, providing valuable information about bone changes associated with disease activity and erosive disease in early RA. DXR is better than DXA for detecting and monitoring periarticular osteoporosis of the metacarpal bone.

The influence of mannose binding lectin polymorphisms on disease outcome in early polyarthritis. TIRA Group.

OBJECTIVE: To determine whether variant alleles of the mannose binding lectin (MBL) gene causing low serum concentrations of MBL are associated with increased susceptibility to rheumatoid arthritis (RA) and erosive outcome in an inception cohort of patients with early polyarthritis. METHODS: MBL and HLA-DRB1 alleles were determined by polymerase chain reaction in 68 Danish patients with incident early polyarthritis observed for one year. The associations between MBL and specific HLA-DRB1 genotypes and disease outcomes were analyzed. RESULTS: Among the patients with early polyarthritis 7.4% (5/68) and 41.2% (28/68) were homozygous and heterozygous for MBL variant alleles, compared with 2.8% (7/250) and 34.4% (86/250) of healthy controls (p = 0.09), while the corresponding figures in the patients with RA were 10% (5/50) and 42% (21/50) (p = 0.03), and in the patients with erosive RA 18.8% (3/16) and 35.3% (6/16), respectively (p = 0.004). Patients with early polyarthritis homozygous for MBL variant alleles had an increased risk of having erosive RA at inclusion by a factor of 4.7 (p = 0.02) and after one year by a factor of 3.6 (p = 0.04). MBL deficiency was associated with increased levels of C-reactive protein (CRP) and IgM rheumatoid factor (RF) at inclusion (p < 0.05). HLA-DRB1 alleles were not found to be associated with disease outcome. CONCLUSION: MBL variant alleles appear to be weak susceptibility markers for RA, and patients with early polyarthritis and homozygous for MBL structural variant alleles have a higher risk of developing early erosive RA. These findings, together with the positive association between MBL variant alleles and the increased serum levels of IgM RF and CRP, point at the MBL gene as a relevant locus in the pathophysiology of RA.

Magnetic resonance imaging, radiography, and scintigraphy of the finger joints: one year follow up of patients with early arthritis. The TIRA Group.

OBJECTIVES: To evaluate synovial membrane hypertrophy, tenosynovitis, and erosion development of the 2nd to 5th metacarpophalangeal (MCP) and proximal interphalangeal (PIP) joints by magnetic resonance imaging in a group of patients with rheumatoid arthritis (RA) or suspected RA followed up for one year. Additionally, to compare the results with radiography, bone scintigraphy, and clinical findings. PATIENTS AND METHODS: Fifty five patients were examined at baseline, of whom 34 were followed up for one year. Twenty one patients already fulfilled the American College of Rheumatology (ACR) criteria for RA at baseline, five fulfilled the criteria only after one year's follow up, whereas eight maintained the original diagnosis of early unclassified polyarthritis. The following MRI variables were assessed at baseline and one year: synovial membrane hypertrophy score, number of erosions, and tenosynovitis score. RESULTS: MRI detected progression of erosions earlier and more often than did radiography of the same joints; at baseline the MRI to radiography ratio was 28:4. Erosions were exclusively found in patients with RA at baseline or fulfilling the ACR criteria at one year. At one year follow up, scores of MR synovial membrane hypertrophy, tenosynovitis, and scintigraphic tracer accumulation had not changed significantly from baseline; in contrast, swollen and tender joint counts had declined significantly (p<0.05). CONCLUSIONS: MRI detected more erosions than radiography. MR synovial membrane hypertrophy and scintigraphy scores did not parallel the changes seen over time in clinically assessed swollen and tender joint counts. Although joint disease activity may be assessed as quiescent by conventional clinical methods, a more detailed evaluation by MRI may show that a pathological condition is still present within the synovium.

Dynamic magnetic resonance imaging of the metacarpophalangeal joints in rheumatoid arthritis, early unclassified polyarthritis, and healthy controls.

