The role of scavenger receptor BI in hepatitis - eTheses Repository ...

More documents

Recommendations

Info

78 5. The level of infection was assessed by enumeration of NS5A positive cells using a fluorescence microscope, data is expressed as infectious units (IU) per ml of harvested HCVcc culture media; 1 infected cell = 1 IU. To ensure reliable data read outs, HCVcc virus was always diluted according to harvest titre so that each well contained 150-300IU. Neutralisation assay. Much of the work covered in this thesis regards the use of antibodies or receptor mimics to inhibit HCVcc infection. These treatments were evaluated using an adapted form of the infection assay. 1. Huh-7.5 cells were seeded for infection, as above. 2. To treat virus particles, titrated HCVcc were diluted in DMEM + 3% FCS + P/S containing the appropriate concentration of inhibitory or control compound. Alternatively, to treat target Huh-7.5 cells, culture media was replaced by 80$l of DMEM + 3% FCS + P/S containing the inhibitory or control compound. In either case pre-treatment was for 1 hr at 37°C. 3. To infect, pre-treated virus was used to inoculate naïve Huh-7.5 cells. If pre-treating target cells, 80$l of appropriately diluted HCVcc was added directly to the culture media containing the treatment. 4. In each case the infection was allowed to proceed for 48 hrs. The original virus inoculum and inhibitory treatment were present for the duration of the assay to ensure evaluation of absolute neutralisation titres.
79 5. Data is expressed as percentage neutralisation; this was calculated by comparison of the level of infection after inhibitory treatment to that after treatment with a control compound. Statistical analysis of infectivity data. To ensure appropriate statistical analysis of infection assays we used a D'Agostino & Pearson omnibus normality test to determine whether typical infectivity data fits a Gaussian distribution. Briefly, the data from n=10 JFH-1 and J6/JFH-1 HCVcc infection assays were analysed using GraphPad Prism (GraphPad Software, CA, USA), both data sets were deemed to be normally distributed as summarised in Table 2-5. We therefore have assumed normal distribution for all infectivity data and statistical analyses were performed using an unpaired t-test. Table 2-5 Normality testing. Analysis of JFH-1 and J6/JFH-1 infectivity data with the D'Agostino & Pearson omnibus normality test following the guidelines in the GraphPad statistics handbook. Iodixanol density gradient centrifugation. The analysis of HCVcc particle density was achieved by iodixanol density gradient centrifugation. This work was carried out in collaboration with Dr Søren Nielsen of Newcastle University Medical School. The preparation and
Page 1 and 2:
The role of scavenger receptor B-I
Page 3 and 4:
i Abstract. With 170 million infect
Page 5 and 6:
iii Acknowledgements. I would like
Page 7 and 8:
v 5 Results: Identification of a re
Page 9 and 10:
vii Figure 5-5 Analysis of JFH-1 wt
Page 11 and 12:
1 Introduction 1.1 The disease. 1 D
Page 13 and 14:
3 predominantly immuno-pathogenic i
Page 15 and 16:
5 Aside from considerations of the
Page 17 and 18:
7 Current attempts to design a HCV
Page 19 and 20:
1.2 An elusive pathogen. 9 The emer
Page 21 and 22:
11 PCR). This technique is highly s
Page 23 and 24:
13 Within six years of the descript
Page 25 and 26:
1.3 Basic virology. 15 Like other m
Page 27 and 28:
17 Figure 1-2 HCV genome and polypr
Page 29 and 30:
19 241). E1 and E2 are anchored to
Page 31 and 32:
21 The characterisation of HCV geno
Page 33 and 34:
23 in the E1 and E2 glycoprotein ge
Page 35 and 36:
25 The solution reached by a great
Page 37 and 38: 27 binding to mouse cells. sE2 inte
Page 39 and 40: 29 density lipoprotein (HDL), allow
Page 41 and 42: 31 blood brain barrier is permeable
Page 43 and 44: 33 act as receptors for viral entry
Page 45 and 46: 1.4.2 Attachment factors 35 The tru
Page 47 and 48: 1.4.3 Endocytosis and fusion 37 Aft
Page 49 and 50: 39 1.4.4 Co-receptor interplay and
Page 51 and 52: 41 inhibition of protein kinase A (
Page 53 and 54: 1.5 Lipoproteins 43 Since 1992 ther
