PhD Thesis Title Page Nayyar Ahmed Identification and...
Transcript of PhD Thesis Title Page Nayyar Ahmed Identification and...
PhD Thesis – Title Page Nayyar Ahmed
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Identification and Characterisation of a Novel Interleukin-4 Receptor Antagonist for Allergy Treatment
___________________
by
Nayyar Ahmed
B.Biomed.Sc. (Honours)
Submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Deakin University
February, 2015
PhD Thesis – Acknowledgements Nayyar Ahmed
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Acknowledgements
Firstly, I would like to thank Associate Professor Cenk Suphioglu for being such a caring and
supportive person throughout my PhD. Your friendliness surpasses your duty as a professor
and no matter how many words I spill on this paper, they will never be enough. Thank you
for being so patient with my work. Thank you once again, for all your efforts and constant
guidance throughout my candidature. I am also grateful to you for giving me the opportunity
to be a part of the NARL laboratory. I wasn’t sure if I wanted to pursue a career as a research
scientist, however after joining your laboratory, you have inspired me and increased my
passion for scientific research. Ever grateful to you!
I would like to thank my co-supervisors Professor Colin Barrow and Professor Leigh
Ackland for serving as my co-supervisors for my candidature. I also want to thank you for
your brilliant comments and suggestions. I would also like to thank Professor Andrew
Sinclair and Dr. Philip Taylor for their unprecedented support for my research.
My sincere thanks to Nadia Sadli, who helped me with tissue culture work. Thanks Nadia for
being such a caring friend and a wonderful lab mate. And thanks to all the members of
NARL, God Bless you all!
My very special thanks to Pathum (MEYA). You have been like a brother to me, thanks a lot
for making my candidature such a joy. I would be privileged, if I ever get the opportunity to
work with you again! I’ll miss the good times we’ve spend together. Thanks for all the
favours you have done for me. You are nothing less than an angel.
PhD Thesis – Acknowledgements Nayyar Ahmed
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My sincere gratitude goes to the Deakin University security guards, Tom, Dayle, Alex, Rohit
and Shiv. Thanks for all the good times and late-night chats. Thank you for making me feel
safe while working in the lab around the clock. I am ever grateful to you.
I would like to express my appreciation to my beloved wife Mashal Pall who spent sleepless
nights with me and was always my support in moments when there was no one to answer my
queries. Thank you for your timely support and your words of encouragement. I would like to
extend my sincere and heart-filled thanks to my wife’s parents (Waseem Pall & Farhat Pall)
and siblings (Mehwish Pall, Sabahat Pall and Zoha Pall). One can only wish for such
wonderful in-laws like yourselves. Thanks for making my wedding a success and for all the
kind favours you did for me. I am forever grateful to you all.
Last but not the least, I want to extend my gratitude and love to my family members, my
father Shams Daud (Abbu), my beloved mother Attiya Shams (Ammi), my brother Nasir
Ahmed and his wife Ajam Ahmed. Thank you Abbu & Ammi for your encouraging words
and unconditional love for which I am ever thankful and indebted to you. Your prayer for me
was what sustained me thus far. Thanks for your emotional support; you guys have stood by
me in my times of weakness. And thank you for waiting so patiently for me to finish my
studies. I have been away from you both far too long.
Finally I thank my God Almighty for letting me through all the difficulties. I have
experienced Your guidance day by day. You are the one who let me finish my degree. I will
keep on trusting You for my future. Thank you, Lord.
PhD Thesis – List of Publications Nayyar Ahmed
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List of Publications
Journal Articles: Ahmed N, Dhanapala P, Suphioglu C. Identification and characterization of a novel IL-4 receptor α chain (IL-4Rα) antagonist to inhibit IL-4 signalling. Cell Physiol Biochem 2015; 36:831-842 (Chapter 2 & 3 results published) Ahmed N, Dhanapala P, Sadli N, Barrow CJ, & Suphioglu C. Mimtags: The use of phage displaytechnology to produce novel protein-specific probes. Journal of Immunological Methods. 2014; 405(0):121-9 (Chapter 2 results published). Ahmed N, Barrow CJ, Sinclair A, Suphioglu C. Discovering the effects of omega-3 and omega-6 fatty acids on allergy using a HEK-Blue cell-line. International Journal of Molecular Sciences (Chapter 4 results submitted for publication). Ahmed N, Barrow CJ, Adcock J & Suphioglu C. Discovering the effects of CoQ10 and fatty acid derived resolvins on allergy using a HEK-Blue cell-line. International Journal of Molecular Sciences (Chapter 5 results under preparation). Taylor PE, Ahmed N, Suphioglu C. Relationship between ryegrass pollen count and weather variables in regional and urban Victoria, Australia. (Chapter 6 results under preparation) Book Chapters: Ahmed N, Dhanapala P, Suphioglu C., (2014). Nothing to be sneezed at: Th2 cytokines as novel therapeutic targets for the production of anti-allergy agents, Bentham Science Publishers (eds), frontiers in clinical drug research anti-allergy agents (Chapter 1 literature review submitted). Sadli N, Ahmed N, Ackland M.L, Sinclair A, Barrow C & Suphlioglu, C., (2011). Effect of zinc and DHA on expression levels and post-translational modifications of histones H3 and H4 in human neuronal cells, in Raymond, Chuen-Chung Chang (eds), Neurodegenerative diseases - processes, prevention, protection and monitoring, pp. 141-164, InTech, Rijecka, Croatia. Conference Papers (abstracts published): Presented at the European Academy of Allergy and Clinical Immunology (EAACI) conference Ahmed N, Dhanapala P, Sadli N, Barrow CJ, & Suphioglu C, 2014, ‘Mimtags: the use of phage display technology to produce novel protein specific probes’, Allergy, vol. 69, p. 298.
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Ahmed N & Suphioglu C, 2014, ‘Discovering the effects of omega-6 fatty acids on allergy using a HEK-Blue cell line’, Allergy, vol. 69, p. 534. Ahmed N, Ramsbottom K, Carr S & Suphioglu C, 2013, ‘Identification and characterization of IL-4 and IL-4 receptor as novel intervention targets for allergy treatment’, Allergy, vol. 68, p. 470. Ahmed N, Barrow CJ, Adcock J, Sinclair A & Suphioglu C, 2013, ‘Importance of CoQ10 and omega-3 fatty acids as novel therapeutics for allergy’, Allergy, vol. 68, p. 344. Ahmed N, Dhanapala P, Carr S & Suphioglu C, 2011, 'Characterisation of a novel IL-4 receptor antagonist for allergy treatment', Allergy, vol. 66, p. 509-10. Presented at the Australasian Society for Clinical Immunology and Allergy (ASCIA) conference Ahmed N & Suphioglu C, 2014, ‘Discovering the effects of omega-3 and omega-6 fatty acids on allergy using a HEK-Blue cell line’, Internal Medicine Journal, vol. 44, p. 1. Ahmed N, Dhanapala P & Suphioglu C, 2013, 'Discovering the effects of Omega-3 fatty acids on allergy using a HEK-Blue cell line', Internal Medicine Journal, vol. 43, p. 1. Ahmed N, Dhanapala P & Suphioglu C, 2013, 'Identification and characterisation of a novel cytokine IL-13 antagonist for allergy treatment', Internal Medicine Journal, vol. 43, p. 1. Ahmed N, Dhanapala P & Suphioglu C, 2012, 'Identification and characterization of a novel IL-4 receptor antagonist for allergy treatment', Internal Medicine Journal, vol. 42, p. 1.
PhD Thesis – Table of Contents Nayyar Ahmed
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Tables of Contents
Page number
Title page……………………………………………………………………….i
Deakin University – ACCESS TO THESIS- A………………………………..ii
Candidate Declaration Form…………………………………………………...iii
Acknowledgements…………………………………………………………....iv
List of Publications…………………………………………………………….v
Table of Contents……………………………………………………………...viii
Abstract………………………………………………………………………..xviii
List of Abbreviations…………………………………………………………..xxi
Chapter 1 – Literature review………………………………………………….1
1.1 Introduction of Allergy and Allergens…………………………………...2
1.1.1 Sources of Allergens………………………………………………….2
1.1.2 Symptoms of Allergy…………………………………………………4
1.1.3 Rising Incidence/Trends in Allergic Disease…………………………4
1.2 Overview of Allergy as a Disease: Atopy in Allergic Individuals………5
1.2.1 Underlying Causes of Allergy in Atopic individuals…………………5
1.2.2 Diagnostic Tests for Allergic Conditions……………………………..9
1.2.2.1 RAST…………………………………………………………....9
1.2.2.2 Skin Prick Tests…………………………………………………9
1.2.2.3 Blood Specific IgE Testing……………………………………..9
1.2.3 Current Available Treatments………………………………………..10
1.3 Immune Response: Allergic vs. Non-Allergic Response………………..11
1.3.1 Non-Allergic Immune Response……………………………………..11
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1.3.2 Allergic Immune Response…………………………………………..12
1.3.2.1 Humoral Response in Allergic Individuals…………………….12
1.3.2.2 Cell-Mediated Response in Allergic Individuals…………………..14
1.4 Role of Cytokines in Immunity…………………………………………..14
1.4.1 Cytokines IL-4 and IL-13…………………………………………….15
1.4.2 Comparative Roles of IL-4/IL-13 with IL-4Rα chain………………..17
1.5 IL-4Rα as an Important Target for Allergy Treatment………………..19
1.5.1 Anti-IL-4R Monoclonal Antibody…………………………………...19
1.5.2 Soluble IL-4 Receptor (sIL-4R)……………………………………...21
1.5.3 Targeting IL-4R with Hybrid Peptides………………………………21
1.5.4 Further Analysis of Blocking IL-4R………………………………....22
1.5.5 Current Available Treatments for Targeting IL-4
and IL-13 Cytokines……………………………………………………….,22
1.5.5.1 Specific Immunotherapy (SIT)…………………………………23
1.5.5.2 Modified Recombinant Vaccines…………………………….…25
1.5.5.3 Allergoids………………………………………………….……25
1.5.5.4 Adjuvants……………………………………………………….26
1.5.5.5 Peptide Immunotherapy………………………………………...26
1.5.5.6 Plasmid Deoxyribonucleic Acid (DNA) Immunotherapy……...27
1.5.5.7 DNA Vaccines………………………………………………….28
1.5.5.8 Toll-Like Receptor 9 (TLR-9)………………………………….28
1.5.5.9 Anti-IgE………………………………………………………...29
1.5.5.11 Chinese Herbal Medicine……………………………………..30
1.5.5.12 Anti-IL-4 and IL-13 Antagonists……………………………..31
1.5.5.13 Piktrakinra………………………………………………….....33
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1.6 Phage Display Technology…………………………………………….....33
1.6.1 Mimtags the Use of Phage Display Technology
to Produce Novel Protein-Specific Probes………………………………….36
1.6.1.1 Mimtags…………………………………………………………38
1.6.2 Testing the Peptide’s Inhibitory Efficiency in Tissue Culture…..........39
1.6.3 HEK Cytokine Reporter Cells………………………………………...39
1.6.4 Comparative Role of Ramos B Cells and HEK-Blue Cells…………..40
1.7 Effects of Dietary Antioxidant and Fatty Acids on Allergy:
Connecting the Dots…………………………………………………………...42
1.7.1 Inhibition of Allergy Pathway Using n-3 Fatty Acids
and Vitamin E Antioxidant…………………………………………………42
1.7.2 Discovering the Effects of Omega-6 AA on Allergy………………...45
1.8 Anti-inflammatory Effects of Resolvins and Coenzyme Q10
on Allergy………………………………………………………………….......47
1.8.1 Role of CoQ10 in Allergy…………………………………………….47
1.8.2 Role of Resolvins in Allergy………………………………………....48
1.8.3 What is the Resolution Phase of Acute Inflammation?........................50
1.8.4 Resolving Inflammation…………………………………………........51
1.9 Pollen Allergy………………………………………………………….......53
1.9.1 Epidemiology of Pollen Allergy………………………………….......53
1.9.2 Overview of Ryegrass Pollen Induced Allergy…………………........54
1.9.3 Diseases Associated with Ryegrass Pollen Allergy……………….....56
1.9.4 Diagnosis of Grass Pollen Protein Extracts……………………….....57
1.9.5 Grass Pollen Count and Meteorological Data……………………......57
1.9.6 Treatment for Pollen Allergy………………………………………...58
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1.9.7 Deakin AIRwatch: Pollen Counting and Forecasting Facility…….....60
1.11 Overall Aims and Objectives of PhD Studies………………………….60
1.12 Expected Outcomes of Study……………………………………….......61
Chapter 2 - Identification and Characterization of a Novel IL-4R
Antagonist for Allergy Treatment…………………………………………...63
2.1 Introduction……………………………………………………………….64
2.2 Materials and Methods…………………………………………………...66
2.2.1 General Description of the M13 Phage Library……………………...66
2.2.2 Biopanning…………………………………………………………...66
2.2.3 Amplification and Identification of Eluted Phage Clones…………....68
2.2.4 Sequencing of Phage DNA: Rapid Purification……………………...68
2.2.5 M13 Binding ELISA………………………………………………....70
2.2.6 Synthesis of the Biotinylated Peptide………………………………...71
2.2.7 Direct ELISA……………………………………………………........71
2.2.8 Inhibition ELISA……………………………………………………..72
2.2.9 Statistical Analysis…………………………………………………...73
2.3 Results……………………………………………………………………..73
2.3.1 Phage Display Biopanning…………………………………………...73
2.3.2 M13 ELISA with Three Homologous M13 phage clones………........75
2.3.3 Direct ELISA……………………………………………………........75
2.3.4 Inhibition ELISA……………………………………………………..79
2.4 Discussion………………………………………………………………….81
2.4.1 Phage Display………………………………………………………...81
2.4.2 M13 Binding ELISA with N4, N5 and N7 Phage Clone…………….82
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2.4.3 Direct ELISA and Inhibition ELISA………………………………...83
2.4.4 Conclusion……………………………………………………………84
Chapter 3 - Expression of HEK-Blue IL-4/IL-13 Reporter Cell-line
and Further Characterization of the IL-4Rα, IL-4 and IL-13
Peptide Antagonist in-vitro……………………………………………….........85
3.1 Introduction………………………………………………………………..86
3.2 Materials and Methods…………………………………………………....88
3.2.1 HEK-Blue™ IL-4/IL-13 Cells.……………………………….............88
3.2.2 Synthesis and Use of Peptides N1, P9 and K1……………….............89
3.2.3 HEK-Blue Cell Line Inhibition Assay with N1 Peptide……………..89
3.2.4 HEK-Blue Cell Line Inhibition Assay with P9
and K1 Peptide……………………………………………………………...90
3.3 Results……………………………………………………………………...91
3.3.1 HEK-Blue Cell Line Inhibition Assay with N1 Peptide…...................91
3.3.2 HEK-Blue Cell Line Inhibition Assay with P9 Peptide……………...93
3.3.3 HEK-Blue Cell Line Inhibition Assay with K1 Peptide……………..95
3.4 Discussion………………………………………………………………….97
3.4.1 Characterization of Novel Peptide Antagonists
Using a HEK-Blue Cell Line……………………………………………….97
3.4.2 Conclusion……………………………………………………............98
Chapter 4 - Discovering the Effects of Omega-3 and
Omega-6 Fatty Acids on Allergy Using a HEK-Blue Cell Line……………..100
4.1 Introduction………………………………………………………………101
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4.2 Materials and Methods…………………………………………………..103
4.2.1 Cell Culture, PUFA and Vitamin E Solutions………………………103
4.2.2 HEK-Blue Cell Viability Test Using Cytokines
IL-4 and IL-13……………………………………………………………..105
4.2.3 Time-course Assay Using Cytokine IL-4 and IL-13………………...106
4.2.4 Statistical Analysis…………………………………………………..107
4.3 Results……………………………………………………………………..107
4.3.1 HEK-Blue Cell Viability Test Using Cytokine
IL-4 and IL-13……………………………………………………………..107
4.3.2 HEK-Blue Time-course with Fatty Acid and
Vitamin E Treatment…................................................................................111
4.4 Discussion…………………………………………………………………114
4.4.1 Fatty Acids and SEAP Secretion…………………………………….114
4.4.2 Importance of Arachidonic Acid in Allergy………………………....115
4.4.3 Conclusion…………………………………………………………...116
Chapter 5 - Discovering the Effects of CoQ10 and
Resolvins on Allergy Using a HEK-Blue Cell Line………………………….117
5.1 Introduction………………………………………………………………118
5.2 Materials and Methods…………………………………………………..120
5.2.1 Cell Culture, Resolvins and CoQ10…………………………………120
5.2.2 HEK-Blue Detection Assay Using Cytokines IL-4 and IL-13……....120
5.2.3 Time-course Assay Using Cytokine IL-4 and IL-13………………...121
5.2.4 Statistical Analysis…………………………………………………..122
5.3 Results…………………………………………………………………….122
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5.3.1 HEK-Blue Detection Assay Using Cytokines IL-4 and IL-13……...122
5.3.2 HEK-Blue Time-course with Resolvins and CoQ10 Treatment…….126
5.4 Discussion…………………………………………………………………129
5.4.1 CoQ10 and Resolvins………………………………………………..129
5.4.2 Significance of Expression of SEAP/IgE Secretion…………………131
5.4.3 Conclusion…………………………………………………………...131
Chapter 6 - Relationship between Ryegrass Pollen Counts
and Weather Variables in Regional Victoria, Australia……………………..133
6.1 Introduction……………………………………………………………....134
6.2 Materials and Methods…………………………………………………..135
6.2.1 Establishment of the Air Sampler…………………………………...135
6.2.2 Preparation of Slides………………………………………………...135
6.2.3 Changing the Burkard Air Sampler Using the 24 hour Drum………138
6.2.4 Preparing Slides for Pollen Counting and Examining
Using an Olympus BX50 Microscope…………………………………….140
6.2.5 Calculations for Pollen per m3 of Air………………………………..141
6.3 Results……………………………………………………………………..143
6.3.1 Ryegrass Pollen……………………………………………………...143
6.3.2 Weather Variables and Grass Pollen Count (2012-2013)…………...145
6.3.3 Weather Variables and Grass Pollen Count (2013-2014)…………...147
6.3.4 Weather Variables and Grass Pollen Count (2014-2015)…………...149
6.4 Discussion…………………………………………………………………151
6.4.1 Seasonal Changes in Grass Pollen Count……………………………151
6.4.2 Thunderstorm Allergy in Victoria, Australia………………………..153
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6.4.3 Ruptured vs. Intact Ryegrass Pollen…………………………………153
6.4.4 Conclusion…………………………………………………………...155
Chapter 7 – General Discussion……………………………………………...156
7.1 General Discussion……………………………………………………….157
7.2 Overview of IL-4Rα……………………………………………………...157
7.2.1 Phage Display……………………………………………………….158
7.3 Mimtags: The use of Phage Display Technology to
Produce Novel Protein-Specific Probes…………………………….............160
7.3.1 The use of Phage Display in Isolating Mimtags as
Protein-Specific Probes…………………………………………………...160
7.3.2 Probing of a Conformational Epitope on Target Protein
with Mimtag................................................................................................162
7.3.3 Mimtag Technology………………………………………………...164
7.3.4 Further Applications of Mimtag Technology……………………….165
7.4 Inhibition of STAT6 Phosphorylation Pathway in
HEK-Blue Cells Using Peptides as Antagonists……………………………166
7.4.1 Preferential Use of Peptides as Antagonists………………………...166
7.4.2 Importance of Inhibitory Values at 50% (IC50) for
Inhibition Studies………………………………………………………….167
7.5 Further Analysis of n-3 and n-6 PUFA…………………………………167
7.5.1 Production of Inflammatory Prostaglandins………………………...168
7.6 The Missing Link: Inflammation and Allergy………………………….169
7.7 Deakin AIRwatch Project………………………………………………..171
7.7.1 Limitations of the Deakin AIRwatch Project………………………..172
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7.8 Future Directions…………………………………………………………174
7.8.1 Future Directions for IL-4Rα, IL-4 and IL-13 Inhibition…………...174
7.8.2 Future Directions for Fatty Acid, CoQ10 and Resolvins……………177
7.8.3 Pollen Sampling……………………………………………………..178
References…………………………………………………………………….179
List of Tables
Table 2.1……………………………………………………………………….74
Table 2.2……………………………………………………………………….75
Table 6.1………………………………………………………………………143
List of Figures
Figure 1.1………………………………………………………………………3
Figure 1.2………………………………………………………………………6
Figure 1.3………………………………………………………………………8
Figure 1.4………………………………………………………………………13
Figure 1.5………………………………………………………………………16
Figure 1.6………………………………………………………………………18
Figure 1.7………………………………………………………………………20
Figure 1.8………………………………………………………………………35
Figure 1.9………………………………………………………………………41
Figure 1.11……………………………………………………………………..43
Figure 1.12……………………………………………………………………..49
Figure 2.1………………………………………………………………………67
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Figure 2.2………………………………………………………………………77
Figure 2.3………………………………………………………………………78
Figure 2.4………………………………………………………………………80
Figure 3.1………………………………………………………………………92
Figure 3.2………………………………………………………………………94
Figure 3.3………………………………………………………………………96
Figure 4.1………………………………………………………………………109
Figure 4.2………………………………………………………………………110
Figure 4.3………………………………………………………………………112
Figure 4.4………………………………………………………………………113
Figure 5.1………………………………………………………………………124
Figure 5.2………………………………………………………………………125
Figure 5.3………………………………………………………………………127
Figure 5.4………………………………………………………………………128
Figure 6.1………………………………………………………………………137
Figure 6.2………………………………………………………………………139
Figure 6.3………………………………………………………………………142
Figure 6.4………………………………………………………………………144
Figure 6.5………………………………………………………………………146
Figure 6.6………………………………………………………………………148
Figure 6.7………………………………………………………………………150
Figure 7.1………………………………………………………………………163
PhD Thesis – Abstract Nayyar Ahmed
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Abstract
In recent times, allergy has become a financial, physical and psychological burden to the
society as a whole. Allergic reactions can result in life-threatening situations causing
morbidity and high economic cost. Therefore, more effective reagents are needed for allergy
treatment. In Chapter 2, IL-4R was targeted by employing a novel molecular method of
phage display technology. A novel synthetic peptide (N1 biopep) was isolated using this
technology, and demonstrated a strong affinity to the target IL-4R . This was followed by
extensive immunoassays such as ELISA. Indeed, the N1 biopep has shown to be very
promising in its capacity to significantly down-regulate the interactions between IL-4R and
the cytokine IL-4.
In Chapter 3, peptides were commercially synthesized and used for in vitro assays and a
HEK-BlueTM IL-4/IL-13 reporter cell line model, transfected with a gene producing an
enzyme SEAP. SEAP acts as a substitute to IgE when cells are stimulated with cytokine IL-4
and IL-13. QUANTI-Blue was added as a substrate that breaks down in the presence of
SEAP producing blue coloration. The blue color confirmed the activation of STAT6 pathway
using a spectrophotometer. We have successfully used peptides N1 to IL-4R , K1 to IL-4
and P9 to IL-13 that demonstrated inhibition of SEAP production in HEK-Blue cells. A
colorimetric analysis showed a >50% inhibition, resulting into less blue color with all three
peptides. A statistical Student’s t-test revealed the significance of the results. Since IL4 and
IL-13 interaction with IL-4R/IL-13R is a common pathway for many allergies, a prophylactic
treatment can be devised by using such novel methods in the future.
In Chapter 4, it was hypothesized that a causal relationship exists between the intake of
omega-3/6 fatty acids such as DHA, EPA, DPA and AA and atopic individuals suffering
PhD Thesis – Abstract Nayyar Ahmed
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from allergies. In allergic cascades, cytokines IL-4 and IL-13 bind to IL-4 receptor and IL-13
receptor (IL-4R and IL-13R), respectively, which activate the STAT6 phosphorylation
pathway leading to gene activation of allergen-specific IgE antibody production by B cells.
The overall aim here was to characterize omega-3/6 fatty acids and their effect on IL-4/IL-13
signaling. The fatty acids were tested in vitro with the HEK-Blue IL-4/IL-13 reporter cell line
model. We have successfully used DHA, EPA and DPA in our studies that demonstrated a
decrease of SEAP secretion as opposed to an increase in SEAP secretion with AA treatment.
A statistical Student’s t-test revealed the significance of the results. We have successfully
identified and characterised DHA, EPA, DPA and AA in our allergy model with varying
potentials. While AA was a potent stimulator, DHA, EPA and DPA were potential inhibitors
of IL-4R signalling, which regulates the STAT6 induced pathway in allergic cascades in
vitro.
As literature suggests, resolvins and CoQ10 have shown promising results with down-
regulation of allergy and inflammation. In Chapter 5, we assessed the effect of CoQ10 and
resolvins on the HEK-Blue cell model of allergy. Through our series of experiments, we have
shown the differential effects of resolvins and CoQ10 on SEAP secretion. A statistical
Student’s t-test revealed the significance of the results. Resolvins, although anti-inflammatory
by nature, showed an increase in SEAP secretion as opposed to CoQ10, which stood out as a
promising candidate in repressing allergy. SEAP secretion was completely inhibited in the
presence of CoQ10 even at lower concentrations, suggesting a novel therapeutic for allergy
treatment.
Lastly, we assessed the distribution of ryegrass pollen in the regional city of Geelong
(Victoria, Australia). The purpose of Chapter 6 was to establish a link between weather
PhD Thesis – Abstract Nayyar Ahmed
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variables and the presence of ryegrass pollen (intact vs. ruptured) in the atmosphere. For this
purpose, a Burkard volumetric air trap was installed at the Deakin University (Waurn Ponds,
Geelong) for pollen collection (known as Deakin AIRwatch). Our results show that ryegrass
pollen distribution is fairly correlated to weather variables such as rain, temperature, humidity
and wind. Pollen counting from three years were incorporated as part of this study. We have
successfully shown the presence of intact and ruptured pollen in air, which is important for
future studies relevant to allergic individuals suffering from pollen allergy.
To sum up, chapters 2-5 have shown the use of different therapeutics to down-regulate
allergic pathways which are common to all allergies. The isolated peptides along with n-3
fatty acids and CoQ10 can serve as novel candidates for allergy treatment. On the contrary,
avoidance of pro-inflammatory products such as n-6 fatty acids and resolvins can prevent
allergic symptoms. In chapter 6, we have conducted a thorough investigation of ryegrass
pollen distribution in regional Victoria which can help alleviate allergy through awareness
and avoidance of pollen allergy hence, improving quality of life. Our work paves a new and
exciting path for the safe and effective treatment of allergies.
PhD Thesis – List of Abbreviations Nayyar Ahmed
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List of Abbreviations
7-mer Amino acid chain consisting of 7 amino acids
12-mer Amino acid chain consisting of 12 amino acids
AA Arachidonic Acid
AHR Airway Hyper-Responsiveness
ALX Lipoxin Receptor
APC Antigen Presenting Cells
Ara h 1 Arachis hypogaea 1
Ara h 2 Arachis hypogaea 2
Ara h 3 Arachis hypogaea 3
ASCIA Australasian Society of Clinical Immunology and
Allergy
Bet v 1 Betula verrucosa 1
Biopep Biotinylated Peptide
BSA Bovine Serum Albumin
C1 Control 1
C2 Control 2
CCR5 Chemokine Receptor Type 5
CD4+T Cluster of Differentiation Expressing T-cells
CD23 Cluster of Differentiation-23
CD55 Complement Decay Accelerating Factor
ChemR23 Chemokine-Like Receptor 1
COX Cyclooxygenase
CpG Phosphorothioate Guanisosine
Da Daltons
PhD Thesis – List of Abbreviations Nayyar Ahmed
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DC Dendritic Cells
DH2O Distilled Water
DHA Decosahexaenoic Acid
DMEM Dulbecco’s Modified Eagle Medium
DMSO Dimethyl Sulfoxide
DNA Deoxyribonucleic Acid
DPA Docosapentaenoic Acid
EDTA Ethylenediaminetetraacetic Acid
ELISA Enzyme Linked Immunosorbant Assay
EPA Eicosapentanoic Acid
FAHF-2 Food Allergy Herbal Formula
FBS Fetal Bovine Serum
FcεRI Fc Epsilon RI
FEV1 Forced Expiratory Volume in 1 Second
Fmoc Fluorenyl-methyl-oxycarbonyl
FOXP3+ Forkhead box P3
GPR32 G Protein Coupled Receptor
H2SO4 Sulphuric Acid
HBTU O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate
HEK Human Embryonic Kidney
HKE Heat-Killed E. coli
HKE MP123 Heat-Killed E. coli modified peanut protein Ara h 1-3
HOBt Hydroxybenzotriazole
hrs Hours
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hr Hour
HPLC High Performance Liquid Chromatography
HRP Horseradish Peroxidase
Hsp65 Heat-Shock Protein 65
IFN Interferons
IFN-γ Interferon- Gamma
IgA Immunoglobulin A
IgD Immunoglobulin D
IgE Immunoglobulin E
IgG Immunoglobulin G
IgG2a Immunoglobulin G subclass 2a
IgG4 Immunoglobulin G subclass 4
IgM Immunoglobulin M
IL-1 Interleukin-1
IL-2 Interleukin-2
IL-4 Intereukin-4
IL-4DM IL-4 Double Mutant
IL-4R Interleukin-4 Receptor
IL-4Rα Interleukin-4 Receptor-Alpha
IL-5 Interleukin-5
IL-10 Interleukin-10
IL-12 Interleukin-12
IL-13 Interleukin-13
IL-13Rα1 Interleukin-13 Receptor-Alpha1
IMO Immune Modulatory Oligonucleotide
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IPTG Isopropyl β-D-1-thiogalactopyranoside
JAK Janus Kinase
kDa Kilo-Daltons
LB Luria Broth LT Leukotrienes
LTB4 leukotriene B4
LOX Lipoxygenase
mAb Monoclonal Antibody
MHC class II Major Histocompatibility Complex class II
Mimtags Mimotopes that can be Tagged
Min Minutes
NaN3 Sodium Azide
NARL NeuroAllergy Research Laboratory
NCBI National Centre for Biotechnology Information
NF-κB Nuclear Factor kappa-light-chain-enhancer of
Activated B cells
NK Natural Killer Cells
OD Optical Density
OPD ortho-Phenyl Diamine
PAF Platelet Activating Factor
PC Positive Control
PCR Polymerase Chain Reaction
PEG Polyethylene Glycol
Pfu Plaque-Forming Units
PG’s Prostaglandins
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PGD2 Prostaglandin-2
PGE2 Prostaglandin E2
PGE3 Prostaglandin E3
Ph.D Phage Display
PKC Protein Kinase C
PMN Polymorphonuclear Leukocyte
PPI Protein-Protein Interactions
PUFA Polyunsaturated Fatty Acids
RAST Radioallergosorbent Test
rAlt a 2 Alternaria Alternata Allergen 2
RH Relative Humidity
RhuIL-4R Recombinant Human Interleukin-4 Receptor
RhuIL-4Rα Recombinant Human Interleukin-4 Receptor Alpha
rpm Revolutions Per Minute
RT Room Temperature
RvD1 Resolvin D 1
RvD2 Resolvin D2
SCIT Subcutaneous Immunotherapy
SEAP Secreted Embryonic Alkaline Phosphatase
shRNA Short-Hairpin Ribonucleic Acid
SIT Specific Immunotherapy
sIL-4R Soluble Interleukin-4 Receptor
sIL-4Rα Soluble IL-4 Receptor Alpha
STAT Signal Transducer and Activation of Transcription
TBST Tris-buffered Saline with Tween-20
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TetR Tetracycline Resistant
Th T helper
Th1 T helper Type 1
Th2 T helper Type 2
TNF Tumor Necrosis Factor
TNFα Tumor Necrosis Factor Alpha
Tr1 Type 1 Regulatory T Cells
Treg Regulatory T Cells
U.S. FDA United States Food and Drug Administration
X-gal 5-bromo-chloro-indolyl-galactopyranoside
PhD Thesis – Chapter 1 Nayyar Ahmed
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Chapter 1 - Literature Review
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1.1 Introduction of Allergy and Allergens
The term “allergy” was first coined by Von Pirquet in 1906. He described the word allergy as
a ‘changed reactivity’ induced by antigens (1). Nearly a century later, the term allergy has
been revised as an exaggerated reaction to foreign substances, which are mostly harmless by
nature but injurious to allergic individuals. These foreign substances are known as allergens
and cause the immune system to respond aggressively. Allergies can be induced by various
allergens such as pollens, dust mite, molds, dander, and food (2). In addition, adjuvant risk
factors such as smoking or air pollution can also enhance an individual’s intolerance to
allergens.
Allergens are a particular type of antigen that are detected by the immune system and
translated as a foreign invader. As outlined in Figure 1.1, when atopic individuals come into
contact with these antigens, the body reacts by producing very large amounts of
immunoglobulin E (IgE) antibodies. These IgE antibodies sensitize effectors cells of allergy
(e.g. basophils and mast cells), which subsequently cross-link with allergens, releasing
chemical mediators causing allergy symptoms (2).
1.1.1 Sources of Allergens
There are many different forms of allergens like house dust mite, birch tree pollens and
pollens from grass such as timothy grass, to name a few. The Arachis hypogaea 1 (Ara h 1),
Arachis hypogaea 2 (Ara h 2) and Arachis hypogaea (Ara h 3) are highly allergenic peanut
proteins, which have become a potential hazard in recent years (3). Peanut allergy is mainly
associated with fatal-food induced anaphylaxis (4). IgE antibody production is induced more
severely by food allergens like milk, beans, nuts, rice, and wheat (5).
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The Immune System
Foreign Substance
(cat dander, pollen, virus, bacteria)
Normal Immune Response IgM,
IgG, IgA, IgD and various
immune cells respond to attack.
Exaggerated Immune Response
IgE is overproduced in
response to cat dander, pollens,
and other harmless allergens.
Foreign substance is eliminated. Subsequent exposure results in
an allergic reaction.
Non-Allergic Individual Allergic Individual
Figure 1.1: When a foreign substance enters the body, an allergic individual responds
by overproducing IgE antibodies, which is the initial sensitizing stage. Secondary
exposure to the same antigen results in the release of inflammatory chemicals like histamines
that cause the clinical symptoms observed in allergy. Other antibodies like immunoglobulin
M (IgM), immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin D (IgD)
are also produced when a foreign substance is introduced into the body but does not result
into an allergic cascade. Figure adapted from (2).
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The presence of allergens in food is mainly due to their high resistance to heating and
enzymatic breakdown rendering them unavoidable in food products (6).
Unfortunately, the only remedial method available is to avoid food products that may contain
these allergens. For example, accidental ingestion of peanut by-products is on the rise; an
inevitable situation at times, especially with children. Thus, the chronic nature of this allergy,
its consequences and its availability in a wide range of products has made it a very important
field of clinical and laboratory research (7).
1.1.2 Symptoms of Allergy
Although there are several symptoms for allergy, the most important clinical symptoms of
allergy are listed below;
(i) Allergic rhinitis: nasal obstruction, sneezing, itching and rhinorrhea (3);
(ii) Asthma: breathlessness due to airway obstruction, which is sometimes partial or
totally reversible (3);
(iii) Anaphylaxis: a severe systemic allergic reaction that causes the release of
inflammatory histamine. This causes symptoms like laryngeal edema and asthma (3);
(iv) Atopic eczema: characterized by a red itchy rash (3).
1.1.3 Rising Incidence/Trends in Allergic Disease
There has been a marked increase in prevalence of atopic disease in regions such as Western
Europe, the US and Australasia during recent years, especially in industrialized nations. A
20-fold increase has been experienced by western countries and as much as 40% of the
population suffers from this disease (8). A recent statistical study carried out (9) has shown
that a rise in allergy trend has taken place over the last 20 years. A rise in trend has also been
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studied by the Australasian Society of Clinical Immunology and Allergy (ASCIA) shown in
Figure 1.2 (10). These studies indicate an alarming situation and require a more thorough and
aggressive approach to resolve this disease.
1.2 Overview of Allergy as a Disease: Atopy in Allergic Individuals
To understand allergic diseases, it is important to know the difference between atopic and
non-atopic individuals. Atopy is an inherited predisposition that renders susceptible
individuals to allergic conditions such as rhinitis, asthma and atopic dermatitis, which is
caused by IgE-mediated responses (11, 12). Atopic diseases can further be sub-divided into
extrinsic allergy, which exhibits high levels of serum IgE antibody, and an intrinsic atopic
response, which exhibits low level of IgE antibody (12).
1.2.1 Underlying Causes of Allergy in Atopic Individuals
In atopic individuals, an immune response is mounted by T cells that are activated by
allergens, promoting T helper Type 2 (Th2) variant of cells. Once stimulated, these cells
subsequently produce cytokines such as Interleukin-4 (IL-4), Interleukin-5 (IL-5) and
Interleukin-13 (IL-13). Unlike the cytokines produced by T helper Type 1 (Th1) cells in non-
atopic individuals (detail in the following section), an excessive production of cytokines takes
place from Th2 cells (13). Figure 1.3 illustrates the predisposition factors that can trigger
atopy in individuals. Briefly, allergen exposure to patients with an already existing genetic
predisposition can become a risk factor for sensitization (more detail in the later sections).
Acknowledgment of this risk is essential (14). An example of genetic factors is
polymorphism in the IL-4 family of cytokine genes.
