Suppression of collagen-induced arthritis by combination cyclosporin A and methotrexate therapy

Louvain (LOU) rats were administered either methotrexate (MTX; 0.3 mg/kg/week or 0.8 mg/kg/week intraperitoneally), cyclosporin A (CSA; 4 mg/kg/day or 10 mg/kg/day continuous infusion via osmotic pump), or a combination of both agents. The rats were immunized with native type II collagen (CII) to determine the effects of these agents on collagen-induced arthritis, an animal model of rheumatoid arthritis. A significant decrease in the incidence (P less than 0.01) and severity of arthritis by clinical (P less than 0.05) and radiographic assessments (P less than 0.05) was found in recipients of combination therapy, compared with controls. Delayed-type hypersensitivity reactions to CII were measured on day 26, and production of IgG antibody to CII was measured on days 7, 14, and 26. IgG antibody was evident by day 7, and titers were near-maximal by day 14. Both delayed-type hypersensitivity and antibodies to CII were reduced in animals that received the higher dosage of CSA. Liver, kidney, and spleen specimens obtained from rats treated with CSA and MTX demonstrated no histologic abnormalities on light microscopy, compared with controls. These studies indicate that CSA and MTX in combination is a safe and effective therapy for collagen-induced arthritis and may be useful in the treatment of rheumatoid arthritis.     

Inhibitory effect of cyclosporin A on erythroid and stromal colonies

Cyclosporin A is used to prevent graft-versus-host disease (GvHD) following bone marrow transplantation (BMT) and it has been implicated in reducing the time to engraftment for leukaemia and aplastic anaemia patients. To evaluate the effect of cyclosporin A on engraftment, the proliferative capacity of bone marrow progenitors (CFU-E, CFU-F and CFU-C) was assessed both in vitro and following treatment with cyclosporin A over a 9-week period using an animal model. Cyclosporin had a differential effect on the haemopoietic progenitors, with the myeloid series unaffected at therapeutic concentrations. Both erythroid and stromal progenitors were significantly inhibited at similar concentrations. The mechanism by which cyclosporin A enhances engraftment remains unclear; however, it is not mediated by enhancing any of the haemopoietic progenitors.     

Infliximab treatment in combination with cyclosporin A in patients with severe refractory rheumatoid arthritis

OBJECTIVE: To investigate whether infliximab can be used in combination with cyclosporin A (CsA) in patients with refractory rheumatoid arthritis (RA) who cannot tolerate methotrexate (MTX). MATERIALS AND METHODS: Eighteen patients with refractory RA receiving low dose CsA (2 mg/kg/day) and prednisone (5 mg/day) were treated with intravenous infliximab. The patients were given infliximab (3 mg/kg weight) at 0, two, six, and every eight weeks thereafter for a total period of 12 months. Clinical improvement was evaluated according to the American College of Rheumatology (ACR) 20% response criteria. RESULTS: Eighty per cent of patients receiving the combination treatment with CsA and infliximab achieved the 20% ACR criteria for response to treatment, whereas 39% satisfied the 50% response criteria. In addition, a 76% reduction in swollen and tender joint count was found. Finally, a reduction in C reactive protein and erythrocyte sedimentation rate was maintained throughout the study. In general, treatment was well tolerated, with minimal adverse drug reactions. Two patients dropped out; one because of an immediate hypersensitivity reaction and the other because of the development of pulmonary tuberculosis. CONCLUSION: Multiple infusions of infliximab and low doses of CsA improve patients with refractory RA. It seems that CsA may be an alternative disease modifying drug to be used in combination with infliximab in patients with refractory RA who cannot tolerate MTX.     

Effects of combination M40403 and dexamethasone therapy on joint disease in a rat model of collagen-induced arthritis

