Pathophysiology and Novel Therapeutic Approaches in Autoimmune Addison's Disease

307 Pages·2015·5.23 MB·English

Pathophysiology and Novel
Therapeutic Approaches in
Autoimmune Addison’s Disease

Earn Hui Gan
MB ChB, MRCP (UK)

Thesis submitted for the degree of Doctor of Philosophy

Institute of Genetic Medicine, School of Medical Sciences,
Newcastle University, UK

September 2014

i
Declaration

This thesis is based on my studies performed in the Institute of Genetic Medicine,
Newcastle University from August 2010- August 2013. The contents of this thesis are
original. . All the studies were performed solely by myself, apart from some of the
immunoblotting and Elisa works, which the research assistant, Katie MacArthur,
assisted me with. No part of this thesis has been submitted for the award of any other
degree.

ii
Acknowledgements
I would like to express my gratitude to everyone who has assisted me throughout the
process of producing this thesis. Firstly, I would like to thank my supervisor Professor
Simon Pearce for his teaching and encouragement, which has enhanced my training and
experience throughout my studies. He has given me a good grounding in both clinical
and laboratory aspects of research and has inspired me to be enthusiastic in medical
sciences. I am also grateful to my co-supervisor, Dr Petros Perros, who has given me
advice and encouragement to make this thesis possible.
Enormous thanks also go to Dr Rachel Oldershaw, for her advice on
immunophenotyping of mesenchymal stromal cells and supplying important reagents. I
am thankful to Professor Robert Pickard, Professor Thomas Lennard and research nurse
Wendy Robson who generously provided me with discarded adrenal tissue that was
crucial to my research.
I would like to thank all the patients who had kindly volunteered for this study, and am
grateful to Dr Petros Perros, Dr Andy James, Dr Steve Ball and Dr Richard Quinton for
allowing me to study their patients.
I would also like to express my gratitude to my colleagues working in the Institute of
Genetic Medicine for advice on aspects of the project and sharing some reagents. In
particular Katie MacArthur and Dr Anna Mitchell, who have taught me many of the
techniques used in this work and supported me during the most difficult of times.
My project could not have been carried out without the financial support from the
Medical Research Council (MRC) who funded the translational stem cell study. Many
research development opportunities including funding for conferences were also
generously provided by the Society for Endocrinology.
Finally, I would like to thank my parents and family for their endless love and support.
In particularly my husband, Nicholas, for always being there whenever I need him.

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Abstract

Autoimmune Addison’s disease (AAD) is a debilitating condition and affected patients
rely on lifelong steroid replacement. Despite treatment, many patients have increased
morbidity and mortality. The disease’s rarity has precluded large scale genomic or
cellular studies in humans, resulting in an incomplete picture of the pathophysiology of
AAD. This thesis details my research on the pathophysiology and novel therapeutic
approaches in AAD.
I performed two candidate gene studies on susceptibility alleles that have been
implicated in other autoimmune diseases to explore potential causal pathways of these
genetic determinants in AAD. The common variant 307*Ser allele of CD226 gene was
found to contribute to AAD susceptibility as part of autoimmune polyendocrinopathy
type 2. Two genetic variants from a panel of rare and functionally defective variants in
the sialic acid acetylesterase (SIAE) gene were identified but they were not significantly
associated with AAD.
I explored new therapeutic approaches in AAD using a therapeutic regimen of
parenteral ACTH . This study demonstrated the presence of residual adrenal function
1-24
in patients with established AAD, which was remediable to ACTH therapy. Clinical
1-24
remission was induced in 2 of 13 study participants. However, some patients developed
cutaneous reaction following ACTH injection, with one failing to sustain clinical
1-24
remission. I proceeded to investigate the potential immunologic effects induced by
ACTH therapy using ELISA and immunoblotting methods. Auto-reactivity against
ACTH and ACTH was detected, which could account for the side effects and
1-24 1-39
treatment resistance observed.
Finally, I attempted to characterise adrenocortical progenitor cells through cell cultures,
immunocytochemistry and flow cytometry. A mesenchymal stem cell-like (MSC)
population was isolated from human adrenals for the first time. My studies have
advanced knowledge relevant to regenerative medicine approaches to adrenal failure,
and the finding of residual adrenal function in some Addison’s disease patient opens an
important therapeutic window for this condition.

