Evaluation of serum MMP-9 as predictive biomarker for antisense therapy in Duchenne.

Duchenne Muscular Dystrophy (DMD) is a severe muscle disorder caused by lack of dystrophin. Predictive biomarkers able to anticipate response to the therapeutic treatments aiming at dystrophin re-expression are lacking. The objective of this study is to investigate Matrix Metalloproteinase-9 (MMP-9) as predictive biomarker for Duchenne. Two natural history cohorts were studied including 168 longitudinal samples belonging to 66 patients. We further studied 1536 samples obtained from 3 independent clinical trials with drisapersen, an antisense oligonucleotide targeting exon 51: an open label study including 12 patients; a phase 3 randomized, double blind, placebo controlled study involving 186 patients; an open label extension study performed after the phase 3. Analysis of natural history cohorts showed elevated MMP-9 levels in patients and a significant increase over time in longitudinal samples. MMP-9 decreased in parallel to clinical stabilization in the 12 patients involved in the open label study. The phase 3 study and subsequent extension study clarified that the decrease in MMP-9 levels was not predictive of treatment response. These data do not support the inclusion of serum MMP-9 as predictive biomarker for DMD patients.

Fast Calibration of Haptic Texture Synthesis Algorithms.

Calibrating displays can be a time-consuming process. We describe a fast technique for adjusting the subjective experience of roughness produced by different haptic texture synthesis algorithms. Its efficiency is due to the exponential convergence of the ldquomodified binary search methodrdquo (mobs) applied to find points of subjective equivalence between virtual haptic textures synthesized by different algorithms. The method was applied to find the values of the coefficient of friction in a friction-based texture algorithm that yield the same perception of roughness as the normal-force variations of conventional texture synthesis algorithms. Our main result is a table giving the perceptual equivalence between parameters having different physical dimensions. A similar method could be applied to other perceptual dimensions provided that the controlling parameter be monotonically related to a subjective estimate.

Synthesis and polymerization of benzyl (3R,4R)-3-methylmalolactonate via enzymatic preparation of the chiral precursor.

beta-methylaspartate ammonia-lyase, EC 4.3.1.2, (beta-methylaspartase) from Clostridium tetanomorphum was used to produce a 40/60 molar ratio of (2S,3R) and (2S,3S)-3-methylaspartic acids, 2a and 2b, respectively, from mesaconic acid 1 as substrate, on a large scale. To prepare (3R,4R)-3-methyl-4-(benzyloxycarbonyl)-2-oxetanone (benzyl 3-methylmalolactonate) 6, 2a and 2b were transformed, in the first step, into 2-bromo-3-methylsuccinic acids 3a and 3b and separated. After three further steps, (2S,3S)-3a yielded the alpha, beta-substituted beta-lactone (3R,4R) 6 with a very high diastereoisomeric excess (> 95% by chiral gas chromatography). The corresponding crystalline polymer, poly[benzyl beta-(2R,3S)-3-methylmalate] 8, prepared by an anionic ring opening polymerization, was highly isotactic as determined by 13C NMR. Catalytic hydrogenolysis of lactone 6 yielded (3R,4R)-3-methyl-4-carboxy-2-oxetanone (3-methylmalolactonic acid) 7, to which reactive, chiral, or bioactive molecules can be attached through ester bonds leading to polymers with possible therapeutic applications. Because of the ability of beta-methylaspartase to catalyse both syn- and anti-elimination of ammonia from (2S,3RS)-3-methylaspartic acid 2ab at different rates, the (2S,3R)-stereoisomer 2a was retained and isolated for further reactions. These results permit the use of the chemoenzymatic route for the preparation of both optically active and racemic polymers of 3-methylmalic acid with well-defined enantiomeric and diastereoisomeric compositions.

Contrast enhanced Gd-DTPA magnetic resonance imaging in the evaluation of rheumatoid arthritis during a clinical trial with DMARDs. A prospective two-year follow-up study on hand joints in 31 patients.

