IMMU E MARKERS A D PROTECTIVE MECHA ISMS I LATE T A D ACTIVE TUBERCULOSIS JOA E KA G SU LI (B. Sci. (Hons.), US) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2008 Table of Contents ACK OWLEDGEME TS I express my deepest gratitude to Dr Seah Geok Teng for her constant guidance, invaluable advice and unfailing patience, not forgetting the many sacrifices she had made for the past years I spent under her supervision. I thank Mrs Thong for her help in acquisition of laboratory reagents and guidance in using of the flow cytometer. Special thanks to all present and past lab colleagues, particularly Eunice, Chai Lian, Irene and Baihui in processing of patient’s samples, Jamie for generating eGFP-BCG and pMV261-BCG and anyone who have supported and helped me get through this long journey. I thank Prof P Andersen and M Doherty (Statens Serum Institute, Denmark) for provision of some of the ESAT-6/CFP-10peptides, mycobacterium sonicate and PPD. I would also like to thank A/Prof YT Wang, Dr C Chee, Dr A Cherian and nursing staff (TBCU), Dr TH Lee and Dr KC Lee (Clifford Dispensary) for their help in patient recruitment and phlebotomy, as well as patients and healthy volunteers who participated in this study. Special thanks also to Fu Ling and Fui Leng (Biopolis Resource Centre) for their kind assistance and guidance in using the Cellomics ArrayScan. ii Table of Contents To my parents, sister and William, I deeply appreciate their support, patience and sacrifices made in one way or another so that I could fulfil my ambitions and dreams. Lastly, I thank everyone else who have provided me with assistance or invaluable advice in one way or another during the course of my graduate studies. I could not have made it without everyone’s help! iii Table of Contents Oral presentation: The 5th Combined Scientific Meeting (Singapore Society for Biochemistry and Molecular Biology, Singapore Society for Microbiology and Biotechnology, Biomedical Research & Experimental Therapeutics Society of Singapore) incorporating the 4th Graduate Students’-Society – Faculty of Medicine Scientific Meeting, Singapore; 12-14 May 2004: Joanne SL Kang and GT Seah. Mycobacterium-specific IFN-γ production is protective in healthy PPD+ TB contacts but not in those with latent TB infection. Poster presentation: Novartis Institute for Tropical Diseases Inaugural Symposium on Dengue Fever and Tuberculosis, Singapore; 22-23 January 2003: JSL Kang, A Cherian, CBE Chee, TM Doherty, P Andersen, GT Seah. Mycobacterial antigen recognition and immune protection against tuberculosis. TB VACCINES FOR THE WORLD - TBV 2003, Montreal, Canada; 17-19 September 2003: JSL Kang, TM Doherty, GT Seah. Host immunity in tuberculosis infection. Keystone symposium: Innate immunity to pathogens, Colorado, USA; 8-13 January 2005: Joanne SL Kang and GT Seah. Mycobactericidal activity of macrophages is associated with distinct T cell cytokine expression profiles in human tuberculosis. iv Table of Contents TABLE OF CO TE TS Page Title Acknowledgements Table of Contents Summary List of Tables List of Figures List of Abbreviations CHAPTER i v ix x xi xv PROJECT OVERVIEW AND AIMS 1-1 Immune responses in tuberculosis patients 1-2 Immune responses in latent tuberculosis infection (LTBI) 1-3 Research aims and project design CHAPTER 2-1 LITERATURE REVIEW . Bacteriology of tuberculosis 2-2 Epidemiology of tuberculosis 2-2.1 Global burden of TB . 