Quantitation and characterization of plasma DNA in normals and patients with systemic lupus erythematosus.

Using the in vitro DNA labeling technique of nick translation on purified plasma DNA, we have estimated the plasma DNA concentration in three normal individuals to be 266 +/- 57 ng/ml (mean +/- SD). This was not significantly different in three patients with a chronic inflammatory disease (209 +/- 14 ng/ml) or in five patients with steroid-inactivated systemic lupus erythematosus (SLE) (293 +/- 57 ng/ml). In two untreated, newly diagnosed, active SLE patients, however, the plasma DNA concentration was considerably higher (4,024 and 2,437 ng/ml, respectively). Characterization of these in vitro labeled DNA preparations by neutral sucrose-gradient sedimentation analysis showed a sedimentation coefficient of 6-8S, corresponding to a molecular weight of similar to or approximately 0.2-0.45 x 10(6). No difference was observed between normal subjects or patients. In addition, the relative size uniformity of these DNA molecules might suggest some form of specific protection of the DNA from blood DNAases. Further characterization in terms of buoyant density in cesium chloride did not reveal a difference between normal or SLE plasma and the human (HEp-2 cell) DNA used as marker. Taking into account the limitations of the method, no indication of a possible exogenous origin of the DNA circulating in SLE patients could be found. The physiological or pathophysiological role of this plasma DNA remains to be determined.

Detection and expression of a cDNA clone that encodes a polypeptide containing two inhibitory domains of human calpastatin and its recognition by rheumatoid arthritis sera.

RA is the most frequent and most destructive inflammatory arthropathy. Rheumatoid factors, in spite of their lack of disease specificity, are important serological markers for RA and appear important in its immunopathogenesis as well. In search of more disease-specific autoimmune systems, we have screened a human placenta lambda gt11 cDNA expression library using selected sera from patients with classical erosive RA. We have identified one clone (RA-1) that is recognized by three of five screening sera. The 950-bp insert shows a complete nucleotide sequence homology to the cDNA encoding the two COOH-terminal domains of calpastatin. The deduced open reading frame of the RA-1 cDNA predicts a 284-amino acid protein, with a calculated mol wt of 35.9 kD. Calpastatin is the natural inhibitor of calpains, which are members of the cysteine proteinases recently implicated in joint destruction in rheumatic diseases. The two domains encoded by the RA-1 clone each contain the structural features associated with the inhibitory activity of human calpastatin. By Western blotting, 45.5% or 21/44 RA sera specifically recognized both the fusion and the cleaved recombinant protein. This is in contrast to 4.7% (2/43) in nonrheumatoid sera and 0/10 in normal sera. Anticalpastatin autoantibodies could represent a disease-associated marker in chronic erosive arthritis of the rheumatoid type and could hypothetically play a dual pathogenic role, directly via an immune interference and indirectly through an immune complex mechanism.

Airway disease in a subset of nonsmoking rheumatoid patients. Characterization of the disease and evidence for an autoimmune pathogenesis.

Previous investigations of airway disease in rheumatoid patients have been oriented toward establishing the prevalence of the disease, but the pathogenesis and the time course of the airflow obstruction in rheumatoid disease are still unclear. In this study, we analysed the clinical, serial pulmonary function and histopathologic data of six rheumatoid patients who had never smoked but who had airflow limitations documented repeatedly up to 10 years previously. We have attempted to characterize the site, nature and evolution of the chronic airway disease in this group of patients. Bronchiectasis was excluded in all patients by bilateral bronchography. Clinical and histopathologic evidence of the Sjörgen autoimmune exocrinopathy was documented in five of the patients, and the sixth patient had lymphoplasmocytic infiltrates of the labial glands without obstruction of the lumen or destruction. By pulmonary function tests and histopathologic examination of four open lung biopsies, the airway disease was found to be located predominantly in the peripheral airways of the lung. On each biopsy, the lesions were in different stages of activity, but on all specimens there was a definite predilection for selective bronchiolar injury. Early stage lesions were characterized by mononuclear cell infiltrates of the peribronchiolar tissue which led to deformation of airway lumen, focal mucosal extension and ulceration. Subsequently, the inflammatory reaction was replaced by fibroblastic proliferation, and in the end stage of the disease, there was complete obliteration of many bronchioles by collagenized fibroblastic tissue. From regression analyses of serial pulmonary function tests of these patients, it was concluded that (1) the airway disease in our patients who did not smoke progressed inevitably but not uniformly and (2) deterioration of pulmonary functions was more rapid in our patients than it was in the cigarette smokers who had chronic obstructive lung disease. This study also documents major dysfunctions of the chest wall mechanics which appear to contribute to the restriction of lung volumes in some rheumatoid patients.

