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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳垣崇 | |
| dc.contributor.author | Tai-Ming Ko | en |
| dc.contributor.author | 柯泰名 | zh_TW |
| dc.date.accessioned | 2021-06-17T00:54:15Z | - |
| dc.date.available | 2021-09-30 | |
| dc.date.copyright | 2012-03-02 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-10-03 | |
| dc.identifier.citation | Aceves-Avila, F. J. & Benites-Godinez, V. (2008). Drug allergies may be more frequent in systemic lupus erythematosus than in rheumatoid arthritis. J Clin Rheumatol 14, 261-263.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66733 | - |
| dc.description.abstract | 藥物過敏仍然是個臨床上的重要問題。史帝文生強生症候群及其相關的毒性表皮溶解症都是嚴重的藥物過敏。這兩種症狀不但會針對特定藥物而產生劇烈的免疫反應還具有生命威脅的危險性。儘管HLA-B和特定的藥物所引起的嚴重過敏有密切關聯性，但負責釋放毒性物質的關鍵T細胞是如何被誘導的依照目前的研究仍無法解釋。現在的假說認為藥物反應的T細胞應有特別的族群，因此是否有特定的T細胞受器認識藥物和HLA-B所形成的複合體進而引發近一步的藥物過敏是一個重要的研究課題。藉由CBZ-SJS和HLA-B*1502的高度相關性, 我們欲以這個病例作為模組來研究藥物過敏的T細胞受器的變異性。在CBZ的體外刺激下，從CBZ-SJS的病人中所分離的CBZ專一性CD8 T細胞可以被放大並且活化進而產生干擾素-γ和毒性顆粒蛋白來毒殺標的細胞。在CDR3的圖譜分析的結果中，我們發現了藉由藥物所增生的CD8 T細胞具有共同並且受限的T細胞使用情形。在其T細胞受器使用中，beta鏈中的變異區第11號 (VB-11) 有明顯的集中表現的情況，這種情形在8個病人都有發生。同樣我們也利用定序分析來確認它的選植株表現情形，並鑑定VB-11-ISGSY為最優勢的選植株。這些選植株在19個人中有16個人有發病，17個人不會發病。為了瞭解VB-11-ISGSY的分布比例，我們也分析了這個基因在HLA-B*1502攜帶者的分佈情形，我們發現有相當低的比例的人帶有這樣的序列 (29個人中有4個人)。除此之外，這些帶有VB-11-ISGSY序列正常人的T細胞可以被CBZ所刺激而增生，這些T細胞也具有毒殺效果並且可以被anti-VB-11抗體所阻斷。更進一步的，我們也以TCR clone和T細胞轉殖株來表現這些特殊的T細胞受器證實其功能。因此這樣的研究建立了T細胞受器在SJS和TEN的致病機轉上的關鍵角色，並且解釋了為何有些帶有HLA-B*1502的人對於CBZ是有耐受性的，甚至提供了SJS和TEN的一個可能的治療標的。 | zh_TW |
| dc.description.abstract | Drug hypersensitivity remains a major clinical problem. Stevens-Johnson syndrome (SJS) and the related disease toxic epidermal necrolysis (TEN) are life-threatening drug hypersensitivities with robust immune responses to drugs. Despite the strong genetic association between HLA-B*1502 and carbamazepine (CBZ)-induced SJS/TEN, it is not known whether particular T-cell receptor (TCR) repertoires participate in the recognition of small drug-peptide-HLA complexes in T cell-mediated drug hypersensitivity. Using the strong HLA predisposition in CBZ-SJS as a model, we studied the diversity of the TCR repertoire involved in drug hypersensitivity. Upon in vitro CBZ stimulation, CBZ-specific CD8+ T cells isolated from CBZ-SJS patients could be expanded, activated with release of interferon-γ and granulysin, and exhibited strong cytotoxicity. CDR3 spectratyping of drug-enriched CD8+ T cells revealed common, restricted TCR repertoire usages that were VB-11 skewed in all 8 CBZ-SJS patients examined. CDR3 sequence analysis confirmed this oligoclonality and identified VB-11-ISGSY as the most predominant clonotype. This clonotype was present in 16 of 19 (84%) SJS patients, absent in all 17 tolerant patients, and present at low frequency in normal individuals (4 of 29, 14.8%); all study subjects were HLA-B*1502 carriers. Moreover, CBZ-specific cytotoxicity could be primed in vitro in those normal individuals with disease-specific clonotypes; this cytotoxicity could be blocked by an anti-TCR VB-11 antibody. Thus, this study establishes the key role of the T cell receptor in the pathogenic mechanism of SJS/TEN, explains why some HLA-B*1502 positive individuals are tolerant to CBZ, and provides a potential therapeutic target for SJS/TEN. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T00:54:15Z (GMT). No. of bitstreams: 1 ntu-100-D94445004-1.pdf: 7651747 bytes, checksum: 143f8c2d99838308c15b3041dbfd8163 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | CONTENTS