OBJECTIVE: To investigate the effects of combination therapy with M40403, a superoxide dismutase mimetic (SODm), and dexamethasone (DEX) on collagen-induced arthritis (CIA) in rats. METHODS: CIA was elicited in Lewis rats by an intradermal injection of 100 mul of an emulsion of bovine type II collagen (CII) in Freund's incomplete adjuvant (IFA) at the base of the tail. On day 21, a second injection of CII in IFA was administered at the base of the tail. RESULTS: Lewis rats developed erosive arthritis of the hind paw when immunized with an emulsion of CII in IFA. The histopathology of CIA included erosion of the articular cartilage at the joint margins and subchondral bone resorption. Immunohistochemical analysis for nitrotyrosine, poly(ADP-ribose) polymerase (PARP), inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX-2) revealed positive staining in inflamed joints of collagen-treated rats. The combination therapy with M40403 2 mg/kg and DEX 0.01 mg/kg significantly reduced the development of the inflammatory process and reduced the degree of staining for iNOS, COX-2, nitrotyrosine, and PARP. No significant difference in the degree of staining between the combination therapy and the higher dose of DEX (0.1 mg/kg) was found. Furthermore, radiographic evidence of protection from bone resorption was apparent in the tibiotarsal joints of rats that received the combination therapy. CONCLUSION: This study shows that combination therapy with M40403 and DEX reduced the degree of chronic inflammation and tissue and bone damage associated with CIA in the rat. It supports the possible use of SODm in combination with steroids to reduce the dose necessary and the side effects related to the use of steroids in the management of chronic diseases such as rheumatoid arthritis.     

白藜芦醇对胶原诱导大鼠关节炎的抗炎作用研究

目的 观察白藜芦醇对胶原诱导大鼠关节炎(CIA)的抗炎作用.方法 选择牛Ⅱ型胶原(CⅡ)为免疫原,建立CIA模型.选取42只造模成功的大鼠随机分为7组:模型组;来氟米特(LEF)对照组;白芍总苷(TGP)对照组;甲氨蝶呤(MTX)对照组;白藜芦醇(Res)小剂量组;白藜芦醇中剂量组;白藜芦醇高剂量组.采用关节炎指数评分(AI)法对大鼠关节炎症程度进行评分;采用酶联免疫吸附试验(ELISA)方法测定各组大鼠血清中抗CⅡ抗体水平.结果 AI评分:低剂量白藜芦醇组与模型组比较差异无统计学意义(12.3±0.5与12.8±0.4);高剂量白藜芦醇组低于中、低剂量组(6.0±0.6与8.2±1.0,12.3±0.5,P＜0.01).高剂量白藜芦醇组低于白芍总苷组(6.0±0.6与8.8±0.8,P＜0.01);LEF组低于TGP组、MTX组、3组白藜芦醇组(4.7±0.5与8.9±0.8,6.4+0.5,P＜0.01);血清抗CⅡ抗体水平:模型组明显高于LEF组、MTX组、TGP组及高、中剂量白藜芦醇组(0.928±0.021与0.391±0.016,0.503±0.010,0.525±0.015,0.507±0.01,0.570±0.021,P＜0.01),而与低剂量白藜芦醇组之间差异无统计学意义(0.928±0.021与0.908±0.026);高剂量白藜芦醇组低于白芍总苷组及低剂量白藜芦醇组(0.507±0.01与0.525±0.015,0.908±0.026,P＜0.01);LEF组低于高、中剂量白藜芦醇组(0.391±0.026与0.507±0.010,0.570±0.021,P＜0.01).结论 ①高、中剂量白藜芦醇能够缓解CIA的炎症症状,而低剂量白藜芦醇则无此作用.②中等剂量白藜芦醇对CIA大鼠的短期疗效与TGP相当,高剂量白藜芦醇的疗效强于TGP,但弱于LEF.③白藜芦醇能够抑制血清抗CⅡ抗体的生成.     

Effect of estrogen replacement therapy on arthritis and bone mineral density in estrogen-replete rats with collagen-induced arthritis

The influence of estrogen therapy on changes in arthritis and bone mineral density (BMD) was evaluated using an estrogen-replete collagen-induced arthritis (CIA) rat model. Seven-month-old female Sprague–Dawley rats (n = 30) were divided into the three groups; control (CONT), collagen sensitization (CIA), and CIA + 17β-estradiol administration for 7 weeks (CIA + E). BMD was measured by peripheral quantitative computed tomography in the proximal tibia every 2 weeks. Eight weeks after the initial sensitization the rats were killed and histomorphometry of tibia was performed. The hind paw thickness increased with time in CIA rats and there was a significant difference between CONT and CIA at 8 weeks after the initial sensitization. Estrogen tended to make the development of arthritis milder. In CIA, BMD at metaphyseal cancellous bone began to decrease with the onset of arthritis and became significantly lower than in CONT after 8 weeks. Compared with the CIA, the deterioration in BMD was inhibited in CIA + E. Histomorphometrical parameters of bone resorption were increased in CIA compared with CONT, and those elevations were reduced by estrogen treatment, but estrogen had no effect on bone formation parameters. In conclusion, estrogen could partially suppress arthritis and bone loss in estrogen-replete rats as well as estrogen-deplete ones.     