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Figure 1.2: Rise in trend of allergies over the next four decades in both sexes. ‘Std’ in the
figure denotes using one standard prevalence rate, rather than a different rate every year -
‘trend’. Figure adapted from (10).
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The two most important reasons of allergy prevalence in the western world are high level of
hygiene maintenance and excessive use of antibiotics at a younger age (Figure 1.3). Both
factors impede the immune system from acclimatizing to environmental conditions, which
causes a release of a Th2 cells cytokine profile in allergic patients (15, 16). This is referred to
as the hygiene hypothesis.
A number of studies have shown that children who are raised on farms have a lower
predisposition to hay fever and atopic sensitization, as opposed to children who are raised in
urban areas. The important environmental changes recognized are animal sheds (exposure to
diverse microbes) and consumption of unpasteurized milk (17).
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Figure 1.3. Genetic and environmental triggers of atopic diseases and its effect on
various parts of the body. Environmental factors such as allergens and genetic factors (as
mutations and polymorphisms) are responsible for a combined atopic effect. Figure adapted
from (11).
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1.2.2 Diagnostic Tests for Allergic Conditions
Allergy testing is an important component of allergy and asthma management. It enables the
identification of avoidable environmental triggers for asthma and allows appropriate
avoidance advice to be provided.
1.2.2.1 RAST
RAST or radio-allergosorbent immunological test system is only useful if the immune
reaction is IgE antibody-mediated (18). It uses an in vitro immunological measuring device to
determine allergen-specific antibodies. The precise measurement of allergen antibodies helps
to verify the diagnosis of asthma, allergies and other pulmonary disorders.
1.2.2.2 Skin Prick Tests
The more popular among the two different methods of skin prick test is the skin prick test.
The allergen is applied directly on skin and the skin is pricked in order to absorb the allergen
through the epidermal surface (19). Measurements are recorded at regular intervals of 10-15
minutes (min) for swollen skin (in mm).
A similar but alternative method is to inject the allergen directly into the skin and monitor for
an inflammatory reaction at the site. This method is known as the intradermal skin test (20).
However, skin prick tests pose some risks (e.g. anaphylactic food allergens such as peanuts)
and appropriate precautions need to be taken in order to perform the necessary tests (21).
1.2.2.3 Blood Specific IgE Testing
Blood specific IgE testing helps to quantify a broad spectrum of allergens that induce
allergen-specific IgE antibodies. In contrast to skin prick test, it is less sensitive to allergens
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but more useful when anaphylaxis is being investigated. Blood specific IgE testing can be
performed on sensitive patients who carry risks of allergy or those taking antihistamines. This
allows an alternative to skin prick test, which can otherwise cause adverse reactions in
patients (21).
1.2.3 Current Available Treatments
Current allergy treatment focuses mainly on counteracting the symptoms of allergic reactions.
Treatment of allergy principally involves three different approaches:
i) To know what the person is sensitive to and avoid exposure to that particular
substance. This is usually called ‘environmental control’. For example, if an
individual is allergic to peanuts, avoiding them would be sufficient to overcome
symptoms (22);
ii) The second approach involves the administration of drugs such as decongestants,
steroid nasal sprays, antihistamines, adrenergic agonists, anti-inflammatory drugs,
leukotrienes (LT) and bronchodilators to relieve allergic symptoms (23);
iii) The third most important therapy involves desensitizing the patient by repeated
administration of a specific allergen in small doses (24). This approach helps the
patient to develop immunity against the allergen; the treatment is known as
specific immunotherapy (SIT). SIT has many functions including, the induction of
Th1 cells in the production of IgG immunoglobulin rather than IgE (24, 25). Th2
cytokines, principally IL-4 and IL-13, are reduced considerably, further alleviating
the patient from the symptoms (25). However, it has been argued that SIT has its
disadvantages such as unwanted effects, poor efficacy and specificity as a result of
poor quality of allergy vaccines; comprised of poor quality of allergen extracts
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(26). Moreover, it is of high risk for patients with anaphylactic allergy (e.g. peanut
allergy).
Although these methods are the only available remedy for allergy, they offer very little
resistance to the growing problem at hand, with problems of efficacy and safety. These
therapies are yet to undergo further development before they can offer any significant change.
Hence new, safer and more reliable methods are required to combat allergic diseases.
1.3 Immune Response: Allergic vs. Non-Allergic Response
This section will demonstrate the involvement of allergen, antigen presenting cells (APC),
lymphocytes, plasma cells, mast cells and cytokines in the allergic immune response (27).
1.3.1 Non-Allergic Immune Response
During a non-allergic immune response, an allergen is engulfed by an APC, which processes
the internalized allergen and subsequently presents a fragment of the allergen on its surface,
via major histocompatibility complex (MHC) Class II proteins (28). The APCs migrate to the
lymph nodes to present the processed antigens to MHC Class II proteins, which further attach
themselves to T-cell receptors (29). This process is called priming. This causes differentiation
in naïve T helper cells (Th cells) also known as cluster of differentiated (CD4+) T cells into
Th1 cells (29). The Th1 cells produce cytokines interleukin-2 (IL-2) and interferon-gamma
(IFN-γ), which evoke cell-mediated immunity and phagocyte-dependent inflammation. As a
result, B-cell proliferation and IgG antibody production takes place (30, 31).
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1.3.2 Allergic Immune Response
The allergic response can be divided into two separate reaction phases. The initial phase is
known as the ‘humoral response’ and occurs as a result of initial sensitization of an allergen
which triggers B cells or B lymphocytes to release IgE antibodies (11). The second phase,
‘cell-mediated response’, involves an exposure to the same allergen but elicits a response by
effector cells, such as mast cells and basophils, that release chemical mediators. These
chemical mediators, namely histamine and heparin, are responsible for allergy symptoms
(32). Figure 1.4 below illustrates a brief summary of these events.
1.3.2.1 Humoral Response in Allergic Individuals
Allergic individuals respond to antigens by differentiating Th cells into Th2 cells, rather than
Th1. This occurs in the presence of the cytokines IL-4 and IL-13, which are also associated
with the production of IgE and eosinophilic response in atopic disease (11, 33). The primary
source of IL-4 in atopy is Th2 lymphocytes and mast cells (Figure 1.4) (11), while the
primary source of IL-13 is activated Th2 cells (34).
In atopic individuals, the APC engulf the allergen and cross link the exposed antigen to naïve
T cells via the MHC class II molecule. The T cells then differentiate into Th2 variant
enhancing cytokines IL-4/IL-13, ensuing production and proliferation of B lymphocytes to
plasma cells via the CD40 ligand expressed on B cells (35-38). The Th2 cells further release
cytokines IL-4 and IL-13 that bind to B lymphocytes, activating them which releases more
IgE into the system (36). These IgE antibodies then bind to high affinity IgE receptor Fc
epsilon RI (FcεRI) on the surface of basophils (not shown) and mast cells. This phase, shown
in Figure 1.4 is alternatively known as the ‘sensitization phase’ (24, 39, 40).
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Figure 1.4. Cellular interaction in the allergy phase of immune response and its
consequences. When allergens come into contact with APC, the allergens are processed and
presented to naïve T cells, which mature into Th2 cells and release cytokines such as IL-4/IL-
13. The cytokines bind to receptors on B cells, which then differentiate into plasma cells and
trigger the release of IgE antibodies (A-F). Secondary exposure to the same allergen cross-
links neighboring membrane-bound IgE antibodies on mast cells (as well as circulating
basophils) and releases the chemical mediators, which cause symptoms like sneezing,
wheezing, eczema etc. (G-H). Figure adapted from (41).
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1.3.2.2 Cell-Mediated Response in Allergic Individuals
This phase, also known as the ‘effector phase’ is initiated by subsequent exposure to the same
allergen, which was the previous sensitizing agent (36). As demonstrated in Figure 1.4, the
allergen cross-links with the IgE on the surface of basophils and mast cells, which leads to
degranulation. As a result, chemical mediators are released, such as proteoglycans, LT’s,
proteases, chemokines, histamine and most importantly cytokines, such as tumor necrosis
factor-alpha (TNF-α), IL-13 and IL-4 (42). This results in a variety of symptoms, such as
asthma (caused by excess mucous production), hay fever, anaphylaxis, itchy rash, dermatitis
etc.
The shift in cytokine production from Th1 cells (IFN-γ and IL-2) to Th2 (IL-4 and IL-13)
variant forms the hallmark of allergic response in atopic individuals. Therefore, the aim of
my project was to reduce the interaction of IL-4 and IL-13 with their common interleukin-4
receptor (IL-4R) (discussed in the later sections). Hence, this will down-regulate the allergic
cascade from progressing to the ‘sensitizing phase’ of the immune response.
1.4 Role of Cytokines in Immunity
Cytokines are secreted proteins that orchestrate a range of innate, adaptive and inflammatory
responses in the body (43). They are important for the development of hematopoietic and
nervous systems. The major cytokine producing cells are macrophages, lymphocytes and
monocytes (43). They stimulate target cells by affecting gene expression (44), subsequently
causing cell proliferation and cell differentiation (43).
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Cytokines play this role by binding to specific receptors on cell surfaces known as Type-1
membrane proteins (45). There are a variety of cytokines, namely chemokines, colony
stimulating factors (also known as transforming growth factors), interferons and interleukins
(45). Interleukin family of cytokines is involved in immune responses and associated
diseases. IL-4 and IL-13 play a more dominant role in the progression of allergic disease.
Studies carried out in the previous decade suggest that IL-4 and IL-13 are extensively used as
therapeutic targets for alleviating allergy (46-48).
1.4.1 Cytokines IL-4 and IL-13
IL-4 and IL-13 are examples of interleukins involved in allergic responses and play
fundamental roles in causing allergic diseases (49). Th2 cells are responsible for the
production of IL-4 and IL-13 (49) and both IL-4 and IL-13 are capable of initiating the Janus
kinase (JAK) signaling pathway and activate signal transducer and transcription factor known
as signal transfer and activation of transcription-6 (STAT6), which triggers gene expression
in the nucleus and results in the production of IgE antibodies (refer to Figure 1.6) (50, 51).
IL-13 (Figure 1.5A) and IL-4 (Figure 1.5B) are pleiotropic cytokines (having multiple
effects), secreted by many cells such as Th2 cells, activated B cells basophils and mast cells,
as well as dendrites (52). Both cytokines share many similar characteristics like inducing the
production of IgE class of immunoglobulins and have 30% amino acid homology with
similar chromosomal locations (52). They share many common functional characteristics as
well, but the most significant difference lies in the ability of IL-4 cytokine to develop CD4+
Th2 cells as opposed to IL-13 which has no effect on T cells (53). IL-4 is also involved in the
differentiation of monocytes to dendritic cells (DC) and is a pioneer of allergic diseases
associated with IgE-mediated response (54). In a study carried out on IL-4 knockout mice,
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the Th2 cell differentiation did not take place and as expected, the production of eosinophils
and IgE antibodies were significantly decreased or abrogated altogether.
1.5A) 1.5B)
1.5C)
Figure 1.5. Crystal structures of cytokines IL-13 (1.5A), IL-4 (1.5B) and IL-4Rα (1.5C).
Reducing the interaction between IL-4/IL-13 and IL-4R complex can ultimately reduce the
IgE concentration in sera, hence, minimizing the allergic threshold. Figure adapted from (55-
57).
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IL-4 and IL-13 are potent inducers of allergic diseases and symptoms of allergy (58).
Therefore, the focus of this study is to reduce the interaction of cytokines IL-4/IL-13 with
their common IL-4Rα (Figure 1.5). This would consequently reduce the production of IgE
antibodies to a level of a non-atopic individual. This approach is particularly appealing, as it
may provide a treatment to all common allergies.
1.4.2 Comparative Roles of IL-4/IL-13 with IL-4Rα Chain
IL-4 and IL-13 play key roles in Th2 immunity and asthma pathogenesis. The function of
these cytokines is partially linked through their shared use of the IL-4Rα chain. The Type I
receptor comprising IL-4Rα and the common γ-chain is expressed by hemopoietic cells and is
exclusively responsive to IL-4. In contrast, the Type II receptor comprising IL-4Rα and
interleukin-13 receptor-Alpha 1 (IL-13Rα1) is responsive to both IL-4 and IL-13 (Figure 1.6)
(59).
The binding of IL-13 to the IL-13Rα1 induces heterodimerization with the IL-4Rα. STAT6
then dimerizes and travels through cell cytoplasm until it reaches the cell nucleus, which
activates genes that encode for another cluster of differentiation protein called CD23 and
MHC Class II (60). Dephosphorylation of STAT6 can result into gene repression and
deactivation. In lymphocytes, STAT6 plays a primary role of proliferation and defense
against apoptosis (60).
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Figure 1.6. Signaling pathways of IL-4 and IL-13, along with their corresponding
receptors. The IL-4 cytokine is not only capable of activating Type I but Type II receptors as
well, since both receptors share a common IL-4Rα chain. By reducing this interaction, it is
predicted to suppress the effect of IL-4 on cell signaling mechanisms. The JAK signaling
molecules activate STAT6 transcription factor that subsequently activates gene expression in
the cell nucleus. Figure adapted from (47).
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In IL-4 and STAT6 knockout mice, it was found that IgE production diminished by a factor
of 100 and the mice lacked T cell development (61). The cytokines appear to have a link in
regulating allergic conditions and in some cases also improve the effects of tissue-damaging
autoimmunity (61). These physiological roles lay down the importance of cytokines IL-4 and
IL-13 with IL-4R signaling mechanisms. For this reason, targeting the IL-4R complex along
with its cytokines would make tangible targets.
Therefore, the concept that IL-4 and IL-13 can be targeted simultaneously is of great
importance because of their shared dependence on the IL-4Rα chain of the receptor. Hence,
this research targets the common receptor with a novel synthetic antagonist (Figure 1.7).
1.5 IL-4Rα as an Important Target for Allergy Treatment
Various researches have been conducted using a range of techniques to down regulate the
interaction between IL-4/IL-13 with IL-4R, namely monoclonal antibodies, antagonists and
soluble receptors, but the efficacy of these procedures still remains in doubt and under a lot of
scrutiny. Therefore, an alternative technology, known as phage display, will be used to carry
out the task of identifying a novel peptide inhibitor as an intervention target for IL-4R.
1.5.1 Anti-IL-4R Monoclonal Antibody
Over the years, monoclonal antibodies (mAb) have been developed and designed for more
complicated procedures for use as therapeutics. Mouse models have been designed to
improve mouse monoclonal antibodies against human IL-4R to study the effects of T and B
cell differentiation (62, 63).
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Figure 1.7. Hypothetical representation of a synthetic peptide inhibitor reducing the
interaction between IL-4 and IL-4R on B cells. The reduction of this binding can
ultimately reduce the IgE concentration in sera, hence, minimizing the allergic threshold.
Process leading from G-H (effector phase) may be blocked (red crosses); alleviating
symptoms of allergy. Figure adapted from (41).
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In one study, an M1 mAb had down-regulating effects on cell secretion of IgG1 and IgE,
whereas another mAb M2 showed no effects. The research in this area is still incomplete and
demands a more thorough and complete investigation to make it a reality (63-65).
1.5.2 Soluble IL-4 Receptor (sIL-4R)
One of the therapeutic applications of soluble receptors was the development of recombinant
human IL-4 receptor (RhuIL-4R). RhuIL-4R only has the extracellular domain of the IL-4R
which has the ability to bind to cytokine IL-4 but simultaneously acts as an antagonist to
block the function of the cytokine (66). As we know that allergic symptoms occur from
combined effects of IL-4 and IL-13 cytokines, the disadvantage of this system is that it does
not affect IL-13 but only IL-4. Therefore, this remains an unreliable method of curing allergy
(67). In a separate study carried out by Holtzman (2003), it was found that administration of
soluble IL-4 receptor alpha (sIL-4Rα) had no clinical efficacy in treating asthma and the
development of this project has been stopped altogether (67).
These results are disappointing and raise concerns regarding therapies for allergy. Therefore,
a need arises to redirect strategies in the favour of phage display technology, in the quest to
identify novel peptide inhibitors for allergy treatment.
1.5.3 Targeting IL-4R with Hybrid Peptides
In a study by Yang et al., (2012), it was found that using L-amino acid peptides as a remedy
to cancer showed detrimental effects in vivo. A hybrid peptide, composed of IL-4Rα binding
moiety, killed normal cell lines at low concentrations in vitro and failed to show antitumor
activity in vivo. However, when the peptide was modified to a DL-amino acid, it was able to
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induce a modest cancer cell death (68). Here, it is important to note that peptides are now
becoming an important study to counter diseases at the cellular level.
1.5.4 Further Analysis of Blocking IL-4R
In an experiment carried out by Konig et al., (1995), IL-4 induced antigen CD23 fragments
were suppressed by sIL-4R and a partial antagonistic mutant IL-4 protein, Y124D was
produced. The target cells showed an initial suppression of IgE synthesis but 4 days later, IgE
synthesis occurred (69). In a study carried out by Lai et al., (1991), blockade of IL-2R and
IL-4R both resulted in the increase of IL-4 (70).
In yet another study, IL-2R and IL-4R were blocked with an MR6 (mAb against IL-4R) and
showed a reduction in interferon gamma production in T cells. The blocking of IL-2R with an
antagonist suppressed the full expression of IL-4R (70).
All of these studies indicate that a new strategy needs to be devised to fight allergy and
overcome allergic symptoms. Such a new strategy includes the innovative method of phage
display technology, where novel peptide inhibitors of a target can be readily and successfully
identified.
1.5.5 Current Available Treatments for Targeting IL-4 and IL-13 Cytokines
Treatment of cytokines IL-4 and IL-13 both result into a decrease of IL-4R signaling. The
NeuroAllergy Research Laboratory (NARL) research group at Deakin University has
conducted experiments using both cytokines and IL-4R as novel targets for allergy
intervention. Hence, it is highly important to highlight the current treatments available for not
only targeting IL-4R but it’s cytokines as well.
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1.5.5.1 Specific Immunotherapy (SIT)
The third most important therapy involves desensitizing the patient by repeated
administration of a specific allergen in small doses (24). This approach helps the patient to
develop immunity against the allergen by temporary loss of responsiveness because of
continuous exposure; the treatment is known as specific SIT. SIT has many functions
including the induction of Th1 cells in the production of IgG immunoglobulin rather than IgE
(24, 71). Th2 cytokines, principally IL-4 and IL-13, are reduced considerably, further
alleviating the patient from the symptoms (71-73). SIT is commonly used for respiratory
allergy but the underlying mechanism is not well understood. Initial desensitization occurs in
mast cells and basophils. Effector cells may also include allergen specific T and B cells. The
aim of allergen SIT is to induce peripheral T cell tolerance, suppress the activation level of
mast cells and basophils and inhibit the secretion of histamines in response to IgE antibodies
(74). The production of peripheral T cell tolerance during allergen SIT plays a vital role in
the generation of allergen-specific T regulatory cells (Treg) that are able to produce anti-
inflammatory cytokines such as interleukin-10 (IL-10) and transforming growth factor-β
(TGF-β). Treg cells regulate allergen-specific IgE and are capable of recruiting
immunoglobulin G 4 (IgG4) and IgA production. The two subsets of inducible Treg cells
known as forkhead box P3 (FOXP3+) and the IL-10-positive type 1 regulatory T cells (Tr1)
play important roles in allergen tolerance. These cells can be induced by allergen SIT in
humans (75-77).
Subcutaneous immunotherapy (SCIT) is being use for well over a century and has been
proved to be very effective in patients. However, the use of SCIT for food allergens has
proved to be lethal for patients suffering from severe allergic symptoms such as an
anaphylactic shock (78). Alternative methods for administering immunotherapy have been
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devised over the last few decades such as oral immunotherapy (OIT). This technique has
proved to be more effective than SCIT with various food allergens (79, 80). The initial aim
for this type of immunotherapy is to increase desensitization and to increase the threshold
dose for exposure to peanut. This reduces the risk of severe allergic symptoms in response to
accidental intake of peanut (80). The risk of such therapies has to be weighed against their
benefits. A study carried out by Longo et al., (2008) showed that highly milk-allergic
population with repeated therapy using OIT reduced the risk of allergy in almost half the
population. 36% of the population became completely desensitized and 54% had become
tolerant to milk-derived products (81). Although some of the patients showed adverse
reaction to OIT, the benefits outweigh the risks since a large number of the population was
cured (80). Overall, OIT has been found to reduce peanut-specific IgE levels and increase
peanut-specific IgG4 levels, while basophil activation is reduced. Down-regulation of Th2
cytokines did not occur with OIT or the up-regulation of Th1 cytokines (80). Sublingual
immunotherapy (SLIT) is another potential treatment for allergic rhinitis and asthma. A
randomized double-blind, placebo-controlled study investigating SLIT for hazelnut was
carried out by Enrique et al., (2005, 2008), in which 12 patients were treated with SLIT for a
period of five months. Significant changes in sensitivity to hazelnut were observed following
SLIT treatment. There was an increase in hazelnut-specific IgG4 and IL-10 cytokine post-
treatment. A decrease in specific IgE antibodies was also observed in patients with
interrupted SLIT treatments (82-84).
However, it has been argued that SIT has its disadvantages such as unwanted effects, poor
efficacy and specificity as a result of low quality of allergy vaccines, comprised of poor
quality of allergen extracts. Moreover, it is of high risk for patients with anaphylactic allergy
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(e.g. peanuts). Additional randomized, placebo-controlled clinical trials need to be carried out
to determine the true efficacy and potential use of these therapies as novel allergy treatments.
1.5.5.2 Modified Recombinant Vaccines
Modified recombinant food proteins are developed to decrease IgE binding capacity but
retain dominant T-cell epitope regions. Currently, many recombinant proteins have been
developed such as peanut, apple and fish allergens. A study carried out by Ferreira et al.,
(1996) used polymerase chain reaction (PCR) to prepare nine isoforms of Betula verrucosa 1
(Bet v 1) from birch pollen. Two isoforms showed low IgE reactivity, however induced
similar T-cell proliferation when compared to other seven IgE-reactive isoforms (85, 86).
Modified peanut allergens using site-directed mutagenesis can stimulate T cells but have
greatly reduced IgE binding capacity (87, 88). A study carried out by Li et al., (2003) showed
the effects of heat-killed E. coli (HKE) producing recombinant peanut proteins on a murine
model of peanut anaphylaxis. The group treated with high doses of HKE modified peanut
protein Ara h 1-3 (HKE MP123) demonstrated reduced clinical symptoms when compared to
the placebo-treated group. A significant decrease in IgE levels and cytokines IL-4, IL-5, IL-
13 and IL-10 were also observed as opposed to an increase in IFN-γ. The study demonstrated
that HKE-MP123 can induce long-term down-regulation of peanut hypersensitivity by
targeting Th2 cytokine production and up-regulating Th1 cytokines (89).
1.5.5.3 Allergoids
Allergoids are extracts of allergen polymerized to form larger aggregates that exhibit reduced
allergenicity but retain their immunogenicity properties. Formaldehyde is commonly used to
polymerize allergenic proteins to produce allergoids and poses less threat of inducing
systemic reactions during immunotherapy. Other polymerizing agents such as gluteraldehyde
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and polyethylene glycol conjugated allergens have been used in clinical studies with
comparable effectiveness to other allergoids (90).
1.5.5.4 Adjuvants
To reduce the risk of IgE-mediated side effects such as anaphylaxis during treatment,
modified allergoids have been used to treat allergic response. A study carried out by
Haydenreich et al., (2014) analysed that adsorbance of allergens to aluminum hydroxide
(alum) had no consistent adjuvant effect but decreased allergenicity in vitro, while showing
an adjuvant effect with regards to IgG production in vivo (91, 92).
1.5.5.5 Peptide Immunotherapy
Peptide fragments containing T-cell epitopes which lack tertiary conformational structure to
bind with IgE antibody can be used to decrease adverse reactions caused by immunotherapy
(84). In a murine model of peanut allergy, mice were treated with immunotherapy using
peptides that contained IgE epitopes to the major peanut allergen Ara h 2. This was done
prior to allergen challenge. Mice only experienced mild allergic reactions as compared to
placebo-treated mice showing severe anaphylaxis (84). Several murine models of allergy
have demonstrated a down-regulation of immune reactions in atopic individuals using
peptides which are designed with T cell epitopes (93). Allergen peptide immunotherapy has
been used in many clinical applications to date. In one of the clinical trials carried out by
Norman et al., (1996), a peptide vaccine for cat allergy showed variable effects. Some
patients suffered a local reaction a few hours (hrs) after administering the peptide (94). Large
peptides can sometimes cause physiological reactions in patients, which is a common
phenomenon. However, many reasons cast a shadow of doubt on the use of peptides as novel
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therapeutics such as peptide stability, susceptibility to degradation, size (usually > 500 Da)
and methods of delivery (95).
1.5.5.6 Plasmid Deoxyribonucleic acid (DNA) Immunotherapy
DNA nanoparticles containing the gene for allergens can be synthesized and administered
orally for immunotherapy. A study carried out by Fronseca et al., (2012) showed
Mycobacterium leprae heat-shock protein 65 (Hsp65) expressed by recombinant DNA
plasmid could reduce earlier established allergic response. Mice used were experimental
models of airway allergic inflammation to test the effects of plasmid DNA immunotherapy.
Allergic mice were previously sensitized and challenged using ovalbumin. Post-challenge,
mice were administered intramuscular doses of recombinant DNA encoding Hsp65. After
treatment, mice were administered a second allergen challenge and the allergic response was
recorded. It was shown that immunotherapy reduced airway inflammation by reducing Th2
cytokine and mucus production. Another important discovery was that cells transferred from
DNA-immunized mice to allergic mice travelled to allergic infected sites and attenuated Th2
response (96). Another study using a murine model demonstrated the use of DNA
nanoparticles containing the gene for Ara h 2, which was administered orally to mice.
Immunized mice displayed less severe anaphylactic response following allergen challenge
compared to unimmunized mice. This was accompanied by an attenuated production of IgE
antibody, histamine and vascular leakage (97).
Although these methods are the only available remedy for allergy, they offer very little
resistance to the growing problem at hand, with problems of efficacy and safety. These
therapies are yet to undergo further development before they can offer any significant change.
Hence new, safer and more reliable methods are required to combat allergic diseases.
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1.5.5.7 DNA Vaccines
DNA-based vaccines can be used to stimulate immune responses against foreign antigens.
The DNA, once injected, can be incorporate into local cells and synthesize the coded protein,
which induces an immune response. The fungus Alternaria was found to cause asthma with
severity, which prompted Sanchez et al., (2009) to experimentally test a rat model of
Alternaria-induced asthma. The major alternaria alternata allergen 2 (rAlt a 2), was targeted
using the DNA-based vaccine with remarkable results. Post-administration, decreased
production of IgE antibody was detected, along with a reduction in IL-13 cytokine secretion
and reduced airway inflammation. Treatment of sensitized animals with DNA-based vaccines
leads to a more balanced production of Th1 and Th2 cytokines, which reduces allergic
symptoms (90, 98).
1.5.5.8 Toll-Like Receptor-9 (TLR-9)
TLR-9s are found on cells of the innate immune system such as DC. TLR-9s are activated by
non-coding sequence of DNA referred to as phosphorothioate guanisosine (CpG), found in
bacteria. Once activated, they lead to the synthesis of cytokines such as IFN-γ, IL-10 and
interleukin-12 (IL-12) and increased expression of CD40 ligands. These responses shift the
balance from a Th2 profile towards Th1. In murine models of asthma, it was found that
treatment with CpG DNA reduced eosinophilic airway inflammation, mucus secretion,
airway remodeling and airway hyper-reactivity (90, 99). A study published by Zhu et al.,
(2007) used a synthetic agonist called immune modulatory oligonucleotide (IMO) for TLR-9
to modulate peanut-induced allergy in mice. Peanut allergen was orally administered to the
mice with or without IMO in a prevention model and in a treatment protocol, mice were
sensitized with peanut allergen at days 0 and 14 and treated 4 times with administration of
IMO starting on 21 days. In the prevention model, mice that were administered with
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IMO/peanut showed decreased IgE and increased immunoglobulin G subclass 2a (IgG2a)
levels in serum and intestinal tissue. In the treatment model, mice treated with IMO showed
attenuation of IgE, IL-5 and IL-13 levels and increased IgG2a and IFN-γ in serum (100).
Subsequently, the experiments showed that IMOs can switch peanut-induced Th2 response
toward a Th1 response.
1.5.5.9 Anti-IgE
Recombinant monoclonal humanized anti-IgE treatment reduces circulating IgE, suppresses
inflammation and provides an enhanced control for allergic diseases (101). 84 patients with a
history of peanut allergy were treated in a double-blind, randomized, dose-dependent trial for
managing food allergy. Patients were randomly given 150, 300 or 450mg of anti-IgE
antibodies or placebo for 4 months. As hypothesized, the patient given the highest dose of
anti-IgE antibody, experienced a significant drop in clinical symptoms for allergy when the
patient was challenged with peanut allergens. The placebo group showed no significant
change to the peanut challenge. Although the anti-IgE treatment was successful in 25% of the
patients, the other 25% showed zero tolerance of allergen challenge, which indicated that this
treatment was effective but variable (102).
Combination therapy of anti-IgE mAb and allergen immunotherapy was recently investigated
by Kuehr et al., (2002). This was a randomized, double-blinded, placebo-controlled, parallel-
group study with a total of 225 patients treated with combination therapy using anti-IgE and
SIT for birch and grass pollen. The results show a reduction of 48% in clinical symptoms for
the entire combined pollen season (average length, 84 days) for SIT + anti-IgE-treated
patients when compared to patients treated with SIT + placebo. The study showed that
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combination therapy with monoclonal anti-IgE antibody and SIT was more effective than
using immunotherapy alone for allergy treatment (103).
1.5.5.11 Chinese Herbal Medicine
A 9-herb formula derived from traditional Chinese medicine is under investigation as a
potential treatment of food allergy. The medicine is referred to as the food allergy herbal
formula (FAHF-2) and is effective in a murine model of peanut-induced anaphylaxis (104).
FAHF-2 treatment protected mice against allergic reactions when peanuts were administered
to mice. These pre-clinical studies showed that clinical effects were persistent for 6 months
after discontinuation of treatment. The underlying effects of these treatments showed a
decrease of peanut-specific IgE antibody and an enhancement in peanut-specific IgG2a
levels. More importantly, Th2 cytokines such as IL-4 and IL-13 were suppressed as well. The
protective effect of FAHF-2 lasted for 6 months post-therapy, which makes it a very effective
treatment in the murine model. Furthermore, the treatment had successfully treated allergic
response in the murine model of multiple food allergies making it a prophylactic treatment
for multiple allergic diseases (105).
Preliminary studies with a human peripheral blood mononuclear cell line taken from peanut-
allergic individuals showed that when cells were treated with peanut extracts in the presence
of FAHF-2, a decrease in antigen-dependent T-cell proliferation was observed. A dose-
dependent reduction in IL-4 and IL-13 cytokines was also noted which indicates that FAHF-2
inhibits the Th2 response in atopic individuals (106).
With the studies mentioned above, it was determined that FAHF-2 was highly effective and
safe to use in a murine model. This led the United States food and drug administration (U.S.
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FDA) to approve FAHF-2 as an investigational drug to be used in clinical trials in 2007. The
first human study was an acute phase 1 trials, which investigated the safety of using FAHF-2
to treat food allergies in atopic individuals. A total of 18 subjects were used in this study
between the age group of 12 and 45 years. Patients suffered from different allergies such as
peanut, tree nut, fish and shellfish. The patients were part of a double-blind, placebo-
controlled, dose-dependent trial. Accordingly, the subjects were administered 4, 6 or 12
tablets three times a day for a week. It was found that all doses for FAHF-2 were tolerated by
the individuals and the patients showed no signs of side-effects. While no adverse effect of
FAHF-2 was observed in the individuals, no suppression or inhibition of IgE antibody and
Th2 cytokines was detected which raises doubts about the efficacy of this drug in a human
model (107).
Studies have also been performed to demonstrate the synergistic effect of all the 9-herbs that
make up the FAHF-2 formula. In a series of experiments it was understood that using
individual herbs did not show the same level of inhibition of peanut-induced anaphylaxis as
FAHF-2. In addition, none of the 9 individual herbs were able to suppress or inhibit Th2
responses and/or enhancing Th1 response (106, 107).
1.5.5.12 Anti-IL-4 and IL-13 Antagonists
A humanized anti-IL-4 neutralizing antibody (pascolizumbab) was designed and used in a
study by Hart et al., (2002), which inhibited IL-4 cytokine from binding to human-derived
cell lines and was tolerated in monkeys (108). Pharmacokinetic studies involving cynomolgus
(Southeast Asian) monkey models have found two humanized anti-IL-13 mAbs, named Ab-
01 and Ab-02, to be effective at suppressing acute airway hypersensitivity (109). Ab-01
blocks contact between IL-13 and IL-4Rα whereas Ab-02 blocks contact between IL-13 and
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IL-13Rα1. Additionally, murine studies have confirmed the efficacy of two unnamed anti-IL-
13-mAbs at not only preventing asthmatic inflammation, but also at reversing mucus
hyperplasia to basal levels under persistent allergen challenge (110, 111). Interestingly, the
IL-13-specific mAb lebrikizumab is more effective for treatment of asthma. Molecular
observations suggest that high-affinity interaction between lebrikizumab and IL-13 sterically
hinder the binding of IL-13 to IL-4Rα, which down-regulates receptor signalling (112, 113).
IL-13 cytokines can be selectively targeted using a naturally occurring soluble IL-13Rα2-Fc
fusion protein. This receptor silences the effect of IL-13 cytokine when administered to mice
post-allergen challenge. This treatment suppressed mucus hyper-secretion and completely
down-regulated airway hyper-responsiveness (AHR) (114). The efficacy of these mAbs on
humans is yet to be elucidated. However, mAbs are large molecules that are not as stable and
as cheaply produced as synthetic peptide inhibitors, as employed in our current studies.
Antagonists are substances that block the signaling action of agonists (i.e. signaling
molecules). IL-13 antagonists disrupt the interaction between IL-13 and IL-13R by binding to
either of the molecules. An example is the IL-13 binding protein, named IL-13BP, binds IL-
13 with 100-300 times higher affinity than IL-13Rα chains in vitro (115). IL-13E13K is
another antagonist, which binds IL-13R with 8-fold affinity than IL-13 in vitro (116). Murine
studies have identified IL-4 double mutant (IL-4DM) as a potent IL-13 and IL-4 inhibitor due
to its ability to bind to IL-4Rα with high affinity and blocks signal transduction (117). IL-
4DM treatment effectively controlled the dermatitis, reduced mast cell and eosinophil
numbers, and inhibited plasma IgE and histamine. In addition, splenocytes produced an
increased level of IFN-γ (117).
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1.5.5.13 Piktrakinra
It has been previously established that IL-4 and IL-13 cytokines have a shared dependency on
the IL-4Rα chain, which makes it important to target the receptor, hence, a prophylactic
treatment can prove to be more useful if it can target the IL-4Rα chain. Piktrakinra is an
example of such a treatment. Piktrakinra is a recombinant form of IL-4 cytokine, which is a
potent inhibitor of cytokine IL-4 and IL-13 binding to the IL-4Rα/IL-13Rα1 complex.
Administration of piktrakinra protected allergic monkeys from AHR when challenged with
allergen (118). In a randomized, double-blind, placebo-controlled experiment carried out by
Wenzel et al., (2007), it was shown that, when compared with placebo, decrease in forced
expiratory volume in 1 second (FEV1) after allergen challenge was significantly reduced after
inhalation of piktrakinra for 4 weeks, which showed that inhibition of IL-4 and IL-13
cytokine can affect late asthmatic response after allergen challenge in subjects. The incidence
of spontaneous asthma attacks were also reduced with piktrakinra administration suggesting a
novel method in controlling asthma symptoms (119).
However, all of these findings require clinical trials prior to application on human allergic
conditions, and their efficacy is questionable. Therefore, more stable synthetic peptide
inhibitors promise to be important alternatives.
1.6 Phage Display Technology
A current strategy in improving allergic conditions involves pharmacotherapy,
immunotherapy and avoidance of exposure to allergens. It is a hope that the strategy of phage
display technology will provide a more swift and efficient method of improving antibody
related diseases with the identification of novel synthetic peptide antagonists/inhibitors. In a
nutshell, this technology allows one to perform protein-ligand interactions (120). Phage
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display is a selection technique in which a library of peptide variants are expressed on the
surface of a phage virion, in this case, an M13 phage (121). Minor coat proteins pIII are
expressed on the M13 phage vector surface. These proteins have 12-mer peptides (amino acid
chain consisting of 12 amino acids) fused to their N-terminal. The library has 2.7 x 109
variants in peptide display, which gives a high probability of peptides showing affinity to the
target molecule. This peptide-displaying clone is then identified, isolated, amplified and
sequenced (Figure 1.8).
Biopanning is carried out by incubating a library of phage-displayed peptides on a micro-titer
plate with an immobilized target. The unbound phage is washed away while eluting the
target-bound phage. The eluted phage is then taken through additional binding/amplification
cycles to get a consensus in binding sequences (Figure 1.8). The amplification of phage is
achieved in an E. coli strain ER2738. The phage is then grown on agar plates containing
isopropyl β-D-1-thiogalactopyranoside (IPTG) and 5-bromo-chloro-indolyl-
galactopyranoside (X-gal). The library cloning vector M13 is a derivative of cloning vector
M13mp19, which carries the lacZα gene. This gives a blue color to the plaques that
successfully grow in this medium. The blue plaques are hand-picked, isolated and sequenced
for further analyses (122, 123).
