Significance of apoptosis in hepatitis B virus (HBV) infection [Elektronische Ressource] / Silke Arzberger

Silke Arzberger

Sujets

• Naturwissenschaften

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 42
• Langue: Deutsch
• Poids de l'ouvrage: 27 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Experimentelle Genetik



Significance of Apoptosis in Hepatitis B Virus Infection

Silke Arzberger


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan
für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. B. Küster
Prüfer der Dissertation:
1. Univ.-Prof. Dr. M. Hrabé de Angelis
2. Univ.-Prof. Dr. U. Protzer-Knolle
3. apl. Prof. Dr. V. Bruss
(Georg-August-Universität Göttingen)


Die Dissertation wurde am 25.06.2009 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 07.12.2009 angenommen.

Contents
Contents
List of Abbreviations I
ABSTRACT II
1 INTRODUCTION 1
1.1. Overview of Hepatitis B 1
1.1.1 Epidemiology 1
1.1.2 Classification 2
1.1.3 Experimental Models for HBV 3
1.1.3.1. Cell Culture Models 3
1.1.3.2. Animal Models 4
1.1.3.3. Applied Model Systems 5
1.2. The Hepatitis B Virus 7
1.2.1 Particle Structures 7
1.2.1.1. Virion 7
1.2.1.2. Subviral Particles (SVP) 8
1.2.2 Genomic Structure and Organization 8
1.2.2.1. Virion 8
1.2.2.2. cccDNA 9
1.2.2.3. HBV Transcripts 9
1.2.2.4. Viral Proteins 9
1.2.2.5. Genomic Organization 11
1.2.3 Viral Life Cycle 13
1.2.3.1. Delivery to Hepatocytes 13
1.2.3.2. Entry 14
1.2.3.3. Intracellular Transport to the Nucleus 14
1.2.3.4. Transcription 15
1.2.3.5. Translation 15
1.2.3.6. Replication 15
1.2.3.7. Morphogenesis and Release 17
1.3. Hepatitis B Infection 19
1.3.1 Pathogenesis of an acute HBV-infection 19
1.3.2 Innate Immunity 20
1.3.2.1. Liver Environment 20
1.3.2.2. Important Cytokines Controlling HBV-Infection 21
1.3.2.3. Recognition of Invaders by Innate Immunity 22
1.3.3 Priming Adaptive Immunity 24
1.3.3.1. Dendritic Cells (DC) 24
1.3.3.2. Macrophages 25
Contents
1.3.3.3. B Cells 25
1.3.3.4. Liver Sinusoidal Endothelial Cells (LSEC) 26
1.3.3.5. Cross-Presentation 26
1.3.4 Cell-Mediated Immunity 27
1.3.4.1. Antigen Recognition by T Lymphocytes 27
1.3.4.2. T Cell Receptor (TCR) and Co-Receptor 27
+1.3.4.3. CD4 T cells 27
+1.3.4.4. CD8 T cells 28
1.3.5 Humoral Immunity 29
1.3.5.1. B Cell Receptor (BCR) and Antigen Recognition 29
1.3.5.2. Activation of B cells and Antibody Production 29
1.3.5.3. Isotype-Switching 30
1.3.5.4. T Cell-Independent Activation of B Cells by HBcAg 30
1.4. Apoptosis 31
1.4.1.1. Initiation of Apoptosis 31
1.4.1.2. Characteristic Features of Hepatocytes 32
1.4.1.3. Extrinsic Pathway 33
1.4.1.4. Intrinsic Pathway 35
1.4.2 Interference of HBx with Apoptotic Cell Signaling Pathways 36

2 EXPERIMENTAL PART I 39
DOES HBV REPLICATION SENSITIZE HOST CELLS TOWARDS APOPTOSIS
TO ENHANCE THE SPREAD OF PROGENY THROUGH LIVER TISSUE? 39
2.1.1.1. HBV-transformed Hepatoma Cell Lines 40
2.2. Results 43
2.2.1 Sensitivity of HBV-replicating hepatoma cells towards apoptosis 43
2.2.2 Microarray Analysis of HBV-replicating hepatoma cell lines 45
2.2.3 Structure and Infectivity of HBV particles released from apoptotic cells 53
2.2.3.1. HBV-transformed hepatoma cell lines 53
2.2.3.2. HBV-infected PHH 55
2.2.3.3. Structure analysis of HBV particles released from PHH 59
2.3. Discussion 63
2.3.1 Apoptosis-Induction of Hepatocytes 63
2.3.2 HBV nucleocapsids are released from apoptotic hepatocytes 64
2.3.3 Apoptosis abolished HBV-propagation 66
2.3.4 HBV-replicating cell lines are partially resistant to apoptosis 66
2.3.5 cation does not sensitize cells towards apoptosis on mRNA level 67
Contents
3 EXPERIMENTAL PART II: 71
CD95-INDUCED APOPTOSIS OF HBV-INFECTED HEPATOCYTES
INFLUENCED THE PRIMING OF B AND T CELLS 71
3.1.1.1. Mouse model of acute self-limiting HBV infection 72
3.2. Progression of acute HBV infection in mice deficient for different stimuli of apoptosis 73
3.3. Results I 75
3.3.1 Serology 75
3.3.2 Viral transcription and replication in liver tissue 77
3.3.3 Intrahepatic T cell response 78
3.3.4 Seroconversion 81
3.3.5 Histology 83
3.4. The Clearance of acute HBV infection in mice harboring a hepatocyte-specific
deficiency of CD95-receptor signaling 85
3.4.1 Hepatocyte-specific FADDhep k/o Mice 85
3.4.1.1. Genotyping 86
3.4.2 Experimental Design 87
3.5. Results II 89
3.5.1 Serology 89
3.5.1.1. Viral transcription and replication in liver tissue 93
3.5.1.2. Intrahepatic T cells response 95
3.5.2 Seroconversion 97
3.5.2.1. anti-HBs / anti-HBc 97
3.5.2.2. IgG Isotype Profiling 97
3.5.3 Histology 99
3.5.3.1. H&E stain 99
3.5.3.2. HBc stain 101
3.5.4 Maturation level of LAL in gld mice 103
3.5.5 The Influence of HBV capsids on seroconversion 105
3.6. Discussion 109
3.6.1 CD95-induced apoptosis of HBV-infected hepatocytes is important in priming
the adaptive immune response 110
3.6.2 Comparing cytolytic pathways during viral infection 112
3.6.3 Contribution of perforin/granzyme in controlling HBV infection 114
3.6.4 Liverpathology and ALT 115
3.6.5 Non-cytopathic effector functions in controlling HBV infection 116
3.6.6 Are mice a suitable model system to analyze acute-HBV infection? 118