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Abbreviations

α-MEM α-modified Eagle’s medium
21-OH 21-hydroxylase
AA Adrenal autoantibodies
AAD Autoimmune Addison’s disease
ACSC Adrenocortical stem cell
ACTH Adrenocorticotropic hormone
Ad4BP Ad4 binding protein
AGP Adrenogonadal primodium
AH Autoimmune hypothyroidism
APS1 Autoimmune polyendocrinopathy syndrome type 1
APS2 Autoimmune polyendocrinopathy syndrome type 2
ARS Alizarin Red S
BCR B cell receptor
BrdU Bromodeoxyuridine
BSA Bovine serum albumin
CEDAM Centre for Diabetes and Metabolism
CFU-Fs Colony forming units-fibroblasts
CM Complete growth medium
CMV Cytomegalovirus
CTLA4 Cytotoxic T lymphocyte antigen 4
CXCL10 CXC chemokine ligand 10
CYP Cytochrome P450
CYP11B1 Cytochrome P450, family 11, subfamily B, polypeptide 1
CYP11B2 Cytochrome P450, family 11, subfamily B, polypeptide 2
DAB Diaminobenzidine
DAPI 4’, 6-diamidino-2-pheny-lindole
DAX1 Dosage-sensitive sex reversal, adrenal hypoplasia congenita
critical region of the X chromosome, gene 1
DdH O Double distilled water
2
DHEA Dehydroepiandrosterone
DHEAS Dehydroepiandrosterone sulphate
DHT 5-α-dihydrotestosterone
DMEM: HamF12 Dulbecco’s Modified Eagle Medium/Nutrient mixture F-12
DNAM-1 DNAX accessory molecule-1
EDTA Ethylenediaminetetraacetic acid
FACS Fluorescence-activated cell sorting
FAdE Fetal adrenal-specific SF1 enhancer
FBS Fetal bovine serum
FCS Forward scatter
FGF2 Fibroblast growth factor 2
GAG Glycoaminoglycan
GC/MS Gas chromatography/mass spectrometry
GR Glucocorticoid receptor
HIV Human Immunodeficiency Virus
HLA Human leukocyte antigen
HRP Horseradish peroxidase
HSD Hydroxysteroid dehydrogenase
v
IL-6 Interleukin 6
INF-γ Interferon γ
MAF Minor allele frequency
MALDI-TOF Matric assisted laser desorption/ionisation-time of flight
MC2R Melanocortin-2- receptor
MCM4 Minichromosome maintenance–deficient 4
MGB Minor grove binder
MGPM Mesenchymal stem cell growth promotion medium
MHC Major histocompatibility complex
MICA MHC class I chain-related gene A
MSC Mesenchymal stem cell
MSH Melanocyte stimulating hormone
MR Mineralocorticoid receptor
MRAP Melanocortin 2 receptor accessory protein
NADPH Nicotinamide adenine dinucleotide phosphate-oxidase
NKG2D Natural killer group 2, member D
NFκB Nuclear factor Κb
NLR NOD-like receptors (NLRs
NNT Nicotinamide nucleotide transhydrogenase
PAGE Polyacrylamide gel electrophoresis
PBS Phophate-buffered saline
PCNA Proliferating cell nuclear antigen
PCR Polymerase chain reaction
PDL-1 Programmed cell death ligand 1
POMC Proopiomelanocortin
PRL Plasma renin level
Ptch1 Patched 1
PTPN22 Protein tyrosine phosphatase non-receptor 22
RE Restriction endonuclease
RFLP Restriction fragment length polymorphism
RoSA Revival of autochthonous adrenocortical stem cells in
autoimmune Addison’s disease
SDS Sodium dodecyl sulphate
SF1 Steroidogenic factor 1
SHH Sonic hedgehog
SIAE Sialic acid acetylesterase
SIM Selected-ion-monitoring
SLE Systemic Lupus Erythematosus
Smo Smoothened
SNP Single nucleotide polymorphism
SSC Side scatter
STAR Steroidogenic acute regulatory protein
STAT1 Signal transducer and activator of transcription-1
STAT4 Signal transducer and activator of transcription-4
TMB 3,3’,5,5’-Tetramethylbenzidin
TNF-α Tumour necrosis factor-α
TLR Toll-like receptor
VEGF Vascular endothelial growth factor
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Table of Contents
Declaration………………………………………………………………………..…......ii
Acknowledgements……………………………………………………………..……....iii
Abstract………………………………………………………………………………….iv
Abbreviations…………………………………………………………………...………..v
Chapter 1. Introduction .................................................................................................. 1
1.1 Background to Addison’s disease ................................................................ 2
1.2 Autoimmune Addison’s Disease (AAD) ..................................................... 3
1.3 Presentation and natural history of AAD ..................................................... 5
1.4 Health burden of AAD ................................................................................. 8
1.5 Normal development of adrenal gland ....................................................... 12
1.6 Adrenal steroidogenesis ............................................................................. 14
1.7 Current concept on the pathogenesis of AAD ........................................... 18
1.7.1 Histopathology…………………………………………………………...18
1.7.2 Immunology ...…………………………………………………………...19
1.7.3 Genetics of AAD ………………………………………………………...28
1.8 Current therapeutic approaches in AAD .................................................... 35
1.9 Adrenal plasticity ....................................................................................... 39
1.10 Adrenocortical stem/progenitor cells ......................................................... 40
1.11 The origin of adrenocortical stem/progenitor cells .................................... 41
1.12 Adrenocortical stem cell niche and mediators for stem cell maintenance,
proliferation and differentiation .................................................................................. 45
1.12.1 Steroidogenic factor-1(SF1) ……………………………………………..45
1.12.2 DAX1( dosage-sensitive sex reversal, adrenal hypoplasia congenital
critical region, on the chromosome X, gene 1) transcription factor……...47
1.12.3 The sonic hedgehog signalling pathway…………………………………48
Aims of the thesis ........................................................................................................ 54
Chapter 2. Genetics of Autoimmune Addison’s disease ............................................. 55
2.1 Background ................................................................................................ 56
2.2 Subjects and Methods ................................................................................ 58
2.2.1 Subjects……………………………………………………………….….58
2.2.2 Methods…………………………………………………………………..59
2.2.3 Polymerase chain reaction (PCR) and gel electrophoresis……………….59
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2.2.4 CD226 genotyping: ……………………………………………………...60
2.2.5 SIAE genotyping ………………………………………………………...63
2.2.6 Primer extension-MALDI-TOF genotyping (SEQUENOM-iPLEX)……63
2.2.7 Restriction Enzyme Digestion (RFLP) …………………………………..73
2.2.8 Direct DNA sequencing …………………………………………………74
2.2.9 Statistical analysis ……………………………………………………….76
2.3 Results ........................................................................................................ 79
2.3.1 CD226 (rs763361) association analysis …………………………………79
2.3.2 SIAE rare variants association studies …………………………………..79
2.4 Discussion .................................................................................................. 88
Chapter 3. Re-establishment of adrenocortical steroidogenesis in autoimmune
Addison’s disease with parenteral ACTH1-24 therapy .................................................. 92
3.1 Background ................................................................................................ 93
3.2 Methods ..................................................................................................... 95
3.2.1 Study participants……………………………………………………… ..95
3.2.2 Study design and treatment regimen …………………………………….95
3.2.3 Objective outcome measures and assessments …………………………100
3.2.4 Methods for objective outcome measurements ………………………...101
3.2.5 Subjective outcome measures- Quality of Life assessments …………...106
3.2.6 Statistical analysis.……………………………………………………...107
3.3 Results ...................................................................................................... 110
3.3.1 Baseline characteristics ………………………………………………...110
3.3.2 Adrenal steroidogenic function ………………………………………...114
3.3.3 Participant 02 and 06 …………………………………………………...125
3.3.4 21-hydroxylase antibodies ……………………………………………...128
3.3.5 Urine steroid metabolites ……………………………………………….130
3.3.6 Quality of life assessments ……………………………………………..135
3.3.7 Safety and tolerability ………………………………………………….141
3.4 Discussion: ............................................................................................... 145
Chapter 4. Spontaneous and tetracosactide-induced anti-ACTHantibodies ............. 150

