DNA methylation regulates the expression of CXCL12 in rheumatoid arthritis synovial fibroblasts.

In the search for specific genes regulated by DNA methylation in rheumatoid arthritis (RA), we investigated the expression of CXCL12 in synovial fibroblasts (SFs) and the methylation status of its promoter and determined its contribution to the expression of matrix metalloproteinases (MMPs). DNA was isolated from SFs and methylation was analyzed by bisulfite sequencing and McrBC assay. CXCL12 protein was quantified by enzyme-linked immunosorbent assay before and after treatment with 5-azacytidine. RASFs were transfected with CXCR7-siRNA and stimulated with CXCL12. Expression of MMPs was analyzed by real-time PCR. Basal expression of CXCL12 was higher in RASFs than osteoarthritis (OA) SFs. 5-azacytidine demethylation increased the expression of CXCL12 and reduced the methylation of CpG nucleotides. A lower percentage of CpG methylation was found in the CXCL12 promoter of RASFs compared with OASFs. Overall, we observed a significant correlation in the mRNA expression and the CXCL12 promoter DNA methylation. Stimulation of RASFs with CXCL12 increased the expression of MMPs. CXCR7 but not CXCR4 was expressed and functional in SFs. We show here that RASFs produce more CXCL12 than OASFs due to promoter methylation changes and that stimulation with CXCL12 activates MMPs via CXCR7 in SFs. Thereby we describe an endogenously activated pathway in RASFs, which promotes joint destruction.

[Web-based learning in musculoskeletal ultrasound]. / Webbasiertes Lernen in der Sonographie des Bewegungsapparates.

Education and training in musculoskeletal ultrasound (MSUS) comprises attendance at theoretical and practical courses and independent study. Web-based learning as a novel teaching method has previously been described. The present study summarizes normal and pathological findings in a web-based approach using widely accepted guidelines. In a prospective study over a period of 3 years normal and pathological images of the musculoskeletal system have been documented and catalogued. Overall 1240 ultrasound images and 183 ultrasound videos were collected. A total of 14.4% were normal and 85.6% were pathological MSUS findings; 61% concerned the upper extremity, while 39% were images and videos of the lower limbs. The most captured conditions included synovitis (33.3%), pathologies of the tendons e.g., tenosynovitis or tendinosis (19.6%) and normal findings (14.4%). The most represented diseases were rheumatoid arthritis (20%), calcium deposition disease (8.2%), gout (7.1%) and osteoarthritis (6.9%). The images and videos were edited and integrated in a web-based tool.

Expression of cathepsin K and matrix metalloproteinase 1 indicate persistent osteodestructive activity in long-standing ankylosing spondylitis.

BACKGROUND: Ankylosing spondylitis (AS) is a common, largely genetically determined, rheumatic disease that is characterised by spinal inflammation and new bone formation. However, the exact pathogenesis and pathology are still not clear. OBJECTIVE: To analyse tissue obtained at spinal surgery by immunohistochemistry and compare the specimen of patients with AS to those with degenerative disc disease (DDD). METHODS: Bony and soft tissue specimens of 30 patients with AS and 20 with DDD were obtained during spinal osteotomy from different anatomic regions including articular and spinous processes, interspinous ligaments and intervertebral disks. Immunohistolochemistry was performed with established markers for cathepsin K, matrix metalloproteinase (MMP)1, MMP3 and receptor activator for nuclear factor kappaB (RANK) ligand. RESULTS: Cathepsin K and MMP1-positive cells were only observed in AS specimens. Cathepsin K-positive multinucleated cells were detected at articular processes adjacent to fibrous tissues. MMP1 was expressed in smaller mononuclear cells attached to bone. Invasion of bone by MMP1 cells was seen at entheseal sites. In the intervertebral disks, most mononuclear cells were cathepsin K-positive. Isolated cells expressing these matrix-degrading enzymes found in DDD never showed signs of invasion. No differences were found for MMP3 between AS and DDD. Clear expression of RANK ligand was only detected in one patient with AS. CONCLUSIONS: Cathepsin K is strongly expressed in different regions of the spine in AS. Cathepsin K was mainly expressed by mononuclear cells, fibroblast-like cells and cells attached to bone and at sites of bone remodelling, suggestive of high osteoclastic activity. This supports the role of persistent inflammation in the pathogenesis of AS. How these changes relate to osteoproliferation remains to be determined.

Severe vitamin D deficiency in Swiss hip fracture patients.