OBJECTIVE: The aim of this prospective 24-month follow-up study was to compare clinical features with radiological and magnetic resonance imaging (MRI) findings in evaluating synovial proliferation in the hand joints of 31 patients with rheumatoid arthritis (RA). A single joint was used for the follow-up of each patient. METHODS: Thirty-one small hand joints were examined by conventional radiography and MRI before and after 24 months of treatment. MRI assessment of disease progression (volume and/or signal intensity of the synovial proliferation on T1 weighted precontrast, T1 weighted postcontrast and T2 weighted images) was compared with a clinical assessment of the chosen joints, and with a plain x-ray film evaluation (Larsen's score). RESULTS: Of 26 joints which clinically improved (14 markedly and 14 slightly) during the study, on MRI 16 showed improvement, 8 showed no change, and 2 showed deterioration. Four clinically unchanged joints appeared improved on MRI. One joint deteriorated clinically and on MRI. Overall, there was a 58% congruence between clinical and MRI findings. On x-ray 23 joints showed no change; nine of these were also unchanged on MRI, while 13 showed improvement and one deterioration. Only in 2 out of 8 joints showing deterioration on x-ray were the MRI findings in accordance. In the remaining six joints MRI showed improvement. The congruence between x-ray and MRI was therefore 36%. CONCLUSION: The long-term follow-up of rheumatoid synovial proliferation of the small joints in the hand using contrast enhanced MRI is feasible and may provide additional information regarding disease activity. Important advantages over conventional radiography methods are its ability to demonstrate qualitative differences of synovial proliferation within bone erosions, and demonstrate not only deterioration, but also the improvement of inflammatory disease.

Prognostic value of contrast enhanced Gd-DTPA MRI for development of bone erosive changes in rheumatoid arthritis.

Conventional radiograms have been used to quantitate the progression of rheumatoid arthritis, mainly through the assessment of bone erosions, but this approach has many limitations. It has been suggested that an advantage of contrast-enhanced Gd-DTPA MRI over radiography may be its prognostic value due to its ability to show the natural history of active destructive to inactive fibrous pannus. The aim of this study was to evaluate the possible prognostic value of MRI for future development of bone erosive changes in small hand joints in patients with RA. The results of the study confirm that in joints in which inflammatory active pannus is shown by contrast-enhanced MRI, progression of bone-destructive changes can be expected.

Dose-range and dose-frequency study of recombinant human interleukin-1 receptor antagonist in patients with rheumatoid arthritis. The IL-1Ra Arthritis Study Group.

OBJECTIVE: To preliminarily evaluate the safety and efficacy of different dose levels and dosing frequencies of recombinant human interleukin-1 receptor antagonist (rHuIL-1Ra) in the treatment of patients with rheumatoid arthritis (RA). METHODS: One hundred seventy-five patients with active RA were enrolled in a randomized, double-blind trial of rHuIL-1Ra administered by subcutaneous injection. There were 9 treatment groups in the trial. During the initial 3-week treatment phase, patients were treated with 20, 70, or 200 mg rHuIL-1Ra, administered either once, 3 times, or 7 times per week, followed by a 4-week maintenance phase, during which all patients received the treatment-phase dose once per week. To maintain the blindness of the study, patients received daily injections of either rHulL-1Ra or placebo on the days rHuIL-1Ra was not administered. RESULTS: Recombinant HuIL-1Ra was well tolerated. The most frequent adverse event was injection-site reactions, which were reported in 62% of patients and caused 8 patients (5%) to withdraw prematurely from the study. Five patients (3%) developed serious adverse reactions unrelated to dose or dosing frequency. Due to the lack of a placebo arm and to the multiple small treatment groups, a definitive statement regarding efficacy could not be made. However, by the end of the 3-week treatment phase, daily dosing appeared more effective than weekly dosing when assessed by the number of swollen joints, the investigator and patient assessments of disease activity, pain score, and C-reactive protein levels. CONCLUSION: These preliminary data suggest that rHuIL-1Ra may be safely administered by subcutaneous injection to RA patients. The frequency of dosing appears to be important in determining clinical response, with daily administration providing the most benefit. A placebo-controlled trial is in progress to further assess the clinical usefulness and to better define appropriate doses of rHuIL-1Ra in patients with RA.