Protective effect of soy protein on collagen-induced arthritis in rat

To evaluate preventive and therapeutic effects of soy protein on collagen-induced arthritis rats. Sprague–Dawley rats immunized with bovine type II collagen emulsified in adjuvant and treated with soy protein (7?g/kg), dexamethasone (1?mg/kg), and casein (in control groups) by daily gavages feedings for 30?days. Score of arthritis recorded every day for each paws of animal. Tumor necrosis factor-alpha, interleukin6, leptin, and adiponectin were measured in serums. Treatment with soy protein resulted in significant delay in time to onset of arthritis as well as significantly decreased arthritis incidence, clinical arthritis severity score, histopathological arthritis severity score, and in vivo cell-mediated immunity to collagen (P?<?0.05). Administration of soy protein significantly suppressed the progression of collagen II-induced arthritis and inhibited the production of tumor necrosis factor-alpha, interleukin6, leptin, and adiponectin. Soy protein appeared to be a potent immunomodulatory inhibitor of collagen II-induced arthritis in rats. It could delay onset of RA and reduced cartilage erosion and synovitis inflammation. Therefore, it may be a useful protein in the prevention and treatment of rheumatoid arthritis patient.     

α黑素细胞刺激素对大鼠胶原性关节炎的保护作用

目的 观察α黑素细胞刺激素(α—MSH)对大鼠胶原性关节炎(CIA)发病过程的影响。方法 经牛Ⅱ型胶原(bcⅡ)诱导建立大鼠关节炎动物模型，随机分为4组：正常对照组，CIA对照组，α—MSH低剂量组和α—MSH高剂量组，每组8只，于致敏当天连续腹腔注射不同剂量的α—MSH或PBS，观察比较4组大鼠体重、足爪容积、关节炎指数、病理评分、X线放射学表现等变化。结果α—MSH可缓解大鼠关节炎症状，减轻滑膜增生和炎性细胞浸润，抑制关节骨质破坏。结论 α—MSH可抑制大鼠胶原性关节炎发病，具有潜在的临床应用价值。     

Cyclosporin A monotherapy versus cyclosporin A and methotrexate combination therapy in patients with early rheumatoid arthritis: a double blind randomised placebo controlled trial

OBJECTIVE: To compare the efficacy and toxicity of cyclosporin A (CsA) monotherapy with CsA plus methotrexate (MTX) combination therapy in patients with early rheumatoid arthritis (RA). PATIENTS AND METHODS: 120 patients with active RA, rheumatoid factor positive and/or erosive, were randomly allocated to receive CsA with MTX (n=60) or CsA with placebo (n=60). Treatment with CsA was started in all patients at 2.5 mg/kg/day and increased to a maximum of 5 mg/kg/day in 16 weeks. MTX was started at 7.5 mg/week and increased to a maximal dose of 15 mg/week at week 16. Primary outcomes were clinical remission (Pinals criteria) and radiological damage (Larsen score), at week 48. RESULTS: Treatment was discontinued prematurely in 27 patients in the monotherapy group (21 because of inefficacy, and six because of toxicity) and in 26 patients in the combination therapy group (14 and 12, respectively). At week 48, clinical remission was achieved in four patients in the monotherapy group and in six patients in the combination therapy group (p=0.5). The median Larsen score increased to 10 (25th, 75th centiles: 3.5; 13.3) points in the monotherapy group and to 4 (1.0; 10.5) points in the combination therapy group (p=0.004). 28/60 (47%) of patients in the monotherapy group v 34/60 (57%) of patients in the combination therapy group had reached an American college of Rheumatology 20% (ACR20) response (p=0.36) at week 48; 15/60 (25%) v 29/60 (48%) of patients had reached an ACR50 response (p=0.013); and 7 (12%) v 12 (20%) of patients had reached an ACR70 response (p=0.11).Their was a tendency towards more toxicity in the combination therapy group. CONCLUSIONS: In patients with early RA, neither CsA plus MTX combination therapy nor CsA monotherapy is very effective in inducing clinical remission. Combination therapy is probably better at improving clinical disease activity, and definitely better at slowing radiological progression. Combination therapy should still be compared with methotrexate monotherapy.     