An affinity selection in this way allows the selection of peptides that mimic IL-4 cytokine,
and hence, will most likely block the interaction between IL-4 and IL-4R. If this is
successfully achieved, production of IgE antibodies by B cells may be effectively reduced,
resulting in a down-regulation of the allergic cascade.
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Figure 1.8. The biopanning strategy of phage display technology. Step 1: A library of
phage (M13), each displaying a different peptide sequence is exposed to a 96-well plate
coated with recombinant human IL-4Rα (RhuIL-4Rα) target molecule. Step 2: Phage M13,
which is unable to bind to the target molecule, is washed away with Tris-buffered saline
containing Tween-20 (TBST). Step 3: Phage M13, which was able to bind to the target
molecule, are eluted using Glycine/HCl by lowering the pH. Step 4: The eluted phage is
immediately neutralized, amplified and taken through additional binding/amplification cycles
to enrich the pool in favor of binding sequences.
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1.6.1 Mimtags the Use of Phage Display Technology to Produce Novel Protein-Specific
Probes
The NARL research group at Deakin University has published an article based on the results
of Chapter 2 (“Identification and characterization of a novel IL-4R antagonist for allergy
treatment”). Although not directly relevant to our research for peptide antagonist, it is
essential to highlight the importance of phage display technology as a useful tool for epitope
mapping and protein tagging. Our paper highlights the use of this technology to detect
different proteins such as IL-4R.
Protein-specific probes used for epitope mapping and protein-tagging studies have been the
focal point of research during recent years employing various methods of detection of
proteins, such as antibodies and labelled proteins. Currently, protein analysis consists of
immunoassay techniques such as enzyme-linked immunosorbant assay (ELISA),
polyacrylamide gel electrophoresis and related blotting techniques that use antibodies for
detection and interactions with antigens (124). The advent of monoclonal antibodies with the
development of hybridoma technology by Kohler and Milstein in 1975 was another
remarkable milestone, which has revolutionized the way we conduct research today (125-
128). Monoclonal antibodies are generally specific to the target antigen and are often used for
epitope mapping of proteins for exploring the human proteome for diagnostics and
therapeutic purposes (129). The epitope region can either be linear or conformational in their
structure and the amino acid sequence can be continuous or discontinuous (130). The ability
of monoclonal antibodies to detect a single antigenic determinant (epitope) has earned it a
major advantage over the use of polyclonal antisera. However, there are disadvantages of
using antibodies where precise recognition of epitopes is seldom accurate (131) and there are
limitations and disadvantages with these techniques (132). The hybridoma technology
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involves many phases from the fusion of the primed B cells with myeloma cell line to the
large-scale production of monoclonal antibodies from these highly selective hybrid cell lines
(125, 133). For researchers working in the field, the use of this available in vitro technique is
often problematic and has found to be very labour intensive and expensive (133). Animal
ethics is a major concern when it comes to development of monoclonal antibodies, which
causes unnecessary suffering of animals (133, 134). Another disadvantage of larger protein-
based-probes, such as biotin binding proteins, is the size of these antibodies that are tagged
with a detectable label. Larger probes present a challenge of inefficiently penetrating the
conformational structures of target proteins as opposed to short peptides. A larger protein,
such as an antibody, can also interact with a random functional groups on a single protein to
the exclusion of thousands of other competing cytoplasmic proteins, nucleic proteins,
carbohydrates and small molecules, which poses an enormous chemical problem (135). It is
therefore imperative to find new techniques that are particularly important for quantitative
proteomics, less tedious and cost-effective.
A more contemporary form of research has brought short peptides sequences under the
spotlight as target specific probes for in vitro and in vivo analysis of protein ligands such as
transmembrane proteins. This type of epitope tagging has led to very successful epitope
mapping of target antigens. The methodology has been used for protein localization,
immunoprecipitation, and protein-protein interaction (136). Briefly, an epitope is a sequence
of amino acids in any structure that is recognized by an antibody. A single protein may carry
multiple epitopes that can be recognized by antibodies. Two decades ago it was realized that
using peptide phage libraries, mimotopes (short peptide sequences) could be isolated which
mimic the epitope on a protein in their physical and chemical properties. Using this
technique, the 3-dimensional structure of the epitope can be determined along with the
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critical residues that make up the epitope (137). Hence, in this case the mimotope acts as a
probe against the desired epitope on a protein or antibody as a therapeutic agonist or
antagonist.
1.6.1.1 Mimtags
A new term introduced in this paper (138) was ‘Mimtags’, which are the same short
sequences of amino acids that play a critical role in the advancement of phage technology
from a very different perspective. These short sequences of peptides are a part of fused
proteins on the surfaces of phage used in phage display libraries. These epitope-mimics or
mimotopes (139) that can be tagged (mimtags) on to protein sequence, can effectively
identify the epitope region of a target antigen and used as protein-specific probes. Since the
methods and results for this paper are part of Chapter 1, the term “mimtag” will now be used
for discussion purposes in section 7.3.
The technique has been used in various studies to prove its effectiveness. A recent study
conducted by the NARL research group at Deakin University has shown remarkable results
(138, 140-142). In our study, random peptide library on M13 phage was used to screen an IL-
4R and IL-13. An affinity selection in this way allows the identification of mimtags that bind
to the cytokine and its receptor, and hence, will most likely target epitopes more effectively
due to the small size and nature of the mimtag (12-mer peptide). The mimtag also has the
ability to conform 3-Dimensional configuration increasing its binding efficiency (143). The
bound peptide was sequenced and synthesized for further characterization by immunoassay
techniques. The study shows the potential use of biotinylated peptide mimtags as examples
and their advantage over complex antigens and antibodies being applied as novel protein
specific probes.
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1.6.2 Testing the Peptide’s Inhibitory Efficiency in Tissue Culture
Once the novel peptide inhibitor is isolated by phage display, the next logical step is to test its
inhibition properties in tissue culture. Human embryonic kidney cells-blue (HEK-Blue) will
be used as model systems in this study to characterize the capacity of the synthetic peptide
inhibitor to down-regulate IL-4 signaling pathway in vitro. This will allow us to establish the
profundity of the peptide in more detail.
1.6.3 HEK Cytokine Reporter Cells
HEK-Blue™ IL-4/IL-13 cells allow the detection of bioactive IL-4 and IL-13 through the
activation of the STAT6 pathway. These cells are derived from a stable transfection of
HEK293 cells with human STAT6 gene to obtain a fully active STAT6 pathway. The cells
are also transfected with another stable STAT6 inducible secreted embryonic alkaline
phosphatase (SEAP) reporter gene. Upon stimulation from either IL-4 or IL-13 cytokine,
instead of producing IgE antibodies (as is the case with human B cells), the cells secrete
SEAP. The levels of SEAP production can be detected using QUANTI-Blue™, which turns
purple/blue in the presence of SEAP (Figure 1.9 (A)). These cells, though very new, have
turned out to be very robust in research activity. Publications as recent as 2011 suggest they
are being extensively used for allergy research (144). The overlapping biological functions of
IL-4 and IL-13 have been thoroughly discussed in section 1.5.2 above with their common
receptor chains; IL-4Rα and IL-13Rα1. The HEK-Blue cells are transfected with recombinant
IL-4Rα/ IL-13Rα1 receptor which can be activated by IL-4 and IL-13.
Using HEK-cells for tissue culture research carries many benefits. To name a few, the cells
have been used as an expression tool for recombinant IL-4R for over 25 years. They remain
highly stable and efficient when transfected with foreign genes. Also, once transfected, the
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translation and processing of the gene product (proteins) is performed with high proficiency.
Finally, their effortless reproduction and maintenance adds to the benefits of using this cell
line as a model system (145). HEK-Blue cells on the other hand, allow a colorimetric
analysis, which carries simplicity, high sensitivity and low cost to conduct the required
experiments.
1.6.4 Comparative Role of Ramos B Cells and HEK-Blue Cells
Ramos, a B lymphocyte cell line derived from Burkitt’s lymphoma, is a very suitable and
optimized model system used to study B lymphocyte behavior and apoptosis (146). The cell
line carries the model system for STAT6 phosphorylation upon induction of IL-4/IL-13
cytokines through their respective receptors. This triggering mechanism leads to the
production of IgE antibodies (147) (Figure 1.9 (B)).
In this project, the identified novel peptide inhibitor from earlier studies will be tested on
HEK-Blue cells to test the peptide’s efficacy in reducing the IL-4/IL-4R signaling pathway.
This method has not been used in previous literature. A dose-dependent relationship will be
established where a higher concentration of peptide will deliver a powerful antagonizing
effect, hence, reducing SEAP in HEK-Blue cells (Figure 1.9). The visual spectrum of color
in HEK-Blue cells will ultimately form the basis of future testing of the peptide in an animal
model system. The transfected model mimics the production of IgE antibody from B
lymphocytes where IgE is replaced with SEAP secretion.
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Figure 1.9A
Figure 1.9B
Figure 1.9. Comparison between the JAK-STAT6 signaling in transfected HEK-Blue
cells that lead to SEAP production (1.9 A) as opposed to Ramos B cells that leads to IgE
production (1.9 B). The HEK-Blue cells mimic the internal mechanism of STAT6
phosphorylation found in Ramos B cells. Figure adapted from (147).
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1.7 Effects of Dietary Antioxidant and Fatty Acids on Allergy: Connecting the Dots
Over recent years, the etiology of allergy has implicated several factors that are responsible
for an increase in prevalence of this disease. However, the most important factor is diet. It has
been suggested that low intake of omega-3 fatty acids along with antioxidants such as
vitamins have resulted in a rapid rise in atopic diseases, especially in children (148, 149).
Hence, a thorough study of this underlying cause will perhaps reveal a more reliable method
of therapy for allergy treatment.
1.7.1 Inhibition of Allergy Pathway Using n-3 Fatty Acids and Vitamin E Antioxidant
Over recent years, the etiology of allergy has implicated several factors that are responsible
for an increase in prevalence of this disease. However, the most important factor is diet. It has
been suggested that low intake of omega-3 fatty acids along with antioxidants such as
vitamins have resulted in a rapid rise in atopic diseases, especially in children (148, 149).
Hence, a thorough study of this underlying cause will perhaps reveal a more reliable method
of therapy for allergy treatment.
Omega-3 or n-3 fatty acids are polyunsaturated fatty acids (PUFA) with a double bond (C=C)
at the third carbon atom from the end of the carbon chain, hence the name (150). Recently, it
has been shown that n-3 and n-6 fatty acids (which have a double bond (C=C) at the sixth
carbon atom from the end of the carbon chain) (150) shown in Figure 1.11. They have
opposite effects on allergic conditions (151). More than a decade ago, this phenomena was
suggested by Black and Sharpe (1997), who indicated that an increase in consumption of
dietary n-6 fatty acids and a decrease in fish oil derived n-3 or omega-3 PUFA leads to an
increase in asthma and atopic disease in allergic individuals (152).
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Docosahexaenoic acid (DHA), 22:6ω3 (22:6n-3) contains 22 carbon atoms and 6 double bonds.
Eicosapentaenoic acid (EPA), 20:5ω3 (20:5n-3) contains 20 carbon atoms and 5 double bonds.
Docosapentaenoic acid (DPA), 22:5ω3 (22:5n-3) contains 22 carbon atoms and 5 double bonds.
Arachidonic acid (AA), 20:4ω6 (20:4n-6) contains 20 carbon atoms and 4 double bonds.
α -tocopherol (Vitamin E) ((2R)-2, 5, 7, 8-Tetramethyl-2-[(4R, 8R)-(4, 8, 12-trimethyltridecyl)]-6-chromanol).
Figure 1.11. The chemical structures of DHA, EPA, DPA and vitamin E (153).
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A decrease in fish oil derived n-3 or omega-3 PUFA leads to an increase in asthma and atopic
disease in allergic individuals (152).
It has been shown that n-3 and n-6 fatty acids have opposite effects on allergic conditions
(151). More than a decade ago, this phenomena was suggested by Black and Sharpe (1997),
who indicated the increase in consumption of dietary n-6 fatty acids and a decrease in fish oil
derived n-3 or omega-3 PUFA leads to an increase in asthma and atopic disease in allergic
individuals (152).
The use of fatty acid supplementation in diets has increased and gained much attention in the
recent years, particularly its effect in developing a strong immune system in early stages of
life (154, 155). Pregnant women are given dietary supplements of fatty acids to maintain a
steady supply of development in the child’s immune system. Current studies have suggested
that such supplements have shown to effect IL-4/IL-13 levels and reduce asthma and other
related allergic disorders during childhood. The three main fatty acids included in the studies
were DHA, EPA and DPA (Figure 1.11). The supplementation was started at 20 weeks of
gestation period (156). No credible studies have been carried out to suggest that DPA has a
link to allergic diseases (157).
Furthermore, a study undertaken recently with the effects of DHA on IL-4/IL-4R signaling
mechanisms in human B cells, which forms the precursor to IgE production, has suggested
that incubating DHA with Ramos B cells resulted in a dose-dependent inhibition of IgE by
hampering the IL-4 signaling pathway via STAT6 dimerization (158).
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In addition to this, vitamin E (Figure 1.11) has been found to dampen IgE levels in animal
models. This suggests a protective role of vitamin E in atopic disease and hence can be used
as a model system for challenging allergy “head on” (151, 159). However, a study carried out
in Germany, in a population of over a thousand people, suggested that IgE serum levels had
no significant change after administration of vitamin E (106). These conflicting results
suggest that the underlying mechanism is still unclear on whether vitamin E is anti-
inflammatory in nature or vice-versa. We will deal with the already existing hypothesis that
an increase in dose of vitamin E and PUFA is inversely proportional to serum IgE levels. In
other words, both of these nutrients will affect the STAT6 signaling pathway in HEK-Blue
cell line, as planned in this study.
The aim of this part of the project is to reduce the IL-4/IL-13 with IL-4R and IL-13R
signaling pathway in HEK-Blue cells using colorimetric analyses. This would open new
vistas to our project and help alleviate allergy in atopic individuals by using natural dietary
remedies such as DHA and EPA coupled with the vitamin E antioxidant.
1.7.2 Discovering the Effects of Omega-6 AA on Allergy
Over recent years, Omega-6 fatty acids have gained much attention because of their effects
on the etiology of allergic disease. Industrialized nations have increased their dietary intake
of Omega-6 fatty acids over the past few years. As a result of these increased consumption of
Omega-6 rich vegetables and oils, the ratio between Omega-3:Omega-6 fatty acids has
increased many fold from 1:1 to 10:1 (160). Eicosanoids have important role in
inflammation. Omega-6 oils are precursors to short-lived but very powerful enzymes known
as eicosanoids which play an important role in the pathophysiology of atopic disease (160).
Eicosanoids derived from n-6 fatty acids are generally regarded as pro-inflammatory.
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Eicosanoids such as LT, thromboxanes (TX) and prostaglandins are all pro-inflammatory
with the latter two causing bronchoconstriction in asthmatic patients. Cyclooxygenase (COX)
and lipoxygenase (LOX) can convert AA to the 2-series of prostaglandins, the 2-series of TX,
and the 4-series of LT. In individuals with asthma, a bronchial allergen leads to enhanced
prostaglandin-2 (PGD2) production. PGD2 production begins with the liberation of AA from
membrane phospholipids by phospholipase A in response to inflammatory stimuli (Th2
cytokines). Thus, during asthmatic attacks in humans, PGD2 is released in large amounts by
mast cells (161). PGD2 is a major product from COX-catalyzed reactions in a variety of
tissues and cells such as T cells, dendritic cells, macrophages, mast cells and basophils and a
major cause of bronchial asthma (162, 163).
A study showed that early supplementation of Omega-6 fatty acids did not prevent the
expression of atopy as suggested by serum IgE levels (164). However, conflicting evidence
suggests that Omega-6 fatty acids do not alleviate allergy, although it did reduce serum IgE
levels in 12 months old infants (165). Although researches on Omega-6 fatty acids and
prevention of allergic diseases are on the rise, there is no systematic evidence to prove that
Omega-6 fatty acids can prevent allergic disease.
Chemoattractant receptor homologous molecule is expressed on the surface of Th2 cells. This
receptor is also known as DP2. This receptor binds PGD2 and stimulates the activation and
recruitment process of Th2 lymphocytes leading to production of Th2 cytokines IL-4, IL-13
and IL-5 (166, 167). Antigen-induced cross-linking of cell-surface IgE on mast cells leads to
the rapid abundant production of PGD2. In addition, PGD2 interacts with another receptor
known as DP1 on DC to activate polarization of Th1 cells to Th2 variant. These agonistic
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effects of AA derived PGD2 prostanoids on Th2 cells make it an important target for
research.
1.8 Anti-inflammatory Effects of Resolvins and Coenzyme Q10 on Allergy
Recently, it was revealed that certain PUFA derived lipid mediators had astonishing effects
on inflammation. The lipid mediators, namely E series resolvins (derived from EPA) and D
series resolvins (derived from DHA) (Figure 1.12), were found to be very specific in their
effects on allergy. Instead of causing immunosuppression, the resolvins use pin-point
targeting mechanism to resolve inflammation and therefore, help to main homeostasis (168).
The effects of coenzyme Q10 (CoQ10) in allergy have also been recognized in recent studies.
The studies are associated with symptoms of allergy such as bronchial asthma and acquired
deficiency in CoQ10 intake (169, 170). However, none of these reports were able to
conclusively demonstrate the inhibitory effects of CoQ10 (Figure 1.12) and resolvins on
allergic models.
1.8.1 Role of CoQ10 in Allergy
Coenzyme Q is well defined as an important component of the oxidative phosphorylation
pathway in mitochondria and converts carbohydrates and fatty acids into ATP (171). Another
important substrate called CoQ10, also known as ubiquinone, plays decisive roles in the
production of cell energy and prevents free oxygen radicals from causing cellular damage.
CoQ10 is normally found in tissues and organs which require high energy consumption
(169). The primary role of CoQ10 is its role as an intermediate in the electron transport chain
system found in all mitochondria. Hence, it is ubiquitous in nature and forms an important
substance in cellular respiration. Due to its antioxidant properties, it has been speculated that
CoQ10 is beneficial for allergic patients and asthmatics. CoQ10 has been implicated in
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allergic conditions for more than two decades (172). Bronchial asthma is a medical condition
where, as a result of CoQ10 deficiency, bronchial mucosal inflammation arises (169, 173).
Elaborating on the asthma condition, it was documented by Gazdik et al., (2002) that
asthmatics had reduced levels of CoQ10, causing disruption in antioxidative processes at the
cellular level. This oxidative imbalance is connected to chronic inflammation of the airway.
In yet another study carried out in 2007, it was found that food intolerance and allergies in
children were due to an acquired CoQ10 deficiency (170). With all the overwhelming
evidence that is available, one can hypothesize that CoQ10 may play an important role in
allergic diseases. Hence, dietary supplementation of asthmatics with CoQ10 may result in a
decrease in allergic prevalence.
1.8.2 Role of Resolvins in Allergy
Resolvins are a family of lipid mediators that are derived from EPA and DHA. The term
resolvins (resolution-phase interaction products) was first coined to signify that these
products were synthesized during the resolution phase of inflammation, possessing very
potent anti-inflammatory and immuno-regulatory actions. These actions include reducing
neutrophil infiltration, regulating cytokine and reactive oxygen species, and lowering the
extent of the reaction (174). Resolvins have been studied and understood as anti-
inflammatory and moreover, promote the resolution of the inflammatory response to a non-
inflamed state (175). Resolvin D1 (RvD1) is produced from the oxygenation of DHA by
lipoxygenase. RvD1 reduces human polymorphonuclear leukocyte (PMN) trans-endothelial
migration, which is the initial phase of acute inflammation.
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Resolvin D1 - (4Z,7S,9E,11E,13Z,15E,17S,19Z)-7,8,17-Trihydroxy-4,9,11,13,15,19-docosahexaenoic acid.
Resolvin D2 - (4Z,7R,8E,10Z,12E,14E,17S,19Z)-7,16,17-Trihydroxy-4,8,10,12,14,19-docosahexaenoic acid.
Resolvin E1 - Sodium (6Z,8E,10E,14Z,16E)-5,12,18-trihydroxy-6,8,10,14,16-icosapentaenoate.
Coenzyme Q10 - 2-[(2E,6E,10E,14E,18E,22E,26E,30E,34E)-3,7,11,15,19,23,27,31,35,39-Decamethyl-2,6,10,14,18,22,26,30,34,38-tetracontadecaen-1-yl]-5,6-dimethoxy-3-methyl-1,4-benzoquinone.
Figure 1.12. The Chemical Structures of Resolvins D1-2, E1 and CoQ10 (153).
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In a study carried out by Sun et al., (2007), it was shown that the 17(R) epimer of RvD1,
which is triggered by aspirin, reduces leukocyte infiltration in a mouse-model of peritonitis
with maximal inhibition of 35% at 100 ng dose (176). The 17(R) and 17(S) D series of
resolvins exhibit potent anti-inflammatory action in vivo as described by Song et al., (2003).
Trout brain cells also produce RvD1 from endogenous stores of DHA which also indicates
that RvD1 carries a conserved evolutionary structure (175). Resolvin D2 (RvD2) is produced
physiologically from the oxygenation of DHA by lipoxygenase and shares the same function
as RvD1 in dampening excessive neutrophil trafficking during acute inflammation. It reduces
zymosan-stimualted PMN infiltration by 70% at doses as low as 10 pg per mouse and
significantly reduces platelet activating factor (PAF) stimulated leukocyte adherence and
migration at 1nM. It also stimulates Nitric Oxide (NO) production, which decreases
leukocyte-endothelial interactions (177).
1.8.3 What is the Resolution Phase of Acute Inflammation?
Inflammation is an important part of the immune system. It is triggered by many different
stimuli that pose a threat to the overall homeostasis of tissue structure. In the acute
inflammatory process neutrophils, eosinophils and basophils rapidly mobilize to the site of
inflammation. This is vital for the removal of harmful agents such as bacteria, fungi, viral
infection and parasitic invasion. Allergic response is another example of such inflammation
that result into severe clinical manifestations in the form of inflammation. Resolution of
inflammation is understood to be a process by which granulocytes (mentioned above) are
eliminated from the site of inflammation whereby reducing the mononuclear cell numbers to
basal levels (178, 179). During spontaneous resolution, neutrophils undergo cell apoptosis,
which is a highly regulated process. Phagocytes travel to the site of inflammation and
mediate effective clearance of dying cells (180). An acceptable theory is that resolution is an
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active process and is controlled by a range of cellular activities. Acute inflammatory phase is
self-limiting and results into tissue restoration and homeostasis. In cases where tissue
restoration or reduction in acute inflammation does not occur, the condition is said to be
chronic inflammation (178, 181).
1.8.4 Resolving Inflammation
Resolvins D1 and aspirin-triggered RvD1 have both been shown to be potent regulators of
neutrophils in a human and mouse model. In microglial cells, RvD1 blocks tumor necrosis
factor (TNF) induced activation of pro-inflammatory cytokine interleukin-1 (IL-1), which is
expressed in high quantities during a neuronal injury. In addition, human macrophages have
shown to be more active in the presence of RvD1, which interacts with the lipoxin receptor
(ALX) and G protein coupled receptor (GPR32) while short-hairpin ribonucleic acid
(shRNA) knockdown of each receptor resulted into decreased rate of phagocytosis of
neutrophils.
RvD2 reduced peritoneal neutrophil infiltration by 70% after the mice were challenged with
zymosan at ultra-low doses (in picograms). In a study carried out by Gaboury et al., (1993), it
was found that 1 nM of RvD2 was sufficient to reduce platelet activating factor-stimulated
leukocyte adherence and reduced cell interaction between neutrophils and endothelial cells.
Additionally, RvD2 was also found to reduce local production of NO, which is a well-known
anti-adhesive mediator (182). In another study carried out by Spite et al., (2009), mice treated
with RvD2 shows lower levels of peritoneal neutrophil numbers and the inflammatory
cytokines such as IL-6, IL-23 and IL-17 were all reduced (177). The inflammatory lipid
mediators’ prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) were both reduced by the
antagonistic effect of RvD2 treatment. In vitro RvD2 showed increase phagocytosis by
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macrophages and treatment of neutrophils showed an increase phagocytosis of E. coli (177,
183).
Resolvin E1 (RvE1) and RvD1 upregulate chemokine receptor type 5 (CCR5) expression in
dying neutrophils. CCR5 expression on apoptotic neutrophils acts as a terminator for
chemokine signaling. In addition to this, RvE1 specifically induces the expression of
complement decay accelerating factor (CD55), which is an anti-adhesion molecule expressed
on the mucosal surface of epithelial cells and therefore promotes the clearance of mucosal
neutrophils from mucosal surfaces (184, 185).
RvE1 is a trihydroxy EPA metabolite that has been identified in murine inflammatory
exudates and in human plasma when subjects are treated with both aspirin and supplemental
EPA. RvE1 is a very potent anti-inflammatory EPA metabolite and has been shown to protect
against experimental colitis in a murine model (186-188). In 2010, a publication suggested
that RvE1, an anti-inflammatory bioactive lipid mediator, reduced airway inflammation and
asthma in a murine model (189). In addition to this finding, ovalbumin-specific IgE
antibodies were also shown to decrease with the administration of RvE1 (189) and hence,
airway inflammation. The Resolvins E1 and D1 have been found to attack the resolution of
inflammation, a vital process that helps to shut off the inflammation cascade that can
eventually lead to tissue scarring and damage (190).
RvE1 also down-regulates the production of neutrophils by interacting with two receptors
known as chemokine-like receptor 1 (ChemR23) and the leukotriene B4 receptor (BLT1).
Upon binding with the ChemR23 receptor, the RvE1 attenuates TNF-stimulated nuclear
factor kappa-light-chain-enhancer of activated B cells (NF-κB), which results into counter-
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regulatory action (186). This is a critical step since TNF is a key mediator during the early
phases of acute inflammation. In the case of BLT1, the RvE1 resolvin interacts in a
stereospecific manner and acts as a potent antagonist to neutrophils. In a similar fashion,
RvE1 selectively interacts with ChemR23 also found on mononuclear and DC leading to
resolution of inflammation (191). The anti-inflammatory action of RvE1 is markedly reduced
in a model of mice deficient in BLT1 (191).
The NARL research group at Deakin University, in collaboration of colleagues at Deakin,
will evaluate the potential usefulness of Resolvins E1 and D1-2 along with CoQ10, in allergy
treatment by using our HEK-Blue cell line. This will determine the underlying mechanisms
of resolvins and CoQ10 on cytokine stimulated allergic response.
1.9 Pollen Allergy
Pollen allergy is caused by pollen grains. The pollen grain is the male part of a flowering
plant, which is necessary to propagate seeds. It is released by one plant and transported by an
insect or wind. Plants that use insects for pollinating their seeds are brightly colored to attract
insects. These plants however pose no threat to allergic individuals. However, plants that
pollinate using wind as transport factors are the ones that cause the real trouble. Windblown
pollen is lifted into the air and in enormous amounts. In particular, green plants such as grass
are the main culprits that cause allergy symptoms such as hay-fever and asthma (192).
1.9.1 Epidemiology of Pollen Allergy
Pollen was first recognized by Charles Harrison Blackley in 1873 which was a major cause of
allergic symptoms in patients and performed a skin prick test on himself to test the theory
(193). Aerobiology is the field of science that includes the dispersion of bacteria, fungal
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spores and pollen in air which are studied by trapping pollen using a volumetric spore trap
(194). The method employed to perform grass pollen counts using a microscope was
addressed by Hirst for the first time in 1952 (195).
Aerobiologists have played a very important role in establishing the correlation between
pollen dispersion in air and allergy symptoms. The most important carriers of allergen in the
air are found within pollen grains, which can range between 15 and 60 μm. However, these
pollen are large in size and cannot penetrate the lower airways, which are smaller in diameter.
Hence, it is important to note that pollen have to be water-soluble in order to penetrate lower
airways, which results in an IgE-specific allergic reaction among sensitized individuals (196).
1.9.2 Overview of Ryegrass Pollen Induced Allergy
Generally, it is found that grass pollen season starts with the release of pollen into the air
when the pollen dehisce at the time of flowering. Plants are considered important if the yield
of pollen grains is high. Proteins and glycoproteins which are derived from pollen and spores
can function as allergens (197). As explained in the previous sections, IgE mediated Type 1
allergy is an exaggerated response by the immune system to substances or particles that are
normally deemed un-harmful by nature (198). In our current study, we have considered
pollens released by ryegrass. It is important to note that almost 20% of the world population
now suffers from symptoms of hay fever or allergic asthma and the prevalence has risen to
more than double in the past few decades (199). In Europe, Australia and America, around
70% of the population is allergic to grass pollen when tested for IgE reactivity (200). The
patient is sensitized upon secondary exposure to the allergens when exposed to airways or
skin. Also, the allergic response in each individual varies in its severity (201). One individual
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may suffer from mild symptoms such as sneezing or hay fever as opposed to anaphylaxis in a
few others.
The final deposition site for allergens within the pollen reveals the mechanism involved in
their reaction in the human body. To study allergens, a fixation method was employed in
order to localize the allergens found within mature pollen. Ryegrasses (Lolium spp.) is the
most dominant source of allergenic pollen because of its abundance during the flowering
season. Ryegrass pollens were first reported by Johnson and Marsh in 1965 (202). Ryegrass
pollen grains have a diameter of 25-30 μm and have one pore. The pollen wall consists of a
multi-layered exine traversed by micro-channels (203). A mature ryegrass pollen typically
contains 700 starch granules. Each amyloplast found inside pollen grains contains one starch
granule with a diameter of 1 μm (197, 204). A range of different allergens have been
identified to date of ryegrass pollen from the perennial family. The total number of ryegrass
pollen allergens identified are 17. The most important proteins identified to date are Lol p 1-5
(197). However, the most allergenic proteins are Lol p 1 (35 kDa glycoprotein) (205) and Lol
p 5 (28-33 kDa glycoprotein) (206) that elicited IgE-mediated reactions when tested in
patients using skin prick tests. Although less abundant than Lol p 1, Lol p 5 showed 90% of
patients with IgE antibody against this allergen (207). Both of these proteins have been
extensively studied and characterized. The amino acid sequences of Lol p 1 and Lol p 5 share
non resemblance to other know grass allergens. Another important allergen from ryegrass
identified by Suphioglu et al., (1992) was Lol p 5 (previously known as Lol p IX), which is
located within the starch granules located inside the pollen (208). Detailed molecular
knowledge of the allergenic epitopes of pollen allergens is very important to design mutants
of these proteins resulting into altered allergenicity (209). These mutants can then be used as
therapeutic agents (209). The IgE binding epitope of Lol p 1 has been characterized and
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described by Esch and Klapper, 1989 (210). The characterization and identification of IgE
binding epitopes for Lol p 5 was done by Suphioglu et al., in 1998 (211).
1.9.3 Diseases Associated with Ryegrass Pollen Allergy
Climate change is increasing the prevalence of atmospheric pollen exposure, which results
into greater incidence of allergic disease in Australia. The incidence of allergic disease in
Australia is one of the highest in the world compared to Europe and North America.
Although asthma incidence doubled in the 1980’s and 1990’s, it plummeted for the next 10
years due to a below average rainfall in the south-eastern region of Australia (Melbourne,
Victoria). While pollen grains are large enough to remain at bay without penetrating the
airway system of the lungs, occurrence of rainfall during pollen season releases starch
granules and allergens, which can penetrate into the lower airways where they can trigger
asthma. Asthma is a chronic inflammatory disease which is marked by symptoms such as
wheezing, chest tightness and repeated coughing (212). A study carried out by Pollart et al.,
(1988) demonstrated a positive correlation between IgE antibody produced in allergic
patients and ryegrass pollen. It also supports the fact that patients suffering from a developed
IgE Ab to an environmental allergen is exacerbated by continued exposure to the same
allergen, which results in acute attacks of asthma (213). Allergic rhinitis, also known as hay
fever, is another pollen induced disease characterized by an inflamed nose in response to IgE-
mediated inflammation. Symptoms of rhinitis include sneezing, nasal obstruction and nasal
itching. Conjunctivitis is also considered a symptom of rhinitis (214, 215). In a study carried
out by Annesi-Maesano et al., (2012) showed a positive correlation between grass pollen
count per m3 of air and allergic rhinitis among allergy sufferers (216). Another study carried
out by Kemp et al., (2009) reported that children at a tender age of 8 years, who were born
during the pollen season, had an increased risk of developing allergic rhinitis but not for
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children born outside the pollen season. This birth cohort study was carried out in the state of
Tasmania, Australia (217).
1.9.4 Diagnosis of Grass Pollen Allergy using Protein Extracts
Clinical tests take into account the total grass pollen protein extracts to establish whether a
patient is suffering from allergy. RAST and skin prick tests are used to determine if the
allergens are binding to IgE antibody, which establishes the basis of primary cause of allergy
in individuals. Detail of these tests has been outlined in sections 1.2.2.1 and 1.2.2.2
1.9.5 Grass Pollen Count and Meteorological Data
Grass pollen counts are highly variable from one pollen season to another. In Melbourne,
seasonal total grass pollen ranged from 1000 in 1980 and 1981 to greater than 8000 grains/m3
in 1993 and 1994. Under suitable conditions, Melbourne experiences high Northerly winds
with high temperatures, which translates to a high count of ryegrass pollen per m3 of air.
Ryegrass is commonly grown in Victoria as a pasture grass (218). Pollen grains are ruptured
in rainwater by osmotic shock, which releases starch granules into the air. A growing body of
evidence suggests that relative humidity (RH) and temperature effect the dispersion of
ryegrass pollen in air. A study carried out by Glovsky et al., (2007) reported that peaks in
either RH values or rainfall is sufficient to rupture or fragment pollen grains (219). As for
wind speed, a minimum of 6 mph was required to release ryegrass pollen from their anthers.
In another study reported by Grote et al., (2000), it was found that ryegrass pollen grains
when exposed to isotonic solutions, remained intact. However, upon exposure to distilled
water or rainwater, they were able to expel starch grains and cytoplasmic debris by pollen
rupture (220). It is also reported by Suphioglu in 1998 that ryegrass pollen ruptures upon
contact with water, which releases 700 starch granules. Evidently, starch granules have been
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captured from air in Melbourne, Australia with an increase of 50-fold after an episode of rain
(204). Another phenomenon associated with meteorological data and pollen dispersion is
thunderstorm asthma. Epidemics of acute asthma association with thunderstorms have been
reported worldwide, such as 1987/1989 in Melbourne and 1994 in London. During
springtime thunderstorms, asthma and the marked elevation of ryegrass pollen counts display
a positive correlation, since ryegrass pollen grains rupture upon contact with water, which
releases starch granules. A thunderstorm is usually accompanied with sudden atmospheric
changes such as fall in temperature and air pressure along with rainfall and humidity, all of
which act as contributing factors for the dispersion of pollen (221, 222). Another
thunderstorm event that took the Canadians by surprise took place on the 31st of July 2000 in
Calgary, Alberta. As reported by Calgary Health Region, 157 people reported symptoms of
respiratory distress as opposed to expected number of people reporting such symptoms over a
period of 48 hrs. A sudden onset of high winds or gusts was attributed to the increase in
pollen into the atmosphere, which caused the epidemic (223). Another thunderstorm-
associated asthma study was carried out by Grundstein et al., (2008), which was conducted
over a period of 10 years between from 1993-2004. Emergency department visits from 41
hospitals in Atlanta, Georgia were taken into account. A total number of thunderstorm days
of 564 was included as part of this study to find the risk associated with asthma. Rainfall and
wind gust levels had the strongest association with emergency department visits confirming a
positive correlation (224).
1.9.6 Treatment for Pollen Allergy
SIT has been discussed thoroughly in the previous sections of this chapter. However, there is
a growing body of evidence that suggests the use of SIT to treat pollen allergy. Although
therapeutic by nature, this treatment has disadvantages as described in section 1.5.5.1.
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A study carried out by Jutel et al., (2005) employed a randomized, double-blind, placebo-
controlled study of SCIT therapy in patients suffering from allergic rhino-conjunctivitis, with
or without asthma. The symptom medication score showed significant reduction of symptoms
in patients receiving recombinant allergens as opposed to placebo (225). In the largest clinical
programme conducted with allergen-specific immunotherapy used Grazax for sublingual
administration to a total of 75,000 patients. Results from this trial showed substantial
inhibition of allergic symptoms in individuals suffering from rhino-conjunctivitis. The trial
was conducted throughout the grass pollen season. Subjects that were treated with Grazax
showed an improved quality of life. Although it was declared safe to use Grazax for treatment
of pollen allergy, there were a number of side-effects associated such as puritis, edema
mouth, ear puritis, throat irritation and sneezing (226). In a similar study carried out by
Winther et al., (2000), it was concluded that allergen-specific immunotherapy involving
grass-pollen extract was associated with a higher number of systemic reactions in atopic
individuals (227). In a separate study, it was reported that SIT prevents the onset of new
sensitizations to other allergens and reduces the development of asthma in patients suffering
from allergic rhinitis. It also reports the development and improvement of SIT by reducing
IgE-mediated reactions in atopic individuals (228, 229). The use of recombinant and
genetically engineered allergens is also being explored by many researchers. The clinical
efficacy of SLIT and SCIT against birch-pollen allergy was also determined by a randomized,
placebo-controlled, double-blind, double-dummy study and showed comparable effects on 71
patients suffering from allergic rhinitis. The study also suggested the safe method of SLIT
over SCIT (230). Many other studies have suggested similar treatments for pollen allergy but
are in the initial stages of development and have yet to undergo several clinical trials to prove
their efficacy (228, 229, 231-234).
