Analysis of changes in acute-phase plasma proteins in an acute inflammatory response and in rheumatoid arthritis using two-dimensional gel electrophoresis.

Two-dimensional (2-D) gel analysis was used to examine differences in the levels of 19 plasma proteins: before and after an acute inflammatory reaction (parenteral typhoid vaccination) in normal subjects, between rheumatoid arthritis (RA) patients and normals and in RA patients treated with tenidap (120 mg) and piroxicam (20 mg). Typhoid vaccination increased levels of SAA, haptoglobin alpha1, haptoglobin alpha2, haptoglobin beta and alpha1-anti-chymotrypsin but decreased transthyretin and apolipoprotein E. In RA patients, serum amyloid A (SAA), haptoglobin alpha2, haptoglobin beta, alpha1-antichymotrypsin and C3 proactivator levels were elevated while apolipoprotein A-I, apolipoprotein A-IV, transthyretin, Gc-globulin, alpha2-HS glycoprotein, alpha2-macroglobulin and alpha1-B glycoprotein levels were decreased, compared to normals. Compared to piroxicam, tenidap lowered levels of alpha1-antiprotease and SAA but raised the levels of transthyretin, Gc-globulin, alpha2-HS-glycoprotein and alpha2-macroglobulin in RA patients. C-reactive protein (CRP) could not be quantified on 2-D gels but, when measured by rate nephelometry, levels were reduced after treatment with tenidap compared to piroxicam. The general pattern of the acute phase protein response to an acute inflammatory response to typhoid vaccination is similar to that in the chronic inflammatory condition, RA. The impact of tenidap on both positive and negative acute-phase proteins in RA patients could clearly be distinguished from that of piroxicam.

Post-capillary venules in the "milky spots" of the greater omentum are the major site of plasma protein and leukocyte extravasation in rodent models of peritonitis.

Intraperitoneal injection of inflammatory agents in the mouse and rat causes plasma protein and leukocyte extravasation into the peritoneal cavity. Following an intraperitoneal injection of zymosan A, the milky spots of the omentum were the only abdominal sites detected where intravenously administered Monastral Blue labeled interendothelial cell gaps responsible for plasma extravasation. In addition, when colored microspheres were intraventricularly administered to quantify blood flow, the omentum was the only abdominal organ which showed an increase in blood flow during zymosan A peritonitis. A combination of light and electron microscopy, plus measurement of myeloperoxidase activity (a marker of neutrophil accumulation) demonstrated that the omental milky spots are the major route through which leukocytes migrate into the peritoneal cavity. Identical structures in the pleura likewise are the sites of protein leakage into the pleural cavity. In contrast, selective sites of protein and cellular extravasation could not be detected in the synovial lining of the inflamed knee joint.

Degradation of cartilage matrix proteoglycan by human neutrophils involves both elastase and cathepsin G.

The granule proteases of human neutrophils are thought to be responsible for the connective tissue destruction associated with certain inflammatory diseases. Using a model system for the degradation of a macromolecular connective tissue substrate, purified neutrophil elastase and cathepsin G were both individually able to degrade cartilage matrix proteoglycan and this degradation was blocked by the appropriate specific inhibitors. Neutrophil granule lysate also produced cartilage matrix degradation but little inhibition of degradation occurred when either elastase or cathepsin G inhibitor was used alone. However, a combination of elastase and cathepsin G inhibitors each at 100 microM or each at 10 microM blocked cartilage matrix degradation by 89% +/- 1 and 65% +/- 9 (mean +/- SEM, n = 3), respectively. The magnitude of the cartilage degradation mediated by neutrophil lysate, and its sensitivity to specific inhibitors, was reproduced using purified elastase and cathepsin G at the concentrations at which they are present in neutrophil lysate. Human neutrophils stimulated with opsonized zymosan degraded cartilage matrix in a dose-dependent manner in the presence of serum antiproteases. Supernatants from stimulated neutrophils cultured in the presence of serum did not degrade cartilage matrix, indicating that neutrophil mediated degradation in the presence of serum was confined to the protected subjacent region between the inflammatory cell and the substratum. A combination of elastase and cathepsin G inhibitors each at 500 microM or each at 100 microM blocked subjacent cartilage matrix degradation by stimulated human neutrophils by 91% +/- 3 and 54% +/- 8 (mean +/- SEM, n = 5), respectively, whereas either the elastase or cathepsin G inhibitor alone was much less effective. These studies demonstrate that neutrophil-mediated cartilage matrix degradation is produced primarily by elastase and cathepsin G. Furthermore, these results support the hypothesis that inflammatory neutrophils form zones of close contact with substratum that exclude serum antiproteases and that this subjacent degradation of cartilage matrix by stimulated neutrophils can be blocked by a combination of synthetic elastase and cathepsin G inhibitors.

