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系統識別號 U0026-1910201712444100
論文名稱(中文) 自體免疫在嚴重登革熱的致病機轉
論文名稱(英文) Autoimmunity in the pathogenesis of severe dengue
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 106
學期 1
出版年 106
研究生(中文) 賓蒂
研究生(英文) Nurhafiza Binti Zainal
學號 S58027010
學位類別 博士
語文別 英文
論文頁數 207頁
口試委員 指導教授-林以行
口試委員-張志鵬
口試委員-余佳益
口試委員-林秋烽
共同指導教授-Sazaly Bin Abu Bakar
口試委員-Justin Chu
口試委員-Robert Anderson
中文關鍵字 none 
英文關鍵字 dengue  autoimmunity  HMGB1  resveratrol  innate immune response  antiviral and antibody 
學科別分類
中文摘要 none
英文摘要 Dengue, an arboviral disease is transmitted between humans by the bite of infected female Aedes mosquitoes. Annually, about 50 million cases of dengue virus (DENV) infections were reported with approximately 500,000 cases of severe dengue and about 5% fatality rate. Understanding the mechanism leading to dengue or severe dengue is crucial, considering the fact that it is not completely comprehended. Autoimmunity has known to be involved in the pathogenesis of dengue. Here, we investigated the association of dengue and autoimmunity by comparing the genetic variant distribution among severe, non-severe dengue and systemic lupus erythematosus (SLE) patients using Ion Torrent sequencing method. Four significant variants were identified; novel variant at chr3:49679737 of BSN gene was associated with susceptibility to severe dengue, while polymorphisms rs76018112 of ABCF1 gene, rs1557370 of Mx1 gene and rs945635 of FCRL3 were associated with protective effects against severe dengue. Theoretical analysis on the effects of novel variant (chr3:49679737) suggests the importance of calcium release in the prevention of severe manifestation, whereas rs76018112, rs1557370 and rs945635 demonstrated that effective early antiviral responses and faster virus clearance associated with protective effects against severe dengue manifestation. SLE patients showed high distribution of all variants related to protection against severe dengue, suggesting that autoimmune disease patients might possess protective factors against acute dengue. We then analysed the protective factor of autoimmunity against dengue by determining the capability of SLE-sera to neutralize DENV using foci reduction neutralization test (FRNT). A total of 82 dengue serology negative sera of SLE patients were collected and FRNT against DENV was performed. Results revealed that 69%, 61% and 52% of the SLE patients’ sera showed FRNT50 neutralization against DENV1, DENV2 and DENV3, respectively. SLE-sera significantly neutralized DENV
in comparison to healthy donors, though tested negative for dengue IgG/IgM antibodies, signifying that SLE patient might have an efficient antiviral response against DENV, perhaps via other antibody isotypes or autoreactive antibodies. We further investigated the association of dengue and autoimmunity by observing the role of high mobility group box 1 (HMGB1), a DNA-binding protein commonly linked to the progression of autoimmune diseases, in the pathogenesis of dengue. HMGB1 resides in the nucleus, but translocated out to the cytoplasm and extracellular milieu during DENV infection. HMGB1-knockdown cells showed higher DENV replication and lower interferon-stimulated genes (ISGs) production than wild-type cells, proposing the novel role of HMGB1 in the antiviral response against DENV, through the regulation of innate immune response. Resveratrol (RESV) treatment inhibits the translocation of HMGB1 via Sirt1, and the accumulation of nuclear HMGB1 consequently increased production of ISGs and reduced DENV replication, verifying the role of nuclear HMGB1 in regulating ISGs in the antiviral response against DENV. Nuclear HMGB1 was found to bind to the promoter region of ISG and positively regulated the expression of ISGs. Our findings highlighted the importance of efficient early antiviral responses and protective effects of autoimmunity against severe dengue progression. We also introduce the novel role of nuclear HMGB1 in the antiviral response and RESV as an antiviral drug against DENV.