2-2.2 Tuberculosis in Singapore . 2-3 Impact of the HIV/ AIDS pandemic . 2-4 Transmission of tuberculosis . 2-5 Pathology of tuberculosis . 2-6 Immunology of tuberculosis 11 2-6.1 Innate immunity 11 2-6.2 Macrophages in control of TB . 12 2-6.3 Humoral immunity 13 2-6.4 Cell-mediated immunity (CMI) and T cells 14 2-6.5 Cytokines in TB 18 2-7 Diagnosis and management of tuberculosis . 21 2-7.1 Diagnosis of active TB 21 2-7.2 Diagnosis of LTBI . 23 2-8 Immune correlates of TB protection 25 2-9 Immune profiles in human LTBI 27 CHAPTER STRONG PPD RESPONSES ASSOCIATED WITH POOR INHIBITION OF MYCOBACTERIUM GROWTH IN LATENT TUBERCULOSIS 30 v Table of Contents 3-1 Abstract . 30 3-2 Introduction 32 3-3 Materials and methods 35 3-3.1 Study subjects 35 3-3.2 Antigens 37 3-3.3 PBMC stimulation and IFN-γ quantitation 38 3-3.4 Mycobacterium inhibition assay . 39 3-3.5 Statistics 40 3-4 Results . 41 3-4.1 PPD, ESAT-6/CFP-10 and TST responses in different clinical groups 41 3-4.2 Strong TST responders in ENR contacts show better mycobacterium inhibition than ER/LTBI contacts 46 3-4.3 IFN-γ responses to PPD correlated with mycobacterium inhibition in ENR, but inversely correlated in LTBI and TB groups . 48 3-5 Discussion 53 CHAPTER REGULATORY FACTORS CORRELATE WITH REACTIVITY TO MYCOBACTERIUM ANTIGENS IN ACTIVE TUBERCULOSIS 58 4-1 Abstract . 58 4-2 Introduction 60 4-3 Materials and methods 62 4-3.1 Study subjects 62 4-3.2 Generation of cRNA standards 63 4-3.3 Measuring cytokine mRNA expression . 65 4-3.4 Mycobacterium inhibition assay . 67 4-3.5 Statistics 67 4-4 Results . 68 4-4.1 LTBI TB contacts but not community controls have different cytokine profiles from ENR subjects 68 4-4.2 TB infected subjects have higher basal IFN-γ and IL-4 mRNA expression than ENR subjects 68 4-4.3 IFN-γ, TNF-α expression positively correlate with TGF-β expression in LTBI but negative correlation seen in TB 72 4-4.4 FoxP3, IL-10 and TGF-β expression are correlated in infected groups 74 4-4.5 In TB group, PPD-specific IFN-γ response associated with low TNF-α but high IL-4 and TGF-β 76 4-4.6 ESAT-6/CFP-10-specific IFN-γ response correlated with IL-4, FoxP3, TGF-β . 79 4-5 Discussion 81 CHAPTER DIFFERENTIAL RESPONSIVENESS TO EARLY SECRETED ANTIGENIC TARGET IN TUBERCULOSIS PATIENTS 89 5-1 Abstract . 89 5-2 Introduction 90 vi Table of Contents 5-3 Materials and methods 93 5-3.1 Study subjects 93 5-3.2 Quantitation of cytokine mRNA expression . 93 5-3.3 Mycobacterium inhibition assay . 93 5-3.4 Statistics 94 5-4 Results . 95 5-4.1 ENR TB patients have higher expression of IFN-γ and TNF-α but ER TB patients have higher expression levels of regulatory biomarkers 95 5-4.2 ER TB patients have higher expression levels of suppressive factors . 97 5-4.3 ER TB patients have poorer mycobactericidal activity . 100 5-5 Discussion 101 CHAPTER A NOVEL HIGH THROUGHPUT METHOD FOR PERFORMING THE MYCOBACTERIUM INHIBITION ASSAY TO EVALUATE HUMAN ANTI-MYCOBACTERIUM IMMUNITY 104 6-1 Abstract . 