Structure of full-length porcine synovial collagenase reveals a C-terminal domain containing a calcium-linked, four-bladed beta-propeller.

BACKGROUND: The collagenases are members of the family of zinc-dependent enzymes known as the matrix metalloproteinases (MMPs). They are the only proteinases that specifically cleave the collagen triple helix, and are important in a large number of physiological and pathological processes. Structures are known for the N-terminal catalytic' domain of collagenases MMP-1 and MMP-8 and of stromelysin (MMP-3). This catalytic domain alone, which comprises about 150 amino acids, has no activity against collagen. A second domain, of 200 amino acids, is homologous to haemopexin, a haem-binding glycoprotein. RESULTS: The crystal structure of full-length MMP-1 at 2.5 A resolution gives an R-factor of 21.7%. Two domains are connected by an exposed proline-rich linker of 17 amino acids, which is probably flexible and has no secondary structure. The catalytic domain resembles those previously observed, and contains three calcium-binding sites. The haemopexin-like domain contains four units of four-stranded antiparallel beta sheet stabilized on its fourfold axis by a cation, which is probably calcium. The domain constitutes a four-bladed beta-propeller structure in which the blades are scarcely twisted. CONCLUSIONS: The exposed linker accounts for the difficulty in purifying full-length collagenase. The C-terminal domain provides a structural model for haemopexin and its homologues. It controls the specificity of MMPs, affecting both substrate and inhibitor binding, although its role remains obscure. These structural results should aid the design of site-specific mutants which will reveal further details of the specificity mechanism.

Transforming growth factor beta stimulates the production of the tissue inhibitor of metalloproteinases (TIMP) by human synovial and skin fibroblasts.

IL-1 stimulates the secretion of metalloproteinases by a variety of connective tissue cells and is thought to be the primary inducing agent of connective tissue breakdown in rheumatoid arthritis. Transforming growth factor-beta (TGF-beta) is known to be capable of inhibiting the synthesis of metalloproteinases and to be able to partially inhibit interleukin-1 (IL-1) induced cartilage degradation. The present paper examines the ability of TGF-beta to modulate the action of IL-1 on fibroblasts of synovial and skin origin and investigates the secretion of the tissue inhibitor of metalloproteinases (TIMP) by these cells after exposure to TGF-beta and IL-1. The principal findings are that when four out of five fibroblast lines were exposed to TGF-beta and IL-1 in combination they displayed a significant increase in TIMP secretion; furthermore, in two of these cell lines a significant stimulation of TIMP secretion was induced by TGF-beta alone.

The secretion of the tissue inhibitor of metalloproteinases (TIMP) by human synovial fibroblasts is modulated by all-trans-retinoic acid.

The matrix metalloproteinases are a family of enzymes involved in the turnover of the connective tissues. The regulation of these enzymes is complex, involving the control of synthesis, the activation of proenzyme forms and the presence of specific inhibitors. Retinoids have been reported to inhibit the production of metalloproteinases by human and rabbit synovial fibroblasts and by human skin fibroblasts. The production of the highly specific tissue inhibitor of metalloproteinases (TIMP) by connective tissue cells may be crucial in the regulation of connective tissue breakdown and this present study was undertaken to determine if retinoic acid (RA) could modulate TIMP and collagenase production by synovial fibroblasts. The results show that RA at concentrations from 10(-7) to 10(-5) M significantly stimulated the secretion of TIMP by two of three human synovial cell lines. The effect of mononuclear cell factor (MCF) on TIMP and collagenase levels was also investigated. The apparent reduction of collagenase levels in the presence of RA, could result from a failure to accurately measure this enzyme in the presence of increased levels of TIMP.

Modulation of plasminogen activator production by interleukin 1: differential responses of fibroblasts derived from human skin and rheumatoid and non-rheumatoid synovium.