OBJECTIVE: To introduce dynamic magnetic resonance imaging (MRI) as an indicator of inflammatory activity in the metacarpophalangeal (MCP) joints of patients with rheumatoid arthritis (RA) or early unclassified polyarthritis, and to compare the results with a healthy control group. MATERIALS AND METHODS: We examined 42 RA and 23 early unclassified polyarthritis patients, and 12 healthy controls in a cross-sectional study. Dynamic MRI (repeated FLASH-MR images after injection of a contrast agent) was performed through the 2nd to the 5th MCP joint. Two methods for identification of the enhancing synovial membrane were compared: 1) outlining of enhancing synovial membrane on subtraction images and 2) automated recognition by principal component analysis (PCA). The early enhancement (EE) rate was calculated on the basis of the first method. RESULTS: Method 1) and 2) were closely associated (P<0.00001). From the healthy control group, an upper limit (mean+2SD) of normal enhancement was established for the 2nd to 5th MCP joints, which served to identify abnormal EE rates in the corresponding joints of patients. The patients had higher EE rates in the 2nd to 5th MCP joints than had the healthy controls (P<0.01). There were no significant differences between the two patient groups (P>0.09). CONCLUSION: PCA seems to be a promising method for automated identification of enhancing tissue. EE rates of the finger joints may be useful in the assessment of the inflammatory activity in the joints of patients with RA and early unclassified polyarthritis and may reflect other aspects of disease activity than clinical evaluation.

Blood levels of CD11b+ memory T lymphocytes are selectively upregulated in patients with active rheumatoid arthritis.

The adhesion molecules CD11b (a beta2-integrin component) and CD54 (ICAM-1) on blood leukocytes were studied by flow cytometry in patients with rheumatoid arthritis (RA). The fractions of CD4+ cells co-expressing CD11b were elevated in 16 patients with active RA compared with those in 16 RA patients who improved during therapy and 8 healthy controls: 0.8+/-0.12% (mean+/-SEM) versus 0.3+/-0.06% (p<0.002) and 0.3+/-0.06% (p<0.005), respectively. Increased levels of CD11b+CD45R0+ cells were observed in patients with active RA compared to those with improved RA and controls: 12.6+/-3.9% versus 4.8+/-2.7% (p<0.002) and 6.1+/-1.2% (p<0.003), respectively. Disease activity, determined by C-reactive protein, correlated with the numbers of CD11b+CD45R0+ cells: r=0.62 (p<0.001). Seven patients were followed during induction of remission with methotrexate and glucocorticoids. The numbers of CD11b+CD4+ and CD11b+CD45R0+ cells fell significantly after clinical improvement. The levels of CD11b+CD14+ cells (monocytes) did not differ between the groups. The number of CD11b+CD15+ cells (neutrophils) was elevated in patients with RA irrespective of disease activity. The levels of CD54+ cells were not different between the RA and control groups. We conclude that the increased numbers of CD11b+ memory T cells may arise from exposure to stimuli outside the synovial compartment.

Human bone bank allografts stimulate bone resorption and inhibit proliferation in cultures of human osteoblast-like cells.

Incorporation of a frozen human bone allograft requires osteoclast activity and ingrowth of recipient osteoblast precursors. We examined the effects of allografts on human osteoblasts. Allografts stimulated a release of factors from normal human osteoblast-like cells, capable of inducing osteoclastic bone resorption in vivo. Further allografts inhibited osteoblast proliferation in cultures. The response was detectable within 4 days of culture and was still present after 3 weeks. Devitalized bone autografts had a similar effect. This suggests that bone bank grafts may induce a resorptive reaction at the recipient site by stimulating release of factors from osteoblasts capable of inducing osteoclastic resorption. The storage temperature was crucial for preservation of the response, since the activity was lower in allografts stored for 6 months at -20 degrees C than in those stored at -80 degrees C.

The effects of cyclosporin A on bone and cartilage.

Cyclosporin A (CyA) is a potent immunomodulatory agent with an increasing number of clinical applications. Although its precise mechanisms of action are yet to be elucidated, one of the most important known properties of CyA is its ability to inhibit the production of cytokines involved in the regulation of T cell activation. There is also evidence for direct effects on other cell types, such as B cells, macrophages, and bone and cartilage cells. The effects of CyA on T cells and on bone, cartilage and synovial cells, which can produce a range of cytokines, are of interest in the study of inflammatory diseases such as RA. It has been shown, for example, that in vitro CyA inhibits bone resorption induced by interleukin-1, 1,25-dihydroxy-vitamin D3, parathyroid hormone and prostaglandin E2. In vivo, it protects against adjuvant arthritis.