Nitric oxide synthesis in the CNS endothelium and macrophages differs in its sensitivity to inhibition by arginine analogues.

Inhibition of nitric oxide production by arginine analogues was examined in three cell systems; macrophages, CNS tissue and endothelial cells. Nitric oxide production was assessed indirectly using in vitro assays measuring nitrite production (macrophages), cGMP elevation (CNS) and acetylcholine-induced relaxation of aortic ring segments (endothelium). NG-monomethyl-L-arginine and NG-amino-L-arginine possessed similar inhibitory activity in all three assays, while NG-nitro-L-arginine displayed a striking selectivity for inhibition of brain and endothelial cell nitric oxide synthesis, with IC50 values of 0.05 microM in the CNS versus 200 microM in macrophages. These results suggest that distinct enzymes are responsible for nitric oxide synthesis in different cell types, and indicate that it may be possible to selectively modulate nitric oxide production in vivo.

S-adenosylhomocysteine levels are not involved in the spasmolytic activity of 3-deazaadenosine in guinea-pig lung parenchyma.

The effects of two S-adenosylhomocysteine (SAH) hydrolase inhibitors, MDL 28,842 and 3-deazaadenosine (3-DAA), on the tissue levels of SAH, cAMP and cGMP and on the contractile responses to leukotriene D4 and histamine were compared in guinea-pig lung parenchyma. MDL 28,842 and 3-DAA (both in the presence of L-homocysteine) increased tissue levels of SAH to the same degree, indicating that both compounds had inhibited SAH hydrolase. However, only 3-DAA inhibited the contractile response of the lung parenchyma. The spasmolytic activity of 3-DAA was inhibited by co-incubation with MDL 28,842, suggesting that 3-DAA must be metabolized by SAH hydrolase, probably to 3-deazaadenosylhomocysteine, in order to exert its effect. 3-DAA did not elevate tissue levels of cAMP or cGMP. Although the mechanism by which 3-DAA exerts its spasmolytic effects is unknown, direct inhibition of methyltransferase is a plausible explanation. These data indicate that SHA levels do not regulate the contractile response of guinea-pig lung parenchyma.

The antinociceptive activity of paracetamol in zymosan-induced peritonitis in mice: the role of prostacyclin and reactive oxygen species.

