Arthritis In Lewis Rats Research Articles

Nanotechnology-based drug delivery for targeted therapy of autoimmune arthritis improves the therapeutic profile of anti-arthritis drugs

Abstract Rheumatoid arthritis (RA) is among the major human autoimmune diseases, and it affects over 1 percent of the population. Although many anti-arthritis drugs are available for RA therapy, their long-term use may lead to severe adverse effects. This, combined with high cost of many newer drugs, poses a hurdle for the effective control of RA as well as patient compliance with therapeutic regimens. Therefore, novel treatment modalities are required to overcome these limitations. In this context, we have developed a nanotechnology-based drug delivery system for targeted therapy of arthritic inflammation. Liposomal entrapment of drugs increases their shelf-life in vivo, whereas the display of a joint-homing peptide ligand on liposomal surface aids in guiding them to the site of inflammation. One such peptide (denoted as ART-1) was previously identified by us by phage peptide-display library screening of arthritic Lewis rats. When administered subcutaneously or intravenously, fluorescence-labeled ART-1 shows preferential homing to arthritic joints compared to normal (control) joints. We exploited this drug delivery system for the treatment of experimental arthritis with dexamethasone (Dex) using the rat adjuvant arthritis (AA) model. At the time of onset of AA, rats were treated with liposomal Dex, unpackaged (free) Dex, or the vehicle on alternate days. All rats were observed and graded regularly for arthritis severity. Sera obtained at the terminal step were tested for a panel of enzymes indicating tissue/organ toxicity. Liposomal Dex showed improved therapeutic profile (efficacy/toxicity ratio) over that of free Dex. We propose that this peptide ligand-targeted drug delivery approach can be adapted for effective control of human RA. "Supported by grants from NIH (R01 AT004321), Veterans Affairs (5 I01 BX002424) and Silo Pharma"

Type II collagen peptide Coll2-1 is an actor of synovitis

We evaluated the ability of Coll2-1, a type II collagen peptide, to activate pro-inflammatory pathways in synovial cells and to induce arthritis in Lewis rats. Human synoviocytes and chondrocytes from knee OA patients were cultured for 24h with/without Coll2-1 and/or purified immunoglobulin G (AS0619) binding specifically this peptide, and/or CLI-095, a TLR-4 signaling inhibitor and/or apocynin and diphenyleneiodonium, Reactive oxygen species (ROS) production inhibitors. The Interleukin (IL)-8 and Vascular Endothelium Growth Factor (VEGF) expression, the IL-8 production, the IκB-α and p65 phosphorylation and ROS were evaluated. Coll2-1 peptide, bovine type II collagen (CIA), streptococcal cell wall (SCW) or saline solution were injected into Lewis rats. The Coll2-1 peptide was injected subcutaneously (SC; 20-200μg/100μl/animal) or intra-articularly (IA; 0.5-5μg/50μl/animal) and compared to CIA injected in SC (200μg/100μl/animal) and SCW in IA (5μg/50μl/animal). The animals were injected on day 0 and monitored for 28 days. Histological lesions assessment was performed using an arthritis score. Coll2-1 peptide significantly increased IL-8 gene expression and production by synoviocytes. AS0619 and CLI-095 significantly decreased IL-8 expression. Coll2-1 induced p65 and IκBα phosphorylation and oxidative stress inhibitors decreased it. In human chondrocytes culture, Coll2-1 significantly increased MMP-3 and VEGF gene expression. In Lewis rats, CIA, SCW or Coll2-1 injection triggered arthritis. Like CIA or SCW, Coll2-1 induced synovitis, loss of cartilage proteoglycans, cartilage structure lesion and subchondral bone remodeling. Coll2-1 activates synoviocytes to produce IL-8 and induces arthritis in rat. These findings suggest that neutralizing Coll2-1 could be a therapeutic approach of arthritis.

The Micro-RNA Expression Profiles of Autoimmune Arthritis Reveal Novel Biomarkers of the Disease and Therapeutic Response.