BACKGROUND: Most clinical guidelines for the prevention of hip fractures recommend 800 IU vitamin D per day. This dose shifted serum 25-hydroxyvitamin D levels (25(OH)D) in previous studies to between 60 and 100 nmol/l. AIM: To measure 25(OH)D levels and prevalence of vitamin D supplementation in individuals age 65+ with acute hip fracture. METHODS: 222 consecutive hip fracture patients were investigated over a 12 month period. Mean age of patients was 86 years and 77% were women. RESULTS: Mean serum 25(OH)D levels were low among hip fracture patients admitted from home (34.6 nmol/l), from assisted living (27.7 nmol/l), and from nursing homes (24 nmol/l). Severe vitamin D deficiency below 30 nmol/l was present in 60%, 80% were below 50 nmol/l, and less than 4% reached desirable levels of at least 75 nmol/l. Consistently, only 10% of hip fracture patients had any vitamin D supplementation on admission to acute care with significantly higher 25(OH)D levels among individuals supplemented with 800-880 IU/day (63.5 nmol/l). Controlling for age and gender, vitamin D supplementation, type of dwelling, and season were independently and significantly associated with 25(OH)D levels. CONCLUSION: These data provide evidence that current guidelines for the prevention of hip fractures need further effort to be translated into clinical practice.

The dual inhibitor of lipoxygenase and cyclooxygenase ML3000 decreases the expression of CXCR3 ligands.

OBJECTIVE: To find previously unknown properties of ML3000, a competitive inhibitor of the cyclooxygenase and the lipoxygenase (LO) pathway. METHODS: Gene expression of ML3000 treated and untreated rheumatoid arthritis synovial fibroblasts were measured with Affymetrix gene arrays. Downregulation of chemokine (C-X-C motif) ligands CXCL9, CXCL10 and CXCL11 was verified with Real-time polymerase chain reaction, CXCL10 protein levels were determined with ELISA. Rheumatoid arthritis synovial fibroblasts were treated with the cyclooxygenase inhibitor naproxen, the 5-LO inhibitor BWA4C and the 5-lipoxygenase-activating protein (FLAP) inhibitor MK886, and consecutive changes in CXCL10 protein levels measured. 5-LO expression was determined by polymerase chain reaction and Western blot. RESULTS: In synovial fibroblasts and monocyte-derived macrophages ML3000 inhibited the tumour necrosis factor induced expression of CXCL9, CXCL10 and CXCL11, which are all ligands of the chemokine receptor CXCR3. No effect was observed in monocytes. Whereas inhibition of the cyclooxygenase pathway or the FLAP protein showed no effect, blockade of 5-LO significantly downregulated CXCL10 protein levels. 5-LO mRNA was detected in monocytes and in monocyte-derived macrophages. All tested cell types expressed 5-LO protein. CONCLUSIONS: ML3000 effectively downregulates CXCR3 ligands. This study confirms that a thorough analysis of the impact of a drug on its target cells cannot only reveal unexpected properties of a substance, but also helps to understand the underlying molecular mechanisms. Accordingly, our data provide the basis for further clinical studies testing the application of ML3000 in diseases such as rheumatoid arthritis or multiple sclerosis.

Effects of a novel tyrosine kinase inhibitor in rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: Biologicals have revolutionised the treatment of rheumatoid arthritis (RA). However, progressive joint destruction can still be observed in many patients and the search for novel molecular therapies targeting specific signalling pathways is ongoing. In the present study, we investigated the effects of GW282974, a novel compound directed against tyrosine kinase activity with respect to the potential suppression of inflammation and destruction. METHODS: Synovial tissue specimens were obtained from RA patients undergoing surgical joint replacement. Rheumatoid arthritis synovial fibroblasts (RASFs) were stimulated with cytokines and GW282974 was added in different concentrations. Gene expression was checked by TaqMan PCR, using 18S as housekeeping gene. Protein analysis was quantified by ELISA. Cell growth and proliferation was measured using the "ViaLight" proliferation assay. RESULTS: EGF had no effect on the gene expression profile of RASFs when used as single stimulatory agent. In combination with pro-inflammatory mediators however, EGF showed a synergistic effect. The expression of matrix metalloproteinases, inflammatory cytokines and cyclooxygenase-2 on mRNA levels was strongly increased, whereas the addition of GW282974 abrogated these effects in a dose-dependent manner. These data could be confirmed on protein/lipid levels analysing the supernatants of RASFs by ELISA. Similarly, cell growth and proliferation of RASFs were inhibited by GW282974 in a dose- and time-dependent manner. By contrast, no cytotoxic effects were seen within the concentrations used. DISCUSSION: GW282974 appears to interfere with the inflammatory and the destructive pathways in RASFs and might therefore be used as novel therapeutic strategy for the treatment of RA.

