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Autoantibodies and autoimmune disorders
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系統性 ＆ 器官特異性自體免疫疾病區分 自體免疫疾病 系統性自體 免疫疾病 器官特異性自 體免疫疾病 內分泌系統 血液系統 心血管系統
系統性紅斑狼瘡 類風濕性關節炎 多肌炎／皮肌炎 硬皮病 強直性脊椎炎 骨關節炎 成人斯蒂爾病 白塞病 賴特綜合症 銀屑病關節炎 復發性多軟骨炎 統性血管炎 結節性動脈炎 嗜酸性肉芽腫性血管炎 韋格內肉芽腫 超敏性血管炎 巨細胞動脈炎 毒性彌漫性甲狀腺重 慢性淋巴細胞性甲狀腺炎 糖尿病 自體免疫性多腺體綜合症 內分泌系統 自體免疫性溶血性貧血 特發性血小板減少性紫癜 自體免疫性中性粒細胞減少症 血液系統 風濕熱 病毒性心肌炎與心肌病 高脂血症與動脈粥樣硬化 多發性大動脈炎 心血管系統 呼吸系統 結節病 特發性肺間質纖維化 自體免疫性慢性活動性肝炎 原發性膽汁性肝硬化 原發性硬化性膽管炎 慢性萎縮性胃炎 炎性腸病 消化系統 自體免疫疾病 腎臟與自體免疫 腎小球疾病 腎小管間質性疾病 泌尿系統 神經系統 急性炎性脫髓髓鞘性多發神經根病 Fisher 綜合症 全植物神經功能不全 POEMS 綜合症 多發性硬化 重症肌無力 交感性眼炎 晶體過敏性葡萄膜炎 蠶蝕性角膜潰瘍 Vogt – Koganagi – Havada 病 白塞病 Sjogren 綜合症 Graves 系統性紅斑狼瘡 Wegener 肉芽腫 多發性硬化 重症肌無力 類風濕性關節炎 自體免疫性感音神經性聽力減退 梅尼埃病 復發性口瘡 器官特異性自 體免疫疾病 五官科相關 婦產科 子宮內膜異位症 免疫性不孕 習慣性流產 妊娠高血壓綜合症 母子血型不合 妊娠與甲狀腺疾病 妊娠合併肝炎 天庖瘡 類天庖瘡 泡疹樣天庖瘡 線狀IgG大庖性皮病 妊娠庖疹 庖疹樣皮炎 獲得性大庖性表皮松解症 自體免疫性大疤性皮膚病 景祥
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Systemic lupus erythematosus (SLE)
台灣發生率約千分之2.5 Ratio F/M: 10/1 Age: 20~40 years old HLA type: DR-2 and DR-3 其會引起全身性系統病變，每個人均不儘相同，發生率也不同。
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SLE –臨床症狀 根據1982年美國風濕醫學會標準，凡具有下列十一項準則中的任四項以上，可診斷為紅斑性狼瘡： 1. 面頰有紅斑
1. 面頰有紅斑 2. 圓盤紅疹 3. 陽光過敏 4. 口腔潰瘍 5. 關結炎 6. 心包膜炎或胸膜炎
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SLE –臨床症狀 7. 腎功能障礙，出現蛋白尿或尿中有圓柱體 8. 神經障礙如抽搐或精神病
9. 血液障礙主要是溶血性貧血或（b）白血球過少（＜4000/cumm） (b)淋巴球過少(<1500/cumm) (c)血小板過少(<100000) 10.免疫系統障礙有右列之一：(a)LE cell (b)DNA抗體(c)anti-Sm(d)梅毒血清假陽性反應半年以上 11. 抗核抗體ANA陽性反應
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LE cell
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SLE- Autontibodies 1. anti-ds-DNA antibodies
2. anti-nuclear antibodies(ANA) 3. anti-extractable nuclear antigens(ENA) 4. anti-leukocyte antibodies 5. anti-phosphlipid antibodies (lupus anti-coagulant)
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SLE – Immunology Immune complex formation
Immune comlexes are formed and become lodged in small vesicle or basement membrane. Immune complexes and inflammation Immune complexes serve as a stimulus for complement activation. Role of complement in inflammation Anaphylatoxins:C3a, C4a, C5a Oposonin:C3b
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SLE – Laboratory diagnosis
ANA tests Indirect fluorescent antibody technique ENA tests(ENA panel) Double immunodiffusion ouchterlony Immunoblot ELISA
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SLE – Laboratory diagnosis
Double stranded DNA IFA, ELISA Others anti-phospholipid test
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Serologic test of SLE Anti-extractable nuclear antigen：利用