Page 55 and 56: 45 Lipoproteins exist in a dynamic
Page 57 and 58: 47 and delivers its cargo via the w
Page 59 and 60: 49 uptake of cholesterol from LDL (
Page 61 and 62: Figure 1-4 SR-BI-lipoprotein intera
Page 63 and 64: 53 In summary the class B scavenger
Page 65 and 66: 55 In 1992 Thomssen et. al. demonst
Page 67 and 68: 57 lipoprotein association promotes
Page 69 and 70: 59 Figure 1-5 Lipo-viro-particle as
Page 71 and 72: 61 In vitro culture medium, into wh
Page 73 and 74: 2 Materials and methods. 2.1 Cell l
Page 75 and 76: Preparation of rat anti-E2 mAbs. 65
Page 77 and 78: 2.4 Basic techniques. Flow cytometr
Page 79 and 80: 69 6. Finally, the cells were washe
Page 81 and 82: 71 (Promega, WI, USA) kits accordin
Page 83 and 84: 73 proteins and are incapable of fu
Page 85 and 86: 75 spectrophotometer (Amersham), we
Page 87: 77 Figure 2-2 Electroporated Huh-7.
Page 91 and 92: 81 (Invitrogen). The samples to be
Page 93 and 94: 83 1. The SR-BII coding region was
Page 95 and 96: 2.7 Cloning and synthesis of JFH-1
Page 97 and 98: The sequence encoding amino acids 3
Page 99 and 100: Figure 2-6 sE2 sequencing. 89 JFH-1
Page 101 and 102: 91 7. Large scale transfections wer
Page 103 and 104: 93 3 Results: Investigations using
Page 105 and 106: 95 3.2 CHO cells expressing human S
Page 107 and 108: Figure 3-3 Ability of anti-E2 mAb t
Page 109 and 110: 99 3.3 The interaction of sE2 with
Page 111 and 112: 101 Figure 3-5 Position of SR-BI mu
Page 113 and 114: 103 Table 3-2 Investigation of cell
Page 115 and 116: 105 3.4 Expression of SR-BII in CHO
Page 117 and 118: 3.5 Discussion. 107 We have demonst
Page 119 and 120: 109 may offer a tool to study the i
Page 121 and 122: 111 Figure 4-1A displays endogenous
Page 123 and 124: 113 4.2 Exogenous expression of SR-
Page 125 and 126: 115 4.3 Evidence of enhanced JFH-1
Page 127 and 128: 117 4.4 SR-BI expression levels lim
Page 129 and 130: 119 4.5 Murine SR-BI does not enhan
Page 131 and 132: 121 4.6 SR-BI/II expression levels
Page 133 and 134: 123 Figure 4-5 Over expression of S
Page 135 and 136: 125 Figure 4-6 Anti-SR-BI serum inh
Page 137 and 138: 127 Figure 4-7 Infection of Huh-7.5
Page 139 and 140:
129 cells is largely manifested in
Page 141 and 142:
131 5 Results: Identification of a
Page 143 and 144:
133 Figure 5-1 JFH-1 G451R has an a
Page 145 and 146:
135 Figure 5-2 CD81 dependence of J
Page 147 and 148:
137 G451R sE2 with hCD81 LEL we com
Page 149 and 150:
139 Figure 5-4 JFH-1 wt and G451R s
Page 151 and 152:
141 demonstrated a lower dependence
Page 153 and 154:
143 Figure 5-6 Relationship between
Page 155 and 156:
145 flow cytometry (data not shown)
Page 157 and 158:
147 5.6 Low density JFH-1 particles
Page 159 and 160:
5.7 Discussion. 149 We have demonst
Page 161 and 162:
151 Numerous reports have used dens
Page 163 and 164:
153 demonstrate that low density JF
Page 165 and 166:
155 to over express SR-BI we were a
Page 167 and 168:
157 5-1), better able to engage CD8
Page 169 and 170:
159 Figure 6-1 An overview of antib
Page 171 and 172:
161 it is believed that ligation of
Page 173 and 174:
163 Given our and other’s finding
Page 175 and 176:
6.3 HCV lipo-viro-particles. 165 As
Page 177 and 178:
167 represent the hyper-variable re
Page 179 and 180:
169 The G451R mutation disrupts the
Page 181 and 182:
7 Bibliography 171 1. Acton, S., D.
Page 183 and 184:
173 include the CD81 tetraspanin an
Page 185 and 186:
175 receptor class B type I and inh
Page 187 and 188:
177 83. Drummer, H. E., K. A. Wilso
Page 189 and 190:
179 KewalRamani, D. R. Littman, C.
Page 191 and 192:
181 King. 1996. Efficient infection
Page 193 and 194:
183 hepatitis C virus envelope glyc
Page 195 and 196:
185 recovered chimpanzees exhibit r
Page 197 and 198:
187 218. Monazahian, M., I. Bohme,
Page 199 and 200:
189 243. Perez-Berna, A. J., J. Gui
Page 201 and 202:
191 single-cycle production assay t
Page 203 and 204:
193 298. Strickland, G. T., S. S. E
Page 205 and 206:
195 Remaley, G. Csako, T. L. Eggerm
Page 207:
197 2007. Scavenger receptor class
show all

The role of scavenger receptor BI in hepatitis - eTheses Repository ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?