[History and clinical examination in rheumatology--key to diagnosis and prognosis]. / Die Schlüssel zur Diagnostik in der Rheuma- tologie--Anamnese und klinische Untersuchung.

History and clinical examination are key to diagnosis and prognosis in rheumatology. In the area of soft tissue rheumatism, there is no other diagnostic possibility which could replace clinical examination. Clinical examination also plays a very important role in the diagnosis of the other rheumatic disorders. With a competent clinical examination one can often eliminate the need for additional costly and/or time consuming investigations. In addition, diagnosing by purely careful and competent clinical examination is a highly gratifying activity for the medical doctor. The competent examiner should also be able to derive prognostic conclusions directly from the clinical examination. This is especially important for the more frequent disorders such as soft tissue rheumatism or back problems, where diagnostic imaging is only helpful if interpreted in the context of the pertinent clinical question.

Weight-bearing exercise, overexercise, and lumbar bone density over age 50 years.

This cross-sectional study of 78 healthy subjects older than age 50 years was designed to examine the association between weight-bearing exercise and lumbar bone mineral content as assessed by quantitative computed tomography. In women, a strong correlation existed between bone density and the amount of exercise for up to 300 min/wk. However, 5 of 28 women exercising 300 min/wk or more had surprisingly low bone density, not explained by other factors. In 50 men, we also found a strong positive correlation between exercise and bone density for those up to age 65 years. Over age 65, 3 men had low bone density despite extremely vigorous exercise. We conclude that moderate weight-bearing exercise may increase lumbar bone density, but we raise the hypothesis that extremely vigorous exercise actually may be detrimental to bone density in individuals after age 50.

Children's thinking in the wake of Challenger.

OBJECTIVE: The Challenger spacecraft explosion in 1986 offered an opportunity to study the thinking of normal children after a sudden and distant disaster, differences in thinking among children of different levels of emotional concern and different ages, and changes in their thinking over time. METHOD: The authors studied six thinking patterns known to characterize childhood posttraumatic stress disorder and four additional hypothesized patterns in 153 randomly selected children of Concord, N.H. (who watched the explosion on television) and Porterville, Calif. (who heard about it later). They compared the structured-interview responses of the more involved (East Coast) and less involved (West Coast) children, of the latency-age children and the adolescents, and of the children initially (5-7 weeks after the explosion) and 14 months later. RESULTS: The children exhibited the 10 predictable thinking patterns. They initially defended themselves, denying the reality of the explosion. They later fantasized about it. They tried to cope by seeking additional information on their own, at home, and at school. Most children talked about Challenger, but a minority of the latency-age youngsters avoided related talk and thoughts. The adolescents experienced more paranormal thinking, philosophical changes, and negative attitudes. Over the year, omens, paranormal experiences, and Challenger-based fantasies tended to disappear, but negative views about institutions and the world's future held steady or increased. CONCLUSIONS: The children's thinking followed predictable patterns. A higher degree of emotional involvement (East Coast children) was strongly linked to these thinking patterns, as was being an adolescent. Distant disasters appear to set up commonalities of thought that might come to characterize certain generations of children.

Children's memories in the wake of Challenger.

OBJECTIVE: The Challenger spacecraft explosion of Jan. 28, 1986, offered an opportunity to study the memories of normal latency and adolescent children of different emotional involvements following one sudden and distant disaster. How would children of various levels of concern express their memories? And if studied over time, how would these narratives change? Would there be developmental differences? And would there be false details of memory? METHOD: The authors set out to compare the memories of 153 children from Concord, N.H. (who watched the explosion on television), and Porterville, Calif. (who heard about it). The structured-interview responses of involved and less involved children; latency-age versus adolescent children; and those seen initially (5-7 weeks after the explosion) versus those same children seen later (at 14 months) were statistically compared. RESULTS: The vast majority of children's memories of Challenger were clear, consistent, and detailed, with highlighting of personal placement, who else was there, and personal occurrences linked to the event. Those children who were less emotionally involved demonstrated significantly less clarity, consistency, and correct ordering of sequences and were less likely to remember personal placement, other people who were there, and related personal incidents. About 30% of all children in this study misunderstood something about Challenger and incorporated these misunderstandings into their memories as false details. Latency-age children continued to harbor false details for 14 months, as opposed to the adolescents. CONCLUSIONS: Childhood memories of the Challenger space shuttle explosion appeared predictable, were related to patterns of memory that have been observed following single, unrepeated traumas, and reflected age and stage differences.

Children's symptoms in the wake of Challenger: a field study of distant-traumatic effects and an outline of related conditions.

