类风湿性关节炎的研究进展吕社民M.D., Ph.D 内容提纲RA 概况研究方法常用的动物模型RA 的研究进展综述RA 的生物学治疗展望临床研究描述性研究：发病率、死亡率、共患率等分析性研究：前瞻性研究和回顾性研究干预研究: 各种诊疗方法的效果观察实验研究Hypothesis-Free study Hypothesis-based study  科学研究的一般思路（PHMS 四步法）发现现象（P henomenon ）提出假说（Hypothesis ）机制研究（Mechanism ）意义升华（Significance ）Rat Arthritis Models  models Susceptible strains	  Resistant strians Induced agents Adjuvant arthritis		  LEW, DA  WF	 CFA Oil-induced arthritis  DA		  LEW.1A	 Oil Avridine-induced arthritis	  DA, LEW	 E3	 Avridine Pristane induced arthritis	  DA		 E3	 Pristane CII induced arthritis 	  DA,BB		 F344, E3 Collagen type II CXI induced arthritis	  LEW.1F	  E3, DA  Collagen type CXI COMP-induced arthritis	  E3		 DA	  COMP 1. Collagen type Ⅱ-induced arthritis (CIA) Most commonly used arthritis model. Intradermally inject 200 μg of collagen type II IFA at the base of the tail at d0, and boost with 100 μg of collagen type II IFA at d7. Latent period is about 2 weeks, followed by a chronic disease course. Affect joints and extra-articular cartilage. High anti-collagen type Ⅱantibody and RF levels in SF and in sera. 2. Pristane induced arthritis(PIA) Intradermal injection of 150 μl pristane. Latent period:7-14d;peak period:17-36d; followed by chronic fluctuation period. Joints inflammation: pannus formation, T lymphocyte infiltration and MHC class II expression increase. T cell dependent process.  DA is susceptive and E3 is resistant to PIA. Mouse arthritis models 四、RA 的研究进展综述流行病学遗传学细胞学TLR 与RA RA 的生物学标志已发现的关键遗传因素MHC region: HLA-DRB1 and HLA-DP Innate adaptive interface Interferon signaling: IRF5 TNF-associated signaling pathway ：TNFAIP3 、TRAF1-C5 Immunomodulatory adhesion molecule: ITGAM Lymphocyte differentiation T cell receptor signalling:PTPN22 （Protein tyrosine phosphatase, non-receptor type 22 ）T helper cell polarization: STAT4 and IL-23R B cell activation Post-translational modification: PADI4 （Protein-arginine deiminase type IV ）一个基因与多种疾病的易感性相关PTPN22: RA, DM-1, SLE, AIT FCRL3: RA, AIT, SLE CTLA4: RA, DM-1, AIT SLC22A4/SLC22A5: RA,CD STAT4: RA,SLE 基因对疾病影响具有人群异质性PTPN22 在欧裔人群可增加RA 易感性，而在东亚人群并不是危险因素；PADI4 则相反，在黄种人群可增加RA 的易感性，而白人则不是危险因素。Th17 细胞与RA 的关系IL-17 可上调关节局部IL-6 、IL-1 和TNF- 的水平，进而促进细胞外基质损伤、抑制胶原和蛋白聚糖的合成；IL-17 可促进破骨细胞分化；在正常小鼠关节内注射IL-17 ，可引起RA 类似的症状；IL-17 封闭抗体可改善由IL-17 引起的骨和滑膜的症状。五、RA 的生物学治疗针对新的靶点开发抑制剂或单抗口服小分子抑制剂的研发原有药物基础上的修饰改进SMIP (small modular immunopharmaceutical), which binds and specifically blocks the CD20 molecule (TRU-015) and induces a peripheral B-cell depletion. A phase II clinical trial is currently in progress, and results should indicate potential advantages of this agent in comparison with other therapeutics targeting B-cells. As this molecule is smaller than a standard monoclonal antibody, it is thought to have better tissue penetration, efficacy and safety profile. Other Potential targets Regulatory T cells (Treg) Toll-like receptors (TLRs) Complement pathway (C5a, adipokines) RNAi (siRNA or microRNA) Epigenetic alteration (DNA methylation and histone modification) RA 的生物学分子标志（一）RA 的生物学标志（二）RA 的生物学标志（三）RA 的生物学标志（四）1. 现已应用于RA 生物学治疗的药物：2、在研的RA 生物学治疗药物：3. 新药开发方向：Two TNF-αblocking agents are likely to be approved in 2009 for the treatment of RA. Golimumab (CNTO148) is a fully human monoclonal anti-TNF-αantibody ；Certolizumab pegol (Cimzia) is a humanized monoclonal anti-TNF-αantibody ；Preliminary data of subcutaneously administered exhibit the same efficacy and spectrum of adverse events as currently available TNF-αinhibitors. TNF-αinhibitors To simplify the dosing, decrease the risks of local adverse events, increase the affinity to IL-1, and thus improve the efficacy compared to anakinra, new inhibitor of IL-1 was developed ；Canakinumab (ACZ885) is a humanized mAb targeting IL-1 ；The preliminary results of clinical trials in patients with systemic juvenile idiopathic arthritis are promising, and a phase II clinical trial in patients with RA began in 2008 。