論文目次 TABLE OF CONTENT
ABSTRACT ii
ABSTRAK iv
ACKNOWLEDGEMENT vi
TABLE OF CONTENT viii
LIST OF TABLES xii
LIST OF FIGURES xiii
LIST OF SYMBOLS AND ABBREVIATIONS xv
CHAPTER 1: INTRODUCTION 1
1.1.0. Dengue pathogenesis and autoimmunity 1
1.2.0. Study objectives 2
CHAPTER 2: LITERATURE REVIEW 3
2.1.0 Dengue 3
2.1.1. Dengue history 3
2.1.2. Clinical presentations of dengue 4
2.1.3. Classification of dengue 5
2.2.0 Dengue Virus 8
2.2.1. Virus structure 8
2.2.2. Virus replication 10
2.3.0. Autoimmunity in dengue 12
2.3.1. Host immune response in dengue 12
2.3.2. Pathogenesis of dengue 16
2.3.3. Autoimmunity in the pathogenesis of dengue 17
2.3.4 Systemic Lupus Erythematosus (SLE) 19
2.4.0. Cross-reactive antibodies and its impact in dengue pathogenesis 22
2.4.1. Antibody immune response in dengue 22
2.4.2. Heterologous immunity and virus infection 24
2.5.0. High Mobility Group Box 1 (HMGB1) 25
2.5.1. Protein characteristics 25
2.5.2 Functions of HMGB1 28
2.5.3. The role of HMGB1 in autoimmune diseases 32
2.5.4. The role of HMGB1 in virus infections 32
CHAPTER 3: METHODOLOGY 34
3.1.0. Aim 1: To verify the association between single nucleotide polymorphism (SNP) in severe dengue, non-severe dengue and autoimmune patients 34
3.1.1. Patient Samples 34
3.1.2. DNA Extraction 34
3.1.3. Quantification of DNA 35
3.1.4. Ion AmpliseqTM Library Preparation 35
3.1.5. Ion Sphere Particles (ISPs) Amplification and Enrichment 36
3.1.6. Ion PGMTM Hi-Q Sequencing 38
3.1.7. Sequencing Data Analysis 38
3.1.8. Statistical Analysis 39
3.2.0. Aim 2: To determine the neutralization capability of SLE patients’ sera against DENV 40
3.2.1. Ethical Clearance 40
3.2.2. Patients Samples 40
3.2.3. Cell Cultures 41
3.2.4. Viruses and Stock Preparation 41
3.2.5. Virus Titration Assay 42
3.2.6. Foci Staining Assay 42
3.2.7. Foci Reduction Neutralization Test (FRNT) 43
3.2.8. Statistical Analysis 43
3.3.0. Aim 3: To investigate the role of HMGB1 in the antiviral mechanism of RESV against DENV 44
3.3.1. Cell Culture 44
3.3.2. Drug 44
3.3.3. shRNA-mediated Knockdown Gene Expression 44
3.3.4. Virus Stock Preparation 45
3.3.5. Virus Infection 45
3.3.6. Virus Titer Analysis 46
3.3.7. Protein Extraction 46
3.3.8. Western Blot Analysis 48
3.3.9. Quantitative Real-Time PCR (qRT-PCR) 51
3.3.10. Immunofluorescence Analysis (IFA) 54
3.3.11. Chromatin Immunoprecipitation (ChIP) Assay 54
3.3.12. Statistical Analysis 55
CHAPTER 4: RESULTS 56
4.1.0. Association of SNPs among non-severe, severe and SLE patients 56
4.2.0. Cross-neutralization of autoantibodies in SLE patients’ sera against DENV 80
4.3.0. The role of HMGB1 in the host antiviral response against DENV 97
CHAPTER 5: DISCUSSION 119
CHAPTER 6: CONCLUSION 138
BIBLIOGRAPHIES 140
LIST OF PUBLICATIONS AND PAPERS PRESENTED 187
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