4.1 Background .............................................................................................. 151
4.2 Patients and methods ............................................................................... 154
4.2.1 Patients …………………………………………………………………154
4.2.2 Methods ………………………………………………………………...154
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4.3 Results ...................................................................................................... 163
4.3.1 Anti-tetracosactide immunoreactivity among patients receiving high-dose
depot tetracosactide …………………………………………………….163
4.3.2 Anti- tetracosactide immunoreactivity among controls vs. patients with
autoimmune diseases (autoimmune Addison’s disease, Graves’ disease and
isolated ACTH deficiency) ……………………………………………..167
4.3.3 Immunohistochemistry study …………………………………………..170
4.4 Discussion ................................................................................................ 172
Chapter 5. Characterisation of adrenocortical stem cell phenotype: Isolation of
mesenchymal stem cell-like cell populations from human adrenal cortex ................... 177
5.1 Background .............................................................................................. 178
5.2 Methods ................................................................................................... 180
5.2.1 Preparation of transport medium, growth medium and digestion
solution …………………………………………………………………180
5.2.2 Preparation of adrenal tissue and cell culture …………………………..181
5.2.3 Maintenance of adrenocortical cell culture …………………………….182
5.2.4 Detachment of monolayer cells from tissue culture plastics …………...182
5.2.5 Cell count ………………………………………………………………183
5.2.6 Population doubling time ………………………………………………183
5.2.7 Freezing and thawing of cells …………………………………………..184
5.2.8 Osteogenic, chondrogenic and adipogenic differentiation of human
adrenocortical cells ……………………………………………………..184
5.2.9 Histological examination of differentiated osteogenic, chondrogenic and
adipogenic cells from human adrenocortical cells ……………………..186
5.2.10 Immunocytochemistry study of adrenal cells ………………………….188
5.2.11 Flow cytometry analysis ……………………………………………….191
5.2.12 Tissue embedding and paraffin wax blocks. …………………………...197
5.2.13 Microtome sectioning …………………………………………………..197
5.3 Statistical analysis .................................................................................... 198
5.4 Results ...................................................................................................... 199
5.4.1 Morphological characterisation of monolayer adrenocytes in the CM vs
MGPM ………………………………………………………………….199
5.4.2 The proliferative capacity of monolayer adrenal cells in CM vs
MPGM…………………………………………………………………..206
5.4.3 In-vitro differentiation capacity of adrenal cortex-derived MSCs-like
cells……………………………………………………………………...208
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5.4.4 Flowcytometric analysis………………………………………………...213
5.4.5 Immunocytochemistry study …………………………………………...223
5.5 Discussion: ............................................................................................... 242
Chapter 6. Final discussion ........................................................................................ 246
References ………………………………..…………………………………….…….252
Appendices …………………………………………………………………….……..287
Publications …………………………………………………………………………..291

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