1. Oral administration of high doses of paracetamol (600 mg kg-1 or more) resulted in inhibition of the writhing and reduced the levels of prostacyclin (PGI2, measured as 6-keto-PGF1 alpha) induced by intraperitoneal administration of zymosan in mice. The high oral doses of paracetamol required were accompanied by behavioural toxicity which may have contributed to the inhibition of writhing. 2. The number of writhes per mouse and the proportion of mice writhing at least once correlated significantly with the levels of 6-keto-PGF1 alpha. However, inhibition of writhing by paracetamol occurred at higher levels of 6-keto-PGF1 alpha than was previously observed with acidic non-steroidal anti-inflammatory agents. 3. When injected i.p., PGI2, carbacyclin and iloprost (agonists at the PGI2 receptor) induced writhing. Intraperitoneal injection of PGI2 reversed the inhibition of writhing induced by indomethacin (1 mg kg-1, p.o.) but not that induced by oral administration of paracetamol. 4. Paracetamol at 800 mg kg-1, p.o., inhibited carbacyclin-induced writhing but indomethacin at 1 mg kg-1 p.o. did not. Paracetamol administered i.p. at 100 mg kg-1 reduced the peritoneal levels of 6-keto-PGF1 alpha and inhibited zymosan-induced but not carbacyclin-induced writhing and did not produce behavioural toxicity. 5. The in vitro potency of paracetamol as a prostaglandin synthesis inhibitor is known to be reduced by the presence of lipid peroxides. However, no lipid peroxides, measured as thiobarbituric acid reactive material, were detected in the peritoneal lavage fluid of zymosan-injected mice. 6. Intraperitoneal administration of a mixture of superoxide dismutase and catalase reduced detectable superoxide anion by 98% without inhibiting the writhing response to zymosan or the antinociceptive potency of paracetamol. 7. The data are consistent with the suggestion that inhibition of PGI2 synthesis in the peritoneal cavity by paracetamol is responsible for only a part of its antinociceptive activity in this test. However, extremely high oral doses of paracetamol were required which produced behavioural toxicity which clearly contributed to the inhibition of writhing. The low potency of paracetamol in this model cannot be attributed to the generation of lipid peroxides via the oxidative burst.

Ex vivo lipopolysaccharide-induced interleukin-1 secretion from murine peritoneal macrophages inhibited by probucol, a hypocholesterolemic agent with antioxidant properties.

Probucol, 4,4'-(isopropylidenedithio)bis(2,6-di-tert-butyl-phenol), has been shown to inhibit atherogenesis in genetically hypercholesterolemic (Watanabe) rabbits. Since atherosclerotic lesions contain macrophages capable of screting interleukin 1 (IL 1) and other cytokines that could contribute to the pathogenesis of the disease, we have investigated whether probucol affects IL 1 secretion. Resident peritoneal macrophages from mice dosed with probucol secreted 40-80% less IL 1 than macrophages from control animals when stimulated in vitro with lipopolysaccharide (LPS). The inhibitory effect of probucol was observed when IL 1 was assayed by the standard bioassay, the thymocyte proliferation assay, or a competitive IL 1 receptor binding assay. Probucol treatment had no effect on LPS-induced membrane IL 1 expression; secretion of tumor necrosis factor (TNF); Con A-induced splenic interleukin 2 (IL 2) and interleukin 3 (IL 3) release; and prostaglandin- or zymosan-induced secretion of prostacyclin, leukotriene C4, acid phosphatase, or superoxide anion. In contrast to the effect of oral administration, direct addition of probucol to macrophage cultures did not inhibit IL 1 release. Probucol administration did, however, inhibit the fall in serum zinc level induced by intravenous injection of LPS in zymosan-primed mice but had no effect on the LPS-induced increase in serum triglyceride levels, which indirectly confirms that probucol administration inhibits IL 1 but not TNF secretion. Paw granuloma induced in mice by heat-killed mycobacteria was inhibited by oral administration of probucol, an effect that may be attributable to inhibition of IL 1 secretion. Probucol neither reduced zymosan-induced liver granulomata in mice nor inhibited adjuvant-induced arthritis in rats. We suggest that inhibition of IL 1 secretion from macrophages by probucol contributes to its therapeutic effects in atherosclerosis and may also result in beneficial activity in some chronic inflammatory diseases.

Probucol does not alter acetylated low density lipoprotein uptake by murine peritoneal macrophages.

