Efficacy and safety of up to 192 weeks of etanercept therapy in patients with ankylosing spondylitis.

OBJECTIVE: Evaluate long-term safety and efficacy of etanercept treatment in patients with ankylosing spondylitis (AS). METHODS: Patients with AS who previously participated in a randomised controlled trial (RCT) of etanercept were eligible to enroll in a 168-week open-label extension (OLE). Safety end points included rates of adverse events (AE), serious adverse events (SAE), infections, serious infections and death. Efficacy end points included Assessment in Ankylosing Spondylitis (ASAS20) response, ASAS 5/6 response and partial remission rates. RESULTS: A total of 257 of 277 patients (92%) enrolled in the OLE. After up to 192 weeks of treatment with etanercept, the most common AEs were injection site reactions, headaches and diarrhoea. The exposure-adjusted rate of SAEs was 0.08 per patient-year. The rate of infections was 1.1 per patient-year, and the rate for serious infections was 0.02 per patient-year. No deaths were reported. Of patients who received etanercept in both the RCT and OLE and were still in the trial, 71% were ASAS20 responders at week 96, and 81% were responders at week 192. ASAS 5/6 response rates were 61% at week 96 and 60% at week 144, and partial remission response rates were 41% at week 96 and 44% at week 192. Placebo patients who switched to etanercept in the OLE showed similar patterns of efficacy maintenance. CONCLUSIONS: Etanercept was well tolerated for up to 192 weeks in patients with AS, with no unexpected AEs or SAEs observed. No deaths were reported. Improvements in the signs and symptoms of AS were maintained for up to 192 weeks.

Psoriatic arthritis: epidemiology, clinical features, course, and outcome.

Psoriatic arthritis (PsA) has been defined as a unique inflammatory arthritis associated with psoriasis. Its exact prevalence is unknown, but estimates vary from 0.3% to 1% of the population. The clinical features described initially are recognised by most experienced clinicians, although they are most distinct in early disease. Initially, PsA typically presents as an oligoarticular and mild disease. However, with time PsA becomes polyarticular, and it is a severe disease in at least 20% of patients. Patients with PsA who present with polyarticular disease are at risk for disease progression. In addition to progression of clinical and radiological damage, health related quality of life is reduced among patients with PsA. It important to note that patients included in recent drug trials resemble patients followed prospectively in a clinic.

Psoriatic arthritis therapy: NSAIDs and traditional DMARDs.

Non-steroidal anti-inflammatory drugs (NSAIDs) and traditional disease modifying antirheumatic drugs (DMARDs) are widely used in the treatment of psoriatic arthritis (PsA), but this is based more upon clinical experience than adequate evidence from clinical trials. This report summarises the results from available trials highlighting evidence of efficacy and deficiencies with respect to effect on joints and to a lesser degree cutaneous disease. The available published data on efficacy of NSAIDs, glucocorticoids, antimalarials, sulfasalazine, gold, methotrexate, azathioprine, and ciclosporin are detailed, as well as new data on leflunomide and other novel agents. The conclusions of this review are that evidence supports marginal efficacy of sulfasalazine and perhaps gold in the treatment of peripheral psoriatic arthropathy, and methotrexate and ciclosporin are effective for treating the skin disease although evidence for improvement of the arthropathy is empirical at best. New trials with standardised and validated outcome measures are required to better assess efficacy. Evaluating newer agents, against and in combination with traditional DMARDS, may further clarify the latter's role in the future management of this condition.

Comparison of sulfasalazine and placebo for the treatment of axial and peripheral articular manifestations of the seronegative spondylarthropathies: a Department of Veterans Affairs cooperative study.