Anti-C5 monoclonal antibody therapy prevents collagen-induced arthritis and ameliorates established disease.

Activated components of the complement system are potent mediators of inflammation that may play an important role in numerous disease states. For example, they have been implicated in the pathogenesis of inflammatory joint diseases including rheumatoid arthritis (RA). To target complement activation in immune-mediated joint inflammation, we have utilized monoclonal antibodies (mAbs) that inhibit the complement cascade at C5, blocking the generation of the major chemotactic and proinflammatory factors C5a and C5b-9. In this study, we demonstrate the efficacy of a mAb specific for murine C5 in the treatment of collagen-induced arthritis, an animal model for RA. We show that systemic administration of the anti-C5 mAb effectively inhibits terminal complement activation in vivo and prevents the onset of arthritis in immunized animals. Most important, anti-C5 mAb treatment is also highly effective in ameliorating established disease. These results demonstrate a critical role for activated terminal complement components not only in the induction but also in the progression of collagen-induced arthritis and suggest that C5 may be an attractive therapeutic target in RA.     

A comparative genetic analysis between collagen-induced arthritis and pristane-induced arthritis

OBJECTIVE: To compare the genetic regulation of collagen-induced arthritis (CIA) with that of pristane-induced arthritis (PIA) in rats. METHODS: A genome-wide linkage analysis of an (E3 x DA)DA backcross of rats with CIA (n = 364 male rats; the same strain combinations as previously used to determine the genetic control of PIA) was performed. The strongest loci in both CIA and PIA (i.e., Cia12/Pia4 and Cia13/Pia7) were isolated in congenic strains. Susceptibility in both congenic strains was tested in rats with CIA and in rats with PIA. RESULTS: We found a striking, although not complete, similarity of the arthritis-controlling loci in CIA and in PIA, as well as the previously defined loci associated with cartilage destruction, antibody production, and the acute-phase response. All major PIA quantitative trait loci (QTLs) identified in early severe arthritis were also strong regulators of CIA. The 2 strongest QTLs, Cia12/Pia4 on chromosome 12 and Cia13/Pia7 on chromosome 4, were also analyzed in congenic strains with DA or E3 as the background genome. Consistent with the results of linkage analysis, the congenic strain experiments showed that the chromosome 4 locus was more penetrant in CIA than in PIA, while the chromosome 12 locus almost completely dominated the control of PIA severity. CONCLUSION: The underlying genetic control of CIA was found to have many, but not all, pathogenic mechanisms in common with PIA, despite the use of a cartilage-specific antigen (type II collagen) to induce CIA but not PIA.     

胶原诱导性关节炎大鼠滑膜病变的蛋白质组学研究

目的探讨胶原诱导性关节炎(CIA)大鼠滑膜病变的蛋白质组学研究,为类风湿关节炎(RA)的发病及治疗提供新的线索。方法30只采用皮下注射牛Ⅱ型胶原与完全弗氏佐剂诱导的SD大鼠CIA诱导性关节炎(CIA)模型,应用双向凝胶电泳(2DE)技术分离正常组、CIA模型组大鼠关节滑膜的总蛋白后,经胶体考染显色、图像分析、识别差异表达的蛋白质点,应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)得到相应的肽质量指纹图谱(PMF),然后搜索数据库鉴定部分差异蛋白质点,运用western blotting方法验证差异表达蛋白质。结果建立了正常组、CIA模型组大鼠滑膜的双向凝胶电泳图谱,平均点数为(1020±40)个蛋白质点,平均匹配率为(93．2±2．1)%；发现并鉴定了两组滑膜差异表达大于2倍的蛋白质点35个,这些与滑膜病变相关的差异表达蛋白质的功能涉及物质代谢、能量产生、物质转运、抗氧化、信号转导及细胞骨架蛋白,随即用western blotting方法验证差异蛋白质annexinⅠ、aldolase A在正常组和CIA模型组中的表达,其结果也显示了类似的表达差异。结论建立了正常组、CIA模型组大鼠关节滑膜的双向凝胶电泳图谱,鉴定了35个与CIA滑膜病变相关的差异表达的蛋白质,这些差异蛋白质可能以不同的方式参与了病变过程,为揭示CIA滑膜病变的机制及RA的发病机制提供了新的线索,而且annexinⅠ、aldolase A的验证结果与蛋白质组结果的一致性表明,AnnexinⅠ、aldolase A可能与CIA的关节滑膜病变有关。     