It has been suggested that the anti-atherogenic effect of probucol (MDL 11,309) in familial hypercholesterolemic rabbits may be due in part to the inhibition of the uptake of modified low density lipoproteins by macrophages in the arterial wall. To test this hypothesis, mice were treated with dietary probucol (0.25%) for fourteen days, peritoneal macrophages were isolated and the uptake of acetylated low density lipoprotein (ALDL) was studied. In addition, peritoneal macrophages from control animals were treated with probucol (200 micrograms/ml) for 24 h in vitro prior to the ALDL uptake assay. The assay involved a fluorescent ALDL probe (dioctadecyltetramethylindocarbocyanine perchlorate-labeled ALDL), and measurement of uptake with a flow cytometer. No differences in ALDL uptake were detected between the control macrophages and macrophages treated with probucol in vitro or macrophages taken from probucol-treated mice.

Novel inhibitors of polymorphonuclear neutrophil (PMN) elastase and cathepsin G: evaluation in vitro of their potential for the treatment of inflammatory connective tissue damage.

Inhibitors of neutrophil proteases may have therapeutic effects in inflammatory diseases. MDL 27,324 (Dansyl-Ala-Ala-Pro-Val-CF3), inhibits human neutrophil elastase and MDL 27,399 (MeOSucc-Ala-Ala-Pro-Phe-COOCH3), inhibits human neutrophil cathepsin G. These compounds individually or in combination, partially inhibited the hydrolysis of fluoresceinated bovine serum albumin and fluoresceinated immune complexes by rat and human neutrophil granule lysate. In contrast, the combination of inhibitors completely prevented the breakdown of a complex connective tissue substrate, azure hide powder. Rat neutrophils phagocytosed and hydrolyzed fluoresceinated immune complexes, a process which was inhibited by cytochalasin B (15 micrograms/ml, 65% inhibition) and chloroquine (200 microM, 80% inhibition). Although MDL 27,324 was taken up by the cells, it had only a modest inhibitory effect on the proteolysis of ingested fluoresceinated immune complexes (200 microM, 20% inhibition); MDL 27,399 had similar limited efficacy. Therefore, these compounds may be effective inhibitors of neutrophil serine proteases secreted into the extracellular space during inflammation without interfering with the normal process of intracellular degradation of phagocytosed material.

Comparison of the anaphylactoid response induced in rats by castanospermine and dextran.

The plant alkaloid castanospermine (10 mg/kg or higher, i.p.; 400 mg/kg, p.o.) induced in some rat strains an anaphylactoid reaction similar to that induced by dextran, i.e. erythema and edema of the snout, ears and paws, for several hours after administration. 86% of the rats from a responsive strain responded to castanospermine while 79% responded to dextran. Rats which responded to castanospermine showed marked, but transient, tachyphylaxis to a second dose of castanospermine or dextran. Rats maintained on a complex-carbohydrate-free diet also responded to castanospermine, excluding the possibility that the effect was due to absorption of dextran-like, dietary, complex carbohydrates. These data raise the possibility that some apparent food allergies in man could be due to the presence in the diet of plant alkaloids with properties similar to those of castanospermine.

Multiple topical applications of arachidonic acid to mouse ears induce inflammatory and proliferative changes.

The response to daily topical applications of arachidonic acid (0.25-4 mg/ear/day) to the ears of outbred CD-1 mice was monitored. The first application produced erythema, extravasation of plasma proteins resulting in an increase in ear weight, and some neutrophil accumulation (detected histologically and quantified by myeloperoxidase content). The second application produced minimal edema but did cause erythema and a greater accumulation of neutrophils. Subsequent daily application caused erythema, neutrophil accumulation, and an increase in ear weight predominantly due to cell proliferation (epidermis and connective tissue). Daily applications of other unsaturated fatty acids did not match the response induced by arachidonic acid. Mast cell deficient mice (W/Wv) exhibited a smaller edema response to the first dose of arachidonic acid compared to either their wild-type controls or CD-1 mice. In addition, W/Wv mice exhibited a smaller ear weight increase and myeloperoxidase accumulation following eight daily doses of arachidonic acid. However, epidermal proliferation was similar in all the strains of mice tested. These data suggest that the edema caused by the first topical application of arachidonic acid is partly mast cell mediated. Mast cells also appear to be involved in the neutrophil infiltration induced by multiple topical applications, but not in the epidermal proliferation.