OBJECTIVE: To determine if the peripheral articular manifestations of the seronegative spondylarthropathies (SNSA) respond differently than the axial manifestations to treatment with sulfasalazine (SSZ). METHODS: This is a reanalysis of a previously reported series of randomized, double-blind, placebo-controlled, multicenter trials comparing the effects of SSZ, 2,000 mg/day, and placebo on the axial and peripheral articular manifestations of ankylosing spondylitis (AS), psoriatic arthritis (PsA), and reactive arthritis (ReA; Reiter's syndrome). Patients were classified as treatment responders on the basis of meeting predefined improvement criteria in 4 outcome measures: namely, patient and physician global assessments in all patients, morning stiffness and back pain in patients with axial manifestations, and joint pain/tenderness scores and joint swelling scores in patients with peripheral articular manifestations. RESULTS: Six hundred nineteen SNSA patients (264 AS, 221 PsA, and 134 ReA) were studied. One hundred eighty-seven of these patients had only axial manifestations of their disease, while 432 patients had peripheral articular manifestations. Of the patients with axial disease, 40.2% of the SSZ group and 43.3% of the placebo group met the predefined response criteria (P = 0.67). Of the peripheral articular group, 59.0% of the SSZ-treated patients and 42.7% of the placebo-treated patients showed a response (P = 0.0007). CONCLUSION: In a large group of affected individuals, the response of SNSA patients to SSZ appears to be related to the articular manifestations of their disease. These data demonstrate that the axial and peripheral articular manifestations of SNSA respond differently to treatment with SSZ. In SNSA patients with persistently active peripheral arthritis, SSZ is safe, well tolerated, and effective.

Baseline relationships between psoriasis and psoriatic arthritis: analysis of 221 patients with active psoriatic arthritis. Department of Veterans Affairs Cooperative Study Group on Seronegative Spondyloarthropathies.

OBJECTIVE: To determine differences in disease onset, extent, and manifestations of psoriasis among patients with active, inflammatory psoriatic arthritis (PsA), and to examine relationships that may exist between psoriasis and PsA. METHODS: Baseline demographic, clinical, and laboratory data were analyzed from 221 patients enrolled in a multicenter cooperative study, and relationships between measures of psoriasis and PsA were determined. RESULTS: Mean percentage of body surface area (BSA) affected by psoriasis was modest (12+/-17), and mean severity of erythema, induration, and scaling was moderate (4.9+/-2.1 on a 0-9 scale). Spanish Americans tended to have a higher mean percentage of BSA (18.5%) than Caucasians (11%; p = 0.067), as well as higher target lesion severity (5.55 vs. 4.84; p = 0.077). Patients with psoriatic nail disease (180/221, 81%) had significantly greater number of involved distal interphalangeal (DIP) joints (p = 0.004). There were no other significant associations of skin pattern or regional involvement with PsA. CONCLUSION: Patients with active PsA have generally mild skin disease, and baseline relationships between psoriasis and PsA tend to be weak except for nail involvement and DIP joint activity.

Modelling the major histocompatibility complex susceptibility to RA using the MASC method.

To explain the association between HLA-DRB1 gene and rheumatoid arthritis (RA), two main hypotheses have been proposed. The first, the shared epitope hypothesis, assumes a direct role of DRB1 in RA susceptibility. The second hypothesis assumes a recessive disease susceptibility gene in linkage disequilibrium with DRB1. To investigate these two hypotheses, we analysed data on the HLA-DRB1 and TNF-LT loci in 49 affected sib-pairs. We used the Marker Association Segregation Chi-square (MASC) method in which the genotype distribution of markers among index cases and the haplotype sharing in affected sib-pairs are jointly taken into account. With DRB1 data alone, both hypotheses were shown to fit but with analysis of TNF data, both hypotheses were strongly rejected. Thus the TNF data provided additional information for a better understanding of genetic susceptibility to RA than was previously possible using only HLA-DR data. A theoretical standpoint is addressed here on the advisability of using different linked markers in a candidate region for modelling the contribution of this region in disease susceptibility.

Structures and dynamic motion of laminin-1 as observed by atomic force microscopy.