OBJECTIVE: The Challenger space shuttle explosion in January 1986 offered an opportunity to determine what, if any, symptoms of posttraumatic stress disorder (PTSD) and bereavement normal latency-age children and adolescents would develop after a distant, horrifying event. METHOD: With a structured interview, the authors assessed the symptoms of 153 randomly selected children from Concord, N.H., and Porterville, Calif. Responses were statistically compared between East Coast children, who saw the event on television and who generally cared more about the teacher aboard Challenger, and West Coast children, who heard about it first; between latency-age children and adolescents; and between children seen 5-7 weeks later and those same children seen 14 months later. RESULTS: More than 60% of the subjects feared at least one stimulus related to Challenger within the first 5-7 weeks of the explosion. The East Coast and latency-age groups appeared significantly more symptomatic than did the West Coast and adolescent groups. Over the 14-month study period, most symptoms dramatically faded. However, adolescents' diminished expectations for the future in general increased, and latency-age children's changed approach to space careers held relatively steady. Three East Coast latency-age children met the DSM-III-R symptom requirements for PTSD in 1986; no children met these in 1987. CONCLUSIONS: Children's symptomatic patterns after Challenger relate to the patterns for PTSD listed in diagnostic manuals and to three symptoms not in the DSM-IV list. To the authors, distant traumas appear to be one of a newly defined spectrum of trauma-related conditions that include relatively evanescent symptoms and a few longer-lasting ones. These symptoms may affect large numbers of normal children.

Nitric oxide and proteoglycan biosynthesis by human articular chondrocytes in alginate culture.

Interleukin-1 alpha and beta induced the production of large amounts of nitric oxide by normal, human articular chondrocytes in alginate culture; at the same time the biosynthesis of proteoglycan was strongly suppressed. In a dose-dependent manner, NG-monomethyl-L-arginine both inhibited nitric oxide formation and relieved the suppression of proteoglycan synthesis. However concentrations of NG-monomethyl-L-arginine which completely prevented nitric oxide production only partially restored proteoglycan biosynthesis, even at low doses of interleukin-1 where suppression of proteoglycan synthesis was modest. The organic donor of nitric oxide, S-nitrosyl-acetyl-D,L- penicillamine also inhibited proteoglycan biosynthesis, but not as extensively as interleukin-1. These data suggest that interleukin-1 suppresses synthesis of the cartilaginous matrix through more than one mechanism, at least one of which is dependent upon the production of nitric oxide.

Nonsteroidal anti-inflammatory drug-associated gastropathy: incidence and risk factor models.

PURPOSE: The most prevalent serious drug toxicity in the United States is increasingly recognized as gastrointestinal (GI) pathology associated with the use of nonsteroidal anti-inflammatory drugs (NSAIDs). The incidence of serious GI events (hospitalization or death) associated with NSAID use was therefore prospectively analyzed in patients with rheumatoid arthritis (RA) and patients with osteoarthritis. PATIENTS, METHODS, AND RESULTS: The study consisted of 2,747 patients with RA and 1,091 patients with osteoarthritis. The yearly hospitalization incidence during NSAID treatment was 1.58% in RA patients and was similar in all five populations studied. The hazard ratio of patients taking NSAIDs to those not taking NSAIDs was 5.2. The incidence in osteoarthritis may be less. The risk of GI-related death in RA patients was 0.19% per year with NSAIDs. Multivariate analyses assessing risk factors for serious GI events were performed in the 1,694 (98 with an event) RA patients taking NSAIDs at the predictive visit. The main risk factors were higher age, use of prednisone, previous NSAID GI side effects, prior GI hospitalization, level of disability, and NSAID dose. A rule is presented that allows estimation of the risk for the individual patient with RA. CONCLUSION: Knowledge of the risk factors for NSAID-associated gastropathy and their inter-relationships provides a tool for identification of the patient at high risk and for initiation of appropriate therapeutic action.

Topical diclofenac patch in patients with knee osteoarthritis: a randomized, double-blind, controlled clinical trial.

OBJECTIVE: To assess the efficacy and safety of a diclofenac hydroxyethylpyrrolidine (DHEP) patch in the treatment of symptomatic osteoarthritis (OA) of the knee joint. METHODS: A double-blind, randomised, placebo-controlled trial was carried out on 103 outpatients for 2 weeks. The main efficacy parameters were spontaneous pain and Lequesne's Index. Secondary endpoints were walking time over a standard distance, global assessment of efficacy and tolerability, and paracetamol consumption. RESULTS: The active treatment group showed a significant improvement in pain, Lequesne's Index, and the physician's and patient's global assessment of efficacy. For these parameters the difference between groups was statistically significant in favour of the DHEP patch. Adverse reactions were seen in a small number of probands and were similar in both groups. CONCLUSIONS: The results of this trial suggest that the DHEP patch appears to be an effective and safe treatment for patients suffering from symptomatic knee OA.