Inhibition by probucol of interleukin 1 secretion and its implication in atherosclerosis.

Intravenous injection of 1.5 mg of acetylated low-density lipoprotein (LDL) or 100 micrograms of lipopolysaccharide (LPS) to zymosan-primed mice induced a decrease in serum zinc levels measured 6 hours after injection, suggesting the release of interleukin 1 (IL-1). Oral administration of probucol, 100 mg/kg once daily for 14 days, inhibited the LPS-induced fall in serum zinc levels, suggesting inhibition of IL-1 release. Direct evidence for inhibition of IL-1 release by probucol was obtained with an ex vivo system in which, compared with controls, peritoneal macrophages from probucol-treated mice (100 mg/kg orally X 3, or 0.25% in the diet for 3 weeks) secreted 80 to 90% less IL-1 upon LPS stimulation, measured by the C3H/HeJ thymocyte proliferation assay. Inhibition of IL-1 secretion by probucol may contribute to the therapeutic effect of probucol in atherosclerosis since as little as 1 unit of recombinant IL-1 beta was found to induce proliferation of aortic smooth muscle cells. With regard to the endogenous stimulus for IL-1 secretion, oxidized LDL is a putative candidate because it is capable of stimulating peritoneal macrophages to secrete IL-1. Because oxidized LDL is involved in the transformation of macrophages to foam cells, our data on IL-1 induction by oxidized LDL and the mitogenic effect of IL-1 on aortic smooth muscle cells suggest that activated macrophages play an important role in atherogenesis.

The role of prostaglandins in the nociceptive response induced by intraperitoneal injection of zymosan in mice.

Intraperitoneal injection of zymosan (1 mg in 0.5 ml saline) in mice induces a transient writhing response accompanied by the synthesis of small amounts of prostaglandin E2(PGE2, less than 2 ng) and larger amounts of PGI2 (200 ng per mouse), measured as its non-enzymatic breakdown product, 6-keto-PGF1 alpha. Although both centrally-acting analgesics (morphine, clonidine) and prostaglandin biosynthesis inhibitors (aspirin, indomethacin, ibuprofen) blocked the writhing response to intraperitoneal injection of zymosan, only the latter reduced prostaglandin levels in the peritoneal cavity. The writhing response correlated equally well with PGE2 levels and 6-keto-PGF1 alpha levels when data from mice treated with centrally-acting analgesics were excluded. However, intraperitoneal injection of PGI2, but not PGE2, reversed the analgesia induced by indomethacin in zymosan-injected mice. Centrally-acting agents, but not ibuprofen, blocked the ability of PGI2 to reverse the analgesic activity of indomethacin. PGI2 (2 micrograms per mouse), injected intraperitoneally in otherwise untreated mice, induced writhing. These data indicate that PGI2 is the prostaglandin involved in mediation of the writhing response to zymosan and that prostaglandin biosynthesis inhibitors, but not centrally-acting analgesics, exert their analgesic activity by reducing the peritoneal level of PGI2. It is possible that PGI2 may have the ability to stimulate pain receptors directly in the mouse peritoneal cavity, in addition to its previously recognized ability to sensitize pain receptors to other pain-producing stimuli.

Intraperitoneal injection of zymosan in mice induces pain, inflammation and the synthesis of peptidoleukotrienes and prostaglandin E2.