Rheumatoid synovial fibroblasts were treated with purified porcine interleukin 1 alpha and recombinant human interleukin 1B, and the production of secreted and cell-associated plasminogen activator activity was measured. No stimulation of plasminogen activator activity was seen in response to either preparation of interleukin 1, and in more than half of the cell cultures interleukin 1 caused a significant decrease in the secreted levels of PA activity. Increased levels of prostaglandin E were produced in the same experiments, indicating that the cells were responsive to the interleukin 1 preparations. Both retinoic acid and unfractionated monocyte conditioned medium were able to stimulate the production of PA activity by the rheumatoid synovial fibroblast cultures. The rheumatoid synovial fibroblasts produced two species of plasminogen activator as indicated by SDS polyacrylamide gel electrophoresis, with apparent Mr of approx. 50,000 and 100,000. The Mr = 50,000 species co-migrates with urokinase-type plasminogen activator. No species is produced which co-migrates with tissue type plasminogen activator. Studies with antibodies also indicate that the activity produced is urokinase-type plasminogen activator. The Mr = 100,000 species may be an enzyme-inhibitor complex. Two non-rheumatoid synovial fibroblast cultures and two out of six human skin fibroblast cultures did produce elevated levels of plasminogen activator activity in response to recombinant human interleukin 1B. The results suggest that fibroblast populations may differ in their response to interleukin 1, in terms of production of plasminogen activator activity.

Synovial fluids from infected joints contain metalloproteinase--tissue inhibitor of metalloproteinase (TIMP) complexes.

Samples of synovial fluids aspirated from patients with septic arthritis prior to the commencement of any treatment contained active metalloproteinases but no proteinase inhibitory activity. We therefore assayed these samples for proteinase-inhibitor complexes. Although no biologically active alpha 2-macroglobulin or tissue inhibitor of metalloproteinase (TIMP) was present in the fluids, immunoassay of the samples clearly showed that high molecular weight proteinase-TIMP complexes were present. It is proposed that high levels of active metalloproteinases are released from neutrophils into septic synovial fluids and that these proteinases complex all the available TIMP, forming metalloproteinase-TIMP complexes.

Multiple changes in gene expression in chronic human Achilles tendinopathy.

Atlas cDNA cell interaction arrays (CLONTECH) were used to examine degenerate tissue from four patients with Achilles tendon disorders, in order to identify changes in expression of genes important in cell-cell and cell-matrix interactions. The greatest difference between normal (post-mortem) and degenerate tissue samples was in the level of MMP-3 (stromelysin) mRNA, which was down-regulated in all the degenerate samples. Quantitative RT-PCR assay of RNA extracted from paired 'normal' and degenerate Achilles tendon tissue samples taken from tendons during surgery mirrored the results of the arrays. Levels of MMP-3 mRNA were lower, whereas levels of type-I and type-III collagen mRNAs were significantly higher, in the degenerate compared to the 'normal' samples. Immunoblotting of proteins extracted from the same tendon samples showed that three of four normal tissue samples taken from individuals without apparent tendon disorder had much higher levels of MMP-3 protein than 'normal' or degenerate samples from patients with tendinosis. We suggest that MMP-3 may play an important role in the regulation of tendon extracellular matrix degradation and tissue remodelling.

A rapid and reproducible method for the analysis of immune complexes using affinity chromatography and Western blotting.

A new procedure which couples different analytical techniques in a format permitting the rapid analysis of immune complex components is described. Complexes obtained from sera by polyethylene glycol (PEG) precipitation were resuspended and then added, using a batch method, to antibody coupled to Sepharose beads. Antibody directed against either human C1q or human C3c were used in the present study. Bound immune complexes were washed and then eluted from the Sepharose by sodium dodecyl sulphate (SDS) treatment and simultaneously reduced with dithiothreitol. Individual components were separated by SDS gradient polyacrylamide gel electrophoresis and then transferred to nitrocellulose by Western blotting. Individual strips of nitrocellulose were investigated using specific antisera and a radiolabelled probe. Immune complexes (IC) isolated from the sera of 7 rheumatoid arthritis (RA) patients were analysed using this method and the results obtained for both affinity adsorbents compared.

Incidence of neoplasms in patients with rheumatoid arthritis exposed to different treatment regimens.

Immunosuppressive drugs have been used during the last 30 years in treatment of patients with severe rheumatoid arthritis. The drugs commonly used are cyclophosphamide and chlorambucil (alkylating agents), azathioprine (purine analogue), and methotrexate (folic acid analogue). There is evidence that all four immunosuppressive drugs can reduce synovitis, but disease activity almost always recurs after therapy is stopped. Since adverse reactions are frequent, less than 50 percent of patients are able to continue a particular drug for more than one year. Since it takes three to 12 months to achieve maximal effects, those patients who are unable to continue the drug receive little benefit from it. Patients treated with alkylating agents have an increased risk of development of acute nonlymphocytic leukemia, and both alkylating agents and azathioprine are associated with the development of non-Hodgkin's lymphoma. Cyclophosphamide therapy increases the risk of carcinoma of the bladder. There have been several long-term studies of patients with rheumatoid arthritis treated with azathioprine and cyclophosphamide and the incidence of most of the common cancers is not increased. Data on the possible increased risk of malignancy in rheumatoid arthritis are still being collected, and until further information is available, the use of immunosuppressive drugs, particularly alkylating agents, in the treatment of rheumatoid arthritis should be reserved for patients with severe progressive disease or life-threatening complications.