Construct validation of the ACR 1991 revised criteria for global functional status in rheumatoid arthritis.

In order to assess the construct and discriminatory validity of the 1991 ACR functional status criteria for rheumatoid arthritis (RA), a cross-sectional analysis of 62 consecutive patients with RA (according to the American Rheumatism Association 1987 revised criteria) who were attending the outpatient clinic of rheumatology, University Hospital Zürich, was carried out. A moderate-to-strong association of the ACR criteria with pain (r = 0.54), the tender (r = 0.54) and swollen joint count (r = 0.31), grip strength (r = -0.49), C-reactive protein (r = 0.35), the HAQ disability index (r = 0.76), self-perceived global health (r = 0.58), and the Larsen radiological score (r = 0.32) was found. The mean scores of most disease parameters and all 8 domains of the health assessment questionnaire were significantly different between, and increased regularly across, the 4 classes. We conclude that the ACR 1991 functional status criteria for RA are a valid measure of the consequences of impairment and discriminate well the physical functional status.

The expression of cyclooxygenase-1, cyclooxygenase-2 and 5-lipoxygenase in inflammatory muscle tissue of patients with polymyositis and dermatomyositis.

OBJECTIVE: To describe cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) expression in muscle tissue in patients with idiopathic inflammatory myopathies (IIM) - dermatomyositis (DM) and polymyositis (PM) and to find out if any differences between affected and non-affected muscles detected by MRI exist. METHODS: Samples of muscle tissue from 7 patients with dermatomyositis (DM) and from 4 with polymyositis (PM) were obtained by needle biopsy from affected and non-affected sites distinguished by magnetic resonance imaging. In situ hybridization with antisense mRNA probes was employed to detect COX-1, COX-2 and 5-LOX mRNA. RESULTS: Expression of COX-1, COX-2, and 5-LOX mRNA was found in all samples - in the muscle cells, inflammatory cells and in vessels. COX-1 mRNA expression predominated in the inflammatory cells and vessels and was higher in affected than in non-affected sites detected by MRI (mean intensity 3.22+/-0.67 vs. 2.0+/-0.87; p = 0.0006). The expression of COX-2 mRNA was high mainly in inflammatory cells and/or vessels and was increased in MRI-detected affected tissues (3.5+/-0.88; 1.9+/-1.1; p = 0.003), as was the expression of COX-2 mRNA in muscle cells (2.1+/-1.0 vs. 1.3+/-1.0; p = 0.021). 5-LOX mRNA was largely expressed in muscle cells from MRI-detected affected sites and the signal intensity was higher in comparison with samples taken from non-affected tissues detected by MRI (3.22+/-0.7 vs. 1.67+/-0.7; p = 0.0007). CONCLUSION: Expression of COX-1, COX-2 and 5-LOX mRNA was observed for the first time in muscle tissues from IIM patients. This expression was increased in affected tissues detected by MRI, which may suggest a role of COX-1, COX-2, and 5-LOX in the pathogenesis of IIM.

Ankylosing spondylitis and sarcoidosis--coincidence or association? Case report and review of the literature.

We report a 25-year-old woman presenting with sarcoidosis and bilateral sacroiliitis. Her sarcoidosis related symptoms (malaise, cough and dyspnoea) improved dramatically under treatment with steroids but severe back pain persisted. Only seven similar cases have been described over the last 40 years and the question of a possible association between the two diseases has been raised. However, prevalence data from the literature and the apparent lack of genetic links are better arguments for coincidence than for association.

Preventing recurrence of reflex sympathetic dystrophy in patients requiring an operative intervention at the site of dystrophy after surgery.

The development of reflex sympathetic dystrophy (RSD) is a common complication after surgery. Exacerbation or recurrence of RSD is a major concern after a second intervention at the site of previous surgery and consecutive RSD. It is unclear whether the risk of recurrent RSD can be reduced by using appropriate precautions. The objective of our study was to examine, in a case series of consecutive patients, whether recurrences in patients with a history of RSD after surgery, who were reoperated at the same location, can be avoided by using a standardised intervention protocol containing perioperative calcitonin prophylaxis. None of the patients experienced a recurrence of RSD. We concluded that the recurrence of RSD in patients requiring operative intervention at the site of former dystrophy after surgery appears to be unlikely with careful perioperative management.