Immunodiffusion, counter immunoelectrophoresis, Western blot 來偵測 antibody，例如：scl-70, Jo-1, SS-B, SS-A, Sm Anti-cardiolipin antibody：也可稱為anti-phospholipid antibody or lupus anticoagulant，常於SLE, RA, Sjőgren’syndrome 中發現；此外，近來發現 plasma β2-glycoprotein I 也會使 antibody 結合上 cardiolipin
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SLE的檢驗流程 先做FANA測定，再配合ds-DNA來進行判讀 FANA連續幾次皆呈陰性反應，即可排除為SLE的可能性
FANA borderline： 可能為正在治療中的患者 藥物誘發性ANA，需配合dsDNA抗體檢查 FANA strong positive： 檢查FANA的titer ，titer越高則SLE的可能性越大 titer高於或是等於1：10即可判定為SLE
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Anti-nuclear antibodies(ANA)
對抗細胞核內成分 診斷SLE的優良指標 IFA staining Homogenous Nucleolar Speckled Peripheral
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Anti-nuclear Antibody (ANA)
Against a variety of Ag within Hep2 cell nucleus No organ or species specificity 可作為screening tool for SLE 可分類為: Ab to DNA Ab to histones Ab to nonhistone proteins Ab to nucleolar antigens
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Diffused or Homogeneous Pattern
Antideoxyribonucleic acid nucleoprotein Ab (Anti-nDNA、Anti-dsDNA、Anti-ssDNA、Anti-DNP、Anti-histones) High titer: SLE Low titer: SLE、RA、Sjogren’s syndrome、MCTD (mixed connective tissue disease)
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homogeneous 均勻型
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Peripheral Pattern Ab to DNA (nDNA、dsDNA、DNP) High titer: SLE
Low titer: SLE、RA、 Sjogren’s syndrome、 MCTD
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Peripheral 周邊型
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Speckled Pattern Anti-non-histone protein、anti-extractable nuclear antigen(ENA) High titer: anti-Sm: SLE anti-RNP(ribonucleoprotein): MCTD anti-Scl-70: scleroderma (progress systemic scleroderma, PSS) anti-SS-A(Ro): SLE(mild)、Sjogren’s syndrome anti-SS-B(La): Sjogren’s syndrome anti-cetromere: CREST syndrome anti-Jo-1:polymyositis (PM)
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Speckle 斑點型
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Nucleolar Pattern Ab to nucleolar RNA (4-6S RNP)
High titer: scleroderma、 sjogren’s syndrome、 polymyositis
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Nucleolar 核仁型
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Antinuclear antibody 自體抗體 Anti-DNA Anti-Sm Anti-RNP Anti-SSA(RO)
SSB(La) Anti-Jo-1 Anti-Scl-70 自體抗原 dsDNA ssDNA snRNAs複合體之 B`/B, 和D protein A和C protein 構成scRNP複合體 Histidyl-tRNA synthetase DNA topoisomerase I 常於哪些疾病中發現 SLE 對於SLE有專一性 MCTD, SLE, Sjőgren’syndrome SLE, Sjőgren’syndrome Polymyositis Scleroderma