Rheumatoid arthritis (RA) is a chronic autoimmune disease of the joints affecting about 0.3–1% of the population in different countries. About 50–60 percent of RA patients respond to presently used drugs. Moreover, the current biomarkers for RA have inherent limitations. Consequently, there is a need for additional, new biomarkers for monitoring disease activity and responsiveness to therapy of RA patients. We examined the micro-RNA (miRNA) profile of immune (lymphoid) cells of arthritic Lewis rats and arthritic rats treated with celastrol, a natural triterpenoid. Experimental and bioinformatics analyses revealed 8 miRNAs (miR-22, miR-27a, miR-96, miR-142, miR-223, miR-296, miR-298, and miR-451) and their target genes in functional pathways important for RA pathogenesis. Interestingly, 6 of them (miR-22, miR-27a, miR-96, miR-142, miR-223, and miR-296) were further modulated by celastrol treatment. Interestingly, serum levels of miR-142, miR-155, and miR-223 were higher in arthritic versus control rats, whereas miR-212 showed increased expression in celastrol-treated rats compared with arthritic rats or control rats. This is the first study on comprehensive miRNA expression profiling in the adjuvant-induced arthritis (AA) model and it also has revealed new miRNA targets for celastrol in arthritis. We suggest that subsets of the above miRNAs may serve as novel biomarkers of disease activity and therapeutic response in arthritis.

Adjuvant-induced rheumatoid arthritis in Lewis rats alters dopamine utilization and cytokine concentration in five brain regions

Abstract Word Rheumatoid arthritis (RA) is a chronic autoimmune disease which can cause inflammation in joints, bone damage, as well as disability. A reported symptom of RA includes cognitive dysfunction. This study aimed to measure brain dopamine utilization and inflammatory markers in an adjuvant-induced Lewis Rat model of RA. Arthritic intensity was scored based on physical examination of the four paws. Twenty-eight days after the adjuvant injection, control and RA group were sacrificed and the frontal lobe of the cerebral cortex (Cx), hippocampus (Hip), hypothalamus (Hypo), amygdala (Amg), and cerebellum (Cbl) dissected using the stereotaxic atlas of Paximos and Watson as a guide. High performance liquid chromatography (HPLC) was used to analyze the concentration of dopamine, DOPAC, and homovanillic acid (HVA). Pro-inflammatory cytokines IL-1β, IL-6, and IL-23, and the anti-inflammatory IL-10 were measured by enzyme-linked immunosorbent assays (ELISA) in tissue homogenates. The results indicate RA significantly (p<0.05) decreased the dopamine content in Cbl, Amg, Hip and Cx; DOPAC significantly decreased in Amg and Cbl; HVA significantly decreased in Cbl. RA increased the concentration of pro-inflammatory cytokines and the anti-inflammatory cytokine IL-10 in the Cbl. IL-1β increased in Hyp, and Amg with a concomitant decrease in IL-10. IL-6 and IL-23 increased in Cx, Hip, Hypo, and Amg with a concomitant decrease in IL-10. This data provides evidence that adjuvant-induced RA alters the inflammation status and dopamine utilization of these brain regions. The results are useful in the development of novel drug therapies for the millions of RA patients at risk of developing mood changes, memory loss, and other cognitive deficits.

APL-1, an altered peptide ligand derived from heat-shock protein, alone or combined with methotrexate attenuates murine collagen-induced arthritis.