Laminins are a family of multifunctional extracellular matrix glycoproteins that play important roles in the development and maintenance of tissue organization via their interactions with cells and other extracellular matrix proteins. To understand the structural basis of laminins' functions, we examined the motion of laminin-1 (Ln-1) in physiological buffers using atomic force microscopy. While many Ln-1 molecules assumed the expected cruciform structure, unexpected dynamic movements of the Ln-1 arms were observed in aqueous environments. These dynamic movements of the Ln-1 arms may contribute to the diversity of laminin functions.

Radiographic results from the Minocycline in Rheumatoid Arthritis (MIRA) Trial.

OBJECTIVE: To assess radiographically determined disease progression in patients in the Minocycline in Rheumatoid Arthritis (MIRA) Trial. METHODS: A double blind, randomized, multicenter, 48 week trial of oral minocycline (200 mg/day) or placebo in 6 clinical centers in the United States. Patients include 219 adults with active RA previously receiving limited treatment with disease modifying drugs. Posteroanterior films of the hands from baseline and final visits, blinded for sequence, were read for erosions and joint space narrowing by trained observers. Outcomes included rate of disease progression (change/month) and percentage of patients with progression from baseline, newly involved joints, and newly erosive disease. RESULTS: Using intent-to-treat analyses, progression rates for erosions (0.11 +/- 0.42 minocycline, 0.17 +/- 0.41 placebo; p = 0.47) and joint space narrowing (0.16 +/- 0.55 minocycline and 0.23 +/- 0.71 placebo; p = 0.14) were similar. (Power 43% to detect a 50% difference.) Newly erosive joints occurred more frequently in the placebo group (44 vs 32%; p = 0.08), not a statistically significant difference. CONCLUSION: Radiographic measurement of disease progression using 4 measures failed to show a significant difference between minocycline and placebo treatment, although for all methods there was a trend toward treatment benefit, consistent with reported clinical results. A one year trial duration, high measurement variability, and slow rate of radiographic progression in this cohort may explain the low power to detect a treatment effect. The measurement that denoted "newly involved" joints was most sensitive in detecting change. In future trials longer term assessment (minimum 2 years) of radiographic changes and further comparison of measures of disease progression are warranted.

Comparison of sulfasalazine and placebo in the treatment of psoriatic arthritis. A Department of Veterans Affairs Cooperative Study.

OBJECTIVE: To determine whether sulfasalazine (SSZ) at a dosage of 2,000 mg/day is effective for the treatment of active psoriatic arthritis (PsA) resistant to nonsteroidal antiinflammatory drug therapy. METHODS: Two hundred twenty-one patients with PsA were recruited from 15 clinics, randomized (double-blind) to SSZ or placebo treatment, and followed up for 36 weeks. Treatment response was based on joint pain/ tenderness and swelling scores and physician and patient global assessments. RESULTS: Longitudinal analysis revealed a trend favoring SSZ treatment (P = 0.13). At the end of treatment, response rates were 57.8% for SSZ compared with 44.6% for placebo (P = 0.05). The Westergren erythrocyte sedimentation rate declined more in the PsA patients taking SSZ than in those taking placebo (P < 0.0001). Adverse reactions were fewer than expected and were mainly due to nonspecific gastrointestinal complaints, including dyspepsia, nausea, vomiting, and diarrhea. CONCLUSION: SSZ at a dosage of 2,000 mg/day is well tolerated and may be more effective than placebo in the treatment of patients with PsA.

Genetic variability in the tumor necrosis factor-lymphotoxin region influences susceptibility to rheumatoid arthritis.