Safety and effectiveness of leflunomide in the treatment of patients with active rheumatoid arthritis. Results of a randomized, placebo-controlled, phase II study.

OBJECTIVE: To assess the safety and effectiveness of leflunomide versus placebo in patients with active rheumatoid arthritis (RA) treated for 6 months. METHODS: Four hundred two patients were randomly assigned to receive placebo or leflunomide at 5 mg, 10 mg, or 25 mg daily. A washout period of 6-12 weeks from prior second-line therapy was required. RESULTS: Statistically significant improvement in primary and secondary outcome measures, as well as by responder analyses, occurred in the 10-mg and 25-mg dosage groups compared to placebo. Twenty-one patients (7.0%) in the active treatment groups withdrew due to adverse events (AEs). The incidence of AEs was higher with leflunomide than with placebo. Gastrointestinal symptoms, weight loss, allergic reactions, skin rash, and reversible alopecia were more common in the 10-mg and 25-mg dosage groups. The incidence of infections was similar between the treatment and placebo groups; no opportunistic infections were seen. Transient elevations in liver function studies were noted in a small number of patients. CONCLUSION: Leflunomide is effective in daily doses of 10 mg and 25 mg in patients with active RA. Improved efficacy at the 25-mg dose was associated with a higher incidence of AEs. Randomized, placebo-controlled trials using daily doses of 10 mg and 20 mg are under way in the US and Europe to confirm these positive results.

Hip fractures in the elderly: a world-wide projection.

Hip fractures are recognized to be a major public health problem in many Western nations, most notably those in North America, Europe and Oceania. Incidence rates for hip fracture in other parts of the world are generally lower than those reported for these predominantly Caucasian populations, and this has led to the belief that osteoporosis represents less of a problem to the nations of Asia, South American and Africa. Demographic changes in the next 60 years, however, will lead to huge increases in the elderly populations of those countries. We have applied available incidence rates for hip fracture from various parts of the world to projected populations in 1990, 2025 and 2050 in order to estimate the numbers of hip fractures which might occur in each of the major continental regions. The projections indicate that the number of hip fractures occurring in the world each year will rise from 1.66 million in 1990 to 6.26 million by 2050. While Europe and North America account for about half of all hip fractures among elderly people today, this proportion will fall to around one quarter in 2050, by which time steep increases will be observed throughout Asia and Latin America. The results suggest that osteoporosis will truly become a global problem over the next half century, and that preventive strategies will be required in parts of the world where they are not currently felt to be necessary.

Metabolism and characterisation of kinins and Hoe 140 (kinin antagonist) in the synovial fluid of patients with inflammatory joint diseases.

Methods have been optimised for the collection of synovial fluid and the chromatographic separation of individual kinins (bradykinin and kallidin) in the fluid by HPLC. In addition, the stability of the kinin antagonist, Hoe 140, in synovial fluid was compared with that of synthetic bradykinin. Although bradykinin was completely degraded after incubation for only 6 h in pooled synovial fluid obtained from patients with rheumatoid arthritis, Hoe 140 was stable for as long as 2 weeks under the same conditions. These studies will provide quantitative information regarding levels of kinins in inflamed joints and an insight into the therapeutic potential of kinin antagonists.

Levels of keratan sulfate in the serum and synovial fluid of patients with osteoarthritis of the knee.