Intraperitoneal injection of zymosan in mice induced rapid extravasation and accumulation of plasma protein in the peritoneal cavity. Neutrophils began to appear in the peritoneal cavity after a lag period of approximately 3 hours. The injected mice exhibited a pain response (writhing) during the first 30 minutes after injection, but writhing ceased before protein or cell accumulation had reached maximum levels. The injection of zymosan induced synthesis of PGE2 (measured by RIA) which reached maximum levels at 30 minutes, then declined slowly. Peptido-leukotriene levels (detected by bioassay, RIA and HPLC) increased rapidly after injection, reached a peak within an hour of injection and declined to undetectable levels within 4 hours. The early peptido-LT was predominantly LTC4, while later, LTE4 was the major component. LTD4 levels remained low throughout and no LTB4 was detected at any time. Indomethacin treatment elevated levels of peptido-LTs, reduced PGE2 levels and inhibited writhing. Phenidone reduced peptido-LT levels. In vitro studies demonstrated that zymosan stimulates LTC4 synthesis by peritoneal cells whereas LTE4, LTD4, LTB4 or monoHETES were not detectable (using HPLC methods). The source of enzymes responsible for the in vivo metabolism of LTC4 to LTD4 and LTE4 could not be identified.

Role of alpha-2 adrenergic receptors in the control of diarrhea and intestinal motility.

Oral administration of clonidine blocked diarrhea induced in mice by castor oil, prostaglandin E2, 5-hydroxytryptophan and bethanechol. The antidiarrheal action of clonidine was blocked by yohimbine but not by naloxone, propranolol, prazosin or cimetidine, indicating involvement of the alpha-2 adrenergic agonist activity of clonidine. Other alpha-2 adrenergic agonists (naphazoline, guanabenz, ergometrine, alpha-methylnorepinephrine) were also effective antidiarrheal agents, but alpha-1 (methoxamine, phenylephrine) or beta (isoproterenol) adrenergic agonists were not. Clonidine also blocked normal defecation in mice, an effect which was antagonized by yohimbine. Intracerebroventricular injection of clonidine produced no antidiarrheal effect, suggesting a peripheral site of action. Clonidine inhibited the distension-induced peristaltic reflex in the isolated guinea-pig ileum, an effect which was antagonized by yohimbine. The profound inhibition of peristaltic flow rate was due largely to inhibition of the rate of peristalsis, although an inhibition of the force of peristaltic contractions also contributed. This suggests that the inhibitory effects of alpha-2 adrenergic agonists on intestinal motility are due to the presence of inhibitory alpha-2 adrenergic receptors located on the pacemaker neurons of the enteric nervous system as well as presynaptically on postganglionic neurons innervating intestinal smooth muscle cells.

The interaction of tilorone and RMI 9563DA with the complement system.

RMI 9563DA inhibited the classical complement pathway in rat serum in vitro with an 150 of 51 micrograms/ml in diluted serum and 750 micrograms/ml in undiluted serum. Tilorone was much less active than RMI 9563DA in diluted serum and was inactive in undiluted serum. No complement inhibition could be detected in vivo following a near lethal dose of RMI 9563DA (25 mg/kg i.v.). Thus, complement inhibition is not the mechanism by which these compounds exert their anti-inflammatory activity. Both compounds increase serum hemolytic complement activity 24 h after administration to rats. With RMI 9563DA, this effect is entirely due to the local irritancy produced by s.c. administration of the compound. Tilorone, however, has a specific effect on complement synthesis which may be related to its ability to induce interferon.

Inhibition of arachidonic acid release as the mechanism by which glucocorticoids inhibit endotoxin-induced diarrhoea.