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抗核仁抗體(ANA)的陽性率 ※ 正常人：5~10% 系統性紅斑性狼瘡(SLE)： 活動期 95~100% 非活動期 80~100%
活動期 95~100% 非活動期 80~100% 葯物性狼瘡 (DLE)：95~100% 混合性結締組織病(MCTD)： 95~100% 硬皮病 (SSC)： 70~90% 乾燥綜合症 (SS)： 60~80% 多發性肌炎／皮膚炎(PM／DM)： 40~60% 類風濕性關節炎 (RA)： 30~50% 幼年型風濕關節炎(JRA)：20~40% 非結締組織病： 10~20% 慢性活動性肝炎(CAH) 重症肌無力(MG) 慢性淋巴性甲狀腺炎 Substrate…. Hep-2 Cell Positive Titer…. 1：40
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ANA Screen Negative Positive PCNA Peripheral Anti-DNA Homogeneous
確認追查 對應抗體 n-DNA ss-DNA ds-DNA 相關疾病 SLE, PSS, SjS Drug induced- lupus Peripheral Anti-DNA n-DNA ss-DNA ds-DNA Homogeneous Anti-Histone SLE ENA-SCREEN ENA(RNP Sm SS-A SS-B Scl-70, jo-1) SLE, PSS, SjS MCTD, PM/DM PSS, Speckled Anti-ENA Positive Discrete speckled Centromere ASMA,AMA PSS, CREST- syndrom, PBC Autoimmune hepatitis Anti-Centromene 呈現型態 Nucleolar Anti-Nucleolus PSS PBC Autoimmune hepatitis Cytoplasm Anti-Cytoplasm ASMA,AMA PCNA Anti-PCNA SLE
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Anti-extractable nuclear antigens (ENA)
測存在於serum中會對抗nuclear proteins 的antibody (對抗細胞核萃取物) 可使用double immunodiffusion、 counter immunoelectrophoresis、 Western blot檢測 ANA為Speckled pattern,再鑑定ENA pannel
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Anti-ds DNA Antibody 檢驗SLE病人是否會併發狼瘡性腎炎 (Lupus nephritis)
抗原:Crithidia luciliae (因為其Kinetoplast中 不含其他核蛋白或單股DNA，只有雙股DNA) 抗體:血清內Anti-ds DNA Positive: Kinetoplast濃染，多數生殖核也染上 測定效價的高低，可作為治療的參考 例外:藥物引起的SLE、 Anti-ds DNA為IgM 非IgG
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Anti-nDNA 只有kinetoplast 染上才算陽性，其餘染上螢光皆不算
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Kinetoplast of Crithidia lucilliae
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Rheumatoid arthritis Ratio:在台灣有將近十萬人口患有類風溼性關節炎 其中有60%的患者是女性
Age:20~45 years old HLA type: DR-4 臨床表徵: 關節腫痛且有對稱性、類風濕性結節、陽性類風濕因子和X光的變化 Antigen: unknown
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Prevalence of Rheumatoid Arthritis
Caucassian (USA) % African % Mainland China % Taiwan % -rural % -urban (Taipei) %
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Rheumatoid arthritis-Immunology
Model for molecular mimicry Cross reactive. Role of T cell derived cytokines Type 1 cytokines (e.g., IFNγ,and TNF). Role of macrophage TNF Role of mast cells and basophils Degranulation
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Rheumatoid arthritis-Immunology
Role of B cell Rheumatoid factor serve as a stimulus for the formation of large immune complexes. Unregulated inflammation Immunological clinical trial Anti-TNF antibodies.