Expression of recombinant human cyclooxygenase isoenzymes in transfected COS-7 cells in vitro and inhibition by tenoxicam, indomethacin and aspirin.

The recent discovery of cyclooxygenase-2 (COX-2), an isoenzyme associated mainly with inflammation created the need to reevaluate cyclooxygenase inhibitors with reliable screening methods. In the present study we standardized a technique to determine the IC50S of cyclooxygenase inhibitors on recombinant human COX-1 and COX-2 expressed in mammalian cells and used it to study the compounds tenoxicam, aspirin and indomethacin. The IC50S of aspirin, indomethacin and tenoxicam for human COX-1 were 0.41 +/- 0.07 microgram/ml, 0.008 +/- 0.003 microgram/ml, and 7.94 +/- 3.28 micrograms/ml, respectively, and for human COX-20.64 +/- 0.16 microgram/ml, 0.09 +/- 0.05 microgram/ml, and 10.61 +/- 1.50 micrograms/ml, for aspirin, indomethacin, and tenoxicam. Tenoxicam had the lowest IC50hCOX-2/IC50hCOX-1 ratio (1.34), followed by aspirin (1.53) and indomethacin (10.82). The system described in the present study provides a simple and efficient way to determine the specificity of NSAID inhibition for each of the human cyclooxygenase isoenzymes separately.

Immunological cross-reactivity of adenovirus structural proteins.

The interserotypic cross-reactivity of adenovirus-specific antisera was examined on Western blots of virion proteins. The pattern of cross-reactivity was very complex. Virion polypeptides III, VI and the core VII were the major reactants. Generally, the degree of reactivity correlated with type and subgroup specificity.

Assessment of salsalate, a nonacetylated salicylate, in the treatment of patients with arthritis.

This study evaluated physicians' use of the occurrence of tinnitus as a tool to establish the optimal dosage of salsalate, a nonacetylated salicylate, in patients with arthritis treated in routine clinical practice. The use of printed educational materials to improve compliance was also studied prospectively. A total of 782 patients were enrolled in this 3-week study by 95 general practitioners in an office setting. Of the 771 assessable patients, 90.0% had osteoarthritis, 9.7% had rheumatoid arthritis, and 0.3% had both types of arthritis. Most patients experienced improvement of symptoms after 3 weeks of treatment. There were no differences in the rates of improvement at the first and third weeks of treatment between patients with osteoarthritis and patients with rheumatoid arthritis. In addition, duration of arthritis had no effect on rates of improvement. Rates of patient satisfaction tended to increase over the study period. Rates of patient satisfaction did not differ significantly at the first and third weeks between patients who did not receive printed educational materials and whose who did not. Treatment was discontinued in 234 patients (30.4%) because of side effects. The most frequent reasons for discontinuation were gastrointestinal symptoms (n = 102; 13.2%) and tinnitus (n = 52; 6.7%). The clinical effectiveness and safety of salsalate were confirmed in patients with arthritis in routine clinical practice settings.

Patient compliance with tenoxicam in family practice.