We examined the relationship between serum and synovial fluid (SF) levels of antigenic keratan sulfate (KS) and the clinical, laboratory, and radiologic features of disease in 125 well-characterized patients with knee osteoarthritis (OA). KS was quantified by enzyme-linked immunosorbent assay, using an antibody specific for a highly sulfated epitope on KS chains; the results were calculated as equivalents of an international standard of KS from human costal cartilage. The mean level of serum KS (393 ng/ml) was significantly higher than those previously reported for populations of adults without OA. There was a wide scatter of serum KS values (range 156-912 ng/ml), with little correlation with clinical or radiologic features. Men had significantly higher levels than women (456 +/- 135 ng/ml versus 368 +/- 110 ng/ml, mean +/- SD), and there was a statistically significant but weak association with indicators of polyarticular involvement (number of symptomatic joints, Heberden's nodes, hip symptoms) in women. Despite the wide scatter of results in the population as a whole, individual levels of KS were stable for up to 4 consecutive years in the 9 patients studied. Levels of KS were much higher in SF (n = 25) than in serum, but the two were not correlated. There was an inverse correlation between radiographic evidence of cartilage loss and the level of KS in SF. The large variations in serum KS values suggest that this measure may not be of diagnostic significance among populations of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of synovial fluid and serum on the growth of calcium hydroxyapatite crystals.

The presence or absence of natural crystal growth inhibitors in joint tissues and fluids may be important in the pathogenesis of several arthropathies. Synovial fluid (SF) has therefore been examined for inhibitors of seeded hydroxyapatite growth rate (Vo) using a pH stat system. Pretreatment of seed crystals with SF reduced growth (Vo = 56 +/- 5, control 117 +/- 141 mol/base/min/g hydroxyapatite, p less than 0.001). Addition of small amounts of serum or SF to the growth medium caused a dose dependent growth inhibition (0.04% SF reduced Vo by 16%). Pretreatment with protease, but not hyaluronidase, abolished this activity and gel filtration localized it to the 55-80 kDa fraction. A macromolecular factor(s) with potent inhibitory activity has therefore been detected.

The Felty syndrome: a case-matched study of clinical manifestations and outcome, serologic features, and immunogenetic associations.

Thirty-two patients with the Felty syndrome, defined by the presence of rheumatoid arthritis, splenomegaly, and neutropenia, have been studied in comparison with 32 patients with rheumatoid arthritis matched for age, sex, and disease duration, and 9 patients with rheumatoid arthritis and idiopathic neutropenia. Patients with the Felty syndrome had severe destructive arthritis, which progressed during follow-up despite little evidence of objective synovitis, and a higher frequency of extra-articular manifestations, including vasculitis. Bacterial infection tended to occur in patients with the lowest neutrophil count but continued to occur in some despite normalization of the WBC. Prognosis was poor and 8 deaths occurred, predominantly from sepsis. Serologic features were prominent. High titers of IgG rheumatoid factor and circulating immune complexes characterized patients with persistent neutropenia. A family history of rheumatoid arthritis was more common in patients with the Felty syndrome. The association with HLA DR4 was very strong; in addition there was an increased frequency of the DQw3 variant, 3b, suggesting that HLA Class II genes in linkage with DR4 may contribute to disease expression.

Serum and synovial fluid osteocalcin (bone gla protein) levels in joint disease.

Osteocalcin (bone gla protein) is a sensitive marker of bone turnover in metabolic disease. Using a well characterized antiserum (R 102M) we have assayed serum and synovial fluid samples from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) and related levels to serological and radiological markers of disease. There were 21 patients with RA (mean age 58.2 years, 15 F) and 33 with OA (mean age 69.2, 28 F). Paired serum and synovial fluids (SF) were available in 19 RA patients and 30 OA patients. Serum osteocalcin levels were related to age-/sex-matched normals and to a small group of elderly disease controls. Serum levels tended to be lower in RA than controls, but not significantly so: RA 5.56 (3.67); control 6.09 (2.54) ng/ml; expressed as mean (SD) and the mean serum/SF ratio was 0.88 (0.86). The results were much more variable in OA (mean serum osteocalcin 6.1 (3.9]. Elevated levels were mainly due to a small number of patients with a destructive form of OA and were higher than those with non-destructive OA (10.3 (3.5), n = 20, versus 3.83 (1.6), n = 10). Patients with non-destructive OA had a lower serum osteocalcin than age-/sex-matched normals. In this study, synovial fluid levels were usually less than serum concentrations, but in two RA and four OA patients the ratio was reversed, suggesting local production. Osteocalcin may be an important marker of bone activity in OA.