Induction of tolerance to autoantigens in vivo is a complex process that involves several mechanisms such as the induction of regulatory T cells and changes in the cytokine and chemokine profiles. This approach represents an attractive alternative for treatment of autoimmune diseases. APL-1 is an altered peptide ligand derived from a novel CD4+T cell epitope of human heat-shock protein of 60kDa (HSP60), an autoantigen involved in the pathogenesis of rheumatoid arthritis (RA). We have shown previously that this peptide efficiently inhibited the course of adjuvant-induced arthritis in Lewis rats and induced regulatory T cell (Treg) in ex vivo assay with PBMC isolated from RA patients. This study was undertaken to evaluate the therapeutic effect of APL-1 and its combination with methotrexate (MTX) in collagen-induced arthritis (CIA). CIA was induced in male DBA/1 mice at 8weeks of age by immunization with chicken collagen. APL, MTX or both were administrated beginning from arthritis onset. Therapeutic effect was evaluated by arthritis and joint pathologic scores. In addition, TNFα and IL-10 in sera were measured by ELISA. Treg induction was assessed by FACS analysis. APL-1 inhibits efficiently the course of arthritis in CIA, similar to MTX. In addition, therapy with APL-1 plus MTX reduced CIA in mice, associated with an increase in Treg. These facts reinforce the therapeutic possibilities of APL-1 as a candidate drug for treatment of RA.

Inhibition of Arthritis in the Lewis Rat by Apolipoprotein A-I and Reconstituted High-Density Lipoproteins

This study questions whether high-density lipoproteins (HDLs) and apolipoprotein A-I inhibit joint inflammation in streptococcal cell wall peptidoglycan-polysaccharide (PG-PS)-induced arthritis in female Lewis rats. Administration of PG-PS to female Lewis rats caused acute joint inflammation after 4 days, followed by remission by day 8. The animals subsequently developed chronic joint inflammation that persisted until euthanasia at day 21. Treatment with apolipoprotein A-I 24 hours before and 24 hours after PG-PS administration reduced the acute and chronic joint inflammation. Treatment with apolipoprotein A-I at days 7, 9, and 11 after PG-PS administration reduced the chronic joint inflammation. Treatment with apolipoprotein A-I or reconstituted HDLs consisting of apolipoprotein A-I complexed with phosphatidylcholine 24 hours before and at days 1, 7, 9, and 11 after PG-PS administration reduced acute and chronic joint inflammation. Treatment with apolipoprotein A-I also reduced the inflammatory white blood cell count, synovial fluid proinflammatory cytokine levels, synovial tissue macrophage accumulation, as well as toll-like receptor 2, and inflammatory cytokine expression. At the molecular level, preincubation of human monocyte-derived macrophages with apolipoprotein A-I or reconstituted HDLs before PG-PS stimulation inhibited the PG-PS-induced increase in toll-like receptor 2 and myeloid differentiation primary response gene (88) mRNA levels, nuclear factor-κB activation, and proinflammatory cytokine production. The effects of apolipoprotein A-I and reconstituted HDLs were abolished by transfecting the human monocyte-derived macrophages with ATP-binding cassette transporter A1 or G1 siRNA. Apolipoprotein A-I and reconstituted HDLs attenuate PG-PS-induced arthritis in the rat. Studies in human monocyte-derived macrophages indicate that this benefit may be because of the inhibition of toll-like receptor 2 expression and decreased nuclear factor-κB activation in macrophages.

Chinese Herbal Formula Huo-Luo-Xiao-Ling Dan Protects against Bone Damage in Adjuvant Arthritis by Modulating the Mediators of Bone Remodeling

Huo-luo-xiao-ling dan (HLXL) is an herbal mixture that has long been used in traditional Chinese medicine for the treatment of rheumatoid arthritis (RA) and other inflammatory disorders. Despite the availability of potent conventionally used drugs for RA, their limited efficacy in a proportion of patients coupled with their high cost and severe adverse effects has necessitated the search for novel therapeutics for this debilitating disease. Further, the control of both inflammation and bone damage is essential for effective management of arthritis. The aim of our study was to evaluate the efficacy of HLXL against arthritic bone damage in adjuvant arthritis (AA) model of RA. Our results show that HLXL treatment suppressed inflammatory arthritis and reduced bone and cartilage damage in the joints of arthritic Lewis rats. HLXL-induced protection against bone damage was mediated primarily via inhibition of mediators of osteoclastic bone remodeling (e.g., receptor activator of nuclear factor kappa-B ligand; RANKL), skewing of RANKL/osteoprotegerin (OPG) ratio in favor of antiosteoclastic activity, reduction in the number of osteoclasts in the arthrodial joint's bone, and inhibition of cytokine production and MMP activity. Our results suggest that HLXL might offer a promising alternative/adjunct treatment for both inflammation and bone damage in RA.