This study evaluated factors that may influence patient compliance and also confirmed tolerability and efficacy of tenoxicam in routine clinical practice. Compliance in 1809 patients was evaluated over a 4-week period by physician pill-counts, patient assessment cards, and, for a subpopulation, by electronically monitored pill vials. In addition, physicians documented patient improvement and side effects after 2 weeks and after 4 weeks of therapy; patients reported satisfaction with therapy and side effects on a weekly basis. A total of 399 physicians provided data on 1809 patients, of whom 84.3% had osteoarthritis, 12.6% had rheumatoid arthritis, and 3.2% had ankylosing spondylitis. The typical patient was a woman (64.9%), white (91.2%), in her 50s (mean age, 57.9 years), with a duration of osteoarthritis of at least 1 year (72.3%). High and similar compliance rates were achieved by patients regardless of age, gender, or diagnostic category. Patient and disease characteristics were similar between compliant and noncompliant patients. Most patients (81.1%) experienced improvement of symptoms after 4 weeks of treatment. A low incidence of side effects (12.6%) was reported, with no significant differences observed among patients with respect to age, gender, or diagnostic category. Product characteristics, such as tolerability, efficacy, and dosing regimen, are more significant factors of compliance than patient or disease characteristics. Tenoxicam's tolerability and clinical effectiveness were confirmed in patients with arthritis in routine clinical practice settings.

Efficacy and tolerability of enteric-coated naproxen in the treatment of osteoarthritis and rheumatoid arthritis: a double-blind comparison with standard naproxen followed by an open-label trial.

One hundred and twenty-three patients with osteoarthritis (n = 50) or rheumatoid arthritis (n = 73) were enrolled in a 6-week, double-blind, randomized, controlled, parallel trial comparing enteric-coated naproxen with standard naproxen. Ninety-eight patients subsequently entered a 20-week, open-label trial of enteric-coated naproxen. The study demonstrated that naproxen in both its standard formulation and its new enteric-coated formulation is a highly effective form of therapy for osteoarthritis and rheumatoid arthritis. The tolerability profiles of the two formulations were similar in terms of the types of complaints reported. It is concluded that enteric-coated naproxen is an efficacious and well-tolerated formulation for the treatment of osteoarthritis and rheumatoid arthritis.

Open-label tolerability study of enteric-coated naproxen in the treatment of osteoarthritis and rheumatoid arthritis.

Two hundred and ninety-six patients were enrolled in a 6-month, open-label tolerability study of enteric-coated naproxen in patients with rheumatoid arthritis (n = 174) and osteoarthritis (n = 122). Thirty percent of the patients were greater than 65 years of age. Under standard clinical prescribing conditions, enteric-coated naproxen 500 mg twice daily and 375 mg twice daily demonstrated an acceptable tolerability profile that was not different from what one would expect with standard naproxen.

Heterogeneity of ANCA sera showing atypical, peripheral and classical cytoplasmic immunofluorescence patterns.

Atypical (A) ANCA immunofluorescence (IF) patterns have been described in several disease groups. We have previously reported a distinct cytoplasmic A-ANCA in 7-10% of patients with SLE and/or RA. Here, we show that these rheumatic disease associated A-ANCAs are best identified using U937 cells as substrate and that they do not target either a serine proteinase or a peroxidase. Furthermore, these sera immunoprecipitate a 40 kDa or a 42 kDa band using in vivo 35S-amino acid labelled HL60 or U937 cell extracts, respectively. Although these bands are the only one seen with pure A-ANCA sera, they can also be found in addition to the expected bands of PR3 or MPO in up to 30% of bona fide C- or P-ANCA sera. These data confirm and extend our previous observations. They also suggest that target heterogeneity of ANCA antibodies is frequent. Care should thus be taken in interpreting in a cause and effect relationship, an IF pattern or a biological effect produced by a serum with ill documented monospecificity. The exact nature and significance of this (these) new antigen(s) have yet to be clarified.

Trace element determination in serum by proton-induced x-ray emission.

We report the use of proton-induced x-ray emission (PIXE) as an analytical method in clinical research. The experimental set-up and target preparation procedures are briefly discussed together with the methods of automatic data acquisition and analysis. Results from a clinical project involving rheumatoid patients receiving chrysotherapy are presented.

Trace elements and acute phase reactants in gold treated rheumatoid arthritis patients.