Contents……ii Figure contents……vi Table contents……viii 中文摘要……ix Abstract……x Abbreviations……xi 1 Chapter 1: INTRODUCTION……1 1.1 Adverse drug reactions…1 1.2 Cutaneous adverse drug reactions (cADRs) …2 1.3 Immune-mediated cADR…3 1.4 CTLs confer major cytotoxicity in SJS and TEN…4 1.5 Hypothesis for drug hypersensitivity …5 1.6 Pharmacogenomics & HLA predisposition in cADR…5 1.7 Other potential pathogenic factors involved in cADR…6 1.8 Pathogenic roles of TCR and its analysis …7 1.9 Evidence of pathogenicity of TCR in immune-mediated disease …9 1.10 Study model in this study: CBZ-SJS/TEN and HLA-B*1502…10 2 Chapter 2: HYPOTHESIS AND SPECIFIC AIMS……11 2.1 Specific Aim 1…11 2.2 Specific Aim 2…12 3 Chapter 3: METHODS……13 3.1 Patients and samples…13. 3.2 Culture medium and reagents…13 3.3 Culture of drug-specific T cells…14 3.4 Generation of T cell-clones…14 3.5 Experimental design for CDR3 analysis…15 3.6 Flow cytometry…15 3.7 RNA extraction and cDNA synthesis…16 3.8 TCR repertoire analysis…17 3.9 Cloning, sequencing and TCR analyzing…18 3.10 Enzyme-linked immunosorbent assay (ELISA)…19 3.11 Antigens titration and IL-2 secretion assay…20 3.12 Determination of drug-specific cytotoxicity in vitro…20 3.13 Blockade of CBZ-specific cytotoxicity with anti-TCR VB mAbs…21 3.14 Quantitative real-time PCR…21 3.15 Generation of TCR transfectant…22 3.16 Antigens titration and IL-2 secretion assay…22 3.17 Experimental design…23 4 Chapter 4: RESULTS……25 4.1 Generation of CBZ-specific T cells…25 4.2 The composition of CBZ-enriched T cells…25 4.3 CBZ-specific T cell responses in a conserved HLA-B*1502 genetic background…25 4.4 Common skewed TCR VB repertoires of drug-specific CTLs…26 4.5 Common skewed TCR VA repertoires of drug-specific CTLs…27 4.6 Upregulation of skewed VB and VA subfamilies in CBZ-SJS/TEN…27 4.7 CDR3 sequence analysis confirms oligoclonality of CBZ-specific T Cells …28 4.8 Identification of the correlation between the immunodominant TCR clonotypes and the severity of drug hypersensitivity in CBZ-SJS…29 4.9 Detection of immunodominant TCR clonotypes in the PBMCs of CBZ-SJS/TEN patients in recovery stage…29 4.10 In vitro priming of disease-specific clonotypes in healthy donors…30 4.11 Blockade of CBZ-specific cytotoxicity with anti-TCR VB mAb…30 4.12 T-cell clones expressing drug-specific TCR showed CBZ and HLA-B*1502-restricted response…31 4.13 The specificity of the transfectant was controlled by the TCR…31 4.14 Cross-reactivity of TCR transfectants to CBZ and OXC…32 4.15 T-cell receptor recognition of CBZ was HLA-B*1502 dependent…33 4.16 T-cell receptor recognition of CBZ was α- and β-Chain-dependent …33 5 Chapter 5: DISCUSSION……34 5.1 Identification of drug-specific TCR clonotype(s) in cADR…34 5.2 The pathogenic role of common CBZ-specific TCR clonotypes…34 5.3 Multiple TCR clonotypes recognizing the complex of CBZ/peptide-HLA-B*1502…35 5.4 Other risk factors in drug hypersensitivity…37 5.5 Cross-reactivity of CBZ and OXC…38 5.6 Application of these restricted TCRs…38 5.7 Other effector T cells in SJS and TEN 5.7.1 Multiple roles of CD4+ T cells in drug hypersensitivities…39 5.7.2 NK cell recognition of particular HLA class I allotypes in drug hypersensitivities…40 5.7.3 Induction of NKT cells in drug hypersensitivities…42 6 Chapter 6: SUMMARY……43 7 Chapter 7: REFERENCE……44 8 Chapter 8: FIGURES & TABLES……52 Figure contents Figure 1. Experimental design for co-culturing CBZ-specific T cells…53 Figure 2. Flow cytometric of