雷公藤甲素对胶原诱导关节炎大鼠滑膜血管新生的调节作用

目的 观察雷公藤甲素(TP)对血管新生的影响,探讨TP治疗关节炎的作用机制.方法 建立胶原诱导关节炎(CIA)大鼠模型,常规苏木素-伊红(HE)染色,测量关节体积,计算关节炎指数和病理积分,分别检测滑膜和血清中血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)和内皮抑素(endostatin)的蛋白表达,并通过CD34给滑膜血管密度计数(MVD).结果 CIA大鼠滑膜和血清中VEGF、bFGF及滑膜MVD明显高于正常组(X2分别为65.3、31.6,q=9.2,三者P＜0.01),endostatin的表达与正常组差异无统计学意义(X2=0.8,P＞0.05).TP治疗后,滑膜和血清中VEGF、bFGF、滑膜MVD均下降(X2分别为19.7、6.0,q=6.5,三者P＜0.01),而endostatin表达却升高(X2=3.9,P＜O.05),甲氨蝶呤(MTX)则主要以endostatin表达升高为主(X2=17.9,P＜0.01).结论 生长因子的表达失衡可能在关节炎的形成及发展过程中起重要作用,TP能够调整生长因子的失衡状态而发挥治疗作用.     

雷公藤多苷对胶原诱导性关节炎大鼠中高迁移率族蛋白B1影响的研究

目的 探讨高迁移率族蛋白B1(HMGB1)在类风湿关节炎(RA)治疗中的作用及雷公藤多苷治疗RA的可能机制.方法 建立胶原诱导性关节炎(CIA)大鼠模型建立后,将造模成功的大鼠随机分为模型组、甲氨蝶呤治疗组、雷公藤多苷治疗组、雷公藤多苷和甲氨蝶呤联合治疗组进行药物干预,4周后取材,采用免疫组织化学法检测HMGB1在正常组、模型组和各治疗组大鼠的滑膜与关节中的表达,采用酶联免疫吸附试验(ELISA)法检测HMGB1在正常组、模型组和各治疗组大鼠血清中的含量.统计方法采用方差分析.结果 HMGB1在模型组大鼠滑膜、关节中的蛋白表达以及血清中的含量[(23.8±2.2)ng/ml]明显高于正常组[(7.4±1.6)ng/ml](P＜0.01),3个治疗组大鼠滑膜、关节中HMGB1的表达和血清中HMGB1的含量[分别为(133±3.1),(17.4±4.9),(11.7±1.5)ng/ml]显著低于模型组(P＜0.01),但又高于正常组(P＜0.05).结论 HMGB1参与了CIA滑膜增生、软骨和骨质破坏的病理过程;甲氨蝶呤与雷公藤多苷治疗RA滑膜炎和骨质破坏的分子机制与其降低HMGB1的表达相关.     

The clinical and immunological effects of cyclosporin A on patients with rheumatoid arthritis

Summary Ten patients with rheumatoid arthritis entered this open study to investigate the clinical and immunological effects of cyclosporin-A (CsA) on rheumatoid arthritis. A dosage of 2.5mg/kg/day was used, and each patient was examined every two weeks. The clinical assessment was performed on the basis of the number of painful and swollen joints, the duration of morning stiffness, and the physician and patient global assessment. The laboratory assessment included blood and urine analysis, liver and renal function, C-reactive protein (CRP), and erythrocyte sedimentation rate. The cell surface markers were determined by flow cytometer. Soluble interleukin-2 (IL-2) receptor and IL-2 production were determined by ELISA. Eight patients continued through the six-month study, and marked improvement was found after three months in all clinical indices except morning stiffness. The laboratory assessment demonstrated significant decrease of CRP af-ter six months treatment. Serum creatinine, though within normal limit, was about 30% above the baseline value. There was no significant change of IL-2R and HLADR expression on T cells during the therapeutic course. However, statistically significant decreases of both IL-2 production from lymphocytes and serum IL-2R concentration were found after 22 weeks treatment. The current studies suggest that CsA is clinically effective in the treatment of rheumatoid arthritis and may act through suppression of T cells.