The major histocompatibility complex class III tumor necrosis factor-lymphotoxin (TNF-LT) region (6p21.3) was investigated as a possible susceptibility locus for rheumatoid arthritis (RA). Inheritance of five TNF microsatellite markers was determined in 50 multiplex families. Overall, 47 different haplotypes were observed. One of these, the TNF a6, b5, c1, d3, e3 (H1) haplotype, was present in 35.3% of affected, but in only 20.5% of unaffected, individuals (P < .005). This haplotype accounted for 21.5% of the parental haplotypes transmitted to affected offspring and only 7.3% not transmitted to affected offspring (P = .0003). The TNF a6 and TNF c1 alleles were individually associated with RA (P = .0005 and .0008, respectively), as were the HLA-DRB1 "shared epitope" (SE) (P = .0001) and HLA-DRB1*0401 (P = .0018). Both univariate and bivariate conditional logistic regression analysis showed significant effects of TNF c1 and SE in increasing risk to RA (P < .001). Stratification by the presence of SE indicated an independent effect of the TNFc1 allele (P = .0003) and the HLA A1, B8, DR3 extended haplotype (always TNFa2, b3, c1, d1, e3) (P = .0027) in SE heterozygotes, while the H1 haplotype was associated with RA in SE homozygotes (P = .0018). The TNF-LT region appears to influence susceptibility to RA, distinct from HLA-DR.

Human SY5Y neuroblastoma cell interactions with laminin isoforms: neurite outgrowth on laminin-5 is mediated by integrin alpha 3 beta 1.

Laminin (Ln) isoforms may play important roles in neuronal development, particularly axon guidance, but neural receptors mediating interactions with Ln are not entirely understood. In this paper, we have compared the adhesive and process outgrowth activities of a human neuroblastoma cell line SY5Y on various laminin isoforms. Cell adhesion and process outgrowth were examined on murine Ln-1 (Englebreth-Holm-Swarm sarcoma laminin), human placental Ln-1 (human Ln-1[p]), human Ln-2 (merosin), human Ln-5 (kalinin/epiligrin/nicein), and human foreskin keratinocyte extracellular matrix extract (human HFK-ECM). Ln-5 was shown to evoke process outgrowth in amounts comparable to other Ln isoforms. Antibody perturbation experiments showed that adhesion and process outgrowth on murine Ln-1 was primarily mediated by the integrin alpha 1 beta 1, whereas adhesion and outgrowth on human Ln-5 and human HFK-ECM were mediated by alpha 3 beta 1. Adhesion to human Ln-1(p) and Ln-2 was not blocked by addition of anti-alpha 1 or anti-alpha 3 antibodies alone, but adhesion was partially perturbed when these antibodies were added in combination. Process outgrowth on human Ln-1(p) was blocked when either anti-alpha 3 or anti-beta 1 antibodies were added, indicating that alpha 3 beta 1 is the primary integrin heterodimer responsible for process extension on this substrate. These results demonstrate that Ln-5 and other Ln isoforms support comparable levels of adhesion and process outgrowth, but different integrin heterodimers, alone and in combination, are used by SY5Y cells to mediate responses.

Functional identification of integrin laminin receptors that mediate process outgrowth by human SY5Y neuroblastoma cells.

Treatment of the human neuroblastoma cell line SY5Y with nerve growth factor (NGF) induces terminal neuronal differentiation of a subpopulation of cells which can be selected by treatment with a DNA synthesis inhibitor. We have examined the interactions of naive (untreated) and NGF-differentiated SY5Y cells with laminin, and identified integrin receptors that mediate laminin-induced process outgrowth. Differentiated cells displayed a greater capacity for process extension, which correlated with increased expression of integrin laminin receptors. Both naive and differentiated cells expressed integrins alpha 1/beta 1, alpha 2/beta 1, and alpha 3/beta 1 but the differentiated population expressed about 5-fold higher levels of alpha 1/beta 1 and about 2-fold more alpha 2/beta 1 and alpha 3/beta 1 on their surface. Function blocking monoclonal antibodies were used to identify integrin receptors mediating process outgrowth. The anti-alpha 1 monoclonal antibody SR84 was shown to block alpha 1 function and inhibit process outgrowth on laminin. Despite the presence of multiple integrins which have been shown to bind laminin in other cell types, alpha 1/beta 1 mediated the majority of process outgrowth in both naive and differentiated cells, with a minor role played by alpha 3/beta 1. These data indicate that alpha 1/beta 1 function is required for process outgrowth on laminin by SY5Y cells and suggest that increased expression may be a crucial aspect of neuronal differentiation.