104 6-2 Introduction 105 6-3 Materials and methods 108 6-3.1 Human donors . 108 6-3.2 Bacteria used . 108 6-3.3 Lymphocyte stimulation 109 6-3.4 BCG infection assay 110 6-3.5 BCG viability assay . 110 6-3.6 ArrayScan : setting detection criteria 111 6-3.7 Computation of parameters and statistical analysis . 112 6-4 Results . 114 6-4.1 Fluorescence intensity of BCG with single copy versus episomal multi-copy vector . 114 6-4.2 Image capture and analysis 114 6-4.3 Optimisation of lysis procedure for BCG viability assay 116 6-4.4 Correlation between fluorescence intensity and spot count . 118 6-4.5 BCG-inhibition activity : dependence on lymphocytes and PPD stimulation . 119 6-4.6 Correlation between parameters 122 6-4.7 Mycobacterium inhibition in latent infection versus non-infected subjects 123 6-4.8 Strong tuberculin reactivity associated with better mycobacterium inhibition 123 6-5 Discussion 127 CHAPTER HOST IMMUNE PROFILES ARE ASSOCIATED WITH MYCOBACTERICIDAL ACTIVITY IN LATENT TUBERCULOSIS . 134 7-1 Abstract . 134 7-2 Introduction 136 7-3 Materials and methods 138 7-3.1 Study subjects 138 7-3.2 BCG inhibition and viability assays 139 7-3.3 Flow cytometry . 141 7-3.4 Statistical analysis . 141 vii Table of Contents 7-4 Results . 142 7-4.1 Significantly different cytokine profiles between polarised groups 142 7-4.2 Effect of cytokine neutralisation on mycobacterium inhibitory activity . 143 . 145 7-4.3 Balanced cytokine profile in ESAT-responders associated with best mycobacterium inhibition . 145 7-4.4 Cell surface expression of T regulatory cell markers 148 7-5 Discussion 150 CHAPTER 8-1 CONCLUSIONS AND FUTURE APPLICATIONS . 155 Summary of key findings . 155 8-2 Future work 156 8-2.1 Investigating if in vitro mycobacterium inhibition and cytokine profiles correlate with longterm protection against TB infection or reactivation . 156 8-2.2 Investigating the link between environmental mycobacterium sensitisation and immune protection . 157 8-2.3 Factors influencing mycobacterium inhibition 158 CHAPTER REFERENCES . 159 CHAPTER 10 APPENDIX . 183 10-1 Middlebrook 7H9 broth (1 L) . 183 10-2 Middlebrook 7H10 Agar (1 L) 183 10-3 Preparation of reagents for in vitro transcription . 184 10-3.1 TE Buffer 184 10-3.2 TE-saturated phenol:chloroform: isoamyl alcohol (25:24:1) 184 10-4 Propidium iodide staining buffer 185 10-5 ESAT-6/CFP-10 peptide sequences used for stimulation . 186 10-6 Chapters 3-5 patients’ demographic profiles . 188 10-7 Chapters and subjects’ immune profiles 189 10-8 Primer sequences and PCR conditions . 190 viii Summary SUMMARY There is a lack of reliable immune correlates of protection in tuberculosis (TB). This study sought to identify differences in the immune profiles of patients with active or treated TB disease, healthy TB contacts with latent tuberculosis infection (LTBI) and uninfected individuals with strong memory responses to mycobacterium antigens. The work was based on analysing a combination of clinical profiles, cytokine mRNA profiles, regulatory factors, specific responses to mycobacterium antigens and in vitro mycobactericidal activity. Tuberculin reactivity, and