IL-1 inhibitors CNTO 136: mAb against IL-6 ; A phase II clinical trial of a subcutaneously administered CNTO 136 was initiated to establish the suitable dosage, and confirm the efficacy and safety of the drug. Tocilizumab is a humanized mAb against IL-6 receptor. Several phase III clinical trials suggest tocilizumab as an agent with extreme potential for the treatment of moderate to severe active RA. IL-6 inhibitors Blocking IL-15 in an experimental model of arthritis led to a significant reduction of cartilage and bone destruction.  HuMaxIL-15 : humanized mAb against IL-15 ; The first open, placebo-controlled, double blind study proved the HuMaxIL-15 to be efficient; AMG 714: a fully human mAb against IL-15; A phase II clinical trial of AMG 714 failed to confirm significant efficacy. IL-15 inhibitors AIN457: a mAb against IL-17; A phase I/II clinical trial of AIN457 in patients with RA is currently in progress. IL-17 inhibitors Ustekinumab: a specific mAb against the p40 subunit of cytokines IL-12 and IL-23; A recent clinical trial evaluating its effect in patients with psoriasis has generated promising results. One proof-of-concept study has shown that ustekinumab treatment can also reduce the signs and symptoms of arthritis in patients with psoriatic arthritis. Recently initiated clinical trials could show if ustekinumab is of any practical use in the treatment of RA. IL-12/IL-23 inhibitors Baminercept (BG9924), an inhibitor of LTβ; Belimumab, a fully human mAb against BAFF; The results of a phase II clinical trials of them have not been encouraging thus far in RA patients.  Atacicept, a recombinant fusion protein of BAFF and the APRIL receptor (TACI-Ig); A phase I clinical trial shows a depletion of peripheral B-cells as well as decrease in RFs and ACPA. TNF superfamily members inhibitor Denosumab: a mAb against receptor activator for nuclear factor κB ligand (RANKL); It is an efficient inhibitor of osteoclastogenesis and is in phase III clinical trial in patients with postmenopausal osteoporosis; Subcutaneous administration of denosumab led to significant retardation of radiographic progression in patients with active RA. Inhibitors of osteoclastogenesis Bevacizumab (Avastin), a mAb against VEGF; A preclinical trial recently demonstrated a prophylactic effect of a VEGF- βmAb in a Murine model of arthritis; however, there has been no significant impact on an established disease. Inhibitors of angiogenesis Over 10 years ago, a strategy for treatment of RA that induced apoptosis by binding the FAS receptor had been introduced in an in vivo model of arthritis.  ARG098: The first monoclonal anti-FAS IgM antibody was subsequently developed, and is being tested as a single intraarticular application into an active knee in a phase I clinical trial in patients with RA. Regulators of apoptosis Clinical trials directly targeting T-cells —alemtuzumab (anti-CD52, Campath-1H), keliximab (chimeric monoclonal anti-CD4 antibody) or clenoliximab (the IgG4 version of the previous antibody) were accompanied by serious adverse events linked with severe CD4+ T lymphocytopenia and a rash, leading to discontinuation of the clinical trials of these agents. The improved knowledge of the costimulation regulatory mechanisms and good efficacy and safety profile in clinical trials facilitated recently abatacept approval for the treatment of RA. T-cell inhibitors Ocrelizumab, a humanized monoclonal antibody against CD20; Atumumab, a fully human monoclonal antibody against CD20; Both are in phase III clinical trials and have a good effect in patients refractory to both MTX and TNF-αinhibitors.  