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Rheumatoid arthritis– Laboratory diagnosis
C reactive protein Latex agglutination Rheumatoid factor Anti-cyclic citrullinated peptide antibody (Anti-CCP Ab)
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C-Reactive Protein One of the acute phase reactants
Secreted by hepatocytes in response to interleukin 6 (IL-6), which is secreted by macrophages Not specific for RA. It is increased in many other inflammatory conditions Measured by latex agglutination method
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Rheumatoid Factors (RF)
Autoantibody directed against antigenic determinants on the Fc fragment of IgG RF may be of any isotype: IgM, IgG, IgA or IgE IgM is the only one routinely measured by clinical laboratories, using nephelometry, ELISA, and latex agglutination techniques RF is positive in around 80% of RA patients. A prognostic factor in RA. High titers is associated with more severe aggressive disease and increased risk for extra-articular manifetations
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Rheumatoid Factor 檢驗RF的方法：
Warrler-Rose test (passive hemagglutination)：利用 rabbit IgG 和 RBC結合，血清中的RF能與血球上IgG的Fc部位結合，而使血球凝集， 此法敏感度較高 Latex RA (passive agglutination)： 利用human IgG 和 RBC結合，此法敏感度較高 Nepherometry
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Anti-cyclic citrullinated peptide antibody (Anti-CCP Ab)
Previously known as anti-perinuclear factor, antikeratin antibodies, antifilaggrin antibodies Target epitope in which arginine residues have been converted to citrulline by posttranslational action of peptidyl arginine deiminase (PAD) enzymes Citrullinated proteins in RA synovium Citrullinated fibrin Citrillinated vimentin (Sa antigen) Anti-CCP Ab: local production in inflamed synovium Play an important pathogenic role?
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Clinical implication of Anti-CCP Ab in RA
Comparable sensitivity to IgM-RF (41%-87.6% vs. 54%-86%) Superior specificity compared with IgM-RF (88.9%-98.5% vs. 81%-87%) Found early in the course of RA Associated with more severe joint destruction and greater disease activities Predictor of a more aggressive disease course
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ACR Diagnostic Criteria for RA (1987)
4 or more of the following symptoms 1.Morning stiffness more than one hour 2.Arthritis of 3 or more joint areas 3.Arthritis of hand joints 4.Symmetrical arthritis 5.Subcutaneous nodules 6.Seropositive for rheumatoid factors 7.Typical radiological changes Criteria 1 to 4 must be present for more than 6 weeks
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Medication for Rheumatoid Arthritis
Non-steroidal anti-inflammatory drugs (NSAID) Disease modifying anti-rheumatic drugs (DMARD) Corticosteroid Biological agents
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DMARD Slow-acting anti-rheumatic drugs Cytotoxic drugs Cyclosporine
Anti-malarial drug Sulfasalazine Gold salts D-penicillamine Cytotoxic drugs Methotrexate Azathioprine Cyclophosphamide Leflunomide Cyclosporine Tetracycline
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Biological Agents TNF-a blocker
Soluble TNF receptor Etanercept (Enbrel) :Two recombinant P75 TNF-receptor fused with Fc of human IgG1 Anti-TNF mAb Infliximab (Remicade) (chimeric) Adalimumab (Humira) (humanized) Soluble IL-1 receptor antagonist (anakinra)
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Sjőgren’syndrome 臨床症狀：淚腺和唾腺分泌減少，會導致 乾性角膜炎和口乾燥症 Prevalence：主要發生於中老年女性
乾性角膜炎和口乾燥症 Prevalence：主要發生於中老年女性 診斷：1. 約90%患者RF呈現陽性反應，FANA 檢查約70%呈斑點型； 2. 沒有RA的患者，anti-SSA(Ro)呈陽性， anti-SSB(La) 約48%呈陽性； 3. 而同時有RA的患者anti-SSA(Ro)約9%呈 陽性，anti-SSB(La)約3%呈陽性 4. anti-Sm抗體則完全不會產生
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Wegener’s granulomatosus and anti-neutrophil cytoplasmic antibody (ANCA)
ANCA are a group of antibodies against components of neutrophil granules and monocyte lysosomes. There are two main antigens: proteinase 3 (PR3) and myeloperoxidase (MPO), both enzymes located in the azurophilic granules of neutrophils and in some granules of monocytes. Autoantibodies to other neutrophil cytoplasmic proteins may also present.