in vitro peripheral blood mononuclear cell responses to purified protein derivative (PPD), were both correlated with mycobactericidal activity only in clinically healthy persons without LTBI. These people had better mycobacterium killing activity than those with LTBI. In TB patients, strong reactivity to Mycobacterium tuberculosis-specific antigens ESAT-6/CFP-10 was associated with relatively lower protective and higher T regulatory cytokine activity, consistent with their poor mycobactericidal activity. The LTBI groups had a better balance of pro- and anti-inflammatory cytokines. A novel high throughput intracellular mycobacterium inhibition assay technique was developed. Using this assay to study TB-unexposed subjects with LTBI, the ones with a balanced profile of pro-inflammatory and regulatory cytokines were found to be more protected than those with a skewed profile of either predominantly pro- or antiinflammatory cytokines. This study has identified biomarker profiles which characterise those with protective mycobactericidal activity, in both clinically healthy people and those with TB. This is relevant to risk stratification of TB contacts and patients, and also in identifying protective responses in human TB vaccine trials. ix List of Tables LIST OF TABLES o. 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WB Saunders Company. 182 Chapter 10: Appendix CHAPTER 10 APPENDIX 10-1 Middlebrook 7H9 broth (1 L) 900 ml 4.7 g 10 ml ml 100 ml Distilled water 7H9 broth powder 50% glycerol 20% Tween 20 OADC All ingredients except OADC were added aseptically to the prepared culture media and autoclaved at 121 °C for 15 min. OADC was added when the broth had cooled. 10-2 Middlebrook 7H10 Agar (1 L) 900 ml 19 g 10 ml ml 100 ml Distilled water 7H9 broth powder 50% glycerol 20% Tween 20 OADC All ingredients except OADC were added aseptically to the prepared culture media and autoclaved at 121 °C for 15 min. The agar media was left to cool to 60 °C before OADC was added just before pouring plates. 183 Chapter 10: Appendix 10-3 Preparation of reagents for in vitro transcription 10-3.1 TE Buffer A stock of 100 mM Tris-HCl, pH 8.0 (Sigma) in 10 ml was prepared and ml of this stock solution subsequently used to make 10 mM Tris-HCl in 50 ml TE buffer. To make mM EDTA in 50 ml TE buffer, ml of 50 mM EDTA (Sigma) was added to the 10 mM Tris-HCl solution. The pH was lowered with 3.7% HCl to pH 4.5 and the TE buffer was topped up to 50 ml with RNAse-free water. 10-3.2 TE-saturated phenol:chloroform: isoamyl alcohol (25:24:1) Glass pipettes were used to aliquot a volume of 9.6 ml of chloroform (Sigma, molecular grade) and 0.4 ml isoamyl alcohol (Sigma) into a glass bottle and mixed well to make up chloroform:isoamyl alcohol (24:1). A volume of ml TE buffer (pH 4.5) and ml phenol (Sigma) was then added together in a separate bottle and vortexed before allowing the phases to separate. Then 2.5 ml of the lower TE-phenol saturated phase was mixed with 2.5 ml of chloroform:isoamyl alcohol (24:1) to make up the TE-saturated phenol:chloroform:isoamyl alcohol (25:24:1). 