Mechanisms of action of cyclosporine and effects on connective tissues.

Cyclosporine is a potent immunomodulatory agent with an increasing number of clinical applications. Its major mode of action is inhibition of the production of cytokines involved in the regulation of T-cell activation. In particular, cyclosporine inhibits the transcription of interleukin 2. Although cyclosporine's major actions are on T cells, there is some evidence that it produces direct effects on other cell types. Its immunosuppressive action is closely linked to its binding of cyclophilin, a member of a family of high-affinity cyclosporine-binding proteins widely distributed in different cell types and in different species. The cyclophilins have been shown to have peptidyl-prolyl cis-trans isomerase enzyme activity that is blocked by cyclosporine. Although this may be a factor in cyclosporine's selective inhibition of cytokine gene transcription, it is still unclear whether inhibition of this activity is the mechanism through which cyclosporine exerts its effects on target cells. The ubiquitous presence of cyclophilins raises the question of why cyclosporine has major effects on T cells. Perhaps the critical proteins affected are transcriptional regulators restricted in their tissue distribution. The effects of cyclosporine on T cells and, directly or indirectly, on connective tissue cells, all of which can produce a range of cytokines, are of interest in relation to the tissue changes that occur in such inflammatory conditions as rheumatoid arthritis.

Cyclosporin A. Mode of action and effects on bone and joint tissues.

Cyclosporin A is an established immunomodulatory agent with an increasing number of clinical applications. Although its precise mechanisms of action remain elusive, one of the most important known properties of CyA is its ability to inhibit the production of cytokines involved in the regulation of T-cell activation. In particular, CyA inhibits de novo synthesis of interleukin 2(IL-2), the major cytokine involved in T-cell proliferation, as well as other cytokines, probably at the level of gene transcription, as shown by the suppression of mRNA levels in activated T-cells. Although the major actions of CyA are on T-cells, there is some evidence for possible direct effects on other cell types e.g. B-cells, macrophages and, from our own work, on bone and cartilage cells. Cyclosporin A is thought to enter cells and to bind to cyclophilins, which are members of a family of high-affinity cyclosporin A-binding proteins, now known as immunophilins. The binding of cyclosporins to such proteins appears to be closely linked to the immunosuppressive action of cyclosporins. The immunophilins possess enzyme activity, ie. peptidyl-prolyl cis-trans isomerase, also known as rotamase, which can regulate protein folding, and may therefore alter the functional state of many cell proteins. Cyclosporin A blocks peptidyl-prolyl cis-trans isomerase activity but it is not clear whether this plays a part in its selective inhibition of cytokine-gene transcription. Moreover, the ubiquitous presence of cyclophilins and immunophilins raises the question of why cyclosporin A has its apparent major effects only on T-cells. Recent proposals regarding the intracellular mode of action of CyA suggest that it interacts with cyclophilin and other regulatory proteins including calmodulin and calcineurin, which is a serine/threonine phosphatase, and thereby affects the functional state of key regulators of gene transcription in its target cells. The effects of CyA on T-cells and directly or indirectly on connective tissue cells, including bone, cartilage and synovial cells, which all can produce a range of cytokines, are of interest in relation to the tissue changes that occur in inflammatory diseases, such as rheumatoid arthritis. Thus, for example, cyclosporin A inhibits in vitro the bone resorbing activity of interleukin 1, 1,25-dihydroxy-vitamin D3, parathyroid hormone and prostaglandin E2 by apparently non-T-cell effects, while in vivo protects against bone and cartilage loss in adjuvant arthritis. More needs to be known about the direct and indirect modulation of cytokine production by cyclosporin A in connective tissues, in order to understand its potential value in clinical disorders.

Interleukin-1 stimulates diglyceride accumulation in the absence of protein kinase C activation.