Contents
4 MATERIALS AND METHODS 121
4.1. Cell Culture 121
4.1.1 Culture Media and Buffers 121
4.1.1.1. Calculation of cell numbers and cell viability 122
4.1.2 Primary Human Hepatocytes (PHH) 122
4.1.2.1. Isolation of PHH using two step perfusion 122
4.1.2.2. PEG-triggered HBV infection 123
4.1.3 HBV-replicating cell lines 124
4.1.3.1. Cell culture conditions 124
4.1.3.2. Culture conditions boosting HBV-production 124
4.1.4 Production of HBV 124
4.1.5 Induction of Apoptosis 124
4.2. Production of Adenoviral Vectors 125
4.2.1.1. Vector Propagation 125
4.2.1.2. CsCl Gradient 125
4.2.1.3. Dialysis 126
4.2.1.4. Titration 127
4.3. Quantitative Real Time PCR 128
4.3.1 Introduction into Real Time qPCR 128
4.3.2 Quantification of qPCR 128
4.3.2.1. Principles of Measurement 128
4.3.2.2. Threshold 129
4.3.2.3. Efficiency of PCR reaction 129
4.3.2.4. Melting curves 129
4.3.3 Protocols of qPCR 130
4.3.3.1. Used thermcyclers 130
4.3.3.2. PCR Mix 130
4.3.3.3. Running conditions 131
4.3.3.4. Primers 132
4.4. Assays Detecting Apoptosis / Cell Viability 133
4.4.1 Viability Measurement by XTT 133
4.4.2 Immunofluorescence Microscopy 133
4.4.3 Caspase 3/7 Assay 133
4.4.4 DNA Fragmentation 133



Contents
4.5. Molecular Biology 134
4.5.1 CsCl Density-Gradient Centrifugation 134
4.5.2 Dot Blot Analysis 134
4.5.3 Northern Blot Analysis 135
4.5.3.1. Agarose Gel with formaldehyde 135
4.5.3.2. Blotting 135
324.5.4 Hybridization Probes ( P) 135
4.5.4.1. HBV Probe 136
4.5.4.2. GAPDH Probe 136
4.5.5 RNA/DNA-Methods 137
4.5.5.1. Calculation of DNA/RNA Concentration 137
4.5.5.2. RNA Isolation 137
4.5.5.3. cDNA Synthesis 137
4.5.5.4. DNA Isolation 137
4.5.6 ELISA 137
4.5.6.1. HBsAg (characterization of HBV particles) 137
4.5.6.2. HBs, HBe, anti-HBs and anti-HBc 138
4.5.6.3. Isotype Profiles 138
4.6. cDNA Microarray 138
4.6.1 Principle of cDNA Microarray 138
4.6.2 Performance of Microarray 139
4.7. FACS 139
4.7.1 Principle of FACS Measurement 139
4.7.2 Cell Surface Staining 139
4.8. Animal Experiments 140
4.8.1 Intravenous Injection (i.v.) 140
4.8.2 Retro-Orbital Blood Collection 140
4.8.3 ALT-Measurement 140
5 REFERENCES 141
6 APPENDIX 155
6.1. Sequencing Data of HBx ORF 155
6.2. Liver Histology of AdHBV-infected Mice 156
DANKSAGUNG 175


Abbreviations
A G
Ab antibodies GFP green fluorescent protein
Ad adenoviral vector
ADCC antibody-dependent cell-mediated H
cytotoxicity
h hour
ALT alanine aminotransferase
HBcAg hepatitis B core antigen
APC antigen presenting cells
HBeAg hepatitis B e antigen
approx approximately
HBsAg hepatitis B surface antigen

HBV hepatitis B virus
B H&E hematoxylin-eosin stain
BCR B cell receptor HIV human immunodeficiency virus
HNF hepatocellular transcription factors
C
cccDNA covalently closed circular DNA I
CD cluster of differntiation IFN interferon
CsCl cesium chloride IFA incomplete Freunds adjuvant
CTL cytotoxic T lymphocytes Ig immunglobulin
IL- interleukine
D i.p. intraperitoneal
i.v. intravenously ddH O double distilled water 2
DHBV duck hepatis B virus
DC dendritic cells K
DMSO dimethylsulfoxid kb kilo base
DNA desoxyribonucleic acid kDa kilo dalton
DR directed repeat
dsDNA double-stranded DNA L

L ligand
E