Suppression of autoimmune arthritis by Celastrus-derived Celastrol through modulation of pro-inflammatory chemokines

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the synovial joints, deformities, and disability. The prolonged use of conventional anti-inflammatory drugs is associated with severe adverse effects. Therefore, there is an urgent need for safer and less expensive therapeutic products. Celastrol is a bioactive component of Celastrus, a traditional Chinese medicine, and it possesses anti-arthritic activity. However, the mechanism of action of Celastrol remains to be fully defined. In this study based on the rat adjuvant-induced arthritis (AA) model of RA, we examined the effect of Celastrol on two of the key mediators of arthritic inflammation, namely chemokines and their receptors, and related pro-inflammatory cytokines. We treated arthritic Lewis rats with Celastrol (200μg/rat) or its vehicle by daily intraperitoneal (ip) injection beginning at the onset of AA. At the peak phase of AA, the sera, the draining lymph node cells, spleen adherent cells, and synovial-infiltrating cells of these rats were harvested and tested. Celastrol-treated rats showed a significant reduction in the levels of chemokines (RANTES, MCP-1, MIP-1α, and GRO/KC) as well as cytokines (TNF-α and IL-1β) that induce them, compared to the vehicle-treated rats. However, Celastrol did not have much effect on cellular expression of chemokine receptors except for an increase in CCR1. Further, Celastrol inhibited the migration of spleen adherent cells in vitro. Thus, Celastrol-induced suppression of various chemokines that mediate cellular infiltration into the joints might contribute to its anti-arthritic activity. Our results suggest that Celastrol might offer a promising alternative/adjunct treatment for RA.

Microarray-based gene expression profiling reveals the mediators and pathways involved in the anti-arthritic activity of Celastrus-derived Celastrol

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints. The prolonged use of non-steroidal anti-inflammatory drugs and other newer drugs is associated with severe adverse reactions. Therefore, there is a need for newer anti-arthritic agents. Celastrol, a bioactive component of the Chinese herb Celastrus, possesses anti-arthritic activity as tested in the adjuvant arthritis (AA) model of rheumatoid arthritis (RA). However, the mechanism of action of Celastrol has not been fully defined. We reasoned that microarray analysis of the lymphoid cells of Celastrol-treated arthritic animals might provide vital clues in this regard. We isolated total RNA of the draining lymph node cells (LNCs) of Celastrol-treated (Tc) and vehicle-treated (Tp) arthritic Lewis rats that were restimulated in vitro with the disease-related antigen, mycobacterial heat-shock protein 65 (Bhsp65), and tested it using microarray gene chips. Also tested was RNA from LNCs of control arthritic rats just before any treatment (T0). Seventy six genes involved in various biological functions were differentially regulated by Bhsp65 in LNCs of Tp group, and 19 genes among them were shared by the Tc group. Furthermore, a group of 14 genes was unique to Tc. When Tc and Tp were compared, many of the Bhsp65-induced genes were related to the immune cells, cellular proliferation and inflammatory responses. Our results revealed 10 differentially expressed genes and 14 pathways that constituted the “Celastrol Signature”. Our results would help identify novel targets for RA therapy.