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1.9.7 Deakin AIRwatch: Pollen Counting and Forecasting Facility
The Deakin AIRwatch project was launched in October of 2012 with a Burkard volumetric
air trap installed on the roof of the library at the premises of Deakin University (Waurn Ponds
and Burwood campuses). The ryegrass pollen season typically runs from October till January
of the following year. Currently, the only operational counting station in Victoria is housed at
the University of Melbourne and provided to the public by the Asthma Foundation of
Victoria (see: http://www.asthma.org.au/Home.aspx). Since there is currently no measure of
atmospheric pollen and spore concentrations in regional Victoria (e.g. Geelong) and eastern
Melbourne (e.g. Burwood), we have established Deakin AIRwatch, incorporating pollen and
spore counting stations at both the Waurn Ponds and Burwood campuses of Deakin
University. This is timely due to ever-increasing allergy and asthma epidemics. Deakin
AIRwatch network will not only directly benefit the public with pollen and spore counting
service to assist in their allergen avoidance programs but also contribute to significant
research and clinical studies, which is lacking for the greater Geelong area. The term Deakin
AIRwatch was coined by Associate Professor Cenk Suphioglu (PhD supervisor).
1.11 Overall Aims and Objectives of PhD Studies
Primarily, the objective of this study is to identify a novel antagonist that can bind to the
cytokine IL-4R and inhibit/down-regulate the interaction between the cytokines IL-4 and
IL-13 with IL-4Rα to potentially reduce the Th2 allergic response. The second aim is to use
PUFA’s, resolvins and CoQ10 as natural dietary remedies for allergic diseases. The third aim
of this project was to evaluate and quantify ryegrass pollen count in regional Victoria
(Australia) with regards to meteorological factors. Below is a list of specific aims and
objectives for this project.
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In a nutshell, this project aims:
1. To identify a human IL-4R inhibitor by screening a 12-mer phage displayed random
peptide library (Chapter 2);
2. To investigate the ability of the novel synthetic peptide inhibitor to bind to the IL-4R
(Chapter 2);
3. To analyse the effectiveness of the identified novel inhibitor in reducing the
interaction between IL-4/IL-13 and IL-4R by performing quantitative immunoassay
techniques (Chapter 2);
4. To test the peptide antagonist in vitro by using a recombinant cell line (HEK-Blue)
(Chapter 3);
5. To utilise fatty acids to inhibit cytokines IL-4 and IL-13 interaction with their
receptors, subsequently blocking STAT6 phosphorylation in a HEK-Blue model of
allergy (Chapter 4);
6. To utilise resolvins and CoQ10 and their effects on IL-4R and IL-13R signalling in a
HEK-Blue model of allergy (Chapter 5);
7. To study and model atmospheric concentration of grass pollen count and its
correlation with weather variables in regional Victoria, which may improve quality of
life (Chapter 6).
1.12 Expected Outcomes of Study
• Identification of a novel synthetic peptide will pave the way to develop a prophylactic
treatment for allergy in the future.
• Cytokine IL-4 and IL-13 interaction with IL4-R is a common pathway for many
allergies, blocking this process may cure allergies in general.
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• The use of fatty acids, resolvins and CoQ10 is expected to suppress allergic symptoms
and offer an alternative dietary route to reduce STAT6 phosphorylation induced by
cytokines IL-4 and IL-13 to tackle the allergic disease.
• The evaluation of ryegrass pollen distribution in regional Victoria will help improve
quality of life in individuals suffering from allergy and also the overall understanding
of pollen distribution and its relationship to changing climate variables.
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Chapter 2 – Identification and Characterization of a Novel IL-4R Antagonist for Allergy Treatment
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2.1 Introduction
There has been a marked increase in prevalence of atopic disease in regions such as Western
Europe, the US, Australasia and Asia Pacific during recent years, especially in industrialized
nations. A 20-fold increase has been experienced by western countries and as much as 40%
of the population suffers from this disease (8, 235-239). A recent statistical study carried out
has shown that a rise in allergy trend has taken place over the last 20 years (9). A rise in trend
has also been studied by ASCIA (18). These studies indicate an alarming situation and
require a more thorough and aggressive approach to resolve this disease.
In atopic individuals, an immune response is mounted by T cells that are activated by
allergens, promoting Th2 variant of cells. Once stimulated, these cells subsequently produce
cytokines such as IL-4 and IL-13. Unlike the cytokines produced by Th1 cells in non-atopic
individuals, an excessive production of cytokines takes place from Th2 cells (13, 240-243).
When the allergen comes into contact with antigen APC, the allergens are processed and
presented to naïve T cells, which mature into Th2 cells and release cytokines such as IL-4,
causing IgE-mediated immune response. Chemical mediators that are released as a result
cause symptoms like sneezing, wheezing and eczema (13, 244-246).
IL-4 and IL-13 play key roles in Th2 immunity and asthma pathogenesis. The function of
these cytokines is partially linked through their shared use of the IL-4Rα chain. The Type I
receptor comprising IL-4Rα and the common γ-chain is expressed by hemopoietic cells and is
exclusively responsive to IL-4. In contrast, the Type II receptor comprising IL-4Rα and IL-
13Rα1 is responsive to both IL-4 and IL-13 (59, 243, 247-249). Therefore, the concept that
IL-4 and IL-13 can be targeted simultaneously is of great importance because of their shared
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dependence on the IL-4Rα chain of the receptor. Hence, this research targets the common IL-
4Rα with a novel synthetic antagonist.
Current strategies to alleviate or avoid allergic symptoms include avoiding the allergens
altogether, administration of drugs such as antihistamines and anti-inflammatory drugs and
SIT to relieve the symptoms (22, 23). SIT has many functions including, the induction of Th1
cells in the production of IgG immunoglobulin rather than IgE (24, 25). Th2 cytokines,
principally IL-4Rα and IL-13, are reduced considerably, further alleviating the patient from
the symptoms (25). However, it has been argued that SIT has its disadvantages such as
unwanted effects of acute allergic reactions (250), poor efficacy and specificity as a result of
poor quality of allergy vaccines, comprised of poor quality of allergen extracts (26, 251-254).
Moreover, it is of high risk for patients with anaphylactic allergy (e.g. peanut allergy).
Current available treatments and results from various researches are disappointing and raise
concerns regarding therapies for allergy. Therefore, a need arises to redirect strategies in the
favour of phage display technology, in the quest to identify novel peptide inhibitors for
allergy treatment, which we report for the first time for human IL-4Rα receptor. Following
successful identification of the novel peptide, a series of immunoassays were carried out to
test the efficacy of the peptide in its binding to IL-4R. Our aim is to identify and characterize
a novel IL-4R antagonist which can successfully inhibit the interaction of IL-4 cytokine with
IL-4R hence, inhibiting the allergic cascade.
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2.2 Materials and Methods
2.2.1 General description of the M13 Phage library
Phage display libraries, Ph.D. 12, in which 12-mer random peptide are expressed at the amino
terminus of protein pIII of the filamentous bacteriophage were purchased from New England
BioLabs Inc. as a kit. The peptide is followed by a short spacer sequence Gly-Gly-Gly-Ser,
followed by the wild-type pIII sequence. Each of the libraries has a complexity of 2x109
virions and was amplified to give a final titer of 2x1011 plaque-forming units (pfu). All
experiments were carried out under strict sterile conditions using a Class II laminar flow
cabinet (Figure 2.1).
2.2.2 Biopanning
Aliquots of the phage libraries were screened with RhIL-4Rα (Abcam®); coated on flat-
bottomed 96 microtiter microplates (Thermo Scientific Co.). In the first and second rounds of
panning, the coating concentration of protein was 50 nM, which was prepared using
NaHCO3. To increase the stringency of the panning, the wells were coated with decreasing
concentrations of protein and increasing doses of Tris-buffered saline and tween 20 (TBST)
(TBS; 50 mM Tris-HCl pH 7.5, 150 mM NaCl + 0.1% [v/v] Tween-20). M13 phage was
selected for further amplification from the wells that tightly bound to the target antigen. In the
fifth round of panning, the concentration of the protein was 10 nM. A binding disruption
buffer 0.2 M glycine HCl (pH 2.2) was used to elute the bound phage along with 1mg/ml of
bovine serum albumin (BSA). 100 μl of this buffer was added to the wells of the microtiter
plate and was rocked gently for 10 min. The eluate was transferred into a microcentrifuge
tube, and immediately neutralized with 150 μl of 1 M Tris-HCl, pH 9.1.
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Figure 2.1. Schematic diagram of the steps taken to elute the phage and for perform
biopanning. Purified recombinant human IL-4Rα was used as the target antigen.
A library of phage (M13), each displaying a different peptide sequence were exposed to IL-4 receptor
M13 phage unable to bind to the target molecule was washed away with TBST
M13 phage which were able to bind to the target molecule were eluted
The eluted phage were amplified and taken through additional binding/amplification cycles
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2.2.3 Amplification and Identification of Eluted Phage Clones
The titer of the unamplified eluate was mixed with a freshly prepared culture of Escherichia
coli (E. coli) ER2738 (Phage Display Kit; New England Biolabs) host strain which is
tetracycline resistant (TetR) and incubated in a rotator shaker at 37°C and 240 revolutions per
minute (rpm), for 4.5 h. The culture was then transferred to a centrifuge tube and spun for 10
min at 10,000xg at 4°C, to pellet bacterial cells. The supernatant (containing the amplified
phage) was transferred to a fresh tube and re-spun for 5 min. Pellet was then discarded. Next,
80% of the upper supernatant was transferred to another fresh centrifuge tube. The phage was
precipitated by using 20% (w/v) polyethylene glycol (PEG)/2.5 M NaCl, or roughly 1/6th of
the total volume of eluate recovered from amplification. Dilutions of the phage were prepared
and grown on plates containing Luria broth (LB) along with IPTG and X-gal (LB medium +
15g/L agar + 1.25g of IPTG + 1g X-gal in 25 ml of dimethyl formamide; Sigma Aldrich). The
library-cloning vector M13 is taken from the common cloning vector M13mp19, which
carries the lacZα gene. This resulted into phage plaques appearing blue when plated on media
containing X-gal and IPTG (255).
2.2.4 Sequencing of Phage DNA: Rapid Purification
Phage DNA was sequenced from randomly selected clones, as described in the phage manual
provided by New England Biolabs Inc. (256-260), using the Micromon DNA Sequencing
Facility at Monash University (Melbourne, Australia). After the plaque is amplified, as
described in the section above, 500 μl of the supernatant containing phage was transferred to
a fresh microfuge tube. 200 μl of 20% PEG/2.5 M NaCl was added to the phage for
precipitation. The mixture was inverted several times and let to stand for 20 min at room
temperature (RT). A centrifugation was carried out on this mixture at 13,000 rpm for 10 min
at 4°C. The supernatant was discarded and the mixture was once again re-spun for 5 min. The
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supernatant was carefully discarded using a pipette. The pellet, which was left behind, was
suspended in 100 μl of iodide buffer (10 mM Tris-HCl at pH 8.0 + 1 mM
Ethylenediaminetetraacetic acid (EDTA) + 4 M NaI) by vigorously tapping the tube, to
generate single-stranded phage DNA for sequencing. Furthermore, 250 μl of 100% ethanol
was added to the mixture and incubated for 10-20 min at RT. This allowed precipitation of
single-stranded phage DNA and isolation of phage proteins in solution. The solution was
spun in a microfuge at 13,000 rpm for 10 min at 4°C and the supernatant discarded. The
pellet was subjected to another wash with 0.5 ml of 70% ethanol, re-spun briefly, the
supernatant discarded. The pellet was allowed to dry in a biological class II cabinet. The
pellet was finally suspended in 30 μl of TE buffer (10 mM Tris-HCl at pH 8.0 + 1 mM
EDTA) followed by quantification of DNA using a Nanodrop 1000 Nano-spectrophotomer
(Thermo Scientific Co.). Once quantified, 5 μl aliquot was taken from each DNA sample and
transferred to smaller 600 μl microfuge tubes and stored at -20°C. These 5 μl DNA samples
(ten in total) along with the -96 gIII sequencing primer (phage display kit) were sent to
Micromon DNA Sequencing Facility at Monash University for DNA sequencing analysis.
Once the DNA sequences for all 10 phage clones were obtained, they were further analyzed
using the FINCH TV sequence analysis software. This helped ascertain which sequences out
of the 10 were most suitable to carry out further immunoassays. The flanking regions were
used to locate the 36 base pairs of DNA of interest, which encoded the 12-mer peptide
sequence displayed on the M13 phage surface. The hybridization positions of the -28 and -96
sequencing primers were indicated as well. Short peptide designated N1 (Primary Sequence:
XXXXXXXXXXXX) along with the biotinylated version were chemically synthesized by
AUSPEP (Melbourne, Australia) and received in a lyophilized condition with >90% purity.
The biotin was added to the N-terminal of the peptide. The quality of all the peptide was
assessed by high performance liquid chromatography (HPLC) and confirmed by mass
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spectrometry analysis (purity >90%). Further analysis included alignment of 12-mer
sequences with IL-4 and IL-13 cytokines using ClustalW2 multiple alignment software. The
sequence of the peptide has not been shown due to intellectual property reasons with Deakin
University (Waurn Ponds, VIC, Australia).
2.2.5 M13 Binding ELISA
Four rows of a 96-well microtiter plate were coated with 90 μl of the target IL-4Rα (10 nM)
protein. The plate was incubated at 4°C with gentle agitation. Following day, the plate was
taken out of the box and excess target solution was removed by inverting the plate facedown
onto a paper towel. The wells containing the target were blocked using blocking solution (5
mg/ml BSA and 0.02% sodium azide (NaN3)). Simultaneously, a second microtiter plate was
also blocked with the blocking solution. Both plates were sealed with a parafilm and
incubated at 4°C for 1-2 hrs. The excess blocking solution was removed by slapping the plate
face down on a paper towel. Each plate was washed 5 times using 0.3% TBST. A second
microtiter plate, without the target, was used to make 4-fold serial dilutions of the phage
clone in 200 μl of 0.3% TBST; starting with 1012 pfu in the first well, down to 2 x 105 pfu in
the 12th well. Phage dilutions from 4th, 5th, 6th, 7th and 8th wells were transferred to the
plate with the target. This was incubated for 1-2 hrs at RT, with agitation. Once again, the
plate was washed 5 times with TBST. 200 μl of primary antibody, namely mouse anti-M13
antibody (Sigma Aldrich) (1:1000 dilution in blocking buffer) was added to each well and
incubated for 1 hour (hr) at RT. Subsequently, the plate was washed 5 times with TBST.
Post-incubation with anti-M13 antibody, 200 μl of secondary antibody, namely anti-mouse
horseradish peroxidise (HRP) conjugated antibody (Sigma Aldrich) (1:2000 in blocking
buffer) was added to each well and incubated for 1 hr with agitation on a rocker. This was
followed by another round of 5 washes with 0.3% TBST. A developing solution was
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prepared, by dissolving a single tablet of phospho-citrate buffer with sodium perborate in 100
ml of distilled water (DH2O), then 1 tablet of HRP substrate ortho-phenyl diamine (OPD;
Sigma Aldrich,) was dissolved in 7.5 ml of this solution. 200 μl of this solution was added to
each well; the plates were wrapped in foil and incubated for 30 min at RT with agitation. The
reaction was stopped by adding 50 μl of 4M sulphuric acid (H2SO4). The color development
on the plates was read by using a multi-scan plate reader (Thermo Labsystems Inc.), set at 492
nm of wavelength. Three phage clones were used for this experiment namely N4, N5 and N7.
Hence, the method shown was repeated in the same exact manner for all three clones.
2.2.6 Synthesis of the Biotinylated Peptide
All 10 phage clones isolated in this study gave a strong amino acid sequence consensus, only
one 12-mer peptide was sent for synthesis, denoted as N1 (AUSPEP, Australia). A
biotinylated version of the peptide was also synthesized (N1 biopep). The purity of the
peptides was >90% and the total peptide synthesized was 5 mg. Dilution of biotinylated
peptide: 1 mg of the N1 biopep was taken from the 5 mg stock and dissolved in 500 μl of
DH20 (filter-sterilized). The peptide dissolved immediately; hence, another 500 μl DH20 was
added to make up to the required 1mg/ml of peptide concentration.
2.2.7 Direct ELISA
Immobilization of 90 μl target IL-4Rα (10 nM) was carried out on a 96-well plate overnight.
The plate was sealed with a parafilm and placed in a humidified container with agitation.
Following day, the plate was slapped face down on a paper towel to get purged of target
solution. 220 μl of blocking buffer (BSA + NaN3 blocking solution) was added to the wells.
The plate was incubated at 4°C for 1-2 h. The blocking buffer was removed by slapping the
plate face down on the paper towel, and gently washed 3-4 times with 280 μl/well of 0.3%
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TBST. Next, 100 μl of biotinylated N1 peptide was added to each designated well of the
ELISA plate. The plate was incubated for 1-2 hrs at RT with agitation on a rocker. Negative
controls were setup in 3 separate wells without the addition of N1 peptide. The wells were
subjected to DH20 only, with the exception of the peptide. This was followed by 3-4 washes
with 280 μl/well of 0.3% TBST. Finally, 150 μl HRP-conjugated streptavidin (Sigma
Aldrich) (1:500 dilution in blocking buffer) was added to the wells and incubated for 1 h with
agitation on a rocker. Once again, the plate was gently washed 3-4 times with 280 μl/well of
0.3% TBST. A developing solution was prepared, by dissolving a single tablet of phospho-
citrate buffer with sodium-perborate in 100 ml of dH2O, then 1 tablet of HRP substrate OPD
was dissolved in 7.5 ml of this solution. 200 μl of this solution was added to each well; the
plates were wrapped in foil and incubated for 30 min at RT, with agitation on a rocker. The
reaction was stopped by adding 50 μl of 4 M H2SO4. The color development on the plates
was read by using a multi-scan plate reader, set at 492 nm of wavelength. Statistical analysis
was performed on the data obtained from the ELISA.
2.2.8 Inhibition ELISA
Initially, the same protocol was followed, as described in the direct ELISA protocol above.
After the blocking of the target IL-4Rα, IL-4 cytokine (Abcam®; diluted in 1:500 filter-
sterilized dH2O) was pipetted into the wells and was incubated for 1-2 hrs at RT, with
agitation on a rocker, followed by 3-4 washes with 250 μl 0.3% TBST. Following this, 120 μl
of N1 biopep (0.2 mg/ml) was added to each designated well of the ELISA plate. This was
then developed as described in the section above. Further, statistical tests were performed on
the results obtained from the ELISA.
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2.2.9 Statistical Analysis
The data were analysed using Microsoft Excel 2013 (Microsoft Inc.). The results were
analysed by Student’s t-test to determine any statistically significant differences between the
positive control (PC) and treatment wells in all of the above experiments. The statistical
significance was set at P < 0.05.
2.3 Results
Selected peptides from 12-mer libraries were analyzed by taking 3 random peptides from the
9 individual phage clones isolated from the 5th round of biopanning, demonstrating identical
sequence motifs (see next section below). Each sample was diluted, plated and individual
clones were isolated, amplified, sequenced and tested for reactivity with the target antigen IL-
4Rα using ELISA immunoassays.
2.3.1 Phage Display Biopanning
A 12-mer phage-displayed random peptide library was screened through a process known as
‘biopanning’. The procedure was performed with five cycles, repeated consecutively.
Initially, the first round of biopanning was completed and the amplified phage was titered
onto IPTG/X-gal plates. A total of 5 rounds of biopanning were repeated. Table 2.1 shows
the calculations done to obtain input volumes for subsequent panning rounds.
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Table 2.1 Input phage titers for subsequent rounds of biopanning and appropriate calculations.
Biopanning rounds
Initial pfu
Input titer of phage
Input volume for next round
Round 1 2.25 x 1011 2 x 1011/2.25 x 1011 0.88 μl Round 2 2.3 x 1010 2 x 1011/2.3 x 1010 8.8 μl Round 3 5.8 x 1011 2 x 1011/5.8 x 1011 0.344μl Round 4 1.3 x 1011 2 x 1011/1.3 x 1011 1.3 μl Round 5
Last round, hence no further calculations required
From the final 5th round of biopanning, 10 clones were collected by stabbing plaques on the
agar plates and amplified as described in the phage display manual. The plaques were
sequenced to reveal the DNA sequence of the single-stranded phage DNA. Upon return, 9 of
the 10 sequences had identical consensus in binding sequence, N1 to N9. Since the peptides
from the clones N1 to N9 showed a consensus in sequence, N10 was discarded and was no
longer used for further analysis. The sequence of the peptide has not been shown due to
intellectual property reasons with Deakin University.
Further, ClustalW2 was used to perform sequence analysis of N1 amino acids with the IL-4
cytokine to observe the similarities between their sequences. This was performed to evaluate
the binding capacity of the peptide to IL-4Rα, and as a result, “mimic” the cytokine IL-4 in
its amino acid sequence. This also revealed the epitope region of specific amino acid
sequence of IL-4Rα that would hybridize with the peptide. Table 2.2 shows a comparison
between IL-4 and the N1 peptide with 6 amino acid residues showing identical residues, and
also, three weakly conserved residues.
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Table 2.2 Results obtained from ClustalW2 alignments, performed using N1 peptide.
2.3.2 M13 ELISA with Three Homologous M13 phage clones
M13 ELISA was performed using 3 selected phage clones N4, N5 and N7 from the 9 clones
with identical sequences selected for mimtag synthesis. This step was performed to further
enhance results showing specific binding of the phage mimtag to the target IL-4R.
Irrespective of which phage clones were used for the M13 ELISA, since all of them displayed
the same amino acid sequence, the ELISA was carried out using an anti-M13 antibody,
followed by an anti-mouse HRP antibody to detect antibody-binding and OPD substrate for
HRP, to develop the solution for absorbance detection (Figure 2.2). The detection was based
on a strong affinity between the displayed mimtag by the phage clones and the target IL-4R.
Overall, all of the three selected peptides (e.g. N4, N5 and N7) showed a binding to the IL-4R
target. With comparable error bars overlapping between the 3 different clones, shown in the
graph (Figure 2.2), the phage clones displaying the mimtag gave higher absorbance readings
when compared to the control. This is an indication of the accuracy and specificity of the
M13 phage binding to its target antigen with in vitro analysis.
2.3.3 Direct ELISA
In view of the fact that N1 amino acid sequence shared a 50% homology when aligned with
cytokine IL-4, it was necessary to conduct further tests on the N1 biopep, to determine its
avidity with IL-4Rα (Figure 2.3). This was performed by using direct ELISA, in which the
Percentage amino acid similarity with 12-mer N1 peptide
With IL-4 With N1 peptide
Identical sequences 6 amino acids (50%) 100%
Strongly conserved None 100%
Weakly conserved 3 amino acids (25%) 100%
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peptide was allowed to interact with the IL-4RΑin the absence of the cytokines to determine
its avidity to the target antigen. In a similar fashion as other ELISA assays, this was carried
out using a HRP conjugated streptavidin, to detect the biotin on the N1 peptide, and OPD
substrate to develop the solution for absorbance detection as described in earlier sections.
Evidently, the differences in absorbance readings obtained showed that N1 biopep had
specifically bound IL-4Rα, when compared to the control. A trend was seen with increasing
concentration of the peptide, where higher absorbance readings were recorded on the
spectrophotometer. This indicated that the streptavidin-HRP had indeed recognized the biotin
on peptide N1, which in turn interacted with the IL-4Rα target antigen.
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Figure 2.2. M13 ELISA performed using 3 selected phage clones of IL-4Rα. The phage
clones were randomly selected from the 9 clones with identical sequences to verify the
binding affinity and efficacy of the mimtag to the target IL-4Rα protein. Figure illustrates the
absorbance readings that were obtained when a range of phage dilutions were used to perform
the experiment. As a result, with increasing phage clone titers, a higher absorbance reading
was obtained at 492 nm, and vice-versa. The results are representing 3 replicates (mean
values) for each bar graph; hence the error bars denote a standard deviation. A statistical
Student’s t-test revealed a significant difference between the mean absorbance values of the
1.5 x 1010 phage dilution and the negative control (P < 0.05).
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Figure 2.3. Direct ELISA of biotinylated N1 peptide interaction with IL-4Rα. This assay
was conducted to verify the affinity and efficacy of the newly synthesised N1 peptide, which
showed considerable binding with the IL-4Rα target. The graph illustrates the absorption
readings that were obtained when a range of N1 peptide concentrations were used to carry out
the experiment in vitro. As a result, with increasing peptide concentrations, a higher
absorbance reading was obtained at 492 nm, and vice-versa. The results represent 3 replicates
(mean values) for each bar graph; hence the error bars denote a standard deviation. A
statistical Student's t-test revealed a significant difference between the mean absorbance
values of the peptide at 0.2 mg/ml concentration and the negative control (P < 0.05).
* = P < 0.05
*
*
*
*
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2.3.4 Inhibition ELISA
Shortly after the results were achieved from the direct ELISA, the next step was to verify
whether the synthesized N1 biopep was capable of inhibiting the interaction between IL-4
and its receptor IL-4Rα. In this case, the IL-4Rα was immobilized on a 96-well plate,
followed by the addition of the IL-4 cytokine. The ELISA was carried out using a HRP
conjugated streptavidin, to detect the biotin on the N1 biopep and OPD substrate for the
development and absorbance. The inhibition was found to be ~73% with respect to the
biopep. These results show that the biopep N1 is able to successfully inhibit IL-4 cytokine
from binding to its receptor, as shown in Figure 2.4.
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Figure 2.4. Inhibition ELISA showed that the biotinylated peptide N1 successfully
fastened to the target IL-4Rα in the presence of IL-4, as the inhibitor. With the pre-
incubation of cytokine IL-4, the peptide showed lower absorbance reading as opposed to in
its absence. However, the peptide was still able to compete with the cytokine and record
significant readings when observed using a spectrophotometer. The negative control had no
biotinylated peptide N1 or cytokine IL-4 (graph bar on far left). The results represent mean
value of 3 replicates for each bar graph; hence the error bars denote a standard deviation. A
statistical Student’s t-test revealed a significant difference between the mean absorbance
values when compared to the control (P < 0.05).
** = P < 0.05
*
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2.4 Discussion
We report here for the very first time a peptide antagonist identified and characterized against
IL-4Rα which successfully inhibits the interaction of cytokine IL-4 with the receptor. The
peptide antagonist identified is 12 amino acid long and binds with strong affinity to the IL-
4Rα. Hence, rendering it as a useful tool for future allergy therapy.
2.4.1 Phage Display
IL-4Rα plays a vital role in allergic diseases since it is able to bind IL-4 and IL-13 cytokines.
Activation of Th cells by APC leads to the production of these cytokines that cause B cells to
produce IgE. Once synthesized, IgE antibodies circulate in the blood before binding to the
high-affinity IgE receptor FcεRI that is present on mast cells in tissue or on peripheral blood
basophils. After re-exposure, allergens cross-link to mast cell-bound specific IgE, thus
causing the release of preformed mediators, such as histamine, the synthesis of prostaglandins
(PGs) and LT’s (261). Thus, it is necessary to find a novel antagonist to IL-4Rα, to inhibit the
interaction of the cytokines with their receptor, hence down-regulating the excess production
of IgE and thus the allergic cascade, as a prophylactic treatment.
Current clinical strategies for allergy treatment involve allergen avoidance by patients,
pharmacotherapy and specific immunotherapy. Only the third strategy has provided a long-
term solution but with little success, since the allergen extracts used are poorly characterized
and suffers from batch to batch variation (262). In addition, natural allergen extracts exhibit
high allergenic activity that often causes side effects and it has been reported that SIT with
allergen extracts can induce IgE sensitization to new allergens (262, 263). The display of
foreign proteins on the surface of bacteriophage M13 has been used to understand protein-
protein interactions at the molecular level. For this reason, phage display technology has been
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used as a novel approach to identify peptide antagonists (264, 265). This technology has
evolved over the last two decades and has been used for drug discovery and identifying novel
synthetic peptides, which can be used as antagonists to many protein-ligand interactions
(266-269). Our findings indicate that a novel synthetic peptide N1 has been identified and
characterized as an antagonist to IL-4α which inhibits the interaction between the cytokine
IL-4 and its receptor. IL-4, as discussed earlier, plays an important role in the allergic cascade,
eventually leading to the release of inflammatory histamines from mast cells and basophils
that lead to allergic symptoms. Hence, targeting the receptor was the main objective of this
study.
2.4.2 M13 Binding ELISA with N4, N5 and N7 Phage Clone
Since N1-N9 sequences were chosen for further analysis, and they all shared an identical
sequence, only N1 peptide was chosen for synthesis. The peptide sent for synthesis was
biotinylated, on the N-Terminal end of the peptide. While this was being carried out, an M13
binding ELISA was performed by randomly choosing 3 phage clones N4, N5 and N7 from
the 9 identical peptides. ELISA protocol is sufficient for rapidly determining whether the
selected phage clones bind to the target IL-4R or not. The method was useful to determine
qualitatively whether the selected clones in parallel have a relative binding to the target when
compared to binding to the plastic support. Hence, this ELISA does not determine whether
the clones are binding with high or low affinity to the target receptor, but rather indicate a
positive or negative response. As can be seen in the results, phage clones N4 and N5 had
overlapping error bars which suggested that they had similar binding affinity towards the IL-
4R. The N7 had shown a slightly higher affinity towards the IL-4Rα. The subtle changes
might have resulted from the phage titer that was determined by growing the phage clones on
X-gal/IPTG plates, similar to biopanning. This preliminary study carried out with the phage
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clones N4, N5 and N7 laid down the hypothesis that the selected phage clones can indeed
bind to the IL-4Rα target, and may therefore, act as a potential inhibitor for interaction with
the cytokines IL-4 and IL-13. A statistical Student’s t-test analysis showed significant
difference between the experimental values and controls (P < 0.05).
2.4.3 Direct ELISA and Inhibition ELISA
A direct ELISA was performed in our recent study to test the ability of the synthesized
peptide to bind with the IL-4Rα target. The biotin on the peptide was detected by a HRP-
conjugated streptavidin protein. This immunoassay was specifically done to test the efficacy
and affinity of the newly synthesized biopep N1 to the IL-4Rα. The trend was clearly visible
via the graphed readings where higher absorption readings are observed with increasing
peptide concentrations. This was an indication that the IL-4Rα was bound to the bottom of
the well and the N1 biopep was interacting with the target. A final aim of this study was to
test the ability of the identified antagonist to not only bind with the target but down-regulate
or inhibit its interaction with the target IL-4Rα. Hence, an inhibition ELISA was performed
using the N1 biopep as the inhibitor. The inhibition ELISA is a highly sensitive and specific
immunoassay method that uses an antigen capture method to bind the analytes present in the
solution. Results are shown in the graph and a Student’s t-test analysis of the values
compared to the control was highly significant (P < 0.05). This proved that the affinity of N1
biopep for the IL-4Rα was much higher than the cytokine itself. Although the IL-4 cytokine
concentration used was lower than the concentration of the biopep N1, it was consistent with
the recommendation of the manufacturer. The results were comparable to many other
researches where peptide-ligand interactions have proved to be very successful (270, 271). As
a result of the small size of the peptide, they tend to show a higher target-to-background
ratios compared to macromolecular compounds.
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2.4.4 Conclusion
We have sucessfully identified and characterized a novel antagonist to IL-4R which down-
regulates the interaction of the IL-4R with the cytokine IL-4. Using a phage display random
peptide library, the biopanning procedure established in identifying and isolating the
synthetic peptide antagonist N1. Furthermore, the second aim was to test the identified
antagonist using immunoassay techniques and tissue culture assays to justify that the novel
peptide was indeed inhibiting the IL-4R from interacting with IL-4. Results from ELISA
immunoassays suggested that the peptide was proficient in binding to the IL-4R with a higher
affinity and hence, was able to retard the overall protein-ligand interaction between the
cytokines and IL-4R. Furthermore, using HEK-Blue IL-4/IL-13 cells a >50% inhibition was
achieved with the peptide with a simple colorimetric analysis. As this peptide targets the two
most clinically important cytokines in allergy, it promises to provide for the future treatment
for all IgE-mediated allergies.
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Chapter 3 - Expression of HEK-Blue IL-4/IL-13
Reporter Cell-line and Further Characterization of the
IL-4Rα, IL-4 and IL-13 Peptide Antagonist in vitro
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3.1 Introduction
The cells of the immune system are peripatetic and therefore require a complex, but efficient,
communication system in order to regulate prompt and integrated immune responses. Such
sophisticated communication networks exploit a diversity of soluble and membrane bound
signaling molecules, including cytokines (272, 273). These cytokines, being non-enzymatic
mediators synthesized by multiple regulatory levels, function by interacting with membrane
spanning receptors on target cells to send secondary intracellular signals, which in turn
stimulate target cell differentiation, proliferation, migration or further production of cytokines
(272-274). There are six types of cytokines involved in immune responses: interleukins (IL),
chemokines, interferons (IFN), transforming growth factor, hemopoietic colony-stimulating
factors, and TNF (272, 275, 276). Of all the cytokines, interleukins play a pivotal role in
allergy by acting as lymphocyte mediators (277).
Interleukins function via type I or type II cytokine receptors that possess different types of
sub units (e.g. γ chain), which are shared by groups of interleukins (278, 279). This provides
an explanation for the superfluous nature of interleukins during inflammation, including
allergy. IL-4 and IL-13, the foremost mediators of atopic and allergic diseases, as well as
their receptors (IL-4R and IL-13R) share molecular features and function cooperatively
during an allergic reaction (280, 281). IL-4 and IL-13 are type I pleiotropic cytokines
synthesized by activated T-cells and are significant allergic mediator that share structural and
functional properties (282).
IL-4 and IL-13 have various pathological effects on a range of hematopoietic and non-
hematopoietic cells during pathogenesis of allergy. In B cells, the cytokines induces B-cell
proliferation, differentiation and class switching of immunoglobulin to produce IgE (283).
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Cytokines IL-4 and IL-13 are known to down-regulate the expression of immune mediators
including IL-1α, IL-1β, IL-6, IL-8, IL-12 and TNF-α by moderately suppressing the DNA
transcription regulator NF-κB (53, 284). The cytokines also contribute to IgE priming of mast
cells, further influencing increased IgE production (53, 284). However, unlike IL-4, IL-13
does not affect T-cell function, differentiation or proliferation (285), therefore IL-13 can be
classified as a mediator of the effector phase (involving B-cells, mast cells and basophils) of
allergy.
IL-4 and IL-13 play key roles in Th2 immunity and asthma pathogenesis. The function of
these cytokines is partially linked through their shared use of the IL-4Rα chain. The Type I
receptor comprising IL-4Rα and the common γ-chain is expressed by hematopoietic cells and
is exclusively responsive to IL-4. In contrast, the Type II receptor comprising IL-4Rα and IL-
13Rα1 is responsive to both IL-4 and IL-13 (Figure 1.6) (59).
A practical approach to treating allergy is the neutralization of the activities of interleukins. It
is imperative to conduct studies on neutralizing IL-4 and IL-13 and their corresponding
receptors. We have previously reported (138) the identification of a peptide antagonist
potentially capable of inhibiting IL-13 interaction with its receptor which is explained in
chapter 2. Here we have conducted in vitro studies to test the ability of the isolated peptides
to inhibit the IL-4 and IL-13 interaction with the receptor type II IL-4Rα/IL-13αR1 using a
HEK-Blue cell-line. This will elucidate the effects of the peptide antagonist on JAK-STAT6
phosphorylation which leads to IgE synthesis in allergic patients via IL-4 and IL-13
signaling. Hence, blocking the interaction of the cytokines with their receptors using the
isolated peptide antagonists is the specific aim of this chapter.
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3.2 Materials and Methods
3.2.1 HEK-Blue IL-4/IL-13 Cells
HEK-Blue™ IL-4/IL-13 (InvivoGen) cells allow the detection of bioactive IL-4 and IL-13 by
monitoring the activation of the JAK-STAT6 phosphorylation pathway. The cells are a result
of a stably transfected human JAK-STAT6 gene. Other pathways linked to the STAT6
pathway are naturally expressed by the cells. The cells are further transfected with a STAT6-
inducible SEAP reporter gene as an alternative to IgE secretion in B lymphocytes when
signaled by cytokines IL-4 and IL-13. Finally, the cells are transfected with a stably
expressed recombinant type II IL-4Rα/IL-13αR1 receptor (Figure 1.9).
Growth medium: HEK-Blue cells were cultured in Dulbecco’s Modified Eagle Medium
(DMEM) (GIBCO), supplemented with 10% fetal bovine serum (FBS), in a humidified
incubator at 37°C with 5% CO2. Antibiotics were added to the medium to avoid
contamination (50 U/ml of penicillin, 50 μg/ml of streptomycin, 100 μg/ml of normocin, 10
μg/ml of blasticidin and 100 μg/ml of zeocin - GIBCO), Trypsin/EDTA (0.05%; GIBCO)
was used for trypsinization.
Experimental medium: Growth medium was replaced with 2mM L-Glutamine and heat
inactivated 10% FBS.