Clinical manifestations of human parvovirus B19 in adults.

Human parvovirus B19 has been associated with various clinical effects in a number of uncontrolled reports. To define the usual manifestations of B19 infection in adults and the factors that influence them we present a clinicoepidemiological study of an outbreak of B19 infection centered on a junior school. Four hundred fifty-three of 475 adults in this community were interviewed and blood was obtained for serological diagnosis. Fifty-four cases of recent infection were identified and were HLA typed. Fourteen of the cases were asymptomatic; 32 had an influenzalike illness; 23 a rash; and 26 an acute-onset polyarthropathy that was more common in women and lasted for up to 7 months. HLA-A, -B, and -C antigen frequencies were similar to a local control population and showed no association with symptoms except that HLA-DR1 was absent in those with persistent arthropathy.

Mixed crystal deposition.

Mixtures of crystals are found in joint tissues more commonly than would be expected by chance. In osteoarthritic joints, for example, mixtures of different calcium salts are more common than any one type of crystal alone. This is partly explained by the predisposing factors and mechanisms of crystal formation, many of which are not salt specific. It is suggested that rheumatic disorders should not be diagnosed or classified by crystal type.

Synovial fluid collagenase in patients with destructive arthritis of the shoulder joint.

We studied synovial fluid (SF) collagenase in 10 women with severe rheumatoid arthritis (RA), 10 with pyrophosphate arthropathy, and 10 with idiopathic destructive disease of the shoulder conforming to a pattern recently described. SF cell counts were highest in the RA group. Particles were detected by polarized light microscopy and alizarin red staining. Crystals were seen in fluids from all 3 groups; pyrophosphate predominated in the pyrophosphate arthropathy group and alizarin red-positive particles in the idiopathic disease group. Collagenase and tissue inhibitor of metalloproteinase levels were estimated in SF after gel filtration. Tissue inhibitor of metalloproteinase activity was detected in all fluids, but tended to be highest in the RA group. Collagenase activity was detected in 3 RA fluids only. In no sample was collagenase found in an active form. These findings support the clinical concept of an aggressive destructive process which sometimes occurs in the shoulder joints of elderly women. Because we were not able to detect free collagenase in SF from any of the patients with idiopathic shoulder disease, the data suggest that high levels of active collagenase are not characteristic of this group of patients.

Idiopathic destructive arthritis of the shoulder.

IDA of the shoulder is a condition found predominantly in elderly females. Although the shoulder is primarily involved, other joints such as the hip and knee can be affected, and concurrent OA is common at other joint sites. Clinical features include voluminous, often blood-stained effusions, together with features of rotator cuff rupture and restriction of shoulder movement. Laboratory parameters are usually normal and examination of the synovial fluid reveals large amounts of basic calcium phosphate crystals. The synovium is hypertrophied and vascular and shows fibrin deposition. It contains calcified material extracellularly. An acute inflammatory infiltrate is absent. Radiographs demonstrate soft tissue swelling and subchondral sclerosis with marked bony attrition involving the acromioclavicular and glenohumeral joints, as well as the humeral head and neck. Although some aspects of the disease seem distinct, many features are shared with other types of destructive arthritis of the shoulder. The pathogenesis of this disorder is at present obscure, but it is clear that an understanding of the processes involved will provide a vital contribution to our understanding of the response of the joint to insult. With a multidisciplinary approach and adequate communication between interested workers this aim could seen be within our grasp.