Celastrus suppresses the progression of autoimmune arthritis in rats by reducing cellular migration as well as production of pro-inflammatory cytokines and chemokines (116.11)

Abstract Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disease that affects the synovial joints. The target organ pathology in arthritis is primarily a result of inflammation caused by T cells and other leukocytes accumulating within the joints. These leukocytes produce pro-inflammatory cytokines and chemokines. The prolonged use of conventional anti-inflammatory drugs by RA patients is associated with severe adverse effects. Therefore, the use of natural plant products is on the rise. However, the mechanism of action of such products is poorly defined. Using the rat adjuvant arthritis model of RA, we assessed whether Celastrus aculeatus Merril (Celastrus), a traditional Chinese medicine, can limit the disease, and also examined the mechanisms underlying its anti-arthritic activity. We tested the draining lymph node cells and synovial-infiltrating cells (SIC) of Celastrus-treated versus control rats using flow cytometry and assays for cytokines and chemokines. Arthritic Lewis rats treated with Celastrus had reduced inflammation and pathological damage in the joints compared to controls. Further, Celastrus treatment reduced the frequency of Th17 in the pathogenic cellular infiltrate within the joint, limited the Th17 cell differentiation in lymphoid tissues, and decreased the production of pro-inflammatory cytokines and chemokines (IL-17, IL-6, IL-1β, and RANTES) by the SIC. We suggest that Celastrus be further evaluated as a complementary therapy for RA.

Modified Huo-Luo-Xiao-Ling Dan Suppresses Adjuvant Arthritis by Inhibiting Chemokines and Matrix-Degrading Enzymes

Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting the joints that can lead to deformities and disability. The prolonged use of conventionally used drugs is associated with severe adverse reactions. Therefore, safer and less expensive therapeutic products are continually being sought. Huo-Luo-Xiao-Ling dan (HLXL), a traditional Chinese herbal mixture, and its modified versions possess anti-arthritic activity. In this paper, we examined the influence of modified HLXL on two of the key mediators of arthritic inflammation and tissue damage, namely, chemokines and matrix-metalloproteinases (MMPs) in the rat adjuvant-induced arthritis (AA) model of RA. We treated arthritic Lewis rats with HLXL (2.3 g/kg) by daily gavage beginning at the onset of AA. The control rats received the vehicle. At the peak phase of AA, rats were sacrificed and their draining lymph node cells (LNC) and spleen adherent cells (SAC) were tested. The HLXL-treated rats showed a significant reduction in the levels of chemokines (RANTES, MCP-1, MIP-1α, and GRO/KC), MMPs (MMP 2 and 9), as well as cytokines (IL-6 and IL-17) that induce them, compared to the control vehicle-treated rats. Thus, HLXL controls arthritis in part by suppressing the mediators of immune pathology, and it might offer a promising alternative/adjunct treatment for RA.

Glucocorticoids in nano-liposomes administered intravenously and subcutaneously to adjuvant arthritis rats are superior to the free drugs in suppressing arthritis and inflammatory cytokines

We have previously shown that intravenous (i.v.) treatment with sterically stabilized nano-liposomes (NSSL) actively remote-loaded with the glucocorticoid (GC) methylprednisolone hemisuccinate (NSSL-MPS) or betamethasone hemisuccinate (NSSL-BMS) significantly decreased severity of adjuvant arthritis in Lewis rats (a model of human rheumatoid arthritis) throughout all disease stages. Here, we compared i.v. or subcutaneous (s.c.) weekly treatment with each of the two NSSL-GC to weekly or daily treatment with the free drugs or with the TNF-α antagonists Infliximab and Etanercept. Therapeutic efficacy and effects on the profile of pro-inflammatory (IL-6, TNF-α, and INF-γ) and anti-inflammatory (IL-10 and TGF-β) cytokines in rat sera and splenocyte tissue culture supernatants were compared to those of the liposomal and free drugs. Both s.c. and i.v. NSSL-GC suppressed arthritis significantly, compared to higher doses of the free drugs or to TNF-α antagonists. NSSL-GC also suppressed the secretion of pro-inflammatory cytokines, but did not change the levels of TGF- β. The highly efficacious anti-inflammatory therapeutic feature of these nano-drugs makes them candidates for treatment of human rheumatoid arthritis.