Three peptides N1, P9 and K1 were used to perform these assays. Peptide P9 and K1 were
both characterized by fellow colleagues Pathum Dhanapala and Kelley Ramsbottom at the
NARL laboratory at Deakin University. The projects were undertaken as part of their
Honours year project as an extension to their undergraduate degrees. Pathum Dhanapala
performed phage display using cytokine IL-13 as a potential target to identify novel peptide
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antagonist P9. Kelley Ramsbottom used cytokine IL-4 as a potential target to identify novel
peptide antagonist K1. Both colleagues identified and characterized the peptides in a similar
fashion as in the case of peptide N1 for IL-4R. However, the peptides were not tested in vitro
and needed further characterization in vitro.
3.2.2 Synthesis and Use of Peptides N1, P9 and K1
All 10 phage clones isolated in this study gave a strong amino acid sequence consensus, only
one 12-mer peptide was sent for synthesis, denoted as N1 for IL-4Rα, P9 for IL-13 cytokine
and K1 for IL-4 cytokine (AUSPEP, Australia). The purity of the peptides was >90% and the
total peptide synthesized was 10 mg of each. Dilution of the peptides was prepared by
dissolving 1 mg in 500 μl of DH20 (sterilized). The peptide dissolved immediately; hence,
another 500 μl DH20 was added to make up to the required 1 mg/ml of peptide concentration.
However, in the case of peptide K1, 5 mg was dissolved in 1 ml of DH20. Subsequent
dilutions for all peptides were prepared to be used for experiments with HEK-Blue cells as
shown below.
3.2.3 HEK-Blue Cell line Inhibition Assay with N1 Peptide
The protocol used was taken from the datasheet provided with the HEK-Blue IL-4/IL-13 kit.
A few adjustments were made in the protocol to perform the assay. Cells were grown in a T-
75 flask and were used for the detection assay. The cells were detached using 3 ml trypsin
which was neutralized with 7 ml of media (FBS - neutralising agent) to give a total volume of
10 ml. The average cells were counted using a TC10 automated cell counter (Bio-Rad
Laboratories Inc.) and cultured at a density of 50,000 cells per well of a 96-well plate. Six
treatment wells were used in total; 4 wells treated with peptide N1 (MW 1354Da) and 2 wells
without peptide as positive and negative controls (shown in results section). Cells in the
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treatment wells were incubated with 50 μl of N1 peptide (75, 150 and 225 μM corresponding
to 0.1, 0.2 and 0.3 mg/ml) at 37°C in a rotator shaker for 1 hr, whereas the control wells were
incubated with filter-sterilized DH2O. Post-incubation, 20 μl of IL-4 cytokine (100 ng/ml)
was added to the PC and three treatment wells. The 96-well plate was sealed using a parafilm
and incubated at 37°C with 5% CO2 for a period of 24 hrs. Post-incubation, QUANTI-Blue
substrate was prepared using the instructions in the HEK-Blue kit and 180 μl of this solution
was added to 6 wells in a fresh 96-well plate. 20 μl of induced HEK-Blue IL-4 cells
supernatant from each of the treatment wells was added to the QUANTI-Blue solution. The
subsequent results were read using an xMark microplate absorbance spectrophotometer (Bio-
Rad Laboratories Inc.) at a wavelength of 640 nm.
3.2.4 HEK-Blue Cell Line Inhibition Assay with P9 and K1 Peptide
The protocol used for this experiment was slightly changed without affecting the overall
nature of the experiment. Concentrations used for P9 (MW 1322Da) peptide were 0.1, 0.2
and 0.3 mg/ml which corresponds to 75, 150 and 225 μM. However, in the case of peptide
K1 (MW 1238 Da) the concentrations used were 0.5, 1 and 1.5 mg/ml which corresponds to
400, 800 and 1200 μM. The following changes made to the protocol are shown below:
- 100 μl of the cytokines IL-4 and IL-13 at a concentration of 100 ng/ml were incubated
with 50 μl of peptides P9 and K1 and incubated for 1 hr;
- Post-incubation, 30 μl of this solution was added to the HEK-Blue cells to maintain
cytokine concentration at 100 ng/ml.
The protocol from here onwards was the same as in the case of peptide N1 inhibition studies.
Absorption readings were taken after 24 hrs of incubation with HEK-Blue cells. The average
cells were counted using a TC10 automated cell counter.
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3.3 Results
3.3.1 HEK-Blue Cell Line Inhibition Assay with N1 Peptide
Once all necessary immunoassays had been carried out to determine the efficacy of the N1
peptide, further analysis was required to confirm the specificity of the peptide in vitro in
inhibiting the IL-4 signaling pathway. As shown in Figure 3.1A, with the addition of the
peptide N1, a dose-dependent relationship was observed with the increasing concentration of
the peptide, which means less SEAP was secreted by the cells, hence less color produced in
the presence of the QUANTI-Blue substrate. All positive and negative controls behaved as
expected with high and low peaks as suggested by the HEK-Blue IL-4/IL-13 kit. As
hypothesized, >50% inhibition of the IL-4 signaling pathway was achieved with the highest
concentration of peptide at 225 μM (Figure 3.1B). Consistency was also observed with cell
viability when tested for all the treatments and compared to the PC.
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3.1(A)
3.1(B)
Figure 3.1. In the presence of N1 peptide, the receptor was successfully antagonized and hence,
denoted lower SEAP levels. A dose-dependent relationship can be observed with the three treatment wells
containing peptide N1 (Figure 3.1A). PC included cells treated with IL-4 cytokine alone. Control 1 (C1)
included cells incubated with peptide only and Control 2 (C2) included cells without any treatment. P < 0.05
denotes significance of optical density (OD) readings compared to PC. % Inhibition of HEK-Blue cells with
N1 peptide. Inhibitory concentration was obtained with 225 μM of peptide N1, which inhibited 58.8% of
SEAP secretion. Hence, IC50 concentration was determined as 178.1 μM to achieve 50% inhibition. PC is
denoted at 0% which is used as a referral point for the standard value at which inhibition begins (Figure
3.1B).
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3.3.2 HEK-Blue Cell line Inhibition Assay with P9 Peptide
Similar to the previous experiment, the efficacy of peptide P9 was further tested in a HEK-
Blue cell line. As shown in Figure 3.2A, with the addition of the peptide P9, a dose-
dependent relationship was observed with increasing concentration of the peptide, hence less
color produced in the presence of the QUANTI-Blue substrate. As hypothesized, >50%
inhibition of the IL-4/IL-13 receptor signaling pathway was achieved with the highest
concentration of peptide at 225 μM (Figure 3.2B). Peptide had no negative impact on cell
viability.
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3.2(A)
3.2(B)
Figure 3.2. In the presence of P9 peptide, the cytokine IL-13 was successfully antagonized and hence,
denoted lower SEAP levels. A dose-dependent relationship can be observed with the three treatment wells
containing peptide P9 (Figure 3.2A). PC included cells treated with IL-13 cytokine. C1 included cells
incubated with peptide only and C2 included cells without any treatment. P < 0.05 denotes significance of
OD readings compared to PC. % Inhibition of HEK-Blue cells with P9 peptide. Inhibitory concentration was
obtained with 225 μM of peptide P9 which inhibited 51.8% of SEAP secretion. Hence, IC50 concentration
was determined as 213.2 μM to achieve 50% inhibition. PC is denoted at 0% which is used as a referral
point for the standard value at which inhibition begins (Figure 3.2B).
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3.3.3 HEK-Blue Cell line Inhibition Assay with K1 Peptide
Similar to the previous experiment, the efficacy of peptide K1 was further tested in a HEK-
Blue cell line. As shown in Figure 3.3A, with the addition of the peptide K1, a dose-
dependent relationship was observed with increasing concentration of the peptide, hence less
color produced in the presence of the QUANTI-Blue substrate. >50% inhibition of the IL-
4/IL-13 receptor signaling pathway was achieved with the highest concentration of peptide at
1200 μM (Figure 3.3B). Peptide had no negative impact on cell viability.
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3.3(A)
3.3(B)
Figure 3.3. In the presence of K1 peptide, the cytokine IL-4 was successfully antagonized and hence,
denoted lower SEAP levels. A dose-dependent relationship can be observed with the three treatment wells
containing peptide P9 (Figure 3.3A). PC included cells treated with IL-13 cytokine. C1 included cells
incubated with peptide only and C2 included cells without any treatment. P < 0.05 denotes significance of
OD readings compared to PC. % Inhibition of HEK-Blue cells with K1 peptide. Inhibitory concentration
was obtained with 1200 μM of peptide P9 which inhibited 51.8% of SEAP secretion. Hence, IC50
concentration was determined as 995 μM to achieve 50% inhibition. PC is denoted at 0% which is used as a
referral point for the standard value at which inhibition begins (Figure 3.3B).
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3.4 Discussion
3.4.1 Characterization of Novel Peptide Antagonists Using a HEK-Blue Cell Line
We have successfully identified and characterized a novel antagonist to IL-4Rα (N1 peptide),
IL-4 (K1 peptide) and IL-13 (P9 peptide) which down-regulates the interaction of the
cytokines with their receptors. Using a phage display random peptide library, the biopanning
procedure established in identifying and isolating the synthetic peptide antagonist N1, K1 and
P9 (explained in Chapter 2). Furthermore, the second aim was to test the identified
antagonists using immunoassay techniques and tissue culture assays to justify that the novel
peptides were indeed inhibiting the cytokines from interacting with IL-4. Using HEK-Blue
IL-4/IL-13 cells a >50% inhibition was achieved with the peptides using a simple
colorimetric analysis. IL-4 and IL-13 cytokines signal via the type II heterodimeric IL-
4Rα/IL-13αR1 and IL-13αR2 receptors. IL-13αR1 and IL-13Rα2 bind IL-13 with sequence
specificity. However, the latter acts as a high affinity decoy receptor which does not initiate
signal transduction (285). Although IL-13Rα1 binds IL-13 with low affinity, pairing with IL-
4Rα increases the affinity by over 40 times which results in the activation of JAK-STAT
(signal transducer and activator of transcription) signaling pathway responsible for B-cell
differentiation (285). Since HEK-Blue cells expresses the heterodimeric receptor IL-4Rα/IL-
13αR1, blocking or inhibiting one of the receptor chains or the cytokine itself may eliminate
the response of STAT6 signalling. As these peptides targets the two most clinically important
cytokines in allergy, it promises to provide for the future treatment for all IgE-mediated
allergies.
Using HEK-cells for tissue culture research carries many benefits. To name a few, the cells
have been used as an expression tool for recombinant IL-4Rα for over 25 years. They remain
highly stable and efficient when transfected with foreign genes. Also, once transfected, the
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translation and processing of the gene product (proteins) is performed with high efficiency.
Finally, their effortless reproduction and maintenance adds to the benefits of using this cell
line as a model system (145). HEK-Blue cells on the other hand, allow a colorimetric
analysis, which carries simplicity, high sensitivity and low cost to conduct the required IL-4
signaling experiments. HEK-Blue detection assay was performed using a HEK-Blue
transfected cell line with the N1, K1 and P9 peptides as inhibitors. As the results suggest, an
increase in peptide concentration reduced SEAP levels that were detected through a
colorimetric analysis. Although some minor changes have been made to the overall protocol
of the detection assay, the absorption readings observed were at peak values in the PC. The
aim of this experiment was to achieve a >50% inhibition of the IL-4/IL-13 signaling pathway
that would allow the severity of allergic symptoms to be lowered in atopic individuals (286-
289). Completely blocking the production of IgE antibodies can lead to other illnesses since
IgE plays an important role in parasitic immunity such as helminthes infections (290-293).
Although it has been criticized that peptides selected against purified recombinant protein
may not be able to access their targets on living cells, it has proved otherwise in our case
(explained in Chapter 7). The positive and negative controls were setup to pinpoint any
discrepancy in the results. More importantly, the detrimental effects of peptide concentrations
were also tested by performing a cell viability test for all the parameters. Consistency in cell
count suggested that even at higher doses, the peptide antagonist did not affect cell viability.
A Student’s t-test analysis of the results from the graph revealed a significant difference
between the PC and the treatments performed (P < 0.05).
3.4.2 Conclusion
To conclude, we have successfully tested three peptide antagonists N1, P9 and K1 as
potential inhibitors of allergy pathway in a recombinant HEK-Blue cell line. The STAT6
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phosphorylation pathway is activated by bioactive IL-4 and IL-13 via the IL-4Rα and IL-
13Rα1 receptors. This pathway leads to a production of SEAP which mimics the production
of IgE antibody from B lymphocytes. Hence, a decrease in SEAP can be translated as a
decrease in IgE antibody. All three peptides were able to successfully bind to their targets and
inhibit the signaling of STAT6 phosphorylation hence reducing SEAP secretion from cells.
However, K1 peptide was used in higher concentrations as opposed to N1 and P9 to achieve
>50% inhibition. Since IL-4 and IL-13 interaction with IL-4Rα is a common pathway for
many allergies, a prophylactic treatment can be devised by inhibiting this interaction for
future treatment of allergies.
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Chapter 4 - Discovering the Effects of Omega-3 and
Omega-6 Fatty Acids on Allergy Using a HEK-Blue
Cell Line
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4.1 Introduction
Over the past few decades, there has been a dramatic rise in allergic individuals worldwide.
New cases are sprouting in almost every continent and the trend appears to be on a steady
rise. This is becoming increasingly difficult for worldwide governments and healthcare
organizations to cope with the financial burden (294, 295). Allergic reactions can be as mild
as a sneeze or in some cases even life-threatening such as an anaphylactic shock (296). Many
studies have been carried out to investigate the causal relationship between allergy and
various factors that result in atopic disease, such as genetic predisposition, lifestyle or
environment (297, 298). The occurrence of allergic disease has become more prevalent in the
industrialized world. There has been a marked increase in prevalence of atopic disease in
regions such as Western Europe, the US and Australasia during recent years. A 20-fold
increase has been experienced by western countries and as much as 40% of the population
suffers from this disease (8). However, the results are skewed and more research is needed at
this stage to enhance a clear image and understanding to exactly how fatty acids interact with
different cells to stimulate or inhibit the effect of allergy. Although it is known that allergies
run in families and that genetic predisposition is the biggest factor, dietary factors have also
become very important in recent times (296). The intake of certain fatty acids at a young age
and their role in determining the outcome of atopic disease in later life has been thoroughly
investigated by many scientists throughout the world, clinical and non-clinical (156, 299-
303).
It has been suggested that an imbalance in dietary intake of essential fatty acids (EFA), such
as omega-3 (n-3 PUFA) and omega-6 (n-6 PUFA), may lead to a predisposition to allergic
disease. This is usually caused by an increased intake of n-6 fatty acids such as AA (C20:4)
with a simultaneously decreased intake of omega-3 fatty acids such as DHA (C22:6), EPA
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(C20:5) and DPA (C22:5) (304). Such an imbalanced diet leads to an insufficiently balanced
T helper cell Type 1 and 2 pathways. Evidently, concentrations of dietary n-6 PUFA
stimulates a Th2 differentiation of naïve T cells whereas the n-3 PUFA stimulates the Th1
variant (304). In atopic individuals, an immune response is mounted by T cells that are
activated by allergens, promoting Th2 variant of cells. Once stimulated, these cells
subsequently produce cytokines such as IL-4, IL-5 and IL-13 that stimulate B lymphocytes to
produce IgE antibody. Subsequently, this reaction triggers the release of histamines and LT
that result in allergic symptoms (296, 305). Unlike the cytokines produced by Th1 cells, such
as IFN-γ and IL-2 in non-atopic individuals, an excessive production of cytokines takes place
from Th2 cells (13). The shift in cytokine production from Th1 cells (IFN-γ and IL-2) to Th2
(IL-4 and IL-13) variant forms the hallmark of allergic response in atopic individuals.
It was recently reported in dietary intervention studies that during pregnancy, n-3 PUFA
show a strong tendency to reduce serum IgE antibody levels. In a different study carried out
by Weise et al., (2011) showed that B cells treated with DHA inhibited IgE production by
inhibiting the STAT6 phosphorylation pathway. It has also been suggested that DHA can
reduce IgE production by human B cells by affecting the IL-4 pathway via an effect on CD40
ligand (158).
These studies along with many others have prompted many researchers across the world to
revisit the effects of fatty acid supplements on allergic individuals. The main objective of this
study was to investigate the effect of n-3 and n-6 fatty acids on STAT6 phosphorylation
pathway that leads to the production of IgE antibodies when stimulated by bioactive IL-4 and
IL-13 cytokines (Figure 1.9).
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The specific aim of this chapter was to test the effects of omega-3 and omega-6 fatty acid on
STAT6 phosphorylation pathway using a HEK-Blue cell model. This will enable us to
understand the direct effect of these fatty acids on SEAP secretion which is triggered by
bioactive IL-4 and IL-13 cytokines. The HEK-Blue transfected model of cells produce SEAP
as an alternative to IgE antibody produced by B cells when signaled by cytokines IL-4 and
IL-13. Since omega-3 fatty acids have been found to inhibit allergy, we hypothesized that
omega-3 fatty acids will inhibit STAT6 signaling in HEK-Blue cells.
4.2 Materials and Methods
4.2.1 Cell Culture, PUFA and Vitamin E Solutions
HEK-Blue cells have been described in detail in Section 3.2.1 (Figure 1.9).
Growth medium: as described in Section 3.2.1.
Experimental medium: Growth medium was replaced with 2 mM L-glutamine and heat
inactivated 10% FBS along with omega-3 and omega-6 fatty acids namely; DHA (MW
328Da), EPA (MW 302Da), DPA (MW 330.5Da) and AA (MW 304.46Da), all of which
were commercially purchased from InvitrogenTM at a concentration of 10 μg/ml. The final
concentrations prepared were 30, 60, 90, 120 and 150 μM using 100% ethanol as solvent.
Vitamin E (InvitrogenTM) was prepared as an antioxidant to prevent oxidation of fatty acids
using growth medium. The final concentrations prepared were 0.05, 0.1, 0.15, 0.2 and 0.25
μM to all corresponding treatments as outlined above.
Initially, cells were tested in the presence of ethanol and vitamin E separately to confirm
whether cell viability and/or STAT6 phosphorylation pathway was affected or whether it
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caused apoptosis in cells. Cells were incubated with experimental medium containing ethanol
and vitamin E over a period of 24 hrs and cell viability was tested at 5 intervals (0 hrs, 6 hrs,
12 hrs, 18 hrs and 24 hrs). Our results confirmed that ethanol and vitamin E had no effect on
cell viability and/or STAT6 phosphorylation pathway with or without the addition of ethanol
and vitamin E (results not shown). Cells were now ready to be tested with fatty acids.
Stock solution of DHA, EPA, DPA and AA in ethanol were stored at -20°C and pre-
incubated in complete growth medium at 37°C overnight to allow protein conjugation. We
tested a range of concentrations on HEK-Blue cells to test the STAT6 phosphorylation
pathway that leads to a production of serum embryonic alkaline phosphatase (SEAP). This is
an alternative product to IgE antibody that is produced by B lymphocytes when triggered by
bioactive IL-4 and IL-13. SEAP breaks down Quanti-Blue which gives a blue coloration. The
blue color can then be detected using a spectrophotometer. Vitamin E stock was stored at 4°C
and was freshly prepared in ethanol when preparing experimental media with fatty acids.
Two sets of experiments were performed to determine the effects of fatty acids on the STAT6
phosphorylation pathway. In the preliminary experiments, a range of different concentrations
of fatty acids and vitamin E were studied to confirm their effect on SEAP production. Once
confirmed, the most optimum concentration of fatty acid was used to perform a time course
experiment. Cell viability and SEAP production were detected at 5 different time points over
a period of 24 hrs. These experiments allowed us to study two different variables; time and
concentration of fatty acid needed to suppress SEAP secretion by HEK-Blue cells by using a
very simple colorimetric analysis.
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4.2.2 HEK-Blue Cell Viability Test Using Cytokines IL-4 and IL-13
The protocol used was taken from the datasheet provided with the HEK-Blue IL-4/IL-13 kit.
A few adjustments were made in the protocol to perform the assay without compromising the
signalling in cells. Cells were grown in a T-75 flask and were used for detection assay. The
cells were detached using 3 ml trypsin, which was neutralized with 7 ml of media containing
FBS to give a total volume of 10 ml. The average cells were counted using a TC10 automated
cell counter (Bio-Rad Laboratories, Inc.). HEK-Blue cells were pipetted into 15 mL tubes,
which were centrifuged at 1200 RPM for 5 min. The supernatant was discarded and
experimental media was added to suspend the cell pellet. If required, cell concentration was
reduced by adding growth medium to achieve optimum cell density. The cells were pipetted
into 24-well plates (Corning, Sigma-Aldrich) at a density of ~200,000 cells per well for a
total volume of 720 μl. A total of 12 treatment wells were setup in triplicates. Cells were
treated with a combination of fatty acids (30, 60, 90, 120 and 150 μM) with and without
vitamin E (0.05, 0.1, 0.15, 0.2, 0.25 μM). 80 μl of cytokine IL-4 (Integrated Sciences) and
IL-13 (Integrated Sciences) were pipetted to each treatment well at a concentration of 100
ng/ml. PC (with cytokine stimulation) and negative controls (without cytokine stimulation)
were included to provide a comparative study. Cells were incubated with the treatments for a
period of 24 hrs. Post-incubation, 20 μl of induced HEK-Blue IL-4/IL-13 cells supernatant
from each treatment well was added to a corresponding plate with 180 μl of Quanti-Blue in
each well. This is incubated for a total of 3 hrs to detect SEAP levels. The blue color was
detected using a xMark microplate absorbance spectrophotometer (Bio-Rad Laboratories
Inc.) at a wavelength of 640 nm. Cell viability was recorded by using the TC10 automated
cell counter.
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4.2.3 Time-course Assay Using Cytokine IL-4 and IL-13
A time course assay was performed with an optimum concentration of n-3 and n-6 fatty acids.
Once again, the cells were cultured in five (one for each time interval) 24-well plates at a
density of ~200,000 cells per well. Cells were counted using the TC10 automated cell
counter. HEK-Blue cells were pipetted into 15 ml tubes, which were centrifuged at 1200 rpm
for 5 min. The supernatant was discarded and experimental media was added to suspend the
cell pellet. If required, cell concentration was reduced by adding growth medium to achieve
optimum cell density. The cells were pipetted into 24-well plates (Corning, Sigma-Aldrich) at
a density of ~200,000 cells per well for a total volume of 720 μl. Concentration of fatty acids
used at this stage was 90 μM, which is within high physiological levels and the most
optimum concentration of fatty acid to inhibit SEAP secretion (established from previous
section). Cells were incubated in a humidified incubator at 37°C with 5% CO2 for a period of
24 hrs. A total of five time points were used to measure the effect of fatty acids on SEAP
production over a period of 24 hrs; 0 hrs, 6 hrs, 12 hrs, 18 hrs and 24 hrs, respectively. PC’s
were also setup for each time interval which was without fatty acid treatment. Post-
incubation, 80 μl of cytokine IL-4 and IL-13 were pipetted to each treatment well at a
concentration of 100 ng/ml and the plates were incubated for 24 hrs to stimulate the STAT6
phosphorylation pathway. Simultaneously, the experimental media in the wells was replaced
with a fresh supply of growth medium to ensure a steady supply of nutrients to the cells. 20
μl of induced HEK-Blue IL-4/IL-13 cell supernatant from each treatment well was added to a
corresponding plate with 180 μl of Quanti-Blue in each well. This was incubated for a total of
3 hrs to detect SEAP levels. The blue color was detected using a spectrophotometer at 640
nm. Cell viability was recorded by using the TC10 automated cell counter.
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4.2.4 Statistical Analysis
The data were analysed using Microsoft Excel 2013 (Microsoft Inc.). The results were
analysed by Student’s t-test using a two-tailed method to determine any statistically
significant difference between the controls and treatment wells in all of the above
experiments. The statistical significance was set at P < 0.05.
4.3 Results
4.3.1 HEK-Blue Cell Viability Test Using Cytokine IL-4 and IL-13
As the results suggest, a total of two sets with 4 assays in each set were performed using
cytokines IL-4 and IL-13 (Figures 4.1 and 4.2). A dose-dependent relationship was observed
between fatty acid concentration and SEAP secretion from HEK-Blue cells. A gradual
increase in fatty acid concentration meant that SEAP levels would decrease hence, reducing
blue coloration once the HEK-Blue cells supernatant was added to Quanti-Blue. All positive
and negative controls behaved as expected with high and low peaks as suggested by the
HEK-Blue IL-4/IL-13 kit. Statistically significant results were achieved with an optimum
concentration of fatty acids at 90 μM for n-3 PUFA. All inhibitions achieved with n-3 fatty
acid treatments were statistically significant with P values being less than 0.05 with a two-
tailed Student’s t-test. While DHA (Figures 4.1A and 4.2A) and EPA (Figures 4.1B and
4.2B) showed gradual decrease in SEAP secretion with both IL-4 and IL-13, DPA (Figures
4.1C and 4.2C) showed a sudden drop in SEAP levels suggesting a more potent inhibition.
However, the treatment of HEK-Blue cells with n-6 PUFA AA showed no significant change
in the IL-4 cytokine assay (Figure 4.1D). However, an increase in SEAP secretion was
observed with AA treatment when cells were tested with the IL-13 cytokine (Figure 4.2D).
OD readings showed no inconsistencies when n-3 fatty acids were incubated with HEK-Blue
cells without the vitamin E. However, in the case of AA, it was noted that SEAP secretions
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were higher than the PC. Consistency was also observed with cell viability when tested for all
the treatments and compared to the PC.
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4.1(A) 4.1(B)
4.1(C) 4.1(D)
Figure 4.1. IL-4 detection assay with n-3 and n-6 fatty acids (μM) with HEK-Blue cells
using Quanti-Blue. 4.1A and 4.1B shows the detection assay performed with DHA and EPA
with a dose-dependent inhibition. However, in Figure 4.1C, a dose-dependent inhibition does
not occur with DPA but rather a dose-response inhibition occurs. N-3 fatty acids showed no
abnormal effects on SEAP secretion without vitamin E. However, AA without vitamin E
caused higher than normal SEAP secretion. P < 0.05 denotes significance of OD readings
compared to PC.
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4.2(A) 4.2(B)
4.2(C) 4.2(D)
Figure 4.2. IL-13 detection assay with n-3 and n-6 fatty acids (μM) with HEK-Blue cells
using Quanti-Blue. Figure 4.2A shows a dose-response inhibition of SEAP by DHA. 4.2B
and 4.2C depict the detection assay performed with EPA and DPA with a dose-dependent
inhibition of SEAP secretion from HEK-Blue cells. However, in Figure 4.2D with higher
concentrations of AA, there is a rise in SEAP levels hence, a higher OD reading. N-3 fatty
acids showed no abnormal effects on SEAP secretion without vitamin E. However, AA
without vitamin E caused higher than SEAP secretion than the PC. P < 0.05 denotes
significance of OD readings compared to PC.
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4.3.2 HEK-Blue Time-course with Fatty Acid and Vitamin E Treatment
As shown in Figures 4.3 and 4.4 a total of 8 assays were performed with 4 assays in each set.
Figure 4.3 shows results with cytokine IL-4 whereas Figure 4.4 shows results with cytokine
IL-13. DHA (Figures 4.3A and 4.4A), EPA (Figures 4.3B and 4.4B) and DPA (Figures
4.3C and 4.4C) all showed a decrease in SEAP secretion and hence produced less blue
coloration at 18 and 24 hr time points when compared to the PC values. All inhibitions
achieved with n-3 fatty acid treatments were statistically significant with P values being less
than 0.05 with a two-tailed Student’s t-test. Since the concentration of fatty acid was constant
at 90 μM, this experiment was a time-dependent inhibition of STAT6 phosphorylation in
HEK-Blue cells. However, as seen in Figures 4.3D and 4.4D, AA has shown to increase
SEAP secretion hence, it shows a higher absorption reading at 18 and 24 hr time points when
compared to the PC values. No significant change was observed at 0 hrs, 6 hrs or 12 hr time
points. Cell viability was performed at the end of the 24 hr incubation to check for any
anomalies. It was found that AA showed a higher cell count when compared to PC’s in both
experiments; with cytokine IL-4 and IL-13. However, the cell viability count was well within
the range of 105 cells.
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4.3(A) 4.3(B)
4.3(C) 4.3(D)
Figure 4.3. HEK-Blue detection assay with 90 μM of omega-3 and omega-6 fatty acids
with 0.15μM vitamin E using cytokine IL-4 bioactive agent. Figures 4.3A, 4.3B and 4.3C
depict a time-dependent inhibition of SEAP secretion from HEK-Blue cells when treated with
DHA, EPA and DPA, respectively. Figure 4.3D shows an increase in SEAP secretion from
HEK-Blue cells when treated with AA at the 24 hr time point. P < 0.05 denotes significance
of OD readings compared to PC depicted in blue color. C1 depicts the OD reading obtained
when HEK-Blue cells are treated with fatty acid only without the cytokine stimulation.
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4.4(A) 4.4(B)
4.4(C) 4.4(D)
Figure 4.4. HEK-Blue detection assay with 90 μM of omega-3 and omega-6 fatty acids
with 0.15 μM vitamin E using cytokine IL-13 bioactive agent. Figures 4.4A, 4.4B and
4.4C depict a time-dependent inhibition of SEAP secretion from HEK-Blue cells when
treated with DHA, EPA and DPA, respectively. Once again, Figure 4.4D shows an increase
in SEAP secretion from HEK-Blue cells when treated with AA at the 24 hr time point. P <
0.05 denotes significance of OD readings compared to PC depicted in blue color. C1 depicts
the OD reading obtained when HEK-Blue cells are treated with fatty acid only without
cytokine stimulation.
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4.4 Discussion
4.4.1 Fatty Acids and SEAP Secretion
Intake of pro-inflammatory omega-6 fatty acid such as AA has increased in the past few
decades along with a decrease in omega-3 fatty acid diet. This has led to an increase in
allergy prevalence as well as clinical manifestations of allergic disease (306-308). A diet
comprising n-3 PUFA is mainly found in fatty fish as opposed to n-6 PUFA, which are
mainly found in margarine and vegetable oils. N-3 PUFA, specially DHA, EPA and DPA,
exert anti-inflammatory effect, whereas n-6 fatty acids do not (309). The purpose of this
study was to investigate the changes that n-3 and n-6 fatty acids have on STAT6
phosphorylation pathway which leads to IgE production in B lymphocytes. The rationale of
the present study was to investigate the authenticity and general observations made in
previous observations. In this study we have shown for the first time that n-3 fatty acids
DHA, EPA and DPA all suppress and down-regulate SEAP secretion from transfected HEK-
Blue cells. The HEK-Blue model uses a very simple colorimetric analysis as an alternative
method to measuring IgE antibody production from B cells. Incubation with all three n-3
PUFA resulted in a dose-dependent decrease in SEAP secretion which in turn gave less blue
color when added to Quant-Blue substrate. This decrease in SEAP secretion was not due to a
higher cell proliferation as demonstrated by trypan blue cell viability count. The reason for
choosing DHA, EPA, DPA and AA for n-3 fatty acid was due to their abundant availability.
Breast milk contains all three fatty acids and they are also found in a variety of meat products
such as beef, lamb and fish (310-312). Secondly, it is well established and documented
through many clinical studies that DHA, EPA, DPA and AA have a strong link with allergy
(157). Recent publications have reported that lower incidence of eczema was observed in
children who consumed fish and whose mothers had higher intake of fish diet during the
gestation period (313, 314). In the case of AA, several papers have reported the rise of
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allergic reactions in individuals consuming a diet that contains high amounts of AA (315-
317). All of these studies lead to a possible explanation of reduced SEAP secretion from
HEK-Blue cells, which in turn avoids clinical symptoms in allergic individuals since SEAP
secretion occurs as a result of JAK-STAT6 phosphorylation. Although many clinical studies
of allergic individuals have been carried out over the past decade, there is still a lack of in
vitro studies to look at the cellular effects of fatty acids.
4.4.2 Importance of Arachidonic Acid in Allergy
The purpose of choosing AA for these studies was due to the abundance of this fatty acid in
body tissues. It is a major component of mammalian cell membranes and accounts up to a
quarter of all phospholipid fatty acid (311, 312), hence making it a very important target for
allergy studies because of its ubiquitous nature. Previous studies on the relationship between
allergy markers such as IgE and AA have not been carried out. No significant change was
observed in the case of AA when using a range of concentrations in our experiments.
However, in the time course experiment an increase in SEAP production was observed and
slightly elevated levels of cell viability count. This evidence is in agreement with the fact that
AA causes cell proliferation in endothelial cells and HEK-Blue cells are derived from
endothelial cells found in kidneys (318). It was also observed that AA incubated with HEK-
Blue cells without vitamin E supplementation gave higher SEAP secretion than the PC. This
is consistent with literature which suggests that vitamin E suppresses the production of IgE
antibody from B cells hence, it can suppress SEAP secretion by the same mechanism. Three
decades ago, in a study carried out by Inagaki et al., (1984), vitamin E was supplemented to
mice in diets and oral administration with sesame oil which resulted in suppression of IgE
antibody production (319). Furthermore, AA showed inhibition of SEAP secretion with IL-4
detection assay which is an anomaly we found in the overall effects of AA. This may be
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explained by the fact that AA incorporated into cell membranes can effect membrane fluidity,
which may consequently effect cell function (309, 320, 321).
4.4.3 Conclusion
In conclusion, treatment of HEK-Blue cells with n-3 fatty acids lead to a repression of SEAP
production through inhibition of IL-4 and IL-13 signaling pathways. These results comprise a
great biological relevance to the possible development of a prophylactic treatment for allergic
diseases. With n-6 fatty acid AA, an increase in SEAP output was recorded which is coherent
with previous studies. Our studies, presented here, opens a new corridor to research with n-3
fatty acids using a transfected model for allergy research. DHA, EPA and DPA
supplementation may offer a molecular basis for a well-developed anti-allergic effect and
therefore, must be an essential part of our regular diet. Although further investigation will
reveal the best allergy markers that can be used for suppression, it is clear that n-3 fatty acids
offer a great opportunity as potential natural inhibitors of allergy.
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Chapter 5 - Discovering the Effects of CoQ10 and
Resolvins on Allergy Using a HEK-Blue Cell Line
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5.1 Introduction
In recent years, it has become very clear that the burden of allergic disorders reflect long-term
consequences of chronic allergic inflammation at sites that face repetitive exposure to
allergens. This alone has led many scientists to test different therapeutic drugs to down-
regulate the effects of allergic disease such as corticosteroids, which increases tolerance in
allergic individuals to a specific allergen (11, 322). IgE-mediated responses in allergic
disorders are the most important ingredients of allergic inflammation (323). Since allergic
reaction can lead to acute inflammation, no such link has been established so far on the
effects of pro-resolving metabolites such as resolvins in allergy. It may be not far-fetched to
hypothesize that resolvins may have a significant effect on allergy since they down-regulate
inflammation or vice-versa. This theory has not been tested on an IgE model of cells such as
on B lymphocytes, which are primarily responsible for the production of IgE antibodies
during an allergic cascade. The NARL research group at Deakin University has taken the
initiative to conduct a string of experiments using CoQ10 and resolvins on a HEK-Blue cell
model to establish the effects of resolvins on SEAP secretion in response to IL-4 cytokine
signaling, as a substitute to IgE production by B cells. The experimental model is based on
our well-characterized model of HEK-Blue cells that mimic the same IL-4 and IL-13
signaling that is used for the production of IgE antibodies by B lymphocytes.
Asthma is characterized by chronic inflammation of the airways with additional episodes of
acute inflammation (324). These exacerbations can be life threatening and are associated with
high health care costs (325). Allergens are mainly responsible for exacerbations of these
responses. During an acute inflammation phase, the recruitment of a significant number of
neutrophils as well as eosinophils can lead to an obstruction of airflow to lungs (326).
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Treatments for asthma symptoms include standard therapies such as SIT (262, 327).
However, they have not been able to show satisfactory results as novel therapeutics.
Resolution of inflammation is now understood to be an actively regulated process (179).
Lipid derived mediators such as resolvins have demonstrated to be anti-inflammatory and
pro-resolving in their ability to reduce allergic symptoms (183). Resolvins such as resolvin
D1-D2 (RvD1 & RvD2) and RvE1 derived from their precursor fatty acids DHA and EPA
via the action of cyclooxygenase enzyme, have been found to inhibit inflammation in several
models of diseases as well as reducing neutrophil infiltration during inflammation (328).
Similarly, it was found in a study that low dietary intake of CoQ10 resulted in an increased
risk of allergy among children (329). These studies has prompted the NARL research group
to conduct experiments using resolvins and CoQ10 as novel therapeutics for treatment of
allergy.
For this chapter, we hypothesized that since resolvins and CoQ10 suppress allergy and
inflammation, the HEK-Blue model will serve best to test this theory. In the present study, we
first assessed the effect of the administration of resolvins and CoQ10 in HEK-Blue cells
using a range of concentrations over a period of 24 hrs. Secondly, a time-course experiment
with the most optimum concentration was performed to assess the effect of treatments using
time as variable. Therefore, we assessed the overall effects of resolvins and CoQ10 on SEAP
secretion from HEK-Blue cells as an alternative to IgE antibody secretion from B
lymphocytes.
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5.2 Materials and Methods
5.2.1 Cell Culture, Resolvins and CoQ10
HEK-Blue cells have been described in detail in section 3.2.1 (Figure 1.9).
Growth medium: as described in section 3.2.1.