The effect of long-term methotrexate therapy on hepatic fibrosis in rheumatoid arthritis.

OBJECTIVE: To evaluate the progression of hepatic fibrosis in patients with rheumatoid arthritis (RA) treated with methotrexate (MTX). METHODS: Fifteen patients receiving MTX for RA were prospectively studied by electron microscopic analysis of biopsy specimens. RESULTS: Five of the 15 patients had evidence of increased hepatic collagen after 2 years of MTX therapy. CONCLUSION: Hepatic fibrosis may progress in a subgroup of RA patients treated with MTX.

Characterization of a beta-nerve growth factor expression vector for mammalian cells.

A mammalian expression vector that directs expression of murine beta-nerve growth factor (beta-NGF) from a murine sarcoma virus long terminal repeat (LTR) promoter element was constructed and characterized. The vector, designated pLTRSNGF, was stably transfected into murine L-cells, and beta-NGF mRNA and protein levels were quantified and compared to endogenous levels in control L-cells. Transfection of pLTRSNGF resulted in an approximate doubling of both beta-NGF mRNA and mature beta-NGF protein secreted into the media. Transfection of pLTRSNGF into rat PC 12 cells resulted in colonies of autocrine-differentiating cells that extended dense networks of neurites in the absence of added NGF, indicating that the beta-NGF produced from the vector is biologically active.

Intracellular Ca2+ stores in chicken Purkinje neurons: differential distribution of the low affinity-high capacity Ca2+ binding protein, calsequestrin, of Ca2+ ATPase and of the ER lumenal protein, Bip.

To identify intracellular Ca2+ stores, we have mapped (by cryosection immunofluorescence and immunogold labeling) the distribution in the chicken cerebellar cortex of an essential component, the main low affinity-high capacity Ca2+ binding protein which in this tissue has been recently shown undistinguishable from muscle calsequestrin (Volpe, P., B. H. Alderson-Lang, L. Madeddu, E. Damiani, J. H. Collins, and A. Margreth. 1990. Neuron. 5:713-721). Appreciable levels of the protein were found exclusively within Purkinje neurons, distributed to the cell body, the axon, and the elaborate dendritic tree, with little labeling, however, of dendritic spines. At the EM level the protein displayed a dual localization: within the ER (rough- and smooth-surfaced cisternae, including the cisternal stacks recently shown [in the rat] to be highly enriched in receptors for inositol 1,4,5-triphosphate) and, over 10-fold more concentrated, within a population of moderately dense, membrane-bound small vacuoles and tubules, identified as calciosomes. These latter structures were widely distributed both in the cell body (approximately 1% of the cross-sectional area, particularly concentrated near the Golgi complex) and in the dendrites, up to the entrance of the spines. The distribution of calsequestrin was compared to those of another putative component of the Ca2+ stores, the membrane pump Ca2+ ATPase, and of the ER resident lumenal protein, Bip. Ca2+ ATPase was expressed by both calciosomes and regular ER cisternae, but excluded from cisternal stacks; Bip was abundant within the ER lumena (cisternae and stacks) and very low within calciosomes (average calsequestrin/Bip immunolabeling ratios were approximately 0.5 and 36.5 in the two types of structure, respectively). These results suggest that ER cisternal stacks do not represent independent Ca2+ stores, but operate coordinately with the adjacent, lumenally continuous ER cisternae. The ER and calciosomes could serve as rapidly exchanging Ca2+ stores, characterized however by different properties, in particular, by the greater Ca2+ accumulation potential of calciosomes. Hypotheses of calciosome biogenesis (directly from the ER or via the Golgi complex) are discussed.

Evidence against a laminin receptor role for calsequestrin.

