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Immunoglobulin and β-glucuronidase levels in joint exudates and homogenates of synovial tissues were measured during the course of bovine serum albumin (BSA) antigen-induced synovitis in rabbits. After intra-articular BSA challenge, the anti-BSA hemagglutinating antibody in tissue homogenates increased with time up to 14 days. Results obtained indicated relatively greater increases in specific anti-BSA activity compared with total IgG and are compatible with in vivo antibody synthesis by inflamed synovial tissue. β-glucuronidase was measured as a reflection of phagocytic cell activity. Tissue enzyme levels increased to a maximum at 1 day and this level persisted up to at least 7 days, despite the fact that exudate enzyme levels reached a maximum at 1 day and then declined significantly over the next 2-3 weeks. These observations reinforce the view that inflamed synovial tissue possesses a substantial potential for perpetuating joint destruction by producing increased levels of inflammatory mediators. [ABSTRACT FROM AUTHOR]

The effects of varying intra-articular (ia) doses of bovine serum albumin (BSA) antigen on immune synovitis in rabbits have been investigated. Chronic synovitis, characterized by mononuclear cell infiltration in synovial tissues, was induced by a single ia challenge with BSA in sensitized rabbits. However, cartilage and bone erosions and pannus formation were rarely observed. By varying the number and magnitude of the BSA challenges, lesions with different characteristics were observed at different times of analysis of joint pathology. In 3- to 10-week studies, multiple ia challenges with BSA produced lesions characterized by severe cartilage and bone changes; polymorphonuclear leukocyte (PMN) exudates; and mononuclear cells and, sometimes, PMNs in synovial tissues. Substantial increases in knee widths and synovial tissue weights also observed. By increasing the frequency of ia injections, more severe changes were produced more rapidly, so that within a 3-week period, the animals also experienced pain and were unable to fully extend their antigen-challenged knees. Some of the lesions observed in immune synovis resembled those in rheumatoid arthritis (RA). However, the presence of large numbers of PMNs in synovial tissue under certain conditions suggests some possible differences between the pathogenesis of experimental synovitis and RA.

Formulations of a number of steroids were evaluated after topical application in a reversed passive Arthus test (RPA) in rabbits. Four 21-chlorosteroids in the same cream base were investigated. The preparations of SQ 18,566 (halcinonide) and SQ 20,811 showed anti-edema activity, but those of SQ 15,361 and SQ 20,589 were less active. Ointment formulations of halcinonide also reduced edema in the RPA. These results, coupled with previously reported clinical data, suggest that the RPA might be utilized to distinguish good from poor formulations of anti-inflammatory steroids prior to screening tests or clinical trials in humans.

The immune complex-induced reversed passive Arthus (RPA) reaction in the rabbit has been investigated as a screening test for detecting anti-inflammatory agents potentially more effective in the treatment of rheumatoid arthritis than those presently available. RPA lesions, characterized by edema, erythema, and hemorrhage, were elicited by intravenous injections of bovine serum albumin (BSA) followed by intradermal injections of rabbit anti-BSA antiserum. The anti-edema activities of compounds (mg quantities required for testing) were evaluated after their administration by the intradermal route (compounds admixed with antiserum) as well as by the intraperitoneal route. Of 14 reference anti-inflammatory agents tested by the intradermal screening procedure, only aurothioglucose and chloroquine were inactive. Other pharmacologically active compounds (e.g. antihistamines, anti-complement agents, cytotoxic-immunosuppressives) were also evaluated after their intradermal administration. Protoporphyrin, phloretin, and hexadimethrine bromide (Polybrene) were active. When whole antiserum, or the antibody fraction of the serum, was used to eliminate nonspecific edema, intraperitoneally administered reference agents were found to be effective in the RPA test.

Primary rabbit adherent synovial cell monolayer cultures were prepared from animals with antigen-induced synovitis. Primary cultures derived from chronically inflamed tissues released elevated levels of collagenase into the culture media compared to cultures derived from either normal or acute inflammatory tissues. No active of latent collagenase activity was detected in media of cultures derived from normal tissues grown under identical conditions. Cultures of acute inflammatory tissues secreted 1/4 to 1/10 the amount of collagenase when compared to cultures from chronically inflamed tissues. Collagenase secreted by cultures from inflammatory tissues was detected only in latent form. Fetal bovine serum enhanced the trypsin activation of latent collagenase produced by these cultures.