Single-blind comparative study of nabumetone (Relafen) versus naproxen in the treatment of rheumatoid arthritis.

Forty-seven patients with active rheumatoid arthritis were entered into a single-blind study to compare the safety and efficacy of nabumetone with naproxen. Patients were randomly assigned to receive a daily dose of 2 g of nabumetone or 1 g of naproxen after a placebo-washout period of at least three days. In addition to the usual clinical and laboratory measurements of disease activity, thermographic assessment was carried out at each visit and the heat distribution index was calculated. In addition, the safety and tolerance were assessed. Patients had improvement in severity of pain, severity and duration of morning stiffness, and articular index, and there was no statistical difference between the two treatment groups. By the end of six months, 13 patients had withdrawn from treatment; five patients in the naproxen treatment group and five in the nabumetone group were withdrawn from the study due to lack of efficacy. In no patient from the nabumetone group did an adverse reaction develop. In one patient in the naproxen group, a severe decrease in white blood cell count developed, and a skin rash and swollen gums developed in another patient. We conclude that nabumetone is well tolerated and of equal efficacy to naproxen in the treatment of active rheumatoid arthritis.

96-well plate-based method for total collagen analysis of cell cultures.

Total collagen assays are often laborious and use large quantities of consumables. We have developed a new method of assaying total 3H-proline-labeled collagen from cultured cells. Cells and media are harvested from 96-well plates directly onto fiberglass filtermats and counted in the Wallac 1205 flat-bed scintillation counter (BetaPlate). The assay was validated by comparison with a traditional total collagen assay. The resulting assay provides a rapid one-step method for quantifying collagen synthesis, which, unlike many collagen assays, does not require extensive dialysis or precipitation of proteins.

Magnetic resonance imaging protocol optimization for evaluation of hyaline cartilage in the distal interphalangeal joint of fingers.

RATIONALE AND OBJECTIVES: To identify a single magnetic resonance imaging (MRI) protocol that will provide optimal signal-to-noise ratio, resolution, and image contrast with minimal susceptibility artifacts and that will allow clear delineation and visualization of cartilage, fluid, bone, tendons, and ligaments within the distal interphalangeal (DIP) joint of the human hand. METHODS: A highly optimized 2.4 T MRI system was constructed from a 31-cm horizontal bore magnet, using a solenoid radiofrequency coil. This was used to study the DIP joints of 16 healthy, asymptomatic volunteers. RESULTS: A range of image contrast protocols were explored, including spin-echo T1 and T2, field echo, chemical shift suppression to give water only images, and magnetization transfer. Susceptibility variations were explored by changing the field strength from 0.6 to 2.4 T. A spin-echo protocol with TR = 1500 msec and TE = 30 msec can routinely produce images with resolution 0.075 x 0.150 for a slice thickness of 1 mm in 13 minutes. That protocol can visualize simultaneously compact and trabecular bone, two layers of cartilage, synovial fluid, and synovium within the joint, tendons and ligaments, and the volar plate. CONCLUSIONS: Although the contrast is not fully optimized for any one tissue, the spin echo protocol (TR = 1500, TE = 30) provides sagittal MR images, which clearly delineate the major structures of interest within the DIP joint, and which will be used in future studies to compare changes in the DIP joint because of aging or osteoarthritis. Experience gained by applying the above methods to a total of 16 healthy, asymptomatic volunteers has enabled a single sequence to be identified, which although not optimized for any one tissue, nevertheless visualized simultaneously and clearly delineated compact and trabecular bone, two layers of cartilage, synovial fluid, and synovium within the joint.

Effects of single dose compared with three days' prednisolone treatment of healthy volunteers: contrasting effects on circulating lymphocyte subsets.