184 Chapter 10: Appendix 10-4 Propidium iodide staining buffer 500 ml 6.055 g 4.383 g 55.5 mg 50.8 mg 500 µl Nanopure water 100 mM Tris (pH 7.4) 150 mM NaCl 1µM CaCl2 0.5µM MgCl2 0.1% Nonidet-P40 All components were dissolved together and then syringe-filtered using a 0.2 µm filter. 185 Chapter 10: Appendix 10-5 ESAT-6/CFP-10 peptide sequences used for stimulation Table 10-1. ESAT-6/CFP-10 peptide sequences. Peptide number Sequence Molecular mass ES1 MTEQQWNFAGIEAAA 1666.81 ES2 WNFAGIEAAASAIQG 1505.631 ES3 IEAAASAIQGNVTSI 1444.587 ES4 SAIQGNVTSIHSLLD 1554.702 ES5 NVTSIHSLLDEGKQS 1627.753 ES6 HSLLDEGKQSLTKLA 1639.85 ES7 EGKQSLTKLAAAWGG 1516.698 ES8 LTKLAAAWGGSGSEA 1418.551 ES9 AAWGGSGSEAYQGVQ 1467.495 ES10 SGSEAYQGVQQKWDA 1653.705 ES11 YQGVQQKWDATATEL 1737.867 ES12 QKWDATATELNNALQ 1702.822 ES13 TATELNNALQNLART 1629.772 ES14 NNALONLARTISEAG 1571.692 ES15 NLARTISEAGQAMAS 1519.678 ES16 ISEAGQAMASTEGNV 1464.553 ES17 QAMASTEGNVTGMFA 1514.677 CFP10-1 MAEMKTDAATLAQEA 1580.778 CFP10-2 TDAATLAQEAGNFER 1593.651 CFP10-3 LAQEAGNFERISGDL 1619.732 CFP10-4 GNFERISGDLKTQID 1692.827 CFP10-5 ISGDLKTQIDQVEST 1633.754 CFP10-6 KTQIDQVESTAGSLQ 1604.716 CFP10-7 QVESTAGSLQGQWRG 1603.692 186 Chapter 10: Appendix CFP10-8 ASGLQGQWRGAAGTA 1430.524 CFP10-9 GQWRGAAGTAAQAAV 1414.525 CFP10-10 AAGTAAQAAVVRFQE 1489.632 CFP10-11 AQAAVVRGQEAANKQ 1630.805 CFP10-12 VRGQEAANKQKQELD 1803.973 CFP10-13 AANKQKQELDEISTN 1688.793 CFP10-14 KQELDEISTNIRQAG 1701.835 CFP10-15 EISTNIRQAGVQYSR 1721.871 CFP10-16 IRQAGVQYSRADEEQ 1749.838 CFP10-17 VQYSRADEEQQQALS 1751.808 CFP10-18 ADEEQQQALSSQMGF 1668.737 187 Chapter 10: Appendix 10-6 Chapters 3-5 patients’ demographic profiles Table 10-2. Patient demographics. Group Actives Treated Contacts Community Total Number 49 61 105 152 Age (Median, yrs) 47 53 39 49 Gender (F:M) Chinese 1:5 1:3 1:1 1:4 69.4 70.5 63.8 76.3 Ethnicity (%) Malay Indian 14.3 21.3 14.3 12.5 0.0 1.6 8.6 10.5 Others 16.3 6.6 13.3 0.7 BCG vaccination status (%) scar scar scar Unknown 49.0 47.5 25.5 3.9 24.5 39.3 55.7 20.4 11.5 17.0 95.4 6.1 1.7 1.8 0.7 Table 10-3. Characteristics of TB Contacts. Group Contacts TST results (%) ≥ 10 ≥ 16 mm mm 65.7 34.3 Index case Smear for AFB (%) +ve -ve 77.1 20.9 Culture for MTC (%) 100 Extent of exposure (%) Close, prolonged 70.8 Regular, intermittent 25.5 188 Chapter 10: Appendix Table 10-4. Characteristics of TB (Actives and Treated) patients. Group Actives Treated Smear for AFB (%) +ve -ve 69.4 16.3 62.3 37.7 Culture for MTC (%) Extent of disease (%) Minimal 18.4 13.1 100 100 Moderate 71.4 63.9 Advanced 10.2 23.0 10-7 Chapters and subjects’ immune profiles Table 10-5. Immunological characteristics of healthy subjects. Group (total number) Healthy (299) TST results (%) < 10mm 10-15 mm 66.3 BCG vaccination status (%) ≥ 16 mm 24.7 scar 14.7 scar 85.3 ESAT-6/CFP-10 response (%) positive negative 24.7 75.3 T-spot response (%) positive 11.2 negative 88.8 189 Chapter 10: Appendix 10-8 Primer sequences and PCR conditions Table 10-6. Primer sequences. Primer sequences Product size (bps) β-actin single round primers 660 Sense: 5’-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3’ Anti-sense: 5’-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG-3’ β-actin appended with T7 RNA polymerase promoter and