Topical Dermal Application of Essential Oils Attenuates the Severity of Adjuvant Arthritis in Lewis Rats

This study was aimed at examining the effect of an ointment containing essential oils (EO) on the severity of adjuvant arthritis (AA), an experimental model of human rheumatoid arthritis (RA), in Lewis rats and to define the underlying mechanisms. At the onset of AA, the rats received topical application twice daily of an ointment containing 20% EO or placebo ointment. The synovial fluid (SF) and synovium-infiltrating cells (SIC) of rats were tested for pro-inflammatory cytokines TNF-α and IL-1β. The hind paws and skin were examined histologically. The activity/level of matrix metalloproteinases (MMPs) and anti-mycobacterial heat-shock protein 65 (Bhsp65) antibodies were tested. Arthritic rats treated with ointment containing EO developed less severe clinical arthritis compared with the controls, and this activity was attributable to EO and not to the carrier oil. The levels of TNF-α and IL-1β, and the activity of MMPs in SF and SIC-lysate were significantly reduced in EO-treated arthritic rats compared with the controls. However, the levels of anti-Bhsp65 antibodies were unaffected by treatment. Thus, topical dermal delivery of EO-containing ointment down-modulates the severity of AA in Lewis rats by inhibiting defined mediators of inflammation. Such ointments should be tested in patients with RA and other arthritic conditions.

An orally active natural product based anti-inflammatory small molecule (RNV-2005) (117.19)

Abstract RNV-2005 is a natural product based molecule originally designed based on its interaction with glucocorticoid receptor (GR). The effective dose of RNV-2005 was found to be in the range of 0.3-10 mg/kg in rodent models. Unlike most analgesic and NSAID compounds, RNV-2005 was non-ulcerogenic up to a tested dose of 200 mg/kg. In an acute toxicity study, RNV-2005 did not show any adverse effect at a highest tested dose of 2000 mg/kg. The LD50 dose of RNV-2005 is therefore not established. A 70-80% reduction in paw edema was observed in animals treated with RNV-2005 at a dose of 10 mg/kg in carrageenan induced paw edema rat model. The analgesic properties of RNV-2005 was measured using tail flick latency and acid induced writhing reflex in Albino mice at doses 0.3-3 mg/kg. In a LPS induced ex-vivo study in SW mice, reduction of cytokines such as TNFα, IL-1β, IL-6 and IL-12 was measured in plasma. Further studies of RNV-2005 in collagen-induced arthritis in DBA/1J mice, adjuvant induced arthritis in Lewis rats, PK studies and in vitro inhibition assays for COX-1 and COX-2 in cell based assays will be presented. Similarly, the molecular mechanisms underlying GR mediated inflammatory responses, with an emphasis on NF-κB and AP-1 and their respective signaling pathways will be discussed. These results with preliminary safety data encourage the clinical potential of RNV-2005 towards development of a novel anti-inflammatory molecule with high therapeutic index.

The Role of Lipid Mediators in the Phathogenesis of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic disease characterized by synovial inflammation and polyarthritis. Inflammatory mediators activate fibroblast-like synovial cells which exhibit very unique characteristics in the process of bone resorption. The rheumatoid synoviocytes produce high levels of prostaglandin E2 (PGE2) production through increased cyclooxygenase (COX)-2 expression. PGE2 is thought to be a major PG species working in RA pathogenesis and PGE2 exhibits various biological actions: for example, PGE2 mediates some inflammatory responses and bone resorption as well as activation of osteoclasts. Prostaglandin E synthase (PGES) is an enzyme that acts downstream of COX-2 and catalyzes the final step of PGE2 biosynthesis. Microsomal PGES (mPGES)-1 shows the coordinated induction with COX-2 under inflammatory conditions. Sphigosine-1-phosphate (S1P) is the final metabolite of the sphingolipid pathway and controls a wide variety of essential cellular processes. S1P is involved in various pathologic conditions and has been implicated as an important mediator in angiogenesis, cancer, and autoimmune diseases such as RA. S1P1 receptor is strongly expressed in RA synovium, and S1P enhances inflammatory cytokine-induced COX-2 expression and PGE2 production. FTY720 (fingolimod) is a high-affinity agonist for S1P receptors and induces internalization of the receptors. FTY720 has been shown to be a useful agent for the prevention of transplant rejection and autoimmune diseases such as multiple sclerosis and RA. Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor and member of the nuclear receptor superfamily. PPARγ is activated by a range of synthetic and naturally occurring substances, including anti-diabetic thiazolidinediones such as troglitazone and 15-deoxy-Δ12-14-PGJ2 (15d-PGJ2). PPARγ ligands suppress arthritis in Lewis rats with adjuvant- induced arthritis, and induce rheumatoid synoviocye apoptosis. This mini-review focuses on the role of lipid mediators in the pathogenesis of RA.