Experimental media: as described in section 4.2.1. Resolvin stocks were purchased from
Cayman Chemicals Company (Ann Arbor, Michigan, USA) in a solution of 100% ethanol
(RvD1 376Da & RvD2 372Da; 10 μg/ml for each). However, RvE1 was in a lyophilized
powder form and the solution was prepared using 100% methanol (250 μg/ml) as suggested
in the datasheet provided by the supplier. A subsequent stock of RvE1 was prepared by
diluting the stock solution to 10 μg/ml using methanol. Experimental solutions were prepared
by mixing 1 μl of the stock solution with 1 ml of experimental media to get a final
concentration of 30 μM. Subsequently, 2 μl of the stock was added to 1 ml of experimental
media to get 60 μM, so on and so forth. CoQ10 was generously provided by Professor
Andrew Sinclair and Professor Colin Barrow at a concentration of 100 μM (50 ml), which
was subsequently diluted down to the required concentrations for experimental purposes. The
CoQ10 solution was prepared in dimethyl sulfoxide (DMSO).
5.2.2 HEK-Blue Detection Assay Using Cytokines IL-4 and IL-13
The protocol used for performing cell-assays with resolvins has already been described in
detail in section 4.2.2. HEK-Blue cells were used to perform this set of experiments without
making any changes to the protocol listed in the datasheet provided by InvitrogenTM. Cells
were treated with a combination of resolvins (30, 60, 90, 120 and 150 μM). 80 μl of cytokine
IL-4 (Integrated Sciences) and IL-13 (Integrated Sciences) were pipetted to each treatment
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well at a concentration of 100 ng/ml. The concentrations used for resolvins were consistent
with the concentrations used for the fatty acids to maintain an overall standard of the
experiments. However, the concentrations prepared for resolvins were not incubated with
vitamin E as in the case for fatty acids in the previous chapter. CoQ10 was initially tested at
10 and 20 μM concentrations which showed significant inhibition of both IL-4 and IL-13
signaling. Further experiments were performed with concentrations of CoQ10 which showed
100% inhibition of STAT6 phosphorylation pathway in HEK-Blue cells (20 μM, 40 μM, 60
μM, 80 μM and 100 μM). While referring to the above sections, cells were incubated with the
treatments for a period of 24 hrs. Post-incubation, 20 μl of induced HEK-Blue IL-4/IL-13
cells supernatant from each treatment well was added to a corresponding plate with 180 μl of
Quanti-Blue in each well. This is incubated for a total of 3 hrs to detect SEAP levels. The
blue color was detected using an xMark microplate absorbance spectrophotometer (Bio-Rad
Laboratories Inc.) at a wavelength of 640 nm. Cell viability was recorded by using a TC10
automated cell counter.
5.2.3 Time-course Assay Using Cytokine IL-4 and IL-13
Time-course assay was performed in a similar fashion as in the case of fatty acids (Section
4.2.3). However, the highest possible concentration with the resovlins at 150 μM was used
for experiments. In the case of CoQ10, 50 μM was used to perform the time-course. This
value was used as a median value and was used in both IL-4 and IL-13 experiments. A total
of five time points were used to measure the effect of resolvins on SEAP production over a
period of 24 hrs; 0 hrs, 6 hrs, 12 hrs, 18 hrs and 24 hrs, respectively. PC’s were also included
for each time interval which was without fatty acid treatment. Post-incubation, 80 μl of
cytokine IL-4 and IL-13 were pipetted to each treatment well at a concentration of 100 ng/ml
and the plates were incubated for 24 hrs to stimulate the STAT6 phosphorylation pathway.
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20 μl of induced HEK-Blue IL-4/IL-13 cells supernatant from each treatment well was added
to a corresponding plate with 180 μl of Quanti-Blue in each well. This was incubated for a
total of 3 hrs to detect SEAP levels. The blue color was detected using a spectrophotometer at
640 nm. Cell viability was recorded by using the TC10 automated cell counter.
5.2.4 Statistical analysis
The data were analyzed using Microsoft Excel 2013 (Microsoft Inc.) The results were
analyzed by Student’s t-test using a two-tailed method to determine any statistically
significant difference between the controls and treatment wells in all of the above
experiments. The statistical significance was set at P < 0.05.
5.3 Results
5.3.1 HEK-Blue Detection Assay Using Cytokines IL-4 and IL-13
As the results indicate, a total of two sets with 4 assays in each set were performed using
cytokines IL-4 and IL-13 (Figures 5.1 and 5.2). A differential effect of resolvins and CoQ10
was observed at different concentrations and SEAP secretion from HEK-Blue cells. A
gradual increase in resolvins and CoQ10 concentration meant that SEAP levels would
decrease or increase hence, reducing or producing blue coloration once the HEK-Blue cells
supernatant was added to Quanti-Blue. All positive and negative controls behaved as
expected with high and low peaks as suggested by the HEK-Blue IL-4/IL-13 kit. All
inhibitions and agonistic results achieved with resolvins and CoQ10 treatments were
statistically significant with P values being less than 0.05 with a two-tailed Student’s t-test
using standard deviations. While RvD1 (Figures 5.1A and 5.2A) and RvD2 (Figures 5.1B
and 5.2B) showed gradual increase in SEAP secretion with both IL-4 and IL-13, RvE2
(Figures 5.1C and 5.2C) showed no change at all, even at 150 μM concentration. However,
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CoQ10 (Figures 5.1D and 5.2D) showed a sudden drop in SEAP levels suggesting a more
potent inhibition at 40 μM for IL-4 signaling and 60 μM for IL-13. OD readings showed no
inconsistencies when resolvins and CoQ10 were incubated with HEK-Blue cells without
cytokine stimulation. Consistency was also observed with cell viability when tested for all the
treatments and compared to the PC.
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5.1(A) 5.1(B)
5.1(C) 5.1(D)
Figure 5.1. IL-4 detection assay using resolvins and CoQ10 (μM) with HEK-Blue cells and Quanti-
Blue. Figures 5.1A and 5.1B shows the detection assay performed with RvD1 and RvD2 with a dose-
dependent increase in SEAP. However, in Figure 5.1C, no inhibition occurs with RvE1 while CoQ10 shows
a rather potent dose-dependent inhibition (Figure 5.1D). No significant change was observed when resolvins
and/or CoQ10 was used in the absence of cytokines IL-4. P < 0.05 denotes significance of OD readings
compared to PC.
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5.2(A) 5.2(B)
5.2(C) 5.2(D)
Figure 5.2. IL-13 detection assay using resolvins and CoQ10 (μM) with HEK-Blue cells and Quanti-
Blue. Figure 5.2A showed no change when RvD1 was used as a treatment in the presence of IL-13 cytokine
whereas 5.2B shows the detection assay performed with RvD2 with a dose-dependent increase in SEAP
secretion. However, in Figure 5.2C, no inhibition occurs with RvE1 while CoQ10 shows a rather potent
dose-dependent inhibition of IL-13 signaling (Figure 5.2D). No significant change was observed when
resolvins and/or CoQ10 was used in the absence of cytokine IL-13. P < 0.05 denotes significance of OD
readings compared to PC.
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5.3.2 HEK-Blue Time-course with Resolvins and CoQ10 Treatment
As shown in Figures 5.3 and 5.4, a total of 8 assays were performed with 4 assays in each
set. Figure 5.3 shows results with cytokine IL-4 whereas Figure 5.4 shows results with
cytokine IL-13. RvD1 (Figures 5.3A and 5.4A), RvD2 (Figures 5.3B and 5.4B) and RvE1
(Figures 5.3C and 5.4C) all showed a significant increase in SEAP secretion and hence
produced more blue coloration at 18 and 24 hrs’ time points when compared to the PC
values. However, CoQ10 once again showed significant decrease in SEAP secretion when
treated with 50 μM concentration. At 24 hrs, an almost 100% inhibition of SEAP signaling
was achieved in the case of both cytokines IL-4 and IL-13 (Figures 5.3D and 5.4D). All
inhibitions and agonistic effects achieved with resolvins and CoQ10 treatments were
statistically significant with P values being less than 0.05 with a two-tailed Student’s t-test
using standard deviation values. Since the concentration of resolvins was constant at 150 μM,
this experiment was a time-dependent assay. No significant change was observed at 0 hr, 6 hr
or 12 hr time points. Cell viability was performed at the end of the 24 hr incubation to check
for any anomalies. However, the cell viability count was well within the range of x105 cells.
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Figure 5.3. HEK-Blue detection assay with 150 μM of resolvins and 50 μM of CoQ10 in the presence
of IL-4 cytokine as a bioactive agent. Figures 5.3A, 5.3B and 5.3C depict a time-dependent increase of
SEAP secretion from HEK-Blue cells when treated with RvD1, RvD2 and RvE1, respectively. Figure 5.4D
shows a decrease in SEAP secretion from HEK-Blue cells when treated with CoQ10 at 12, 18 and 24 hr time
points. P < 0.05 denotes significance of OD readings compared to PC depicted in blue color. C1 depicts the
OD reading obtained when HEK-Blue cells are treated with resolvins and CoQ10 only, without the cytokine
stimulation.
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Figure 5.4. HEK-Blue detection assay with 150 μM of resolvins and 50 μM of CoQ10 in the presence
of IL-13 cytokine as a bioactive agent. Figures 5.4A, 5.4B and 5.4C depict a time-dependent increase of
SEAP secretion from HEK-Blue cells when treated with RvD1, RvD2 and RvE1, respectively. Figure 5.4D
shows a decrease in SEAP secretion from HEK-Blue cells when treated with CoQ10 at 12, 18 and 24 hr time
points. P < 0.05 denotes significance of OD readings compared to PC depicted in blue color. C1 depicts the
optical OD obtained when HEK-Blue cells are treated with resolvins and CoQ10 only, without the cytokine
stimulation.
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5.4 Discussion
5.4.1 CoQ10 and Resolvins
Resolvins and CoQ10 have not been previously tested on a HEK-Blue model of cells, which
mimics IL-4 and IL-13 signaling for IgE production by B lymphocytes. No previous study to
date has tested the effects of resolvins and CoQ10 on an allergy cell model. In this chapter,
we report for the first time that resolvins exacerbate the production of SEAP production from
HEK-Blue cells when stimulated by bioactive IL-4 and IL-13. On the other hand, CoQ10
showed very promising results as a potential antagonist to IL-4 signaling for allergy
treatment, showing statistically significant inhibition of SEAP secretion with results
achieving up to 100% inhibition.
Although it was hypothesized by Ye et al., (1988), approximately 3 decades ago that CoQ10
can play an important role in patients suffering from autoimmune conditions such as allergy
(172), CoQ10 has gained much attention in recent years for its potential inhibitory effects on
inflammation (330). In a study carried out by Lee et al., (2013), it was reported that CoQ10
showed anti-inflammatory activity in coronary artery disease when human subjects were
supplemented with 300 mg/day of CoQ10 (330). Inflammatory cytokines such as TNF-α
were significantly reduced as well when patients were supplemented with CoQ10 (330). In
another study, Schmelzer et al., (2008), demonstrated that CoQ10 wielded anti-inflammation
effects during inflammation via the inhibition of NF-κB dependent gene expression (331).
When cytokines IL-4 and IL-13 bind to its receptor IL-4R, this activates protein tyrosine
kinases JAK1/JAK3 which results in STAT6 phosphorylation. Following this, dimerized
STAT6 modulates the transcription of IL-4 responsive genes. NFκB and STAT6 work in
collaboration for IgE transcription and IgE production in B cells. Although not tested in our
current study, it has been previously established by Weise et al., (2011) that NF-κB gene
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expression is suppressed by supplementing n-3 fatty acids, which significantly reduces IgE
production of B lymphocytes (158) and forms a critical step in the allergic cascade. This in
turn influences TNF-α gene expression (331). Since IL-4 and IL-13 cytokines mediate the
same response via JAK-STAT signaling pathway, it can be concluded that CoQ10 inhibits
SEAP secretion via this route without effecting cell viability. This helps to preserve
homeostasis in cells, which is key to various biochemical pathways.
The recent discovery of resolvins has exposed molecular and cellular mechanisms for the
resolution of acute inflammation. In response to acute inflammation, these pro-resolving
mediators inhibit the inflammatory response by reducing neutrophil infiltration at the site of
inflammation. During an allergic response, resolvins recruit natural killer (NK) cells to enable
T cells and macrophages in the removal of allergen deposits in the lung (332). The resolution
of acute inflammation is an active process which is crucial for the establishment of
homeostasis and tissue protection. ChemR23 was the first receptor identified which interacts
with RvE1. ChemR23 is expressed on a range of white blood cells (WBC’s), such as
monocytes, macrophages and dendritic cells. Treatment with RvE1 inhibits the prominent
transcription factor NF-κB (333). Although this is in sharp contrast to the effects of CoQ10
on NF-κB inhibition, the results achieved with HEK-Blue suggest otherwise. SEAP secretion
remained unchanged with RvE1 when cells were treated with a range of different
concentrations. However, during the time-course experiment, RvE1 showed to increase
SEAP production. Although no previous study has tested this model of allergy, more
information needs to be gathered to form an overall picture of its effects at the molecular
level. RvD1 and RvD2, in contrast to RvE1, have shown to increase SEAP from HEK-Blue
cells in all the assays performed. This is consistent with published data by Li et al., (2014)
where RvD1 potentiated the effects of IL-4 on STAT6 phosphorylation pathway, which leads
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to activation of gene expression of SEAP (334). RvD2 shares the same distinct features of
resolvins in its ability to resolve acute inflammation. In a mouse-model study of sepsis, RvD2
sharply decreased bacterial burden, cytokine production and neutrophil recruitment while
increasing macrophage phagocytosis (335). The differential results obtained with resolvins
and their effects on SEAP secretion are evidently surprising. Although literature very
strongly supports resolvins to be anti-inflammatory by nature, their effects on IgE secretion
appear to be profound and agonistic.
5.4.2 Significance of Expression of SEAP/IgE Secretion
Generally, a negative notion exists with an over-expression of SEAP secretion from HEK-
Blue cells which in turn means a more severe allergic reaction, however it must be noted that
IgE antibody secretion is part of a normal response by the immune system to invading hosts.
Ige is an important component of host-protective immune responses against the helminthic
parasites in the majority of the world population, specifically in undeveloped countries (336).
Hence, we speculate that an increase in IgE response by resolvins may be considered as a
normal response by tissues to fight against helminthe infection and maintain homeostasis.
5.4.3 Conclusion
In conclusion, treatment of HEK-Blue cells with CoQ10 lead to a repression of SEAP
production through inhibition of IL-4 and IL-13 signaling pathways. These results comprise a
great biological relevance to the possible development of a prophylactic treatment for allergic
diseases. With resolvins, an increase in SEAP output was recorded which is not coherent with
the anti-inflammatory properties of resolvins. Our studies, presented here, opens a new
corridor to research with the effects of resolvins on a transfected model for allergy research.
CoQ10 supplementation may offer a molecular basis for a well-developed anti-allergic effect
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and therefore, must be an essential part of our regular diet. Although further investigation will
reveal the best allergy markers that can be used for suppression, it is clear that CoQ10 as
opposed to resolvins offer a great opportunity as potential natural inhibitors of allergy
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Chapter 6 - Relationship Between Ryegrass Pollen
Counts and Weather Variables in Regional Victoria,
Australia
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6.1 Introduction
Allergic diseases such as asthma, rhinitis and atopic dermatitis induced by ryegrass pollen are
on the rise all over the world. Developed countries are facing a rise in prevalence of allergic
diseases which has an additional effect on healthcare costs (337). This trend is clearly evident
in south-eastern state of Victoria in Australia (204, 338-341). Awareness and thus avoidance
of pollen exposure is the first line of defense in the treatment of allergic disease.
For diagnosis and treatment of allergic disease, the pollen map in an area plays an important
role in helping clinicians to perform the appropriate tests and treatments on allergic patients
such as immunotherapy (342, 343). High prevalence of perennial ryegrass (Lolium perenne)
pollen in south Victoria with a characteristic climate of sunny days, high humidity and
thunderstorms (344) in summer make it important to map pollen distribution in regional
Victoria (Geelong) (211). The coastal city of Geelong is located 75km (47 miles) south-west
of the state capital, Melbourne.
In the last 30 years or so, aerobiological studies in Victoria have been developed rapidly in
the city of Melbourne, Australia, which is well known as the capital of thunderstorm allergy
(340, 341). In the region of Geelong, ryegrass pollen distribution and its relationship to
climatic changes have not been studied previously. In our current study the NARL research
group has investigated the study of ryegrass pollen counts and their changes during the
summer months (October-January). Secondly, the relationship of meteorological variables
and pollen distribution were also studied and examined. Thirdly, we examined the presence
of ruptured pollen which evidently releases allergens such as Lol p 1 and Lol p 5 that are
responsible for allergic reactions in atopic individuals (204, 211).
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In this chapter, we hypothesized that ryegrass pollen distribution in air is positively correlated
to weather variables such as rain, temperature, humidity etc. we aim to establish a positive
correlation of weather variables with ryegrass pollen and the presence of ruptured pollen in
the atmosphere by using a Burkard volumetric air trap as our sampling method. Once this is
established, it will open new exciting pathways for future treatment of pollen allergy
improving quality of life.
6.2 Methods and Materials
6.2.1 Establishment of the Air Sampler
The Burkard volumetric spore trap (Burkard Agronomics Inc.) purchased from Burkard
Scientific Ltd. was installed on the rooftop of the Deakin University library in Waurn Ponds,
Geelong, Australia (Figure 6.1). The trap was positioned in a way to not be obstructed by
any other university building surrounding the pollen trap and no trees that would obstruct
wind or act as a bias to the trap. This was primarily done to avoid missing any pollen released
from grass and trees that are mixed by the turbulent boundary layer. As a result, all pollen
released will be dispersed considerably in air before being sampled by the volumetric trap.
6.2.2 Preparation of Slides
Microscopic slides were prepared using the following solution:
1. Pollen collecting adhesive (10 g gelatine + 155 ml filter-sterilised water + 40 ml
glycerol + 0.5 g phenol).
500 ml glass bottle: Gelatine and water were mixed and warmed at 60°C (hot plate
with gentle stirrer). Once cooled down to RT, glycerol was added along with phenol
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(anti-bacterial agent). 50 ml was decanted into smaller bottles. Bottles were stored at
RT.
2. Coverslip adhesive (35 g Mowiol + 100 ml distilled water + 50 ml glycerol + 0.5 g
phenol).
500 ml glass bottle: Mowiol (gelvatol Burkard Agronomics Inc.) was added to water
and was slowly heated to 95°C. A stirring rod on a magnetic heater/stirrer plate was
used to heat the mixture and then allowed to cool for a few hrs. Once cooled, glycerol
and phenol were added to prepare the coverslip adhesive. The adhesive prepared was
in a volume of 500 ml. Aliquots were prepared in separate bottles of 50 ml and stored
at -20°C for 12 months.
Microscope slides were prepared with the date written on one end of the slide using a marker.
A brush was dipped in the pollen collecting adhesive and the bristles were applied gently to
the slides from end to end. To provide a more uniform application of pollen adhesive, a razor
blade was gently passed over the slide to even out the adhesive and get rid of any bubbles.
However, care was taken not to apply the adhesive on the date written on the slide to avoid
the mixing of marker ink with the adhesive. The slides were placed into the slot of the 24 hr
drum and ready to be placed in the sampler.
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Figure 6.1. An aerial view of the spore trap on the roof of the library at Deakin
University, Waurn Ponds, Geelong. The area immediately surrounding the university
premises is covered with green pastures that allow the dispersal of grass pollen hence making
it a very suitable site for the installation of Burkard volumetric trap to sample air. The photo
was taken using Google Maps (Google Inc.). The Figure on top is an actual photo of the
sampler installed on the rooftop.
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6.2.3 Changing the Burkard Air Sampler Using the 24 hour Drum
1. Stop the trap from rotating with restraining pin on the chain to avoid injury;
2. The locking arm was pressed down and rotated to open the hatch of the trap. The
drum was pulled out straight from the body of the sampler.
3. The slide from the previous day was removed and replaced with a freshly prepared
slide (Figure 6.2). During the 24 hrs sampling period, the clock moves the slide up 48
mm across the orifice of the trap. Used the knurled ring to reorient the carrier;
4. New slide inserted for the next 24 hrs. Slides were prepared using a brush dipped in
pollen adhesive and applied on the slide to ensure uniform distribution;
5. The clock was wound (anti-clockwise) using a key provided in the kit;
6. The 24 hr drum was placed on the grooves located on the inside of the sampler and
the locking arm was rotated back into position to secure the head of the drum;
7. Check the flow rate with the flowmeter; adjust if necessary. It is advisable to check
the flow rate once a week. To change the flow rate, remove the sampling head and
adjust the screw in the white valve at the base of the chamber;
8. Remove the restraining pin on the chain to allow the trap to rotate freely with caution
to avoid injury.
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At start position After 24 hrs
Figure 6.2. The orientation of the slide before and after a period of 24 hrs during which
the slide moves along the orifice of the air sampler for collection of pollen.
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6.2.4 Preparing Slides for Pollen Counting and Examining Using an Olympus BX50
Microscope:
1. The slide was removed from the sampler and coverslip adhesive was applied down
the entire length of the slide (avoiding the date written on one end of the slide) using a
micropipette. Care was taken not to allow the adhesive to dry (Figure 6.3);
2. Immediately, a coverslip was placed at a 45° angle and dropped carefully so as to fall
on the coverslip adhesive covering the entire width of the slide. Care was taken not to
move the coverslip;
3. Gentle pressure was applied using forceps to evenly spread the adhesive;
4. The slides were allowed to dry for a period of 24 hrs;
5. Any access adhesive that may have oozed out from the edges of the coverslip was
carefully removed using a razor blade;
6. Slides were stored away in boxes specifically designed for storing microscope slides
for long-term storage;
7. Slides were examined using an Olympus BX50 microscope (Olympus Australia) at
40x magnification and gradually increasing magnification to 200x. To confirm the
identification of ryegrass pollen, the magnification used was 400x;
8. Pollen were counted using a line scan method which is counting the pollen in one
longitudinal transect of the slide (345). It must be acknowledged that the Burkard
volumetric trap tends to show an instrumental variation of 25% shown in Table 6.1
(345).
9. Photos of the pollen were taken using a Canon DS126291 camera (Canon Inc.) which
was connected to a computer;
10. Pollen data was uploaded on a daily basis on the Deakin AIRwatch webpage which
also included a thunderstorm associated asthma risk;
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11. All weather parameters including, temperature, humidity, precipitation and wind
speed were recorded courtesy of the Geelong Weather website. The weather station
installed is Davis Vantage Pro2 which collects weather information every 5 seconds.
The station comprises of an anemometer, rain gauge and a thermo-hydro sensor
situated in optimal positions for highest accuracy possible.
6.2.5 Calculations for Pollen per m3 of Air
An Olympus BX50 microscope has a field of view of approximately 1 mm at 200x
magnification (micro-meter was used for measurement). The Burkard volumetric trap has a
vacuum pump which takes in air at a rate of 10 litres/min. Hence, total air sampled over a
period of 24 hrs was 14.4 m3. The width of the orifice on the air sampler is 14 mm. Since a
line scan method was used to count daily pollen, the entire length of the slide is taken into
account at 48 mm since the 24 hr drum moves 2mm/hr hence 48 mm over a period of 24 hrs.
Formula applied = Field of view/width of slide x (Total volume of air)
= 1/14 x (14.4) = 1 m3
Correcting count per m3 of air
Formula = final count of pollen per line scan / 1 m3 of air = Total pollen count per m3 of air
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Figure 6.3. Diagrammatic representation of the coverslip adhesive placed on the slide
surface where the pollen is captured using the pollen adhesive.
Coverslip adhesive
Coverslip
Coverslip adhesive
Glass slide
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6.3 Results
6.3.1 Ryegrass Pollen
Pollen captured on the surface of microscope slides was prepared as shown in methods
section. This was followed by an examination of the slides using an Olympus BX50
microscope that allowed us to take photos of ryegrass pollen. It should be elucidated that
pollen are much more visible without the use of stain. The photos taken were at random from
slides to show the distinct shape and rupturing of ryegrass pollen. All pollen was counted
using a line-scan method along the longitudinal traverses of the slide. For results purposes, all
pollen, whether partial or fully ruptured, were taken into the same category. Intact pollen
showed granules within the shell and were identified by the presence of a single distinct pore
on the wall of the pollen (Figure 6.4).
Table 6.1. Weather Variables and Grass Pollen Count. Annual Season 2012-2013 (Oct-Jan) 2013-2014 (Oct-Jan) 2014-2015 (Oct-Jan) Mean maximum temperature °C
23.7202381
22.004
21.09°C
Average daily temperature °C
17.38°C 16.35°C
17.36°C
Average high wind speed km/hr
31.5 km/hr
32.25 km/hr
32.14 km/hr
Average wind speed km/hr
6.7 km/hr
4.41 km/hr
4.58 km/hr
Average RH Max. Values (%)
82 %
77.70 %
78 %
Total precipitation in mm
60.4 mm
117.1 mm
145.4 mm
Intact pollen per m3 of air (+/- 25%)
1,066 per m3 of air
1,397 per m3 of air
1,027 per m3 of air
Ruptured pollen per m3 of air (+/- 25%)
249 per m3 of air
364 per m3 of air
295 per m3 of air
Total pollen for season per m3 of air (+/- 25%)
1,315 per m3 of air 1,761 per m3 of air 1,322 per m3 of air
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A) B) C) D) E)
Figure 6.4. Micrographs of ryegrass pollen identified by using an Olympus BX50
microscope at magnification of 400x. Figure 6.4A shows the ryegrass pollen when stained
with safranin. Figure 6.4B shows intact ryegrass pollen. Figure 6.4C shows an empty shell of
ryegrass pollen which has released all starch granules and allergens. Figure 6.4D shows
partially ruptured ryegrass pollen with starch granules released from the pore. Figure 6.4E
also shows partially ruptured ryegrass pollen.
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6.3.2 Weather Variables and Grass Pollen Count (2012-2013)
For the purpose of determining the effects of weather variables on grass pollen count, we
considered 4 parameters: temperature, wind speed, RH values and total precipitation during
the whole season (Figure 6.5). The start of the pollen season was determined by the
appearance of ryegrass pollen when examining slides. The first day with pollen appearing for
2012-2013 took place on the 18th of October, 2012 and the last day was the 10th of January
2013. Looking at Figure 6.5, three prominent peaks can be found when considering intact
and ruptured grass pollen count. For results purposes, these three peaks will be considered as
significantly higher than other peaks hence showing important events during the season. The
first day denoted 19th of November of 2012 showed the first high peak after an episode of
rain a day earlier and an elevated temperature high in the 20’s. The second most important
peak of the season fell on the 29th of November 2012 followed by smaller peaks, where an
episode of rain along with higher average temperatures immediately translated into a higher
pollen count and ruptured pollen as well. The third most important peak of the season fell on
the 13th of December 2012 when higher than average temperature and wind gave a higher
pollen count. It is worthy to note that although the intact grass pollen count on this day was
equally high to other peaks, very few pollen resulted in rupture due to a dip in RH on that
particular day. The initial peaks also showed lower intact grass pollen counts but had a higher
ratio of ruptured pollen when compared to other peaks.
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Figure 6.5. Weather variables for the grass pollen season 2012-2013 (Oct-Jan).
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6.3.3 Weather Variables and Grass Pollen Count (2013-2014)
The first day with pollen appearing for 2013 took place on the 18th of October, 2014 and the
last day was the 31st of December 2013. Looking at Figure 6.6, three prominent peaks can be
seen when looking at intact and ruptured grass pollen count. For results purposes, these three
peaks will be considered as significantly higher than other peaks hence showing important
events during the season. The first day denoted 8th of November of 2013 did not show a
higher than average temperature, however high gusts of wind in excess of 30km/hr may also
have played an important role in the dispersal of pollen on this particular day. The second
most important peak of the season fell on the 18th of November 2013 followed by a sudden
drop in pollen counts which coincides with an episode of rain. The sudden rise can be
attributed to a rise in average daily temperature which may have resulted into the release of
pollen and rupturing. Despite of the high peak, this day can be regarded as an anomaly when
looking at weather parameters and ruptured pollen. There was no rain prior to this peak and
RH values were within normal range, which are the main triggers of pollen rupturing. The
third most important peak of the season fell on the 8th of December 2013 when higher than
average temperature and an episode of rain a day earlier resulted in a higher pollen count.
Referring to data in Table 6.1, a higher level of precipitation was found when compared to
the previous year. This explains a higher grass pollen count. All rainy days coincided with no
release of pollen. However, the peaks of ruptured pollen between the 23rd and 25th of
December 2013 showed a higher ratio of ruptured pollen, which coincides with a rainy day.
However, with less precipitation, the pollen were able to dehisce since a higher temperature
peak was attained and dispersed by wind.
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Figure 6.6. Weather variables for the grass pollen season 2013-2014 (Oct-Dec).
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6.3.4 Weather Variables and Grass Pollen Count (2014-2015)
The first day with pollen appearing for 2014 took place on the 12th of October, 2014 and
ended on the 31st of December 2014. Looking at Figure 6.7, two prominent peaks and a
cluster of peaks can be found when looking at intact and ruptured grass pollen count. For
results purposes, these peaks will be considered as significantly higher than other peaks hence
showing important events during the season. The first day denoted 22nd October of 2014,
which appears to be an anomaly. With lower average temperature and no rain episodes on
previous days and lower wind speed all indicate a lower grass pollen count. The spike may
have been a result of a sudden gust of wind which causes pollen to be airborne. The second
most important peak of the season fell on the 29th of October 2014, which was followed by an
episode of rain on the previous day. Also to be noted, a higher ratio of ruptured pollen can
also be seen on this day because of a rise in RH max values. The other important peaks of the
season fell in the first week of November of 2014 when an episode of rain along with higher
wind gusts resulted into higher pollen count. However, it can be noticed that the ruptured
pollen may have resulted from the rain on the 1st of November. Referring to data in Table 6.1,
a higher level of precipitation was found when compared to the previous year. This explains a
higher grass pollen count. With the exception of the first week of November, all rainy days
coincided with very few or no release of pollen. However, the peak of pollen on the 25th of
November 2014 showed a higher ratio of ruptured pollen, which coincides with a rainy
episode on the prior day and a higher average daily temperature. The season of 2014-2015
experienced the most amount of precipitation but this cannot be correlated with the total
amount of pollen released and ruptured since a lower mean maximum temperature for the
season was recorded (Table 6.1).
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Figure 6.7. Weather variables for the grass pollen season 2014-2015 (Oct-Dec).
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6.4 Discussion
6.4.1 Seasonal Changes in Grass Pollen Count
In our study of ryegrass pollen distribution in Waurn Ponds, Geelong we report for the first
time the collection of daily pollen counts and their relationship to meteorological conditions
prevailing in regional Victoria.
Ryegrass is grown in pastures for livestock grazing and can be viewed in Figure 6.1 around
the Waurn Ponds campus of Deakin University. Analyzing the overall collection of pollen
samples over a three-year seasonal period shows a differential correlation between weather
parameters and grass pollen albeit with exceptions. Referring to Table 6.1, it was revealed
that the season 2012-2013 has received the least amount of precipitation which coincides
with the theory of fewer grass pollen being released and ruptured. In contrast, the following
seasons had higher pollen counts due to a significant amount of precipitation and higher
temperatures. However, it must be stressed that high temperature can have negative effects on
the maturing and development of pollen grains (346). Although the highest precipitation
occurred in the season of 2014-2015, fewer pollen counts were reported when compared to
2013-2014. This may be a result of a lower average peaks in temperature which is a
contributing factor for the dehiscence of pollen (346). Also, it must be emphasized that grass
pollen count fluctuations were not significantly correlated with changes in weather variables
in some instances.
The pollen peaks achieved during 2012-2013 coincide with high temperature peaks which
cause anthesis. Following anthesis, pollen dispersal is dependent on a variety of physiological
and meteorological factors such as wind speed and wind turbulence, which causes
dehydration of the anther that results in dehiscence. Hence, the dispersal of pollen may
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continue a few days after anthesis (347). Also, it can be observed from Figure 6.5 that the
three major peaks in 2012-2013 occurred in the month of November and December. This
may have been a result of rain episodes that peaked in these months along with higher than
average temperature peaks when compared to October. Rain episodes seemed to have
clustered during this season rather than frequent episodes. The peaks in the initial part of
December of 2012-2013 also coincides with higher temperatures and stronger winds that
disperse pollen. Rainfall is the single most important factor in determining plant growth,
hence making the observations important. Peaks in RH max values and rainfall are sufficient
to cause ryegrass pollen fragmentation.
In the following season of 2013-2014, higher than average temperature along with relatively
higher wind speeds resulted into the highest pollen count. Referring to Table 6.1, it was
revealed that the season 2013-2014 has received the second highest rainfall, which coincides
with the theory of higher grass pollen being released and ruptured. The rain distribution
during this season is more evenly distributed throughout the season with higher than average
rain peaks in October, November and December. This explains a more concomitant release of
pollen when temperatures reached high into the 30’s. This resulted into the drying of the
anthers and thus releasing pollen on the days that followed.
In the latest season of 2014-2015, it was determined that despite of receiving the heaviest
rainfall out of the three seasons, the pollen count failed to achieve higher numbers.
Temperature played an important role in this regard. Referring to Table 6.1, it can be noted
that a lower mean maximum temperature for the season inhibits anthesis and less pollination
of grass by wind and other factors. Another observation can be seen for the month of October
which received the least amount of rainfall and yet showed significant peaks. They may have
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been a result of higher than average wind speeds and temperatures recorded at the initial days
of the season. The highest peaks were recorded in the last week of October and first week of
November, which also coincides with high wind speeds and a hot day prior to these peaks.
6.4.2 Thunderstorm Allergy in Victoria, Australia
Although severe thunderstorm events have been reported previously in the city of Melbourne,
Victoria, such as the thunderstorm event in the spring of 2010 and the 1987/1989
thunderstorms (204, 339-341, 348), no such event has been reported in the three consecutive
seasons we have reported so far. Although some rainfall episodes were accompanied by few
thunders, we are yet to report any unusual increase in pollen count and pollen rupture
associated with thunderstorms.
6.4.3 Ruptured vs. Intact Ryegrass Grass Pollen
Grass pollen develops inside the anther sac. With high temperatures and dry conditions, the
anther splits open releasing approximately 5000 pollen (dehiscence). However, the dispersal
of pollen from the anther sac depends on other variables such as wind. This has been reported
by Taylor et al., (2002) that pollen remained on the anthers despite the fact that anther sacs
were open on wind-pollinated plants such as ryegrass. Hence, it is important to recognize that
anther dehiscence and pollen release are two different events that do not occur
simultaneously (349).
It is a well-documented fact that allergens within pollen grains cause allergy rather than the
pollen itself. Since pollen can’t penetrate respiratory airways, it is the allergens such as Lol p
1 and Lol p 5 which are located on and within the micro-particles found in a pollen grain that
are small enough in size to penetrate lower airways and trigger the allergic symptoms (350,
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351). It has been reported by several papers that ryegrass pollen, upon contact with water
molecules, results in the bursting of the intine layer of the aperture pore. This results in the
release of ~700 starch grain particles (0.6 to 2.5 μm in length) that form the part of respirable
allergen aerosols (208, 338, 349). An experiment designed by Taylor et al., (2002) describes
the harvesting of flowering plants such as ryegrass in a plexiglass chamber. The plants had
their anthers open which releases pollen inside the chamber. A particle counter then collects
the pollen and cytoplasmic debris to give an overall number of particles released by each
flowering plant. Repeated drying and wetting of the chamber did result into the rupturing of
pollen. Gentle airflow inside the chamber also resulted into the dispersal of the cytoplasmic
debris and micronic particles released from the dehisced anthers. It is established that these
micronic particles are responsible for triggering asthma responses in individuals (204, 349).
Hence, a few important observations with regards to pollen rupture were made during the
entire pollen seasons as can be seen in Figures 6.5, 6.6 and 6.7. A typical example can be
seen in the recent pollen season on the 1st of November of 2014. A prior sunny day with a
high temperature of >30°C, followed by rain on the 1st of November that causes rupturing of
pollen. Although rainfall can result into the washing off of pollen grains and micronic
particles (352), we report that ruptured ryegrass pollen incidence increased with rainfall
episodes. Wind speed was also reported to be higher than average on the 1st of November.
These events make it a ‘perfect storm’ for the release of ruptured pollen from anthers that
may have dehisced on the previous sunny day and ruptured upon contact with water during
the rainfall episode followed by wind. Hence, it is important to note that although some days
may appear to have higher than average wind speeds, the pollen counts are reported as low
since anther dehiscence may not have taken place due to low temperatures and lack of rain on
previous days. Similarly, it is not necessary that a rainy day during the pollen season may
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result into a high ruptured pollen count since exposure to water must take place after
dehiscence of the anthers.
6.4.4 Conclusion
To conclude, in the regional city of Victoria (Geelong), pollen distribution and its
relationship with weather variables have not been studied before. In our study, we
investigated the distribution of ryegrass pollen and their changes during the spring/summer
season in Victoria. It is important to establish a pollen concentration in this region where
atopic individuals suffer from pollen allergy. In this three year study, it was observed that
there was a significant increase of pollen counts on days that had high temperatures and wind
gusts. High pollen count days were preceded by rainy days in some instances. Pollen
rupturing was also added into the mix as a more rational approach to understanding pollen
debris that carries allergens. However, more seasons of pollen counting will need to be
carried out in the future which will increase our understanding of the effects of weather
variables on ryegrass pollen distribution.