AIMS: To investigate the effects of longer term corticosteroid treatment on circulating lymphocyte subsets. METHODS: Prednisolone (20 mg daily) was given to 12 healthy volunteers in a single morning dose for three days. Circulating lymphocyte subsets were measured by flow cytometry after whole blood lysis. RESULTS: Seven hours after the first dose of prednisolone there was a significant fall in absolute numbers of lymphocytes, T cells, CD4+ and CD8+ cells, and B cells. The percentage of T cells fell significantly, due to a fall in percentage of CD4+ cells. In contrast to the seven hour findings, at 72 hours there was a significant rise in absolute numbers of lymphocytes, T cells, CD4+, CD8+, and B cells. This trend was already apparent by 24 hours. The percentage of CD4+ cells was significantly raised at 72 hours, while that of CD8+ cells had fallen significantly. The percentage of natural killer cells had fallen at 72 hours; that of B cells remained increased at 72 hours. CONCLUSIONS: These findings show that corticosteroid treatment causes significant changes in lymphocyte subsets, and that such changes must be considered when designing studies of lymphocyte subsets during illness.

Fenbufen and indomethacin: a comparative study in rheumatoid arthritis.

Fenbufen and indomethacin were compared in the treatment of 30 patients with rheumatoid arthritis. In a randomized, double-blind crossover study patients received 75 mg of indomethacin daily or 600 mg of fenbufen daily for six weeks and then were treated with the alternate regimen for a similar period. The drugs were equally efficacious. The erythrocyte sedimentation rate of patients who had received fenbufen was significantly lower than that of patients who had received indomethacin. More side effects were reported by the indomethacin-treated patients. A further 24-week open evaluation of fenbufen in rheumatoid patients indicated that fenbufen is a useful anti-inflammatory analgesic in the long-term management of mild and moderate rheumatoid arthritis.

Cerebrospinal fluid concentrations of the complement MAC inhibitor CD59 in multiple sclerosis and patients with other neurological disorders.

Rodent oligodendrocytes have a unique susceptibility among glia to the lytic effects of complement, due in part to a deficiency in CD59 (protectin), a key surface inhibitor of the complement membrane attack complex (MAC). The possibility that shedding of CD59 by human oligodendrocytes contributes to complement-mediated oligodendrocyte injury in inflammatory demyelinating disease has been investigated by estimating levels of CD59 in cerebrospinal fluid samples from 12 patients with demyelinating disease of the central nervous system and 13 with other neurological diseases. No significant differences were found between patients and controls, or between patients with active and those with clinically inactive demyelinating disease, providing no direct support for oligodendrocyte shedding of CD59 in multiple sclerosis.

Extracorporeal shock wave therapy for plantar fasciitis. A double blind randomised controlled trial.

BACKGROUND: Extracorporeal shock wave therapy (ESWT) is an increasingly popular therapeutic approach in the management of a number of tendinopathies. Benefit has been shown in calcific tendinitis of the rotator cuff, but evidence for its use in non-calcific disorders is limited. AIMS: To perform a double blind randomised controlled trial of moderate dose shock wave therapy in plantar fasciitis. METHODS: Adults with plantar fasciitis for at least 3 months were randomised to receive either active treatment (0.12 mJ/mm(2)) or sham therapy, monthly for 3 months. Pain in the day, nocturnal pain and morning start-up pain were assessed at baseline, before each treatment and 1 and 3 months after completion of therapy. RESULTS: Eighty-eight subjects participated and no differences existed between the groups at baseline. At 3 months, 37% of the subjects in the ESWT group and 24% in the sham group showed a positive response (50% improvement from baseline) with respect to pain. Positive responses in night pain occurred in 41% and 31% in the ESWT and sham groups, respectively. Positive responses in start-up pain occurred in 37% and 36% in the ESWT and sham groups, respectively. Both groups showed significant improvement over the course of the study, but no statistically significant difference existed between the groups with respect to the changes were seen in any of the outcome measures over the 6-month period. CONCLUSIONS: There appears to be no treatment effect of moderate dose ESWT in subjects with plantar fasciitis. Efficacy may be highly dependent upon machine types and treatment protocols. Further research is needed to develop evidence based recommendation for the use ESWT in musculoskeletal complaints.

Extracorporeal shock wave therapy for lateral epicondylitis--a double blind randomised controlled trial.