(Z)-5-(4-methoxybenzylidene) thiazolidine-2, 4-dione ameliorates the adjuvant-induced arthritis via inhibiting the migration of macrophage and down-regulating the cytokine mRNA expression

In our previous study, we have demonstrated that (Z)-5-(4-methoxybenzylidene) thiazolidine-2, 4-dione (SKLB010), a novel analogue of thiazolidinediones, showed protection against concanavalin A (Con A)-induced hepatitis via inhibiting the migration of macrophages and the expression of pro-inflammatory mediators. In present study, Transwell assay was applied to study the effects of SKLB010 on macrophage-like Raw264.7 cell migration and we investigated the effects of SKLB010 on NO generation in vitro. We also studied in vivo effects of SKLB010 on adjuvant-induced arthritis in Lewis rats by oral administration. It was proved that SKLB010 depressed cells migration and the production of NO in Raw264.7 cells in a dose-dependent way. Our results also indicated that oral administration of 50 mg/kg SKLB010 markedly resulted in a clear suppression of clinical signs compared to untreated groups, showing as markedly reduction of paw swelling and inhibition of body weight decreasing. The improvement in disease severity was accompanied by suppression of CD68-positive cells in knee joint and by inhibition of production in pro-inflammatory cytokines of TNF-α, IL-1β and IL-6 in serum. The elevated TNF-α, IL-1β and iNOS mRNA expression in tissue were down-regulated after treatment with SKLB010. SKLB010 also significantly inhibited the histological progress of RA. These data indicate that SKLB010 is a potent anti-inflammatory therapeutic agent targeting the inflammatory response of macrophages.

Intricate regulation of IL-17 response by IFN-γ and IL-27 determines the outcome of the pathogenetic events in experimental autoimmune arthritis (143.18)

Abstract Recent advances in defining the new subsets of T cells and the related newer cytokines have stimulated new undertakings in examining the pathogenesis of autoimmunity beyond the traditional Th1-Th2 paradigm. We report here novel findings regarding the roles of IFN-γ, IL-17 and IL-27 in the adjuvant arthritis (AA) model of human rheumatoid arthritis that challenge the traditional viewpoints. We hypothesized that the temporal kinetics and the relative levels of antigen-induced cytokine responses determined both the propagation of AA as well as resistance to the disease. We determined the cytokine responses of arthritic Lewis rats at different phases of the disease and that of the arthritis-resistant, MHC-compatible Wistar Kyoto (WKY) rats given a potentially arthritogenic challenge. In Lewis rats, IL-17 dominated the early (incubation phase) response, whereas IFN-γ and IL-27 appeared late (peak phase). Intriguingly, in WKY rats, all these 3 cytokines appeared concomitantly and early, showing that IFN-γ and IL-27 successfully pre-empted the pathogenicity of IL-17. This cross-regulation, along with the novel finding of upregulation of IL-27 by IFN-γ, was validated through mechanistic experiments, including the AA-protective effect of IFN-γ injected in vivo, and the differential modulation of specific signaling biomolecules and inflammatory mediators in the joints. These results provide a novel IL-17-IL-27-centered perspective on the pathogenesis of autoimmune arthritis.