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Chapter 7 – General Discussion
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7.1 General Discussion
This chapter entails an overall discussion of Chapters 2-6 with more emphasis on IL-4R
inhibition for allergy treatment. However, in latter sections the inhibition of cytokine
signaling is discussed with respect to fatty acids, resolvins and CoQ10 as novel therapeutics
for allergy. Lastly, we have discussed the mapping of ryegrass pollen distribution in regional
Victoria (Geelong) with links to weather variables.
7.2 Overview of IL-4R
The IL-4R plays a vital role in allergic diseases since it is able to bind IL-4 and IL-13
cytokines. Activation of Th2 cells by APC leads to the production of cytokines that cause B
cells to produce IgE. Once synthesized, IgE antibodies circulate in the blood before binding
to the high-affinity IgE receptor Fc RI that is present on mast cells in tissue or on peripheral
blood basophils. After re-exposure, allergens cross-link to mast cell- and basophil-bound
specific IgE, thus causing the release of preformed mediators, such as histamine, the
synthesis of prostaglandins (PGs) and LT’s (353).
Thus, it is necessary to find a novel antagonist to IL-4R , to block the interaction of the
cytokines with their receptor, hence blocking or down-regulating the allergic cascade as a
prophylactic treatment.
Current clinical strategies for allergy treatment involve allergen avoidance by patients,
pharmacotherapy and specific immunotherapy. Only the third strategy has provided a long-
term solution but with little success, since the allergen extracts used are poorly characterized
and suffers from batch to batch variation. The display of foreign proteins on the surface of
bacteriophage M13 has been used to understand protein-protein interactions at the molecular
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level. For this reason, phage display technology has been used as a novel approach to identify
peptide antagonists (354).
Also, we have discovered the effect of n-3 and n-6 PUFA on a HEK-Blue model of allergy.
This model is versatile and mimics a simple colorimetric analysis of IgE antibody production
from B cells in response to cytokine signaling. Another important finding discussed in this
thesis is the effect of resolvins and CoQ10 on IL-4 and IL-13 signaling in the same HEK-
Blue cell-line. As our results suggest, resolvins although anti-inflammatory by nature, have
been found to be pro-inflammatory with respect to JAK-STAT6 signaling.
In addition, we have investigated the distribution of ryegrass pollen during three
spring/summer seasons in regional Victoria. This will help us evaluate a more clear
understanding of allergy prevalence in Victoria. A broader concept to allergy will be to
understand the cause of allergy before tackling the problem on a molecular basis. Grass
pollen has been implicated in allergy by various scientists all over the world. Hence, it is
highly important to map pollen distribution in a given area and conduct aerobiological
analysis of the pollen data.
7.2.1 Phage Display
In Chapter 2, it was shown that five successful rounds of biopanning were completed using
IL-4R as the potential target. It should be stressed here that effective aseptic techniques are
required to achieve more than 3-4 biopanning rounds as wild-type M13 phage contamination
can easily take over the peptide-displaying recombinant M13 phage. The recombinant phage
has reduced infectivity when compared with wild-type M13. The repeated 5 cycles of
biopanning meant, selecting phage that binds with the highest affinity to the target IL-4Rα.
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Maintaining a constant input phage concentration in each round, incurred a consensus in
binding sequences.
The exposed peptides which showed higher binding affinity to the IL-4Rα target were
collected and washed using TBST. The detergent Tween-20 added to the binding and
washing buffers reduced non-specific hydrophobic interactions between the phage and
blocking/target proteins. Lower concentrations of TBST were used in the earlier rounds,
which resulted into lower stringency but higher eluate titers, and the stringency was gradually
increased from 0.1% to 0.5% in the final round, selecting for highly specific interactions.
Another purpose of using TBST was to favour ionic interactions rather than hydrophobic
ones between the target receptor and the peptide. Target concentration was also reduced
during subsequent rounds of biopanning, as this would allow a higher stringency. By
decreasing the concentration from 50 nM to 10 nM allowed for selection of ligands with high
binding affinity.
Phage-display has become a method of choice for epitope mapping and has been successfully
used in numerous published studies (131, 355-357). For this reason, it was suggested that
sequences N1-N9 that showed consensus in binding represented an epitope that strongly
bound with the IL-4Rα target. This was determined from the ClustalW2 alignments that were
conducted using the IL-4 and IL-13 amino acid sequences with the N1-N9 amino acid
sequence. 6 amino acid residues were exactly identical with the IL-4 (153 amino acids) along
with 3 weakly conserved regions. In essence there was a 50% similarity between the selected
peptide sequence and IL-4 cytokine. When the same 12-mer sequence was aligned with IL-13
(146 amino acids), only two amino acids were identical while the strongly and weakly
conserved amino acids had 3 each. This confirmed that the isolated peptide mimicked the IL-
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4 cytokine more than it did IL-13. This is consistent with the study that IL-4Rα subunit binds
both IL-4 as well as IL-13. Homologous sequences have blocks of alignments separated by
gaps (not shown for intellectual property reasons). The gaps usually indicate loop regions
with no conserved core secondary structure and hence, explain that the epitope of the peptide
is conformational in shape rather than linear (358). Further, a nucleotide BLAST search was
conducted on national centre for biotechnology information (NCBI) using the amino acid
sequence of the identified peptide. It was used to compare the peptide sequence with those
contained in nucleotide databases by aligning the sequence with previously characterized
proteins, therefore assisting to identify homologues that are linked with the IL-4R protein.
However, an analysis of the homologues revealed no relevant protein that might be linked to
the interleukin family of proteins.
7.3 Mimtags: The Use of Phage Display Technology to Produce Novel Protein-Specific
Probes
As part of our overall discussion for Chapter 2, we have demonstrated that phage display can
also be used as an effective tool to study protein structures and the recognition of epitope
regions. The aim of this work was to isolate phage-displayed mimtags (section 1.6.1) against
IL-4R and to investigate the ability of mimtags to bind target proteins and act as protein-
specific probes. High-quality mimtags were isolated during the study, which recognized IL-
4R . Hence, the term peptide will be replaced with mimtag for discussion purposes.
7.3.1 The Use of Phage Display in Isolating Mimtags as Protein-Specific Probes
Once again, for discussion purposes, only mimtag N1 will be emphasized throughout this
section.
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New strategies have been devised in the last few years to characterize regions with epitope
binding capacity. Phage display has shown to be a powerful tool to discover and unravel
conformational mimic epitopes or mimotopes from random peptide libraries (359, 360).
In this study, phage display was successfully used to identify mimtags for target proteins IL-
4R . 12-mers are long enough to fold into short structural elements, which may be useful
when panning against targets that require structured ligands. The exposed random peptides
were allowed to interact with the sequences of the IL-4R . The purity and efficacy of the
target was determined by the company (Sigma-Aldrich), which certified the RhIL-4R to be
pure and biologically active. A 12-mer library was used in the assay because of it renders
peptides that include highly reactive mimotopes as opposed to 7-mer libraries (260). The
additional 4th and 5th round of selective biopanning discarded almost completely the low
affinity binders and 9 out of the 10 mimtags isolated had a consensus in their amino acid
sequences.
The use of phage display has many advantages over the conventional system of using
antibodies for the detection of epitopes. Many studies over the years have shown the
usefulness of phage display in the field of research. The possibility of obtaining mimtags of
interest without learning the three-dimensional structures of proteins avoids the need for
complicated cloning, crystallizing, and modelling procedures, which had to be carried out
first (361-364). The in vitro analysis using a 12-mer phage displayed peptide library
eliminates the use of animals for the production of antibody libraries, which subsequently
eliminates any issues relating to animal ethics and keeps animals out of harm’s way. Mimtags
are also smaller in size when compared to antibodies, which increases their effectiveness and
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efficiency to penetrate large protein fragments or antigens with conformational epitopes
(139).
7.3.2 Probing of a Conformational Epitope on Target Protein with Mimtag
The mimtag sequence N1 had shown to share a homology with the cytokine IL-4 at specific
amino acid sequences. Before stepping into further analysis of the synthesized mimtag, it was
important to understand the interaction between IL-4 and its receptor IL-4R relative to the
binding of mimtag N1 to IL-4R . The multiple sequence alignment in ClustalW2 showed a
50% identity of amino acid sequences of N1 mimtag with amino acid sequence of IL-4
cytokine. There were gaps in the sequence alignments, which indicated a loop region with no
secondary structure conserved. Based on hydrogen bonding, there are three clusters that form
ligand receptor interface. In the IL-4R , two out of three clusters have been identified as the
main binding determinants (Figure 7.1), Glu9 (IL-4) and Arg88 (IL-4). The Glu9 makes
several hydrogen bonds with IL-4R at positions Tyr13, Ser70 and Tyr183. The second
cluster involves Arg88 of IL-4, which binds with Asp72 of IL-4R (365). The critical binding
residues are distant in the primary sequence but close in the folded native conformation of the
protein hence forming a discontinuous chain of amino acid residues (269). However, the N1
mimtag showed binding to IL-4R in the binding ELISA assay which did not overlap the
binding sequence of IL-4 cytokine and its receptor when cross-checked with ClustalW2
alignment software. Hence, it may be possible that the N1 mimtag may have identified a
novel epitope of IL-4R , which remains unreported (55, 366).
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Figure 7.1. Cluster domains of the IL-4R indicated by the dotted Y shape. These clusters
are responsible for binding with the IL-4 cytokine. Two clusters denoted Cluster I and Cluster
II have shown to bind to IL-4 cytokine with about 80% of the total binding energy. At amino
acid positions 13, 70 and 183 lays the first binding region with amino acid at position 9
(glutamate residue). At amino acid position D72 lays an aspartic acid that binds with arginine
at position number 88 of the IL-4 cytokine. Figure adapted from (365).
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7.3.3 Mimtag Technology
Mimotope tagging is a very versatile procedure to identify and tag epitope regions of proteins
using biotinylated versions of the mimotopes. Mimotopes isolated from phage display
libraries enable alignments with the target antigen and subsequent localization of structural
epitope regions (367). Another useful outcome of such a study is the use of such mimotopes
to tag target proteins, which allows them to be protein-specific probes as in the case of
mimtag N1.
There are several advantages of this system over the conventional use of antibodies as protein
specific probes. Mimtags can be stored over long periods of time without the fear of
deteriorating and are cheaper to produce compared to monoclonal antibodies. They are
relatively free of contaminants, such as toxic substances from expression systems.
Application based advantages include the high-throughput of peptide mimtags, which allows
the simultaneous bombardment of the target antigen with several billion mimtags and this
allows the interaction of the target protein with the best possible sequence of mimtag. No re-
probing is required as in the case of antibodies because all immunoassays can be performed
with the new mimtag as demonstrated with the M13 phage ELISA and direct ELISA.
Antibodies are difficult to produce employing cell-based and in vivo methods, not to mention,
the purification of antibodies (368, 369), which are highly expensive methods and difficult to
handle leaving very little margin for errors. The specificity is another factor that most
scientists fail to report when using antibodies, which has to be determined first, making it
ever more difficult to use in immunoassays and detection systems (370, 371). However,
mimtags can conform 3-dimensional structures and can specifically bind and localize an
epitope. Hence, we can propose that mimtags may eventually replace antibodies for their use
in immunoassays and detection systems.
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Another important feature of using biotinylated mimtags is the spacer between the mimtag
and biotin, which reduces steric hindrance to binding with the target epitope as opposed to a
labelled antibody where conjugation reaction has shown to cause deleterious effects on
antibody avidity (372) and in some cases, labelling index in antibody was negatively
correlated with the binding affinity for its target antigen (373). A HRP-conjugated
streptavidin protein can detect biotin on the mimtag with high affinity and avidity. This
immunoassay was specifically conducted to test the efficacy and affinity of the newly
synthesized biotinylated mimtag N1 to the IL-4R (direct ELISA in Figure 2.3). The trend is
clearly visible via the graphed readings where higher absorption readings are observed with
increasing peptide concentrations. A Student’s t-test analysis of the results from the graph
revealed a significant result when compared to the control (P < 0.05). This was an indication
that the IL-4R was bound at the bottom of the well and the N1 mimtag was interacting with
the target.
7.3.4 Further Applications of Mimtag Technology
Apart from their ability to probe and map protein sequences, mimtags can potentially be used
for probing and identifying gene sequences and clusters; using a phage library of exon-sized
inserts (374-376). Mimtags can also be used for the detection of antibodies in a given sera
which is by far an inexpensive, rapid, sensitive, specific and reusable method to detect a
humanized therapeutic antibody (377). Phage displayed synthetic mimtags have also been
used for biosensor analysis by fluorescently labelling the mimtags that could be detected onto
a surface of a sensor chip designed for ligand interaction and replacing larger antibodies as
probes (378, 379). Single point substitution in mimtag sequences can be performed to
improve the avidity of the mimtag enhancing its bioactivity and interacting capability with
the target epitope (380, 381). All of these applications are just a start into replacing larger
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antibodies with smaller mimtag protein-specific probes for the detection and labelling of key
epitope regions on a given protein with higher specificity and low costs making it a more
sophisticated and effective method.
7.4 Inhibition of STAT6 Phosphorylation Pathway in HEK-Blue Cells Using Peptides as
Antagonists
In Chapter 3, it was shown that peptides isolated from phage display showed profound
inhibition of protein-ligand interaction. In the case of peptide N1, there was downregulation
of IL-4 cytokine signaling of SEAP secretion from HEK-Blue cells which can be translated to
be analogous to a decrease of IgE antibody production by B cells. Similarly, peptide K1
binding to cytokine IL-4 showed inhibition of IL-4 signaling suggesting a profound inhibition
of SEAP secretion. Finally, P9 peptide isolated from phage display was used as an antagonist
to IL-13 and reduced cytokine signaling and hence SEAP secretion. All of the above
experiments conducted showed a >50% inhibition of signalling (discussed below in section
7.4.2). Peptide inhibition of biological factors has been studied previously with much success.
Many clinical studies involving peptide inhibition have been previously conducted at the
cellular level as therapeutic agent of tumour growth (382-384). Hence, it is worthy to note
that inhibition studies of allergy have not been conducted previously using a peptide
antagonist for allergy treatment.
7.4.1 Preferential Use of Peptides as Antagonists
Many pharmacological companies believe that peptides are a menace because of their poor
agile properties like poor tissue penetration, serum resistance and quick elimination.
However, recent studies suggest that such problems can be tackled with modification of the
peptide while other proteins or molecules are more difficult to deal with, due to their larger
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sizes. There are potential benefits of using peptides as drugs, which come into play when
dealing with protein-protein interactions (PPI’s). Binding pockets of proteins are sometimes
small and unable to fit an antagonist large in size (antibodies or molecules) and hence, using
peptides is a more viable option; ranging from two to fifty amino acids in length. Second
advantage of using peptides is that it’s unlikely to cause any adverse immunological reactions
in the subject since they are too small to render any physiological reaction. Thirdly, the
chemical diversity found in peptides, as opposed to large molecules, allows more specific
interaction with the protein, increasing the antagonist’s efficiency to penetrate the
conformational structure of the protein (385, 386).
7.4.2 Importance of Inhibitory Values at 50% (IC50) for Inhibition Studies
It is highly important to elucidate the IC50 values in biochemical reactions and inhibitory
studies. Many studies involve inhibition of regulatory receptors with antagonists achieving a
50% inhibition in in vitro and ex vivo applications (387-391). IgE antibody plays an
important role in parasite immunity. Human platelets and eosinophils use IgE in vitro to kill
blood fluke Schistosoma mansoni (290, 392). Infection with the parasite Trichinella spiralis
is supplemented by intestinal mastocytosis and increased IgE responses. T. spiralis requires
expulsion of adult worms and destruction of larval cysts that are deposited in muscle tissue. It
was found that IgE accelerated the removal of worms from the intestine and reduced viability
of larval parasites (393). These are some important functions of IgE antibody in the immune
system (392). Hence, it is vital not to inhibit the IgE response in atopic individuals
completely and down-regulate this vital antibody in immune reactions.
7.5 Further Analysis of n-3 and n-6 PUFA
In Chapter 4 of the thesis, we have investigated the effects of omega-3 and omega-6 fatty
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acids on IL-4 signaling in HEK-Blue cells. It is important to further elucidate and verify our
results with previously published literature. In a study carried out by Weise et al., (2011), it
was found that DHA inhibited IgE production by B lymphocytes, which are derived from
human peripheral blood mononuclear cells. So far, the underlying mechanism is unknown.
Since this study was already established in the case of DHA, we tested other n-3 PUFA in
addition to DHA to improve the overall picture of omega-3 and omega-6 fatty acids. It is also
known that IL-4R (CD124) is suppressed when B lymphocytes are incubated with n-3 fatty
acids, which leads to a suppression of STAT6 phosphorylation pathway (158, 309). DHA,
EPA and DPA all behaved very similarly in the time course experiments in suppressing
STAT6 signaling in HEK-Blue cells. The JAK-STAT6 transcription factor subsequently
activates gene expression in cell nucleus expression. Activity of protein kinases can also be
altered by different fatty acids. A study carried out by VanMeter et al., (1994) suggested that
EPA and DHA can suppress the activity of protein kinase C (PKC) which leads to inhibition
of STAT6 dimerization and gene suppression (394, 395). This is a possible explanation for
less SEAP production by HEK-Blue cells. Furthermore, DHA did not exhibit a dose-
dependent inhibition in the IL-13 detection assay but rather a dose-response inhibition. With
the highest concentration of DHA, it was observed that inhibition was the highest. Literature
suggests that high dose of DHA supplementation changes the outcome of IgE production
from B cells in response to cytokine stimulation which may explain the sudden drop in SEAP
secretion at 150 μM (158, 396).
7.5.1 Production of Inflammatory Prostaglandins
An important explanation of pro-inflammatory effects of AA can be the production of
inflammatory prostaglandins from HEK-Blue cells. AA is found esterified at the sn-2
positions of membrane phospholipids. AA can be released by cytoplasmic phospholipase A2
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and converted by COX 1 and 2 to prostaglandin intermediates known as PGH2. Prostaglandin
synthases then convert PGH2 to 2-series prostanoids known as PGE2, which are
inflammatory. AA can also be converted to TX of the 2-series for e.g. PGD2) or by 5-, 12-,
and 15-LOX which yield inflammatory LT of the 4-series. For the same reason, it was found
that AA was suppressing IL-13 cytokine stimulation of HEK-Blue cells when tested with
higher doses of AA. A study suggests that IL-13 cytokine suppresses COX-2 gene expression
(397). By contrast n-3 PUFA such as DHA and EPA can replace AA in cell membranes and
can be converted by the same enzymes to anti-inflammatory intermediates such as the 3-
series prostanoids known as prostaglandin E3 (PGE3) (309, 398, 399). In fact, EPA and DHA
are poor COX substrates and are potent antagonist to COX activity. Also, gene expression of
proteins involved in AA metabolism can be suppressed by the action of EPA and DHA (399).
All kidney derived cells are capable of producing the COX series of enzymes hence,
incubating cells with fatty acids can alter gene expression through modification of
transcription factor activity (400, 401). This may effect SEAP secretion from HEK-Blue
cells. Although not part of this study, it is important to mention that PGE2 receptors regulate
activation and differentiation of mouse B lymphocytes to IgE production (402).
7.6 The Missing Link: Inflammation and Allergy
In recent years, it has become very clear that the burden of allergic disorders reflect long-term
consequences of chronic allergic inflammation at sites that face repetitive exposure to
allergens. This alone has led many scientists to test different therapeutic drugs to down-
regulate the effects of allergic disease such as corticosteroids, which increases tolerance in
allergic individuals to a specific allergen (11, 322). IgE-mediated responses in allergic
disorders are the most important ingredients of allergic inflammation (323). Since allergic
reaction can lead to acute inflammation, no such link has been established so far on the
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effects of pro-resolving metabolites such as resolvins in allergy. It is not far-fetched to
hypothesize that resolvins may have a significant effect on allergy since they down-regulate
inflammation or vice-versa. For this purpose, in Chapter 5 we have established that although
resolvins are anti-inflammatory in their ability to suppress acute inflammation, it is worthy to
note that they act as agonists in the case of IL-4 and IL-13 signaling in HEK-Blue cells with
SEAP secretion. Evidently, in the case of CoQ10 no previous studies have been conducted at
the cellular level to establish a direct link of CoQ10 and allergy.
Omega-3 fatty acids have been implicated in allergy in the past few decades. Higher intake of
omega-6 fatty acid combined with a low intake of omega-3 diet can subsequently result into
allergy development in children and adults alike (160) (403). Resolvins are precursors of fatty
acid that are produced as a result of acute inflammation (404). Resolvins derived from DHA
and EPA have been shown to signal through specific receptors such as ChemR23, BLT1 and
in the case of RvD1, with PMN transendoethilial migration (186). This consequently reduces
neutrophil infiltration on the site of inflammation (177). Hence, it is important to not only
assess PUFA in allergy but also the bi-products, which are produced as a result of allergic
inflammation. For this purpose, we used the same model of HEK-Blue cells, which showed
remarkable inhibition of STAT6 phosphorylation when using omega-3 fatty acids. It was
hypothesized that since omega-3 fatty acids were able to reduce SEAP secretion in a
remarkable fashion, resolvins would behave in a similar way. However, in the case of
resolvins, it was shown that SEAP production from HEK-Blue cells was increased as a
consequence of the treatments performed. However, in the case of CoQ10 it was shown that
SEAP was reduced even at lower concentrations, which have been established in previous
studies (170, 172).
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7.7 Deakin AIRwatch Project
Grass pollen count is a highly effective technique to investigate the distribution of pollen in
air per m3 of air (216, 227, 338, 405). In Chapter 6 of the thesis, we have used aerobiology to
study and map distribution of ryegrass pollen in regional Victoria (Geelong) using a Burkard
volumetric air trap. In addition, we have studied weather variables linked to grass pollen
count. The purpose of this project was three-fold; firstly to get an accurate measure of
ryegrass pollen per m3 of air, secondly to evaluate ruptured pollen which is caused by water
contact and thirdly to analyze meteorological data in relation to intact and ruptured ryegrass
pollen. Although this project is in the initial stages, it opens new vistas to study pollen
distribution in regional Victoria where grass pastures are a common sight, suggesting a higher
prevalence of pollen in the air (218, 219, 406). A growing body of evidence suggests that
weather variables such as temperature affect the grass pollen in air. Wind gusts pick up pollen
from dehisced anthers and carry them great distances, which results in allergic symptoms
such as hay fever and asthma in genetically predisposed individuals. As the results in Chapter
6 indicate, we were able to show some correlation between weather variables and ryegrass
pollen distribution. Higher temperatures, wind and rain are very important factors (219).
Higher temperatures lead to dehiscence of pollen anthers, which may remain intact on the
anthers. However, wind causes the pollen to be lifted from the anthers and distributed in air
(349). Although rain has a wash-off effect on flowering plants (352), it is shown that water
causes rupturing of pollen (349). The subsequent release of pollen cytoplasm and debris can
become airborne due to wind and can be inhaled by allergic individuals (349). Since the size
of the pollen is large enough not to penetrate lower airways of atopic individuals, the
cytoplasmic debris particles are small enough to penetrate lower airways and cause severe
allergic reactions. Hence, it is important to map pollen distribution to establish a link between
pollen distribution and allergy. The overall aim of our project was to map pollen data that has
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been conducted for 3 consecutive years so far. However, a link between allergy and pollen
distribution in regional Victoria remains to be established.
7.7.1 Limitations of the Deakin AIRwatch Project
Chapter 6 of the thesis discussed the importance of the Deakin AIRwatch project. However, it
is important to note the disadvantages of our current system, which can be improved for
future work and improve the baseline of our project. The disadvantages and future work is
listed below:
a. Several disadvantages of Burkard volumetric trap are associated with poor
collection of data. The trap is sensitive to the size of the particles that are being
inhaled through the orifice of the trap. Some of the pollen collected can be adhesive-
sensitive and are unable to bind themselves to the adhesive being used. The method
used for pollen counting is time-consuming and subject to error during observation.
Also, since grass pollen appear similar when observing a slide under microscope, a
qualified person is needed to identify the pollen and spores. During rain periods, it
becomes increasing difficult to get an accurate idea of pollen distribution since they
are unable to be dispersed by wind. Hydrated anthers of pollen are inhibited from
anthesis or washing off effect (407). The static position of the pollen trap also adds to
the list of disadvantages since a primary location can only be setup without moving
the pollen trap to other surrounding areas to allow slight differences between
microclimates and topography (discussed below). Thus, making it important to install
multiple pollen traps at relevant distances to map out the variation between pollen
counts (408).
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b. Another limitation of using daily counts is the study of hourly structures associated
with a 24 hr pollen count. The days can be broken into hours and observed on a slide
that would allow the study of weather variables and pollen distribution. This will
improve the correlation between meteorological data and grass pollen count
dispersion.
c. Perhaps the most important factor in increasing our understanding of relationship
between grass pollen distribution and weather variables is the length of time. Multiple
years and accumulated data are necessary to have an overall picture of the complex
variables and effects of meteorological conditions on pollen dispersal.
d. Difference in microclimates and ecozones can exist in a particular city causing
minor variations in weather variables such as temperature, wind speed, moisture and
rainfall (409). In our study, the location of the pollen trap was in Waurn Ponds,
Geelong. However, the location of the weather station is in Norlane, Geelong at a
distance of approximately 18 km from the location of the pollen trap. Also, the suburb
of Norlane is located 20 m above sea level as opposed to Waurn Ponds which is 75 m
above sea level. These minor differences in geography may have profound effects on
weather parameters. Perhaps this may reduce the chances of any anomalies that may
have resulted due to weather differences at these sites. Hence, it is important to install
a local weather station at the site of pollen trap.
e. Asthma and hay fever studies have been associated with the rise in grass pollen
count in Melbourne (Australia) during the pollen season (208, 213, 222, 410, 411).
Clinical data to measure the full extent of ryegrass pollen count correlated to allergy
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sufferers can also be incorporated for future studies. This will allow in understanding
the effect of ryegrass pollen on public health and the costs associated with it on the
population of Geelong.
f. Another important investigation can be the correlation of ruptured ryegrass pollen
with clinical data. A rise in ruptured grass pollen may result in an increase in asthma
sufferers.
7.8 Future Directions
Future directions entail work that can be undertaken after the completion of all the
experiments shown in Chapters 2-6. A list of all future directions can be found in the topics
listed below.
7.8.1 Future Directions for IL-4Rα, IL-4 and IL-13 Inhibition
Chapter 2 and 3 discuss the possibility of using peptide antagonists as a novel method to
down-regulate IgE secretion for allergy intervention. However, more work needs to be done
to establish a thorough understanding of the complex nature of using phage display. A list of
future work is itemized below:
a. The Ph.D. 12 library of phage display has proven to be very successful in a range
of experiments carried out. The NARL research group has used this technique to
identify 3 important peptides that bind to IL-4R , IL-4 and IL-13 cytokines. We
have successfully established the effects of the peptide antagonists in vitro.
However, for future studies, the use of Ph.D. 7 library to identify peptide
analogues that can bind to proteins as antagonists. Ph.D 7 library has M13 phage
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that display a hepta-peptide (7-mer) fused to the N-terminus of PIII sequence. The
length of the peptide is shorter than Ph.D. 12 (12-mer) library making it more
suitable to penetrate protein structures (explained in section 7.4.1).
b. A combination of peptide antagonists: for future studies, a combination of peptide
analogues can be used to look at the overall effect of inhibition assays on a HEK-
Blue cell-line.
c. Testing peptide analogues for future studies: peptide analogues can be produced
using Fluorenyl-methyl-oxycarbonyl (Fmoc) chemistry (412, 413), a routinely
performed technique, on PEG polysterene resin. Couplings can be performed
using O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate
(HBTU) and Hydroxybenzotriazole (HOBt) peptide coupling reagents. Further,
peptide purity can be established using HPLC and identity can be confirmed using
mass spectrometry and amino acid analysis. Preparing peptide analogues involves:
d. Alanine scan: amino acid residues can be sequentially replaced in the peptide
inhibitor with Alanine to identify specific amino acid residues responsible for the
peptide’s activity and has been previously performed by our lab (211). This
molecular dissection technique will allow identification of residues with the
highest binding ability to the target IL-4Rα.
e. Truncation studies: This involves systematically removing flanking amino acid
residues from the N- and C-terminus of the peptide, one amino acid residue at a
time (414). This method allows the successful identification of the shortest
sequence needed for activity of the peptide that will bind to the IL-4Rα/IL-13Rα1
and inhibit its interaction with IL-4 and IL-13.
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Analogues prepared using these techniques can allow the development of a very refined form
of the peptide inhibitor, which will bind with the target with the minimum amino acid
residues but maximum binding capacity.
f. Further analysis using a biosensor: The NARL research group has recently
purchased a biosensor (General Electric Co.). However, due to time-
constraints we were unable to perform experiments using the biosensor. For
future studies, cytokine-receptor interaction experiments can be conducted
either with or without pre-incubation of the chip-immobilized IL-4Rα with the
different concentrations of the peptide analogues, to assess the inhibitory
capacity of the peptides. Kinetic parameters can be calculated using the
Biacore evaluation software.
g. Ramos.2G6.4C10 or Burkitt’s lymphoma B cell line: In a series of
experiments carried out with cell-lines, post-completion of experiments with
HEK-Blue cell model, another cell model can be used to carry out the same set
of experiments. Study with Ramos B cells will give a comparison between
recombinant IL-4R found in HEK-Blue cells as opposed to natural IL-4R
found in human Ramos B cells.
h. In vivo testing: The peptide inhibitors can finally be tested in an animal model
system. Tissue and blood serum samples will be taken from a mouse-model
post-allergen challenge and tested for the peptide’s ability to block the allergic
pathway. Following this, the peptide’s ability to inhibit cytokine signaling can
be tested among human subjects in clinical trials.
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177
7.8.2 Future Directions for Fatty acid, CoQ10 and Resolvins
Currently, our work with omega-3 and omega-6 fatty acids was to evaluate their effects on
STAT6 signaling pathway in HEK-Blue cells. We established our results based on a range of
concentrations of DHA, EPA, DPA and AA, along with vitamin E, to test the hypothesis that
fatty acids reduce IgE production by inhibiting IL-4/IL-4R interaction in HEK-Blue cells. In
the time-course experiment, the most optimum concentration of fatty acid was used to
establish the effects of fatty acid on STAT6 signaling using time as a variable. At the
indicated time points, different concentrations of DHA and EPA will be incubated with the
cells. The results generated as a result of these experiments opens new vistas for research of
fatty acids and their correlation to allergy. A study carried out by Kitz et al., (2010)
demonstrated the effect of erythrocyte membrane n-3 fatty acid (DHA) in response to pollen
challenge among asthmatic patients. It was shown that ratios of n-3/n-6 fatty acid were
significantly lower in patients suffering from bronchial asthma. Hence it was concluded that
pharmacological use of n-3 PUFA as an intervention for allergy studies could be established
for the treatment of allergy (415). Hence, for our future studies, it can incorporate the effects
of various fatty acids such as EPA, DPA and AA in response to allergen challenge in patients
or perhaps look at the effects on IgE production from B cells. A combination of fatty acids
can also be used as treatments in a HEK-Blue model. A study carried out by Weise et al.,
(2011) showed that treatment of ovalbumin sensitive mice with a combination of dietary
DHA and AA diminished the symptoms of allergen-induced dermatitis (416). Thus, it can be
concluded that although fatty acids behave differently when used individually for research,
they may have synergistic effects when used in combination with other fatty acids. Perhaps
our future study can include a range of different combinations of fatty acids and their effect
on HEK-Blue cells. Although our study showed that AA induces SEAP secretion in HEK-
Blue cells, further study will elucidate the combinatorial effect of fatty acid at the cellular
PhD Thesis – Chapter 7 Nayyar Ahmed
178
level. All of the above strategies can be used in the case of CoQ10 and resolvins as novel
antagonists for allergy treatment. For example, since CoQ10 had inhibitory effect on SEAP
secretion, it can be used in combination with n-3 fatty acids as a dietary supplement to reduce
allergy. A similar pathway for fatty acid, resolvins and CoQ10 can be performed as shown in
section 7.8.1. In vivo testing can be undertaken with the range of concentrations using an
animal model followed by testing in human subjects.
7.8.3 Pollen Sampling
For pollen sampling, all of the limitations listed in section 7.7.1 must be rectified to enhance
the results for aerobiological studies.
------------------------------------X------------------------------------X------------------------------------
THE END
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References
1. Galli SJ, Tsai M, Piliponsky AM. The development of allergic inflammation. Nature. 2008;454(7203):445-54.
2. Szeftel A. Allergy/Allergies: eMedicine; 2010 [cited 2010 23 March]. Available from: http://www.medicinenet.com/allergy/article.htm.
3. Kay AB. Overview of 'Allergy and allergic diseases: with a view to the future'. Br Med Bull. 2000;56(4):843-64.
4. Sampson H, Mendelson L, Rosen J. Fatal and near-fatal anaphylactic reactions to food in children and adolescents. N Engl J Med. 1992;327(6):380-4.
5. Matsuda T, Nakamura R. Molecular structure and immunological properties of food allergens. Trends Food Sci Tech. 1993;4(9):289-93.
6. Asarnoj A, Moverare R, Ostblom E, Poorafshar M, Lilja G, Hedlin G, et al. IgE to peanut allergen components: relation to peanut symptoms and pollen sensitization in 8-year-olds. Allergy. 2010;65(9):1189-95.
7. Otsu K, Dreskin SC. Peanut allergy: an evolving clinical challenge. Discov Med. 2011;12(65):319-28.
8. Graham-Rowe D. Lifestyle: When allergies go west. Nature. 2011;479(7374):S2-S4.
9. Prescott S, Allen KJ. Food allergy: riding the second wave of the allergy epidemic. Pediatr Allergy Immunol. 2011;22(2):155-60.
10. ASCIA. Economic impact of Allergies: The Australasian Society of Clinical Immunology & Allergy; 2007 [cited 05/06/2012] http://www.allergy.org.au/health-professionals/report-economic-impact-of-allergies.
11. Kay AB. Allergy and Allergic Diseases- First of Two Parts. N Engl J Med. 2001;344(1):30-7.
12. Novak N, Bieber T. Allergic and nonallergic forms of atopic diseases. J Allergy Clin Immunol. 2003;112(2):252-62.
13. Ngoc PL, Gold DR, Tzianabos AO, Weiss ST, Celedon JC. Cytokines, allergy, and asthma. Curr Opin Allergy Clin Immunol. 2005;5(2):161-6.
14. Bessot J, de Blay F, Pauli G. From allergen sources to reduction of allergen exposure. Eur Respir J. 1994;7(2):392-7.
15. Rook GAW, Brunet LR. Give us this day our daily germs. Biologist (London, England). 2002;49(4):145-9.
PhD Thesis – References Nayyar Ahmed
180
16. Semic-Jusufagic A, Simpson A, Custovic A. Environmental exposures, genetic predisposition and allergic diseases: one size never fits all. Allergy. 2006;61(4):397-9.
17. Riedler J, Braun-Fahrländer C, Eder W, Schreuer M, Waser M, Maisch S, et al. Exposure to farming in early life and development of asthma and allergy: a cross-sectional survey. The Lancet. 2001;358(9288):1129-33.
18. ASCIA. Australian Society of Clinical Immunology and Allergy: Australian Society of Clinical Immunology and Allergy; 2012 [cited 2012 23/3]. Available from: http://www.allergy.org.au/content/view/162/293/.
19. Rancé F, Abbal M, Lauwers-Cancès V. Improved screening for peanut allergy by the combined use of skin prick tests and specific IgE assays. J Allergy Clin Immunol. 2002;109(6):1027-33.
20. MedlinePlus. Allergy Testing, US National Library of Medicine, retrieved at 23 March, 2010, <www.nlm.nih.gov/medlineplus/ency/article/003519.htm> 2009 [cited 2010 23/3]. Available from: http://www.nlm.nih.gov/medlineplus/ency/article/003519.htm.
21. Douglass JA, O'Hehir RE. Diagnosis, treatment and prevention of allergic disease: the basics. Med J Aust. 2006;185(4):228-33.
22. Todd T. Pollen Nation. Indianapolis Monthly. 1995: 78-83.
23. Simons FER. Anaphylaxis: Recent advances in assessment and treatment. J Allergy Clin Immunol. 2009;124(4):625-36.
24. Akdis M, Akdis CA. Mechanisms of allergen-specific immunotherapy. J Allergy Clin Immunol. 2007;119(4):780-9.
25. Larche M, Akdis CA, Valenta R. Immunological mechanisms of allergen-specific immunotherapy. Nature Rev Immunol. 2006;6(10):761-71.
26. Focke M, Swoboda I, Marth K, Valenta R. Developments in allergen-specific immunotherapy: from allergen extracts to allergy vaccines bypassing allergen-specific immunoglobulin E and T cell reactivity. Clin Exp Allergy. 2010;40(3):385-97.
27. Epton MJ, Smith W, Hales BJ, Hazell L, Thompson PJ, Thomas WR. Non-allergenic antigen in allergic sensitization: responses to the mite ferritin heavy chain antigen by allergic and non-allergic subjects. Clin Exp Allergy. 2002;32(9):1341-7.