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Proteinase 3 (PR3) A single chain, 229-amino acid serine protease with four disulfide bridge and two potential glycosylation sites. Cationic protein with pI=9.1 A triple band around 29 kDa on SDS-PAGE. Can be inhibited by a1-antitrypsin
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Myeloperoxidae (MPO) MPO is important enzyme to produce OCl- H2O2 and Cl- A heterodimer consisting of two light (b) and two heavy (a) chains, two heme groups and four potential glycosylation sites. Highly cationic, pI>11. M.W. about 140kDa.
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Methods used to detect ANCA and their interpretation
Indirect immunofluorescent staining of ethanol or formalin fixed leukocytes PR3-ANCA produce a granular cytoplasmic pattern (cANCA). MPO-ANCA give rise to an artifactual perinuclear/nuclear pattern (pANCA) due to MPO redistribution after ethanol fixation. ELISA-use purified antigen
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Clinical indications for ANCA
Most in necrotizing glomerulonephritis and primary vasculitic syndromes but also others diseases such as SLE, RA and ulcerative colitis. cANCA (anti-PR3) is a sensitive marker for Wegener’s granulomatosus (necrotizing granulomatous lessions of the respiratory tract, generized arteritis and focal glomerulitis) about 90% (+). Change in titers of ANCA seem to reflect changes in disease activity.
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Serological analysis of acute glomerulonephritis (AGN)
ANCA (+) with pulmonary necrotizing granulomas-Wegener’s granulomatosis. Anti-glomerular basal membrane (+) with lung hemorrhage-Goodpasture’s syndrome. Immunocomplex Lupus, Post-strep, cryoglobulin...
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Differentiation of acute glomerulonephritis
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The Anti-Phospholipid Syndrome (APS)
When abnormal clotting and miscarriage in patients are associated with significant titers of anti- phospholipid Ab (aPL), which include anti-cardiolipin Ab (aCL) and/or Ig that inhibit in vitro phospholipid-restricted blood clotting, termed the "lupus anticoagulants" (LAC). > 40 GPL
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Laboratory criteria Lupus anticoagulant (LA) present in plasma, on two or more occasions at least 12 weeks apart, detected according to the guidelines of the International Society on Thrombosis and Haemostasis (Scientific Subcommittee on LAs/phospholipid-dependent antibodies). Anticardiolipin (aCL) antibody of IgG and/or IgM isotype in serum or plasma, present in medium or high titer (i.e. >40 GPL or MPL, or >the 99th percentile), on two or more occasions, at least 12 weeks apart, measured by a standardized ELISA. Anti-β2 glycoprotein-I antibody of IgG and/or IgM isotype in serum or plasma (in titer > the 99th percentile), present on two or more occasions, at least 12 weeks apart, measured by a standardized ELISA, according to recommended procedures.
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Anti-cardiolipin antibody (ACA)
Also called anti-phospholipid antibody or lupus anticoagulant (LAC) is the antibody responsible for the biologic false-positive for VDRL , found in SLE, RA, SjÖgren's syndrome and other diseases. The presence of LAC and elevated titers of ACA have been correlated with an increased incidence of thrombosis, thrombocytopenia, and fetal loss (antiphospholipid syndrome). Recently, plasma b2-glycoproten I has been found to been important cofactor to let antibodies bind to cardiolipin.
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Anti-cardiolipin antibody detection
ELISA: Ab bind to immobilized Ag (cardiolipin or b2-glycoproten I )→ add enzyme-labled anti-human IgM (or IgG) → add chromogenic substrate → measure the intensity of color that develops In the case of cardiolipin ELISA, washing buffer should not contain detergent, otherwise will cause false negative
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b2GPI: the cationic V domain binds to anionic PL
Physiological EC surfaces are anti-coagulant and do not have negatively charged PL APL cross-link b2GPI > increase the avidity dimerized b2GPI to negatively charged PL > inhibit PL-dependent coagulation reactions, such as thrombin activation, protein C activation, and anti-coagulant activity of activated protein C. Rand, Circ Res. 2002, 90:29.
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