Abnormal concentrations of trace elements, sulfhydryl groups, amino-acids and serum proteins were found in patients with rheumatoid arthritis similar to other chronic inflammatory conditions. These changes have been called the phase reaction. In this study, a systematic profile of the phase reactants was established in rheumatoid arthritis at the onset and during its modulation by chrysotherapy (gold sodium thiomalate 25 mg i.m. weekly). It was shown that this treatment, if successful, is capable of reverting to normal measures of the phase reaction. It was also found that the pattern of the profile varied in individuhe eventual beneficial or toxic effects. In these preliminary studies, no parameters or group of parameters of the phase reaction were predictive of the therapeutic outcome. Extended observations (over 6 months) and more individual profiles are being analyzed to gain insight into these features of inflammation and their modulation by chrysotherapy.

Gold therapy in rheumatoid arthritis. Interim report of the Canadian multicenter prospective trial comparing sodium aurothiomalate and auranofin.

One hundred and twelve patients with classical or definite rheumatoid arthritis (RA) were randomly assigned to receive either sodium aurothiomalate (GSTM) or auranofin (AF). Monthly clinical assessments (morning stiffness, grip strength, articular index, pain, quality of life) and concurrent hematological, biochemical, and urine studies were performed to monitor the efficacy/toxicity (E/T) ratio. Ninety-two patients have completed 3 months; 65, 6 months; 47, 9 months; and 30, 12 months. The groups were numerically balanced at each time period. Analysis of the 0-6 month period suggests that both drugs were equally and significantly beneficial after 3 months and that this was maintained at 6 months. Toxicity was as frequent in both groups but more serious in the GSTM group. The main side effects were gastrointestinal (diarrhea) in the AF group and mucocutaneous in the GSTM group. Half of the withdrawals (14 in each group) were because of side effects in the GSTM group and for inadequate therapeutic efficacy in the AF group. This study suggests that after 6 months of treatment the E/T ratio of AF is greater than or equal to that of GSTM. These conclusions will need to be confirmed during the ongoing longer observation period. A significant clinical difference between the 2 drugs is that in a given patient treated with GSTM, the onset of toxicity coincides with a good therapeutic effect. This relationship does not appear to exist during AF treatment.

Use of a hapten-carrier system in experimental immune arthritis in the rabbit.

Animals immunized systemically with dinitrophenylated-bovine serum albumin (DNP-BSA) in complete Freund's adjuvant, show delayed hypersensitivity skin responses to DNP-BSA and to BSA, but not to other DNP-conjugates. These animals show high levels of antibodies to BSA and to DNP to radioimmunoassay. When injected with the carrier (BSA), the right knee joint develops a chronic arthritis. The left knee, when injected with the DNP hapten conjugated to another carrier (human serum albumin), shows an acute arthus response of the same magnitude as the right knee, but fails to develop a chronic synovitis. It appears that in this model, chronicity is not mediated primarily by a humoral mechanism to the initiator.

Clinical significance of anti-Ro(SSA) antibody in rheumatoid arthritis.

A group of 12 patients with rheumatoid arthritis (RA) with anti-Ro antibody were compared to an age and sex matched group with RA. Both had similar articular manifestations but the anti-Ro positive patients had more severe extraarticular as well as serological abnormalities. Vasculitis, liver abnormalities, neutropenia and circulating immune complexes were prominent while sicca features were not. Treatment with remission inducing drugs failed frequently without increased toxicity. Two patients with classical RA with anti-Ro developed subacute cutaneous lupus erythematosus and anti-dsDNA. Our observations suggest that the presence of anti-Ro in RA sera may be a marker for a small but significant subset of rheumatoid disease.

Experimental immune arthritis: host factors.

An experimental chronic mono-arthritis was induced in rabbits using adjuvants and bovine serum albumin. The results suggest that unidentified host factors (possibly genetic) influence the induction of delayed hypersensitivity to the antigen. The data further show a clear correlation between systemic cell mediated immunity and chronic synovitis. When this condition is absent, there is a poor correlation between the humoral response and the arthritis. The results make difficult the interpretation of local immune complex deposition as the sole, chronic phlogistic stimulus. (J Rheumatol 2: 373-383, 1975).