Calsequestrin, a muscle calcium binding protein, has been shown to bind the extracellular matrix protein laminin and evidence has been presented that CAL (initially called aspartactin) is on the cell surface, consistent with a role as a laminin receptor (1). In this report, we present evidence that does not support a laminin receptor function for CAL. We found that CAL immunoreactivity could not be detected on live cultured chick myotubes unless they were permeabilized with detergent. Furthermore, polyclonal anti-CAL antibodies did not perturb myotube adhesion to laminin or the rate of myoblast fusion on laminin. Expression of the CAL cDNA in a melanoma cell line that was poorly adherent to laminin did not increase adhesion to laminin. In these cells, CAL could not be detected on the cell surface, and the majority of CAL was found to be secreted into the media.

Liver histology in patients receiving low dose pulse methotrexate for the treatment of rheumatoid arthritis.

The liver histology of 52 patients treated with intermittent low dose pulse methotrexate for rheumatoid arthritis was evaluated using a modification of the Roenigk grading system. Patients studied had had an average of 3.2 years of treatment or had received 1.7 g methotrexate. No patient had cirrhosis; 15 (29%) patients had evidence of mild fibrosis. Histological abnormalities were not predicted by liver function test changes, with the exception that hypoalbuminaemia occurred in 60% of those with grade IV (modified criteria) findings. The need for liver biopsy in patients with rheumatoid arthritis treated with methotrexate before two years or 1500 mg of treatment has not been established. Whether serial liver biopsies will be needed beyond this time has yet to be determined.

Efficient transfection and expression of heterologous genes in PC12 cells.

The PC12 pheochromocytoma cell line has been a favorite model system for cell and neurobiologists, but has proven relatively refractory to standard DNA transfection methods. We have found that the cationic lipid "lipofectin" provides a simple, gentle, and nontoxic procedure that vastly improves transfection efficiencies in PC12 cells. Transient expression of chloramphenicol acetyl transferase (CAT) driven by a Rous sarcoma virus long terminal repeat (LTR) is much more efficient using lipofectin when compared with calcium phosphate as a transfection procedure. Additionally, transient transfection of nerve growth factor (NGF)-differentiated PC12 cells proceeds with equal efficiency relative to naive, uninduced cells. Using the lipofectin procedure, the frequency of stable transfection is 100-fold higher than that reported with standard calcium phosphate precipitation protocols. To examine the effectiveness of different promoters for efficient expression of heterologous DNA in PC12 cells, three different promoter-bearing constructs were utilized. Each construct contains a different promoter sequence upstream from a chicken calsequestrin cDNA. A human cytomegalovirus (CMV) immediate early promoter construct produced the highest level of expression, followed by a human beta-actin promoter construct. Expression from a mouse Moloney sarcoma virus LTR construct could not be detected. These results overcome the previous transfection problems of low efficiency and low viability that have plagued many PC12 cell investigations.

Amyloidosis and Reiter's syndrome: report of a case and review of the literature.

Reiter's syndrome is classically described as the triad of urethritis, conjunctivitis, and arthritis, It has many manifestations and has rarely been reported to occur in association with amyloidosis. Four cases of systemic amyloidosis have previously been reported. This case describes a patient who developed progressive renal amyloidosis after a 17-year history of severe Reiter's syndrome. Immunofluorescent staining of the renal biopsy was strongly positive for AA protein, the type of protein found in secondary amyloidosis. This is the first case in which amyloidosis has been proven to be secondary to Reiter's syndrome and not merely the coincidental occurrence of two rare diseases.

Acute, reversible hepatic failure associated with methotrexate treatment of rheumatoid arthritis.

We describe 2 patients who developed reversible decompensated liver disease while taking pulse dosed methotrexate (MTX) for rheumatoid arthritis. One of the patients was available for biopsy and had chronic active hepatitis--a lesion not previously described with MTX. This appears to be a unique and unusual manifestation of MTX hepatotoxicity.