The inhalation of atropine sulfate was investigated in a randomized, 4-period, rising-dose study. Atropine sulfate 2, 4, and 6 mg by inhalation, and atropine free base 1.67 mg (equivalent to 2.0 mg atropine sulfate) by intramuscular (IM) injection were given to 8 healthy, nonsmoking subjects. Serum atropine sulfate concentrations were monitored during an 8-h period by radioimmunoassay. Mean serum concentrations and area under the serum concentration-versus-time curves (AUC) increased as the inhaled dose increased. Peak concentrations (mean +/- SD) were 11.5 +/- 3.4, 16.4 +/- 6.2, and 18.0 +/- 3.1 ng/ml for the 2, 4, and 6 mg doses, and 11.7 +/- 2.5 ng/ml for the IM dose. The time to peak concentration for each dose was similar (mean, 0.8 to 1.9 h). The AUC ratio of the 2-mg inhaled and IM doses was 1.11 +/- 0.41. The observed bronchodilating, anticholinergic, and other pharmacologic effects were seen after all dose concentrations and were typical of atropine. This study showed that inhalation is an efficient way to administer atropine sulfate for systemic use.

In order to better define antiinflammatory activity in new agents, a test was devised utilizing both carrageenan induced paw edema and the reversed passive Arthus reaction in the same animal. The model of carrageenan induced rat paw edema is a standard laboratory assay used to predict classical "aspirin-like" antiinflammatory molecules. The reversed passive cutaneous Arthus reaction involves precipitating antigen-antibody complexes, complement and infiltrating polymorphonuclear leukocytes (PMN's) and can be used to identify agents that affect one or more of these factors specifically. Antiinflammatory compounds were given orally one hour prior to the administration of carrageenan and goat anti-rat serum. Comparisons were made between several non-steroidal compounds and the steroid hydrocortisone. All of the compounds tested gave good carrageenan activity, but only hydrocortisone produced significant Arthus lesion inhibition in this assay.

Immune synovitis in rabbits was investigated as a potential in vivo model for evaluating new antiinflammatory agents. Antigen-induced increases in knee width as well as beta-glucuronidase and acid phosphatase activities in exudates were observed. Histologically, polymorphonuclear leukocytes appeared within hours in synovial tissues and reached maximum infiltration at about 24 hours. Subsequently, mononuclear cells, including plasma cells, appeared. The 6-hour Arthus-like phase of synovitis can be depressed by some antiinflammatory agents, colchicine and steroids being particularly effective. It is suggested that this model can be utilized to define potentially more effective antiinflammatory drugs.

Normal animal sera inhibit at least one Clostridium histolyticum proteinase. An assay procedure based on immune hemolysis was developed for the estimation of this inhibition. This inhibitory activity occurs in various levels in the sera of different animal species. The highest titers have been obtained with rat sera. The inhibitory activity from human serum was isolated and purified 16- to 27-fold by Sephadex G-200 gel filtration and diethylaminoethyl cellulose or hydroxylapatite chromatography. The properties of the human serum inhibitor of the clostridial proteinase were compared with a trypsin inhibiting factor found in the partially purified preparations. Identical behavior of the two inhibitory factors was observed when measured by heat inactivation, beta-mercaptoethanol sensitivity, pH stability, and sucrose gradient centrifugation. The inhibitory factor has an approximate sedimentation coefficient (S(20,w)) of 17. Goat anti-alpha-2-macroglobulin specifically precipitated the clostridial proteinase inhibitor from a partially purified preparation.

A proteinase fraction inhibiting the hemolytic activity of guinea pig complement was obtained from supernatant fluids of Clostridium histolyticum cultures and purified 150- to 350-fold by ammonium sulfate precipitation, Sephadex G-75 gel filtration, and diethylaminoethyl cellulose chromatography. An assay was developed based on the inactivation of hemolytic complement. Partially purified anticomplementary preparations were active against casein and were capable of "solubilizing" Escherichia coli endotoxin. Two components were found by differential heat inactivation, with complement and casein as substrates, but only one of these components was active against endotoxin. The more heat-stable activity, showing 50% inactivation at about 47 C, was characterized as to pH and ionic strength optima and sensitivity to reagents such as cysteine, beta-mercaptoethanol, ethylenediaminetetraacetate, and heavy metals.