poly(dT) sequence 694 T7 Sense: 5’-TAA TAC GAC TCA CTA TAG GTG ACG GGG TCA CCC ACA CTG-3’ dT Anti-sense: 5’-TTT TTT TTT TTT TTT CTA GAA GCA TTT GCG GTG GAC-3’ IL-4 outer primers 281 Sense: 5’-CGA GTT GAC CGT AAC AGA CAT-3’ Anti-sense: 5’-CGT CTT TAG CCT TTC CAA GAA G-3’ IL-4 inner primers 165 Sense: 5’-TGT GCT CCG GCA GTT CTA CAG-3’ Anti-sense: 5’-TGG CTT CCT TCA CAG GAC AGG-3’ IL-4 appended with T7 RNA polymerase promoter and poly(dT) sequence 315 T7 Sense: 5’-TAA TAC GAC TCA CTA TAG GCG AGT TGA CCG TAA CAG ACA T-3’ dT Anti-sense:5’-TTT TTT TTT TTT TTT CGT CTT TAG CCT TTC CAA GAA G-3’ 190 Chapter 10: Appendix IL-6 outer primers 430 Sense: 5’-CCA GCT ATG AAC TCC TTC TC-3’ Anti-sense: 5’-CTC TGG CTT GTT CCT CAC TA-3’ IL-6 inner primers Sense: 5’-GCC ACT CAC CTC TTC AGA AC-3’ 204 Anti-sense: 5’-ACC AGG CAA GTC TCC TCA TT-3’ IL-6 appended with T7 RNA polymerase promoter and poly(dT) sequence T7 Sense: 5’-TAA TAC GAC TCA CTA TAG GCC AGC TAT GAA CTC CTT CTC- 464 3’ dT Anti-sense: 5’-TTT TTT TTT TTT TTT CTC TGG CTT GTT CCT CAC TA-3’ IL-10 outer primers 597 Sense: 5’-ATC AAG GCG CAT GTG AAC TC-3’ Anti-sense: 5’-TGG TCA GGC TTG GAA TGG AA-3’ IL-10 inner primers 222 Sense: 5’-CCG TGG AGC AGG TGA AGA AT-3’ Anti-sense: 5’-TCT CAA GGG GCT GGG TCA GCT ATC CCA-3’ IL-10 appended with T7 RNA polymerase promoter and poly(dT) sequence 631 T7 Sense:5’-TAA TAC GAC TCA CTA TAG GAT CAA GGC GCA TGT GAA CTC3’ dT Anti-sense:5’-TTT TTT TTT TTT TTT TGG TCA GGC TTG GAA TGG AA-3’ 191 Chapter 10: Appendix IL-17 outer primers 576 Sense: 5’-GCC ATA GTG AAG GCA GGA AT-3’ Anti-sense: 5’-GGC CAA GTG TTA CCT CTG AA-3’ IL-17 inner primers 230 Sense: 5’-CTA CAA CCG ATC CAC CTC AC-3’ Anti-sense: 5’-CCA CGG ACA CCA GTA TCT TC-3’ IL-17 appended with T7 RNA polymerase promoter and poly(dT) sequence 610 T7 Sense: 5’-TAA TAC GAC TCA CTA TAG GGC CAT AGT GAA GGC AGG AAT-3’ dT Anti-sense: 5’-TTT TTT TTT TTT TTT GGC CAA GTG TTA CCT CTG AA-3’ IL-23p19 outer primers 436 Sense: 5’-GAG CTG TAA TGC TGC TGT TG-3’ Anti-sense: 5’-AGG CTT GGA ATC TGC TGA GT-3’ IL-23p19 inner primers 195 Sense: 5’-CAC TGG CCT GGA GTG CAC AT-3’ Anti-sense: 5’-CCG ATC CTA GCA GCT TCT CA-3’ IL-23p19 appended with T7 RNA polymerase promoter and poly(dT) sequence 470 T7 Sense: 5’-TAA TAC GAC TCA CTA TAG GGA GCT GTA ATG CTG CTG TTG-3’ dT Anti-sense: 5’-TTT TTT TTT TTT TTT AGG CTT GGA ATC TGC TGA GT-3’ 192 Chapter 10: Appendix FoxP3 outer primers 307 Sense: 5’-CAG TCT CTG GAG CAG CAG CT-3’ Anti-sense: 5’-TAG GTG AAA GGG GGT CGC AT-3’ FoxP3 inner primers 228 Sense: 5’-CAG GCC CAC CTG GCT GGG AA-3’ Anti-sense: 5’-ACT TGA AGT AGT CCA TGT TGT G-3’ FoxP3 appended with T7 RNA polymerase promoter and poly(dT) sequence 341 T7 Sense-5’ TAA TAC GAC TCA CTA TAG GCA GTC TCT GGA GCA GCA GCT-3’ dT Anti-sense: 5’-TTT TTT TTT TTT TTT TAG GTG AAA GGG GGT CGC AT-3’ IFN-γ outer primers 437 Sense: 5’-GGC TGT TAC TGC CAG GAC CCA TAT GT-3’ Anti-sense: 5’-GAT GCT CTT CGA CCT CGA AAC AGC AT-3’ IFN-γ inner primers 306 Sense: 5’-GCA GGT CAT TCA GAT GTA GCG GAT-3’ Anti-sense: 5’-AGC CAT CAC TTG GAT GAG TTC ATG-3’ IFN-γ appended with T7 RNA polymerase promoter and poly(dT) sequence T7 Sense: 5’-TAA TAC GAC TCA CTA TAG GGG CTG TTA CTG CCA GGA 471 