Despite advances in the knowledge of the intracellular signalling in response to extracellular messengers, the mechanism of action of interleukin-1 (IL-1) has remained an enigma. In the present study, we have employed human dermal fibroblasts (Detroit 532 cells) to investigate IL-1 beta-induced changes in intracellular signals. Both recombinant human IL-1 beta and a native preparation purified from human placental tissue were employed. Cyclic AMP levels in cell monolayers were unaltered by IL-1 beta. Also, IL-1 beta did not influence significantly the levels of phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate in the membrane, nor the water-soluble inositol phosphates, inositol monophosphate, inositol bisphosphate and inositol trisphosphate, in cells prelabelled with myo-[3H]inositol. In addition, intracellular calcium as measured by Quin2 was unaffected by interleukin-1. However, in cells labelled with [3H]glycerol or [3H]arachidonic acid, IL-1 beta caused an immediate rise in diglyceride (DG) accumulation. As the effects of IL-1 beta have been reported to be mimicked by tumour-promoting phorbol esters, this rise in DG suggested the involvement of protein kinase C (PKC). However, repeated experiments failed to reveal any acute effect of IL-1 beta on the activity of this enzyme. Furthermore, IL-1 beta did not cause the translocation of PKC between the membrane and the cytosol as has been found in response to other extracellular signals. Rather, IL-1 beta appeared to increase the synthesis of PKC in both membrane and cytosol preparations, an effect which could be prevented by coincubation with cycloheximide. These findings suggest that the diglyceride formed in response to IL-1 beta does not activate protein kinase C.

Constitutive and inducible expression of HLA class II determinants by human osteoblast-like cells in vitro.

Activated immune cells release cytokines which modulate the activity of bone cells in vitro. Expression of major histocompatibility complex (HLA in humans) class II determinants on bone surface cells may be important in local immune cell activation. In this study, expression of HLA-DR and DQ by cultured human bone cells (HBC) derived from normal trabecular bone surfaces was assessed by fluorescence-activated cell sorter (FACS) analysis and immunoperoxidase techniques using monoclonal antibodies. A subset of HBC (10-30%) expressed DR constitutively while 5-15% displayed DQ during long-term culture. HBC lacked a number of monocyte and lymphocyte markers. In addition, both DR+ and DR- HBC (FACS separated) produced osteocalcin stimulated by 1,25-dihydroxyvitamin D2 (1,25(OH)2D3). This suggests that both phenotypes belong to the osteoblast lineage. The number of DR+ HBC was increased by interferon-gamma (IFN gamma; 40-95% DR+ cells) whereas DQ+ HBC remained unchanged or was slightly increased (5-20% DQ+ cells). Moreover, 1,25(OH)2D3 enhanced IFN gamma-induced DR expression and at high concentration (10(-7) M) augmented DR expression by itself. Other major osteotropic factors, parathyroid hormone, interleukin 1, and calcitonin, did not affect HBC DR expression. The findings suggest that HBC may participate in activation of the immune system and that some osteotropic factors may regulate this function.

Human osteoblast-like cells expressing MHC class II determinants stimulate allogeneic and autologous peripheral blood mononuclear cells and function as antigen-presenting cells.

We have previously shown that a subset of human osteoblast-like bone surface cells (HBC) derived from explants of trabecular bone constitutively express class II major histocompatibility complex (MHC) determinants in long-term culture. We now examine the capacity of HBC to stimulate allogeneic and autologous peripheral blood mononuclear cells (PBMC) and to function as antigen-presenting cells (APC). HBC consistently stimulated a strong proliferative response in allogeneic PBMC after 6 days co-culture. Stimulation of autologous PBMC was weaker and less consistent. HBC and adherent PBMC were equally active in stimulating a PPD response in autologous non-adherent PBMC. These observations suggest that immune cells initiated by HBC may play a role in normal and disordered bone remodelling processes.

Jejunoileal bypass and electrolytes. A follow-up study of intra- and extra-cellular electrolytes with special emphasis on magnesium.

It is generally believed that patients operated on for gross obesity with jejunoileal shunt develop electrolyte malabsorption. In follow-up studies electrolyte abnormalities have been reported in 6-37% of the cases. We have not been able to find any description of simple diagnostic tools to help indicate which patients should be treated with electrolyte supplements. The aim of this study was to evaluate different diagnostic tools to determine whether they would identify which patients to treat. Ten patients with end-to-side jejunoileostomies were investigated. Our attempt failed. We were not able to identify the patients who needed supplementary therapy. The reason for this may be absence of severe electrolyte abnormalities or insufficient diagnostic methods.