1 Dexamethasone blocked endotoxin-induced diarrhoea in mice, but not that induced by arachidonic acid or prostaglandin E2. 2 Indomethacin blocked endotoxin and arachidonic acid-induced diarrhoea, but not that induced by prostaglandin E2. 3 Codeine blocked all three forms of diarrhoea. 4 The above data, when considered in relation to literature reports that endotoxin induces prostaglandin synthesis, suggest that dexamethasone blocks diarrhoea by preventing the release of arachidonic acid, the substrate for prostaglandin biosynthesis. 5 The activities of indomethacin and dexamethasone in castor oil diarrhoea support the above conclusion and their inactivity in 5-hydroxytryptophan-induced diarrhoea confirms the absence of 'codeine-like' direct effects on the gut. 6 Other glucocorticoids (hydrocortisone, prednisolone) were also able to block endotoxin diarrhoea, but oestradiol, testosterone and progesterone did not. 7 The inhibitory action of dexamethasone on endotoxin diarrhoea could not be blocked by the protein synthesis inhibitor, cycloheximide, nor by the glucocorticoid receptor antagonist, progesterone. Thus, involvement of glucocorticoid receptor-mediated gene activation could not be demonstrated.

Selective effects of immunosuppressive agents against the delayed hypersensitivity response and humoral response to sheep red blood cells in mice.

The effects of cyclophosphamide, methotrexate, procarbazine, and azathioprine on the delayed-type hypersensitivity (DTH) response and antibody response to sheep red-blood cells were studied in mice. All the compounds inhibited the antibody response. Using appropriate dosage schedules, cyclophosphamide, methotrexate and procarbazine either potentiated or had no effect on DTH. In contrast, azathioprine inhibited the DTH response as measured 24 h after challenge but potentiated the DTH response measured 48 h after challenge. This system provides a convenient means of searching for drugs with selective effects on either DTH or antibody production.

Complement-inhibiting and anti-inflammatory properties of chlorazole fast pink 2BL.

Chlorazole fast pink 2BL inhibited the classical complement pathway in rat serum both in vivo and in vitro. The in vitro potency of chlorazole fast pink against the alternative pathway could not be assessed accurately but it was clearly less than that against the classical pathway. The compound appears to bind strongly to albumin with a resulting decrease in potency in undiluted as compared to diluted serum. Anti-inflammatory activity was observed in models of inflammation known to be highly complement-dependent (direct passive Arthus, zymosan oedema) but not in models in which complement is not involved (dextran oedema) or plays a minor role (carrageenan oedema).

An investigation of the effect of anti-inflammatory and anti-rheumatoid drugs in cell-mediated immune arthritis in guinea-pigs by microfocal radiography.

Guinea-pigs were sensitized by intra-articular injection of M. tuberculosis (2.0 mg) into one knee joint and arthritis induced in the opposite knee 21 days later by intra-articular injection of antigen (0.2 mg). The time course off the arthritic changes was followed for 25 days by assessment of knee swelling and hind-limb flexion. Twenty-eight days after challenge the experiment was terminated and radiographic changes evaluated by means of a microfocal X-ray unit. The effect of treatment with the anti-rheumatic drugs, D-penicillamine (100 mg/kg by mouth), dexamethasone (0.1 mg/kg i.p.), aspirin (100 mg/kg by mouth), chloroquine phosphate (30 mg/kg by mouth) and sodium aurothiomalate (2 mg/kg i.m.) given daily from 10 days after sensitization until 28 days after challenge was assessed. Changes in joint swelling and hind-limb flexion were maximal 1-3 days after challenge. None of the drug treatments influenced these parameters. Microfocal radiography showed marked changes in arthritis animals of all X-ray parameters measured. It was possible readily to identify joint erosion, trabecular loss and associated osteoporosis, the latter occurring proximal to and relatively remote from the affected joint. None of the treatments prevented the radiographic changes but exacerbation of trabecular number in the area of the epiphysis was seen with aspirin and D-penicillamine and of trabecular density further up the shaft of the femur was seen with D-penicillamine. The changes with D-penicillamine may reflect the potentiation of cell-mediated hypersensitivity with this drug reported by other workers. It was concluded that the model is not suitable for the detection of clinically active anti-rheumatic drugs but that microfocal radiography provides a sensitive index for the assessment of joint damage in small animals.