the CD8+ subsets of CBZ-stimulated T cells…54 Figure 3. IFN-g and granulysin release of T cells upon CBZ-stimulation…55 Figure 4. CBZ-specific cytotoxicity of CBZ-SJS patients and tolerant controls…56 Figure 5. Pattern of CDR3 spectratyping analysis in control groups…57 Figure 6. TCR CDR3 Analysis Procedure…58 Figure 7. CDR3-VB spectratyping of T cells (cycle 1) in patients and controls…59 Figure 8. Examples of CDR3 spectratype patterns…60 Figure 9. CDR3-VB spectratyping of T cells (cycle 5) in CBZ-SJS patients…61. Figure 10. CDR3-VB spectratyping of T cells (cycle 5) in tolerant controls…62 Figure 11. CDR3-VA spectratyping of T cells (cycle 5) in CBZ-SJS patients…63 Figure 12. Spectratype profiles of skewed TCR VB-11 with identical CDR3 length…64 Figure 13. CDR3 analysis of T cells stimulated by CBZ and OXC…65 Figure 14. Upregulation of skewed VB subfamilies in CBZ-SJS/TEN…66 Figure 15. Upregulation of skewed VA subfamilies in CBZ-SJS/TEN…67 Figure 16. The correlation between CBZ-specific clonotypes and severity of SJS… 68 Figure 17. The correlation between CBZ-specific clonotypes and onset of SJS…69 Figure 18. Association between CBZ-reactivity and the CBZ-specific clonotypes…70 Figure 19. Blockade of CBZ-specific cytotoxicity with anti-TCR antibodies…71 Figure 20. Procedures of generating the CBZ-specific T cell clones…72 Figure 21. CBZ-specific cytotoxicity of single T cell clones…73 Figure 22. Procedures of generating the TCR transfectants in Jurkat T cell lines…74 Figure 23. Expression of CBZ-specific T cell receptor in Jurkat T cells…75 Figure 24. The IL-2 release of TCR transfectants upon CBZ stimulation…76 Figure 25. Patterns of cross-reactivity with CBZ and OXC (TF-10)…77 Figure 26. Patterns of cross-reactivity with CBZ and OXC (TF-2, 4, 8, 11)…78 Figure 27. Both α- and β-chain of the TCR were needed for drug recognition…79 Figure 28. TCR recognition of CBZ was HLA-B*1502-dependent…80 Figure 29. No expansion of CD4+CD25high Treg cells upon CBZ-treatment…81 Figure 30. The hypothetical model for CBZ-induced SJS…82 Table contents Table 1 Oligoclonality and amino acid sequences of VB-11 CDR3 clonotypes of CBZ-stimulated CD8+ T cells from patients with CBZ-SJS/TEN……83 Table 2 Oligoclonality and amino acid sequences of VA-22 CDR3 clonotypes of CBZ-stimulated CD8+ T cells from patients with CBZ-SJS/TEN……84 Table 3 The HLA-B genotype and clinical information of study subjects……85 Table 4 Primers for TCR VB Q-PCR analysis and cloning……86 Table 5 Primers for TCR VA Q-PCR analysis and cloning……87 Appendix Table A1. Primers for TCR VA CDR3 spectratyping analysis……………………88 Table A2. Primers for TCR VA CDR3 spectratyping analysis……………………89 | |
| dc.language.iso | en | |
| dc.title | 探討T細胞在嚴重藥物副作用中的致病角色:卡巴西平所引起史帝文生強生症候群的T細胞受器分子分析和功能性分析 | zh_TW |
| dc.title | The pathogenic role of T cells in severe adverse drug reactions: A molecular and functional analysis of T cell receptor repertoire in patients with Carbamazepine-induced Stevens-Johnson Syndrome | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.coadvisor | 洪舜郁 | |
| dc.contributor.oralexamcommittee | 張子文,江伯倫,顧家綺 | |
| dc.subject.keyword | 藥物過敏,T細胞受器,人類白血球抗原,互補決定區段,史帝文生強生症候群,毒性表皮壞死鬆解症, | zh_TW |
| dc.subject.keyword | Drug hypersensitivity,TCR,HLA,CDR3,Stevens-Johnson syndrome,toxic epidermal necrolysis, | en |
| dc.relation.page | 89 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-10-03 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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