CCC-3’ dT Anti-sense: 5’-TTT TTT TTT TTT TTT GAT GCT CTT CGA CCT CGA AAC3’ 193 Chapter 10: Appendix TGF-β outer primers 285 Sense: 5’-CGA GCC CTG GAC ACC AAC TAT T -3’ Anti-sense: 5’-GCG GCC CAC GTA GTA CAC GAT G-3’ TGF-β inner primers 142 Sense: 5’-CGT GCG GCA GCT GTA CAT TG-3’ Anti-sense: 5’-GGC CAG GAC CTT GCT GTA CT-3’ TGF-β appended with T7 RNA polymerase promoter and poly(dT) sequence 319 T7 Sense-5’ TAA TAC GAC TCA CTA TAG GCG AGC CCT GGA CAC CAA CTA TT -3’ dT Anti-sense-5’ TTT TTT TTT TTT TTT GCG GCC CAC GTA GTA CAC GAT G-3’ TNF-α outer primers 353 Sense: 5’-CGG GAC GTG GAG CTG GCC GAG GAG-3’ Anti-sense: 5’-CAC CAG CTG GTT ATC TCT CAG CTC-3’ TNF-α inner primers 216 Sense: 5’-TCT TCT CCT TCC TGA TCG TGG CAG-3’ Anti-sense: 5’-TTA TCT CTC AGC TCC ACG CCA TTG GC-3’ TNF-α appended with T7 RNA polymerase promoter and poly(dT) sequence 387 T7 Sense-5’ TAA TAC GAC TCA CTA TAG GCG GGA CGT GGA GCT GGC CGA GGA G-3’ dT Anti-sense-5’ TTT TTT TTT TTT TTT CAC CAG CTG GTT ATC TCT CAG CTC-3’ 194 Chapter 10: Appendix Table 10-7. Optimised PCR conditions. Cytokine β-actin IL-4 IL-6 IL-10 IL-12p35 IL-12p40 IL-17 IL-18 IL-23p19 FoxP3 IFN-γ T-bet TGF-β TNF-α dT, T7 PCR annealing conditions 68°C 60°C 50°C 66°C 55°C 55°C 60°C 55°C 60°C, 52°C 50°C 60°C 50°C 68°C 63°C Number of cycles 30 30 35 40 35 35 45 35 15, 35 45 30 45 35 45 First round PCR annealing conditions 60°C 60°C 50°C 60°C 64.5°C 55°C 50°C 55°C 50°C 50°C 60°C 50°C 55°C 50°C Number of cycles 25 20 20 21 18 21 22 18 20 19 20 22 30 20 Second round PCR annealing conditions N.A 60°C 50°C 60°C 60°C 55°C 50°C 55°C 50°C 50°C 60°C 50°C 60°C 50°C Number of cycles N.A 25 28 25 28 30 27 26 25 26 25 26 20 20 195 [...]... workers from these countries, and reactivation of latent infection in elderly persons who lived in Singapore during the high incidence period may be factors involved in maintaining the TB incidence in Singapore 2-3 Impact of the HIV/ AIDS pandemic The pandemic of the human immunodeficiency virus (HIV) has changed the epidemiology of TB Due to its ability to destroy the immune system, HIV has emerged... Mycobacterium reactivity and mycobacterium inhibition in different clinical groups 50 Figure 3-6 Mycobacterium reactivity and mycobacterium inhibition in different sub-groups of TB Contacts 51 Figure 3-7 Regression lines showing opposite correlation trends between mycobacterium inhibition and PPD-specific IFN-γ responses in infected and uninfected groups 52 Figure 4-1 Differences in cytokine expression between... overview and aims living in a TB endemic area These research questions are relevant, not only to understanding the immunology of host-pathogen interactions in this disease, but also clinically for risk stratification of TB contacts and in assessing vaccine efficacy in clinical trials The project was conducted in Singapore, where BCG is widely given at birth and there is a moderate incidence of TB Two... responses to purified protein derivative (PPD) and ESAT-6/ CFP-10 in different clinical groups 43 Figure 3-2 Tuberculin skin test readings and correlations with in vitro mycobacterium antigen-stimulated responses in TB Contacts 45 Figure 3-3 TST outcomes in relation to