A phase I clinical trial of a fully human monoclonal anti-CD19 antibody (MDX-1342) is in progress in patients with active RA. B-cell inhibitors (1) B-cell inhibitors (2) A number of pharmaceutical companies have developed inhibitors of MAP kinase p38 that predominantly impact its alpha subunit; Most of the therapeutics are in phase II clinical trials; the therapeutic effects have not yet reached expectations. Moreover, the latest data has shown that pamapimod was even less effective than MTX in patients with active RA. Based on these data, one can speculate that other signaling pathways are predominantly involved in the pathogenesis of RA. Inhibitors of MAP kinase p38 Pfizer developed a JAK3 inhibitor (CP-690,550), which has already been in a phase II clinical trial in patients with moderately- to severely-active RA. Remission was achieved in approximately one-third of the individuals, and a relatively good tolerance and safety profile of the therapy was observed. A multicentre study is currently in progress comparing the effect of five dosing regimens of JAK-3 inhibitor with TNF inhibitor (adalimumab) and DMARDs in patients with active RA. Inhibitors of the JAK/STAT signaling pathway. * * 西安交通大学医学院遗传学与分子生物学系2010 年4月10 日一、概况：类风湿性关节炎（rheumatoid arthritis ，RA ）是一种以关节滑膜炎为特征性改变的慢性自身免疫性疾病。好发于手、腕、足等小关节，呈对称分布。滑膜炎持久反复发作，引起关节内软骨和骨的破坏，导致关节结构畸形、关节功能障碍，甚至残废。RA 可引起巨大社会经济负担，已经成为重要的公卫问题。二、研究方法：三、动物模型Avoid risks of clinical experiments Easily obtaining disease models Strictly controlling of experimental conditions Simplifying sampling and operating procedures Fully understanding the nature of diseases 动物实验的优点：“There are, and must be, several different models with different symptoms and different pathogenic mechanisms, controlled by different genes.”Rikard Holmdahl, 2007 Hansson et al.,2002; Soto et al., 2008 hydropsia Hou et al., unpublished data Vingsbo et al,.1998 6-12W DA rats d0 immunization observation and scoring DTH sacrifice 150ul pristane 150ul Col Ⅱ150ul PBS d26/d70 d25/d69 Hou et al,. Unpublished data * * Pristane-induced Arthritis Proteoglycan-induced arthritis Zymosan-induced arthritis Immune complex arthritis Serum transfer models Collagen-induced arthritis K/BxN NZB/NZW HuTNF Tg TNF gene mutation in AUUUA motif Tristetraprolin / IL-1RA / Induced: Genetic: Annual incidence of rheumatoid arthritis in Rochester, Minnesota. Shown is the annual incidence rate per 100,000 population by sex: 1955 to 1995. Each rate was calculated as a 3-year centered moving average. Incidence of RA Male Female Mortality in rheumatoid arthritis by sex. Observed mortality in (a) female and (b) male patients with rheumatoid arthritis and expected mortality (based on the Minnesota white population). Observed is solid line, expected is dashed line, and the gray region represents the 95% confidence limits for observed. Mortality of RA Incidence of silent myocardial infarction: RA versus non-RA. Shown is the cumulative incidence of silent myocardial infarction in a population based incidence cohort of 603 RA patients and a matched non-RA comparison group of 603 non-RA individuals from the same underlying population. Comorbidity in rheumatoid arthritis Cardiovascular diseases Diabetes Malignancy Lung disease Infection Gastrointestinal ulcer disease Anemia Osteoporosis Depression 全基因组关联研究（Genome-Wide Association Studies，GWAS）是指在全基因组层面上，开展多中心、大样本、反复验证的基因与疾病的关联研究，全面揭示疾病发生、发展与治疗相关的遗传基因。遗传学参与RA 发病过程的细胞滑膜细胞软骨细胞成骨细胞破骨细胞T 细胞B 细胞树突细胞巨噬细胞Synovial fibroblasts: key players in RA CIA 500um 正常关节500um 早期RA 就有滑膜炎；滑膜炎是RA 的中心病理改变；RASF 具有侵袭周围软骨和骨的行为。辅助性T细胞在RA 发病中的研究进展IL-17 在PIA 踝关节的表达RA 病人关节滑液中IL-17 表达升高；RA 病人滑膜组织可自发性分泌IL-17 ；Th22 细胞与RA 的关系IL-22 在PIA 踝关节的表达IL-22-/- 小鼠的关节炎发病率、病情轻重以及血管翳均减少，滑膜中的IL-1β、IL-6 、TNF-α和MMP9 的mRNA 表达均下降。IL-22 可促进破骨细胞生成。IL-22 、IL-22R1 mRNA 在RA 患者的滑膜组织中表达上调；重组IL-22 可促进RASF 的增殖，促进其分泌MCP-1 ，并激活MAPK 途径中的ERK-1/2 与p38 。在小鼠CIA 中，血清中的IL-22 的水平显著高于正常对照组。Toll 样受体与RA 的关系Toll 样受体(Toll like receptor ，TLR ）是一类重要模式识别受体，参与识别多种非特性的、保守的微生物组分以及相应内源性配体。TLR 是Ⅰ型膜受体，胞内约含20 个氨基酸残基，结构保守，胞外区含15 一31 个富含亮氨酸的重复序列。不同TLR 的胞外区氨基酸组成的差异较大，决定了它们各自有其特异性的配体。TLR 的生物学效应主要由MyD88 依赖型和非依赖型传导途径介导。人TLR 分类及其配体TLR 在RA 发病中的作用RA 患者TLR 表达升高TLR 调节参与RA 的关键细胞的功能某些TLR 的配体可诱导关节炎的发生