Prevention of adjuvant arthritis in rats by a nonapeptide from the 65-kD mycobacterial heat-shock protein

Adjuvant arthritis in Lewis rats is a model of T cell-mediated autoimmune arthritis resembling human rheumatoid arthritis. A nonapeptide from the 65-kD heat-shock protein of Mycobacterium bovis BCG, amino acid sequence 180-188, has been described to carry the dominant immunogenic epitope(s) for both arthritis-protective and arthritogenic T cell clones. Here we demonstrate that immunizations with the synthetic nonapeptide completely protected rats against adjuvant arthritis induced by M. tuberculosis. Interestingly, deletion of the N-terminal threonine of the nonapeptide resulted in loss of the protective activity. Pretreatments with the nonapeptide resulted in an immune response to the nonapeptide and to M. tuberculosis. After immunizations with the synthetic nonapeptide, only low titres of nonapeptide-specific antibodies were produced, whereas a significant cellular immune response to the nonapeptide was observed. In addition, the protection was transferable to naive rats by spleen T cells. These findings document the requirement of a T cell-specific immune response to the dominant epitope of the 65-kD mycobacterial heat-shock protein for the protection against adjuvant arthritis and suggest the feasibility of immune intervention in autoimmune arthritis through the use of synthetic peptides.

Modeling Corticosteroid Effects in a Rat Model of Rheumatoid Arthritis I: Mechanistic Disease Progression Model for the Time Course of Collagen-Induced Arthritis in Lewis Rats

A mechanism-based model was developed to describe the time course of arthritis progression in the rat. Arthritis was induced in male Lewis rats with type II porcine collagen into the base of the tail. Disease progression was monitored by paw swelling, bone mineral density (BMD), body weights, plasma corticosterone (CST) concentrations, and tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and glucocorticoid receptor (GR) mRNA expression in paw tissue. Bone mineral density was determined by PIXImus II dual energy X-ray densitometry. Plasma CST was assayed by high-performance liquid chromatography. Cytokine and GR mRNA were determined by quantitative real-time polymerase chain reaction. Disease progression models were constructed from transduction and indirect response models and applied using S-ADAPT software. A delay in the onset of increased paw TNF-alpha and IL-6 mRNA concentrations was successfully characterized by simple transduction. This rise was closely followed by an up-regulation of GR mRNA and CST concentrations. Paw swelling and body weight responses peaked approximately 21 days after induction, whereas bone mineral density changes were greatest at 23 days after induction. After peak response, the time course in IL-1beta, IL-6 mRNA, and paw edema slowly declined toward a disease steady state. Model parameters indicate TNF-alpha and IL-1beta mRNA most significantly induce paw edema, whereas IL-6 mRNA exerted the most influence on BMD. The model for bone mineral density captures rates of turnover of cancellous and cortical bone and the fraction of each in the different regions analyzed. This small systems model integrates and quantitates multiple factors contributing to arthritis in rats.

(118) CG100649, a novel dual-acting COX-2 and carbonic anhydrase inhibitor: Preclinical pharmacology

CG100649 is a novel dual-acting cyclooxygenase-2 (COX-2) and carbonic anhydrase (CA) inhibitor which is being developed for the treatment of osteoarthritis, rheumatoid arthritis, and acute pain. CG100649 has moderate COX-2 selectivity over COX-1, ranging from 15-fold in human cells (whole blood, platelets and macrophages) to 45-fold in mouse peritoneal macrophages. In the ex vivo COX-1 activity assay, CG100649 showed weaker COX-1 inhibitory activity than indomethacin in rat whole blood. CG100649 was a potent inhibitor of inflammation in adjuvant-induced arthritis and collagen-induced arthritis in Lewis rats (paw swelling ED50s were 0.10 and 0.22 mg/kg/day, respectively).