mycobacterium inhibition in TB Contacts 47 Figure 3-4 Mycobacterium inhibition by 3H-uridine uptake in different clinical groups 48 Figure... of remaining cases were from South-East Asia, mainly India (WHO 2008) In 2006, 357 Singapore residents were diagnosed with HIV and 3.6% of total notified TB cases (n = 1,256) had HIV co-infection (MOH 2006; Ministry of Health, Singapore Jan-March 2007) 8 Chapter 2: Literature review 2-4 Transmission of tuberculosis Inhalation of aerosolised infectious particles containing Mtb results in primary infection... in murine and human tuberculous granulomas respectively could be important for interaction with different inflammatory cell types (Tsai et al., 2006) 2-6.4 Cell-mediated immunity (CMI) and T cells In murine tuberculosis, both activated CD4+ and CD8+ T cells accumulate in lung-draining lymph nodes within 1 week after Mtb infection (Feng et al., 1999; Serbina et al., 2000) Between 2 to 4 weeks post-infection,... of latent infection in childhood, the increased TB incidence at older age is also likely to be due to increased risk of reactivation with declining immunity Of all reported Singapore resident cases in 2006, 1,256 were new TB cases while 137 were relapsed cases The mortality rate of TB in Singapore in 2006 was 1.7 cases per 100,000 population (61 deaths) and accounted for 0.4% of all deaths in Singapore... 100,000 in 2006 Given the rapid decline in incidence, older local residents had experienced a period of significantly higher TB incidence in their youth – it is unsurprising, therefore that in 2007, the incidence rate in Singaporeans aged 60 and above was 106 per 100,000 whereas for those below 20 years, the rate was only 6 per 100,000 (Ministry of Health, Singapore April-June 2007) In addition to the increased... any organ in the body, including the meninges, bone, joints, reproductive organs, renal system, gut and skin Lymphadenitis and pleuritis are the most common extra-pulmonary forms of TB It has been suggested that in LTBI, the bacteria may survive in a state of nonreplicating persistence in the lesions due to the anoxic conditions, reduced pH, and the presence of inhibitory fatty acids (Poole and Florey... account the heterogeneity in their prior immune exposure to mycobacteria, both pathogenic and non-pathogenic Moreover, there is difficulty in clinically defining a ‘protected’ individual, as exposure does not always lead to disease, yet it is unclear if latent infection represent a state of good protective immunity There is also a lack of a gold standard for diagnosis of latent TB infection (LTBI) 1 Chapter . 8-2.1 Investigating if in vitro mycobacterium inhibition and cytokine profiles correlate with long- term protection against TB infection or reactivation 156 8-2.2 Investigating the link between. CHAPTER 1 PROJECT OVERVIEW AND AIMS 1 1-1 Immune responses in tuberculosis patients 1 1-2 Immune responses in latent tuberculosis infection (LTBI) 2 1-3 Research aims and project design 2 . Regression lines showing opposite correlation 52 trends between mycobacterium inhibition and PPD-specific IFN-γ responses in infected and uninfected groups. Figure 4-1 Differences in cytokine