UNLABELLED: Extracorporeal shock wave therapy (ESWT) is an increasingly popular therapeutic approach to the treatment of a number of soft tissue complaints. Whilst benefit has been demonstrated in calcific tendinitis, evidence is lacking for benefit in the management of non-calcific rotator cuff disorders. AIMS: To perform a double-blind placebo controlled trial of moderate dose ESWT in chronic lateral epicondylitis. METHODS: Adults with lateral epicondylitis were randomised to receive either active treatment (1500 pulses ESWT at 0.12 mJ/ mm2) or sham therapy, monthly for three months. All were assessed before each treatment and one month after completion of therapy. Outcome measures consisted of visual analogue scores for pain in the day and at night. RESULTS: Seventy-five subjects participated and there were no significant differences between the two groups at baseline. The mean duration of symptoms was 15.9 and 12 months in the ESWT and sham groups, respectively. Both groups showed significant improvements from two months. No significant difference existed between the groups with respect to the degrees of change in pain scores over the study period. In the ESWT group the mean (SD, range) pain score was 73.4 (14.5, 38-99) at baseline and 47.9 (31.4, 3-100) at three months. In the sham group the mean (SD, range) pain score was 67.2 (21.7, 12-100) at baseline and 51.5 (32.5, 3-100) at three months. At three months, 50% improvement from baseline was noted in 35% of the ESWT group and 34% of the sham group with respect to pain. CONCLUSIONS: There appears to be a significant placebo effect of moderate dose ESWT in subjects with lateral epicondylitis but there is no evidence of added benefit of treatment when compared to sham therapy.

Acemetacin in the long-term therapy of rheumatoid arthritis.

An open label study was carried out to assess the long-term use of acemetacin in patients suffering from rheumatoid arthritis. One hundred and two patients were treated with acemetacin, some patients for up to a 12-month period. Analysis of efficacy showed that acemetacin therapy was associated with statistically significant sustained improvements in the target variables of duration of morning stiffness, Ritchie articular index and left and right grip strength. With regard to tolerability, a number of adverse events were recorded but were generally reported as either mild or moderate. In total, 28,988 patient days' treatment with acemetacin were completed and acemetacin proved an effective and well-tolerated long-term therapy in patients with rheumatoid arthritis.

Alclofenac in ankylosing spondylitis.

Eighteen patients with definite ankylosing spondylitis were treated with 3 to 4 g. alclofenac daily for up to 8 months in an open trial against previous therapy. Therapeutic efficacy was greater than or equivalent to previous therapy in 10 of 18 patients after 1 month and 8 patients remain on alclofenac. There was a significant increase in symptoms, particularly morning stiffness after 1 month, but they tended to improve with time. Measurement of spinal movements was not found useful. Side-effects were commoner than expected but not serious. Alclofenac may prove a useful alternative drug in the management of ankylosing spondylitis and further trials are indicated.

A comparative study of the long-term efficacy of flurbiprofen and indomethacin in the treatment of rheumatoid arthritis, with special reference to iron metabolism.

Preliminary results are reported for the first 23 rheumatoid arthritis patients entered in a long-term, double-blind trial to compare the efficacy of flurbiprofen and indomethacin. It was planned that, unless withdrawn, patients from matched pairs received either flurbiprofen (150 mg to 300 mg daily) or indomethacin (75 mg to 150 mg daily) over a minimum period of 6 months, dosage being adjusted to suit exacerbations and remission of disease. In addition to clinical assessments of severity of pain, duration and severity of morning stiffness, joint size and joint score, routine laboratory measurements were carried out, including estimates of serum iron and total iron binding capacity, rheumatoid factor and immunoglobulin levels. This interim report gives the statistical analysis of results from the 17 patients completing from 2 to 4 months of treatment and shows that both drugs were equally effective in controlling disease activity. Withdrawals due to side-effects or exacerbations of disease were similar for both drugs.

Long-term evaluation of naproxen suspension in juvenile chronic arthritis.

Twenty-six children suffering from juvenile chronic arthritis were entered into a 6-month open evaluation of naproxen suspension. Sixteen patients completed 6-months' treatment, 6 were lost to follow-up and 4 dropped out, 2 because of side-effects (rash, and burning on swallowing the formulation), 1 for lack of efficacy and 1 because of no further need of treatment. Pain severity scores were significantly reduced from admission levels at all monthly follow-up visits and significant overall improvement since the last visit was noted up to third month of treatment, as assessed by doctor and parent/patient. Transient indigestion was reported by 2 patients. No clinically significant trends were noted in any of the laboratory investigations carried out. The results add to the accumulation of data on the use of naproxen in children and underline its long-term efficacy and tolerance.