28. Chaplin DD. Overview of the immune response. J Allergy Clin Immunol. 2010;125(2, Supplement 2):S3-S23.
PhD Thesis – References Nayyar Ahmed
181
29. Roitt IM, Brostoff J, Male D. Immunology. Sixth Edition ed: Elsevier Science Limited, Spain; 2002.
30. Romagnani S. Human TH1 and TH2 subsets: doubt no more. Immunology Today. 1991;12(8):256-7.
31. Romagnani S. T-cell subsets (Th1 versus Th2). Ann Allergy Asthma Immunol. 2000;85:9-21.
32. MacDonald S, Rafnar T, Langdon J, Lichtenstein L. Molecular identification of an IgE-dependent histamine-releasing factor. Science. 1995;269(5224):688-90.
33. Berger A. Science commentary: Th1 and Th2 responses: what are they? BMJ. 2000;321(7258):424.
34. Minty A, Chalon P, Derocq JM, Dumont X, Guillemot JC, Kaghad M, et al. lnterleukin-13 is a new human lymphokine regulating inflammatory and immune responses. Nature. 1993;362(6417):248-50.
35. Noben-Trauth N, Shultz LD, Brombacher F, Urban JF, Gu H, Paul WE. An interleukin 4 (IL-4)-independent pathway for CD4+ T cell IL-4 production is revealed in IL-4 receptor-deficient mice. Proc Natl Acad Sci U S A. 1997;94(20):10838-43.
36. Wedemeyer J, Galli SJ. Mast cells and basophils in acquired immunity. Br Med Bull. 2000;56(4):936-55.
37. Plaut M, Pierce JH, Watson CJ, Hanley-Hyde J, Nordan RP, Paul WE. Mast cell lines produce lymphokines in response to cross-linkage of Fc[epsi]RI or to calcium ionophores. Nature. 1989;339(6219):64-7.
38. Seder RA, Paul WE, Dvorak AM, Sharkis SJ, Kagey-Sobotka A, Niv Y, et al. Mouse splenic and bone marrow cell populations that express high-affinity Fc epsilon receptors and produce interleukin 4 are highly enriched in basophils. Proc Natl Acad Sci U S A. 1991;88(7):2835-9.
39. Untersmayr E, Bises G, Starkl P, Bevins CL, Scheiner O, Boltz-Nitulescu G, et al. The High Affinity IgE Receptor FcεRI Is Expressed by Human Intestinal Epithelial Cells. PLoS ONE. 2010;5(2):e9023.
40. Bradding P, Feather IH, Howarth PH, Mueller R, Roberts JA, Britten K, et al. Interleukin 4 is localized to and released by human mast cells. J Exp Med. 1992;176(5):1381-6.
41. Sabban S. Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high- affinity FcεRI receptor United Kingdom: The University of Sheffield; 2011.
PhD Thesis – References Nayyar Ahmed
182
42. Stone KD, Prussin C, Metcalfe DD. IgE, mast cells, basophils, and eosinophils. J Allergy Clin Immunol. 2010;125(2, Supplement 2):S73-S80.
43. Hopkins SJ. The pathophysiological role of cytokines. Legal Medicine. 2003;5(Supplement 1):S45-S57.
44. Igaz P, Salvi R, Rey J-P, Glauser M, Pralong FP, Gaillard RC. Effects of Cytokines on Gonadotropin-Releasing Hormone (GnRH) Gene Expression in Primary Hypothalamic Neurons and in GnRH Neurons Immortalized Conditionally. Endocrinology. 2006;147(2):1037-43.
45. Richard MR, Benveniste EN. Cytokines and The CNS. Edition 2, Taylor and Francis Group, USA2002.
46. Foster PS, Martinez-Moczygemba M, Huston DP, Corry DB. Interleukins-4, -5, and -13: emerging therapeutic targets in allergic disease. Pharmacol Ther. 2002;94(3):253-64.
47. Oh CK, Geba GP, Molfino N. Investigational therapeutics targeting the IL-4/IL-13/STAT-6 pathway for the treatment of asthma. Eur Respir Rev. 2010;19(115):46-54.
48. Kasaian MT, Miller DK. IL-13 as a therapeutic target for respiratory disease. Biochem Pharmacol. 2008;76(2):147-55.
49. Kelly-Welch AE, Hanson EM, Boothby MR, Keegan AD. Interleukin-4 and Interleukin-13 Signaling Connections Maps. Science. 2003;300(5625):1527-8.
50. Khaled WT, Read EK, Nicholson SE, Baxter FO, Brennan AJ, Came PJ, et al. The IL-4/IL-13/Stat6 signalling pathway promotes luminal mammary epithelial cell development. Development. 2007;134(15):2739-50.
51. Jiang H, Harris MB, Rothman P. IL-4/IL-13 signaling beyond JAK/STAT. J Allergy Clin Immunol. 2000;105(6):1063-70.
52. Ahn JS, Agrawal B. IL-4 is more effective than IL-13 for in vitro differentiation of dendritic cells from peripheral blood mononuclear cells. Int Immunol. 2005;17(10):1337-46.
53. de Vries JE. The role of IL-13 and its receptor in allergy and inflammatory responses. J Allergy Clin Immunol. 1998;102(2):165-9.
54. Den DE, Grefte S, Huijs T, ten DGB, Versteeg EMM, van den Berk LCJ, et al. Monocyte Cell Surface Glycosaminoglycans Positively Modulate IL-4-Induced Differentiation toward Dendritic Cells. J Immunol. 2008;180(6):3680-8.
PhD Thesis – References Nayyar Ahmed
183
55. Moy FJ, Diblasio E, Wilhelm J, Powers R. Solution structure of human IL-13 and implication for receptor binding. J Mol Biol. 2001;310(1):219-30.
56. Hage T, Sebald W, Reinemer P. Crystal structure of the interleukin-4/receptor alpha chain complex reveals a mosaic binding interface. Cell. 1999;97(2):271-81.
57. Walter MR, Cook WJ, Zhao BG, Cameron RP, Jr., Ealick SE, Walter RL, Jr., et al. Crystal structure of recombinant human interleukin-4. J Biol Chem. 1992;267(28):20371-6.
58. Finkelman FD, Boyce JA, Vercelli D, Rothenberg ME. Key advances in mechanisms of asthma, allergy, and immunology in 2009. J Allergy Clin Immunol. 2010;125(2):312-8.
59. Webb DC, Cai Y, Matthaei KI, Foster PS. Comparative Roles of IL-4, IL-13, and IL-4R{alpha} in Dendritic Cell Maturation and CD4+ Th2 Cell Function. J Immunol. 2007;178(1):219-27.
60. Kelly-Welch A, Hanson EM, Keegan AD. Interleukin-13 (IL-13) Pathway. Science. 2005;2005(293):p. cm8.
61. Nelms K, Keegan AD, Zamorano J, Ryan JJ, Paul WE. THE IL-4 RECEPTOR: Signaling Mechanisms and Biologic Functions. Ann Rev Immunol. 1999;17(1):701-38.
62. Fukuda K, Fujitsu Y, Kumagai N, Nishida T. Characterization of the Interleukin-4 Receptor Complex in Human Corneal Fibroblasts. Invest Ophthalmol Vis Sci. 2002;43(1):183-8.
63. Lindqvist C, Lundstrom H, Oker-Blom C, Akerman K. Enhanced IL-4-mediated D10.G4.1 proliferation with suboptimal concentrations of anti-IL-4 receptor monoclonal antibodies. J Immunol. 1993;150(2):394-8.
64. Beckmann M, Schooley K, Gallis B, Vanden Bos T, Friend D, Alpert A, et al. Monoclonal antibodies block murine IL-4 receptor function. J Immunol. 1990;144(11):4212-7.
65. Maliszewski CR, Sato TA, Bos TV, Beckmann MP, Grabstein KH. Induction of B cell activities by interleukin 4 is inhibited by a receptor-specific monoclonal antibody in vitro. Eur J Immunol. 1990;20(8):1735-40.
66. Negri JA, Steinke JW, Enelow R, Baramki DF, Borish L. Soluble recombinant human interleukin-4 receptor (rhulL-4R) inhibition of T lymphocytes from antigen-specific cell lines and segmental allergen challenges. J Allergy Clin Immunol. 2003;111(1, Supplement 2):S149-S.
PhD Thesis – References Nayyar Ahmed
184
67. Holtzman MJ. Drug Development for Asthma. Am J Resp Cell Mol. 2003;29(2):163-71.
68. Yang L, Horibe T, Kohno M, Haramoto M, Ohara K, Puri RK, et al. Targeting Interleukin-4 Receptor α with Hybrid Peptide for Effective Cancer Therapy. Mol Cancer Res. 2012;11(1):235-43.
69. Konig B, Fischer A, Konig W. Modulation of cell-bound and soluble CD23, spontaneous and ongoing IgE synthesis of human peripheral blood mononuclear cells by soluble IL-4 receptors and the partial antagonistic IL-4 mutant protein IL-4 (Y124D). Immunology. 1995;85:604-10.
70. Lai KN, Leung JC, Li PK, Larche M, Ritter MA. The effects of blockade of interleukin 2 receptors and interleukin 4 receptors on cytokine production. APMIS. 1991;99(5):434-42.
71. Larche M, Akdis CA, Valenta R. Immunological mechanisms of allergen-specific immunotherapy. Nat Rev Immunol. 2006;6(10):761-71.
72. Suarez-Fueyo A, Ramos T, Galan A, Jimeno L, Wurtzen PA, Marin A, et al. Grass tablet sublingual immunotherapy downregulates the TH2 cytokine response followed by regulatory T-cell generation. J Allergy Clin Immunol. 2014;133(1):130-8.e1-2.
73. Xiao C, Li H, Li H, Cheng Z, Qin J, Zhou W, et al. The effect of specific immunotherapy on the regulation of Th1/Th2 cell ratio of the patients with allergic rhinitis in serum. J Clin otorhinolaryngology Head Neck Surg. 2010;24(20):924-7.
74. Akdis CA, Akdis M. Mechanisms of allergen-specific immunotherapy. J Allergy Clin Immunol. 2011;127(1):18-27; quiz 8-9.
75. Akdis M, Blaser K, Akdis CA. T regulatory cells in allergy: novel concepts in the pathogenesis, prevention, and treatment of allergic diseases. J Allergy Clin Immunol. 2005;116(5):961-8; quiz 9.
76. Palomares O, Yaman G, Azkur AK, Akkoc T, Akdis M, Akdis CA. Role of Treg in immune regulation of allergic diseases. Eur J Immunol. 2010;40(5):1232-40.
77. Fujita H, Soyka MB, Akdis M, Akdis CA. Mechanisms of allergen-specific immunotherapy. Clin Transl Allergy. 2012;2(1):2.
78. Oppenheimer JJ, Nelson HS, Bock SA, Christensen F, Leung DY. Treatment of peanut allergy with rush immunotherapy. J Allergy Clin Immunol. 1992;90(2):256-62.
79. Wang J, Sicherer SH. Immunologic therapeutic approaches in the management of food allergy. Expert Rev Clin Immunol. 2009;5(3):301-10.
PhD Thesis – References Nayyar Ahmed
185
80. Nurmatov U, Venderbosch I, Devereux G, Simons FE, Sheikh A. Allergen-specific oral immunotherapy for peanut allergy. Cochrane Db Syst Rev. 2012;9:Cd009014.
81. Longo G, Barbi E, Berti I, Meneghetti R, Pittalis A, Ronfani L, et al. Specific oral tolerance induction in children with very severe cow's milk-induced reactions. J Allergy Clin Immunol. 2008;121(2):343-7.
82. Enrique E, Malek T, Pineda F, Palacios R, Bartra J, Tella R, et al. Sublingual immunotherapy for hazelnut food allergy: a follow-up study. Ann Allergy Asthma Immunol. 2008;100(3):283-4.
83. Enrique E, Pineda F, Malek T, Bartra J, Basagana M, Tella R, et al. Sublingual immunotherapy for hazelnut food allergy: a randomized, double-blind, placebo-controlled study with a standardized hazelnut extract. J Allergy Clin Immunol. 2005;116(5):1073-9.
84. Sicherer SH, Sampson HA. Food allergy: recent advances in pathophysiology and treatment. Ann Rev Med. 2009;60:261-77.
85. Rolland JM, Douglass J, O'Hehir RE. Allergen immunotherapy: current and new therapeutic strategies. Expert Opin Investig Drugs. 2000;9(3):515-27.
86. Ferreira F, Hirtenlehner K, Jilek A, Godnik-Cvar J, Breiteneder H, Grimm R, et al. Dissection of immunoglobulin E and T lymphocyte reactivity of isoforms of the major birch pollen allergen Bet v 1: potential use of hypoallergenic isoforms for immunotherapy. J Exp Med. 1996;183(2):599-609.
87. King N, Helm R, Stanley JS, Vieths S, Luttkopf D, Hatahet L, et al. Allergenic characteristics of a modified peanut allergen. Mol Nutr Food Res. 2005;49(10):963-71.
88. Bannon GA, Cockrell G, Connaughton C, West CM, Helm R, Stanley JS, et al. Engineering, characterization and in vitro efficacy of the major peanut allergens for use in immunotherapy. Int Arch Allergy Immunol. 2001;124(1-3):70-2.
89. Li XM, Srivastava K, Grishin A, Huang CK, Schofield B, Burks W, et al. Persistent protective effect of heat-killed Escherichia coli producing "engineered," recombinant peanut proteins in a murine model of peanut allergy. J Allergy Clin Immunol. 2003;112(1):159-67.
90. Warrington R. Immunotherapy in asthma. Immunotherapy. 2010;2(5):711-25.
91. Pollock KGJ, Conacher M, Wei X-Q, Alexander J, Brewer JM. Interleukin-18 plays a role in both the alum-induced T helper 2 response and the T helper 1 response induced by alum-adsorbed interleukin-12. Immunology. 2003;108(2):137-43.
PhD Thesis – References Nayyar Ahmed
186
92. Heydenreich B, Bellinghausen I, Lund L, Henmar H, Lund G, Adler Wurtzen P, et al. Adjuvant effects of aluminium hydroxide-adsorbed allergens and allergoids - differences in vivo and in vitro. Clin Exp Allergy. 2014;176(3):310-9.
93. Hoyne GF, O'Hehir RE, Wraith DC, Thomas WR, Lamb JR. Inhibition of T cell and antibody responses to house dust mite allergen by inhalation of the dominant T cell epitope in naive and sensitized mice. J Exp Med. 1993;178(5):1783-8.
94. Norman PS, Ohman JL, Jr., Long AA, Creticos PS, Gefter MA, Shaked Z, et al. Treatment of cat allergy with T-cell reactive peptides. Am J Resp Crit Care Med. 1996;154(6 Pt 1):1623-8.
95. Miller MJ, Foy KC, Kaumaya PT. Cancer immunotherapy: present status, future perspective, and a new paradigm of peptide immunotherapeutics. Discov Med. 2013;15(82):166-76.
96. Fonseca DM, Wowk PF, Paula MO, Campos LW, Gembre AF, Turato WM, et al. Recombinant DNA immunotherapy ameliorate established airway allergy in a IL-10 dependent pathway. Clin Exp Allergy. 2012;42(1):131-43.
97. Roy K, Mao HQ, Huang SK, Leong KW. Oral gene delivery with chitosan--DNA nanoparticles generates immunologic protection in a murine model of peanut allergy. Nature medicine. 1999;5(4):387-91.
98. Sanchez H, Bush RK, Sorkness RL, Tuffaha A, Rosenthal LA, Phillips L. Effects of a DNA vaccine in an animal model of Alternaria alternata sensitivity. Revista iberoamericana de micologia. 2009;26(2):121-8.
99. Shirota H, Sano K, Kikuchi T, Tamura G, Shirato K. Regulation of murine airway eosinophilia and Th2 cells by antigen-conjugated CpG oligodeoxynucleotides as a novel antigen-specific immunomodulator. J Immunol. 2000;164(11):5575-82.
100. Zhu FG, Kandimalla ER, Yu D, Agrawal S. Oral administration of a synthetic agonist of Toll-like receptor 9 potently modulates peanut-induced allergy in mice. J Allergy Clin Immunol. 2007;120(3):631-7.
101. Milgrom H. Anti-IgE therapy in allergic disease. Curr Opin Pediatr. 2004;16(6):642-7.
102. Leung DY, Sampson HA, Yunginger JW, Burks AW, Jr., Schneider LC, Wortel CH, et al. Effect of anti-IgE therapy in patients with peanut allergy. N Engl J Med. 2003;348(11):986-93.
103. Kuehr J, Brauburger J, Zielen S, Schauer U, Kamin W, Von Berg A, et al. Efficacy of combination treatment with anti-IgE plus specific immunotherapy in
PhD Thesis – References Nayyar Ahmed
187
polysensitized children and adolescents with seasonal allergic rhinitis. J Allergy Clin Immunol. 2002;109(2):274-80.
104. Srivastava KD, Kattan JD, Zou ZM, Li JH, Zhang L, Wallenstein S, et al. The Chinese herbal medicine formula FAHF-2 completely blocks anaphylactic reactions in a murine model of peanut allergy. J Allergy Clin Immunol. 2005;115(1):171-8.
105. Srivastava KD, Zhang T, Qu C, Sampson HA, Li XM. Silencing Peanut Allergy: A Chinese Herbal Formula, Fahf-2, Completely Blocks Peanut-induced Anaphylaxis for up to 6 Months Following Therapy in a Murine Model Of Peanut Allergy. J Allergy Clin Immunol. 2006;117(2):S328.
106. Sausenthaler S, Loebel T, Linseisen J, Nagel G, Magnussen H, Heinrich J. Vitamin E intake in relation to allergic sensitization and IgE serum concentration. Cent Eur J Public Health. 2009;17(2):79-85.
107. Wang J, Li XM. Chinese herbal therapy for the treatment of food allergy. Curr Allergy Asthma Rep. 2012;12(4):332-8.
108. Hart TK, Blackburn MN, Brigham-Burke M, Dede K, Al-Mahdi N, Zia-Amirhosseini P, et al. Preclinical efficacy and safety of pascolizumab (SB 240683): a humanized anti-interleukin-4 antibody with therapeutic potential in asthma. Clin Exp Immunol. 2002;130(1):93-100.
109. Vugmeyster Y, Szklut P, Tchistiakova L, Abraham W, Kasaian M, Xu X. Preclinical pharmacokinetics, interspecies scaling, and tissue distribution of humanized monoclonal anti-IL-13 antibodies with different IL-13 neutralization mechanisms. Int Immunopharmacol. 2008;8(3):477-83.
110. Yang G, Volk A, Petley T, Emmell E, Giles-Komar J, Shang X, et al. Anti-IL-13 monoclonal antibody inhibits airway hyperresponsiveness, inflammation and airway remodeling. Cytokine. 2004;28(6):224-32.
111. Yang G, Li L, Volk A, Emmell E, Petley T, Giles-Komar J, et al. Therapeutic dosing with anti-interleukin-13 monoclonal antibody inhibits asthma progression in mice. J Pharmacol Exp Ther. 2005;313(1):8-15.
112. Corren J, Lemanske RF, Hanania NA, Korenblat PE, Parsey MV, Arron JR, et al. Lebrikizumab treatment in adults with asthma. N Engl J Med. 2011;365(12):1088-98.
113. Ultsch M, Bevers J, Nakamura G, Vandlen R, Kelley RF, Wu LC, et al. Structural basis of signaling blockade by anti-IL-13 antibody Lebrikizumab. J Mol Biol. 2013;425(8):1330-9.
PhD Thesis – References Nayyar Ahmed
188
114. Grunig G, Warnock M, Wakil AE, Venkayya R, Brombacher F, Rennick DM, et al. Requirement for IL-13 independently of IL-4 in experimental asthma. Science. 1998;282(5397):2261-3.
115. Zhang JG, Hilton DJ, Willson TA, McFarlane C, Roberts BA, Moritz RL, et al. Identification, purification, and characterization of a soluble interleukin (IL)-13-binding protein. Evidence that it is distinct from the cloned Il-13 receptor and Il-4 receptor alpha-chains. J Biol Chem. 1997;272(14):9474-80.
116. Oshima Y, Puri RK. Characterization of a powerful high affinity antagonist that inhibits biological activities of human interleukin-13. J Biol Chem. 2001;276(18):15185-91.
117. Morioka T, Yamanaka K, Mori H, Omoto Y, Tokime K, Kakeda M, et al. IL-4/IL-13 antagonist DNA vaccination successfully suppresses Th2 type chronic dermatitis. Brit J Dermatol. 2009;160(6):1172-9.
118. Tomkinson A, Tepper J, Morton M, Bowden A, Stevens L, Harris P, et al. Inhaled vs subcutaneous effects of a dual IL-4/IL-13 antagonist in a monkey model of asthma. Allergy. 2010;65(1):69-77.
119. Wenzel S, Wilbraham D, Fuller R, Getz EB, Longphre M. Effect of an interleukin-4 variant on late phase asthmatic response to allergen challenge in asthmatic patients: results of two phase 2a studies. The Lancet. 2007;370(9596):1422-31.
120. Azzazy HM, Highsmith WE, Jr. Phage display technology: clinical applications and recent innovations. Clin Biochem. 2002;35(6):425-45.
121. Sidhu SS. Phage display in pharmaceutical biotechnology. Curr Opin Chem Biol. 2000;11(6):610-6.
122. Rodi DJ, Makowski L. Phage-display technology - finding a needle in a vast molecular haystack. Curr Opin Chem Biol. 1999;10:87-93.
123. Scott J, Smith G. Searching for peptide ligands with an epitope library. Science. 1990;249(4967):386-90.
124. Westermeier R, Marouga R. Protein Detection Methods in Proteomics Research. Biosci Rep. 2005;25(1):19-32.
125. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256(5517):495-7.
126. Borrebaeck CA. Antibodies in diagnostics - from immunoassays to protein chips. Immunol Today. 2000;21(8):379-82.
PhD Thesis – References Nayyar Ahmed
189
127. Kennett RG. Monoclonal antibodies. Hybrid myelomas--a revolution in serology and immunogenetics. Am J Hum Genet. 1979;31(5):539-47.
128. Chames P, Van Regenmortel M, Weiss E, Baty D. Therapeutic antibodies: successes, limitations and hopes for the future. Brit J Pharmacol. 2009;157(2):220-33.
129. Rockberg J, Lofblom J, Hjelm B, Uhlen M, Stahl S. Epitope mapping of antibodies using bacterial surface display. Nature methods. 2008;5(12):1039-45.
130. Siddiqui MZ. Monoclonal antibodies as diagnostics; an appraisal. Indian J Pharm Sci. 2010;72(1):12-7.
131. Rowley MJ, O'Connor K, Wijeyewickrema L. Phage display for epitope determination: a paradigm for identifying receptor-ligand interactions. Biotechnol Annu Rev. 2004;10:151-88.
132. Attarwala H. Role of antibodies in cancer targeting. J Nat Sci Biol Med. 2010;1(1):53-6.
133. Falkenberg FW. Monoclonal antibody production: problems and solutions. Res Immunol. 1998;149(6):542-7.
134. Festing S, Wilkinson R. The ethics of animal research. Talking Point on the use of animals in scientific research. EMBO reports. 2007;8(6):526-30.
135. Chen I, Ting AY. Site-specific labeling of proteins with small molecules in live cells. Curr Opin Biotechnol. 2005;16(1):35-40.
136. Hernan R, Heuermann K, Brizzard B. Multiple epitope tagging of expressed proteins for enhanced detection. BioTechniques. 2000;28(4):789-93.
137. Xu Y, Ramsland PA, Davies JM, Scealy M, Nandakumar KS, Holmdahl R, et al. Two monoclonal antibodies to precisely the same epitope of type II collagen select non-crossreactive phage clones by phage display: implications for autoimmunity and molecular mimicry. Mol Immunol. 2004;41(4):411-9.
138. Ahmed N, Dhanapala P, Sadli N, Barrow CJ, Suphioglu C. Mimtags: the use of phage display technology to produce novel protein-specific probes. J Immunol Methods. 2014;405:121-9.
139. Knittelfelder R, Riemer A, Jensen-Jarolim E. Mimotope vaccination--from allergy to cancer. Expert Opin Biol Ther. 2009;9(4):493-506.
140. Ahmed N, Dhanapala P, Suphioglu C. Identification and characterisation of a novel cytokine IL-13 antagonist for allergy treatment. Intern Med. 2013;43:1.
PhD Thesis – References Nayyar Ahmed
190
141. Ahmed N, Dhanapala P, Suphioglu C. Identification and characterization of a novel IL-4 receptor antagonist for allergy treatment. Intern Med. 2012;42, p. 1.
142. Ahmed N, Dhanapala P, Carr S, Suphioglu C. Characterisation of a novel IL-4 receptor antagonist for allergy treatment. Allergy. 2011;66, p. 509-10.
143. Bredehorst R, David K. What establishes a protein as an allergen? J Chromatogr B Biomed Sci Appl. 2001;756(1-2):33-40.
144. Nofziger C, Vezzoli V, Dossena S, Schonherr T, Studnicka J, Nofziger J, et al. STAT6 Links IL-4/IL-13 Stimulation With Pendrin Expression in Asthma and Chronic Obstructive Pulmonary Disease. Clin Pharmacol Ther. 2011;90(3):399-405.
145. Thomas P, Smart TG. HEK293 cell line: A vehicle for the expression of recombinant proteins. J Pharmacol Toxicol Methods. 2005;51(3):187-200.
146. Siegel JP, Mostowski HS. A bioassay for the measurement of human interleukin-4. J Immunol Methods. 1990;132(2):287-95.
147. InvivoGen. HEK-Blue™ IL-4/IL-13 Cells, retrieved on 27/05/2012, <http://www.invivogen.com/hek-blue-il4-il13>. 2011.
148. McKeever TM, Britton J. Diet and Asthma. Am J Resp Crit Care Med. 2004;170(7):725-9.
149. Patel S, Murray CS, Woodcock A, Simpson A, Custovic A. Dietary antioxidant intake, allergic sensitization and allergic diseases in young children. Allergy. 2009;64(12):1766-72.
150. Scorletti E, Byrne CD. Omega-3 fatty acids, hepatic lipid metabolism, and nonalcoholic fatty liver disease. Ann Rev Nutr. 2013;33:231-48.
151. Devereux G, Seaton A. Diet as a risk factor for atopy and asthma. J Allergy Clin Immunol. 2005;115(6):1109-17.
152. Black P, Sharpe S. Dietary fat and asthma: is there a connection? Eur Respir J. 1997;10(1):6-12.
153. ChemSpider. Chemical Structure Database 2015 [updated retrieved on 2/02/2015]. Available from: http://www.rsc.org/.
154. Yaqoob P. Fatty acids and the immune system: from basic science to clinical applications. Proc Nutr Soc. 2004;63(01):89-105.
155. Ergas D, Eilat E, Mendlovic S, Sthoeger ZM. n-3 fatty acids and the immune system in autoimmunity. Isr Med Assoc J. 2002;4(1):34-8.
PhD Thesis – References Nayyar Ahmed
191
156. Klemens CM, Berman DR, Mozurkewich EL. The effect of perinatal omega-3 fatty acid supplementation on inflammatory markers and allergic diseases: a systematic review. BJOG. 2011;118(8):916-25.
157. Johansson S, Wold AE, Sandberg AS. Low breast milk levels of long-chain n-3 fatty acids in allergic women, despite frequent fish intake. Clin Exp Allergy. 2011;41(4):505-15.
158. Weise C, Hilt K, Milovanovic M, Ernst D, Rühl R, Worm M. Inhibition of IgE production by docosahexaenoic acid is mediated by direct interference with STAT6 and NFκB pathway in human B cells. J Nutr Biochem. 2011;22(3):269-75.
159. Fogarty A, Lewis S, Weiss S, Britton J. Dietary vitamin E, IgE concentrations, and atopy. The Lancet. 2000;356(9241):1573-4.
160. Anandan C, Nurmatov U, Sheikh A. Omega 3 and 6 oils for primary prevention of allergic disease: systematic review and meta-analysis. Allergy. 2009;64(6):840-8.
161. Arima M, Fukuda T. Prostaglandin D2 and TH2 Inflammation in the Pathogenesis of Bronchial Asthma. Korean J Intern Med. 2011;26(1):8-18.
162. William L S, Elizabeth A M, David L D. Pharmacology of Prostaglandin Endoperoxide Synthase Isozymes 1 and 2a. Ann Ny Acad Sci. 1994;714(1):136-42.
163. Patterson E, Wall R, Fitzgerald GF, Ross RP, Stanton C. Health implications of high dietary omega-6 polyunsaturated Fatty acids. J Nutr Metab. 2012;2012:539426.
164. van Gool CJ, Thijs C, Henquet CJ, van Houwelingen AC, Dagnelie PC, Schrander J, et al. Gamma-linolenic acid supplementation for prophylaxis of atopic dermatitis--a randomized controlled trial in infants at high familial risk. Am J Clin Nutr. 2003;77(4):943-51.
165. Kitz R, Rose MA, Schonborn H, Zielen S, Bohles HJ. Impact of early dietary gamma-linolenic acid supplementation on atopic eczema in infancy. Pediatr Allergy Immunol. 2006;17(2):112-7.
166. Pettipher R, Hansel TT, Armer R. Antagonism of the prostaglandin D2 receptors DP1 and CRTH2 as an approach to treat allergic diseases. Nat Rev Drug Discov. 2007;6(4):313-25.
167. Xue L, Gyles SL, Wettey FR, Gazi L, Townsend E, Hunter MG, et al. Prostaglandin D2 causes preferential induction of proinflammatory Th2 cytokine production through an action on chemoattractant receptor-like molecule expressed on Th2 cells. J Immunol. 2005;175(10):6531-6.
PhD Thesis – References Nayyar Ahmed
192
168. Zhang MJ, Spite M. Resolvins: Anti-Inflammatory and Proresolving Mediators Derived from Omega-3 Polyunsaturated Fatty Acids. Ann Rev Nutr. 2012;32(1):null.
169. Gazdík F, Gvozdjáková A, Nádvorníková R, Repická L, Jahnová E, Kucharská J, et al. Decreased levels of coenzyme Q10 in patients with bronchial asthma. Allergy. 2002;57(9):811-4.
170. Miles MV, Putnam PE, Miles L, Tang PH, DeGrauw AJ, Wong BL, et al. Acquired coenzyme Q10 deficiency in children with recurrent food intolerance and allergies. Mitochondrion. 2011;11(1):127-35.
171. Crane FL. Biochemical functions of coenzyme Q10. J Am Coll Nutr. 2001;20(6):591-8.
172. Ye CQ, Folkers K, Tamagawa H, Pfeiffer C. A modified determination of coenzyme Q10 in human blood and CoQ10 blood levels in diverse patients with allergies. Biofactors. 1988;1(4):303-6.
173. Young AJ, Johnson S, Steffens DC, Doraiswamy PM. Coenzyme Q10: a review of its promise as a neuroprotectant. CNS Spectr. 2007;12(1):62-8.
174. Serhan CN, Hong S, Gronert K, Colgan SP, Devchand PR, Mirick G, et al. Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals. J Exp Med. 2002;196(8):1025-37.
175. Hong S, Gronert K, Devchand PR, Moussignac RL, Serhan CN. Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid in murine brain, human blood, and glial cells. Autacoids in anti-inflammation. J Biol Chem. 2003;278(17):14677-87.
176. Sun YP, Oh SF, Uddin J, Yang R, Gotlinger K, Campbell E, et al. Resolvin D1 and its aspirin-triggered 17R epimer. Stereochemical assignments, anti-inflammatory properties, and enzymatic inactivation. J Biol Chem. 2007;282(13):9323-34.
177. Spite M, Norling LV, Summers L, Yang R, Cooper D, Petasis NA, et al. Resolvin D2 is a potent regulator of leukocytes and controls microbial sepsis. Nature. 2009;461(7268):1287-91.
178. Nathan C. Points of control in inflammation. Nature. 2002;420(6917):846-52.
179. Serhan CN, Brain SD, Buckley CD, Gilroy DW, Haslett C, O'Neill LA, et al. Resolution of inflammation: state of the art, definitions and terms. FASEB J. 2007;21(2):325-32.
PhD Thesis – References Nayyar Ahmed
193
180. Savill JS, Wyllie AH, Henson JE, Walport MJ, Henson PM, Haslett C. Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. J Clin Invest. 1989;83(3):865-75.
181. Savill JS, Henson PM, Haslett C. Phagocytosis of aged human neutrophils by macrophages is mediated by a novel "charge-sensitive" recognition mechanism. J Clin Invest. 1989;84(5):1518-27.
182. Gaboury J, Woodman RC, Granger DN, Reinhardt P, Kubes P. Nitric oxide prevents leukocyte adherence: role of superoxide. Am J Physiol. 1993;265(3 Pt 2):H862-7.
183. Zhang MJ, Spite M. Resolvins: anti-inflammatory and proresolving mediators derived from omega-3 polyunsaturated fatty acids. Ann Rev Nutr. 2012;32:203-27.
184. Campbell EL, Louis NA, Tomassetti SE, Canny GO, Arita M, Serhan CN, et al. Resolvin E1 promotes mucosal surface clearance of neutrophils: a new paradigm for inflammatory resolution. FASEB J. 2007;21(12):3162-70.
185. Ariel A, Fredman G, Sun Y-P, Kantarci A, Van Dyke TE, Luster AD, et al. Apoptotic neutrophils and T cells sequester chemokines during immune response resolution through modulation of CCR5 expression. Nature Immunol. 2006;7(11):1209-16.
186. Arita M, Bianchini F, Aliberti J, Sher A, Chiang N, Hong S, et al. Stereochemical assignment, antiinflammatory properties, and receptor for the omega-3 lipid mediator resolvin E1. J Exp Med. 2005;201(5):713-22.
187. Flower RJ, Perretti M. Controlling inflammation: a fat chance? J Exp Med. 2005;201(5):671-4.
188. Arita M, Yoshida M, Hong S, Tjonahen E, Glickman JN, Petasis NA, et al. Resolvin E1, an endogenous lipid mediator derived from omega-3 eicosapentaenoic acid, protects against 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Proc Natl Acad Sci U S A. 2005;102(21):7671-6.
189. Aoki H, Hisada T, Ishizuka T, Utsugi M, Ono A, Koga Y, et al. Protective effect of resolvin E1 on the development of asthmatic airway inflammation. Biochem Biophys Res Commun. 2010;400(1):128-33.
190. Calder PC. Omega-3 Fatty Acids and Inflammatory Processes. Nutrients. 2010;2(3):355-74.
191. Arita M, Ohira T, Sun YP, Elangovan S, Chiang N, Serhan CN. Resolvin E1 selectively interacts with leukotriene B4 receptor BLT1 and ChemR23 to regulate inflammation. J Immunol. 2007;178(6):3912-7.
PhD Thesis – References Nayyar Ahmed
194
192. Wahl R. Allergy Made Simple: For Those who are Allergic - and for Those who Might Become So. Germany: Hogrefe & Huber Publishers; 2002.
193. CH B. Experimental researches on the causes and nature Catarrhuss Aestivus (hay-fever and hay-asthma). London: Balliere Tindall and Cox; 1873.
194. Abbas S, Katelaris CH, Singh AB, Raza SM, Khan MA, Rashid M, et al. World Allergy Organization Study on Aerobiology for Creating First Pollen and Mold Calendar With Clinical Significance in Islamabad, Pakistan; A Project of World Allergy Organization and Pakistan Allergy, Asthma & Clinical Immunology Centre of Islamabad. World Allergy Organ J 2012;5(9):103-10.
195. Hirst JM. AN AUTOMATIC VOLUMETRIC SPORE TRAP. Ann App Biol. 1952;39(2):257-65.
196. Behrendt H, Becker WM. Localization, release and bioavailability of pollen allergens: the influence of environmental factors. Curr Opin Immunol. 2001;13(6):709-15.
197. Staff IA, Schäppi G, Taylor PE. Localisation of allergens in ryegrass pollen and in airborne micronic particles. Protoplasma. 1999;208(1-4):47-57.
198. Greaves MW, Yamamoto S, Fairley VM. New In-vitro Test for IgE-mediated Hypersensitivity in Man. BMJ. 1972;2(5814):623-4.
199. Traidl-Hoffmann C, Kasche A, Menzel A, Jakob T, Thiel M, Ring J, et al. Impact of pollen on human health: more than allergen carriers? Int Arch Allergy Immunol. 2003;131(1):1-13.
200. Freidhoff LR, Ehrlich-Kautzky E, Grant JH, Meyers DA, Marsh DG. A study of the human immune response to Lolium perenne (rye) pollen and its components, Lol p I and Lol p II (rye I and rye II). I. Prevalence of reactivity to the allergens and correlations among skin test, IgE antibody, and IgG antibody data. J Allergy Clin Immunol. 1986;78(6):1190-201.
201. Al-Muhsen S, Clarke AE, Kagan RS. Peanut allergy: an overview. Can Med Assoc J. 2003;168(10):1279-85.
202. Johnson P, Marsh DG. Allergens from common rye grass pollen (Lolium perenne). II. The allergenic determinants and carbohydrate moiety. Immunochemistry. 1966;3(2):101-10.
203. Staff IA, Taylor PE, Smith P, Singh MB, Knox RB. Cellular localization of water soluble, allergenic proteins in rye-grass (Lolium perenne) pollen using monoclonal and specific IgE antibodies with immunogold probes. Histochem J. 1990;22(5):276-90.
PhD Thesis – References Nayyar Ahmed
195
204. Suphioglu C. Thunderstorm asthma due to grass pollen. Int Arch Allergy Immunol. 1998;116(4):253-60.
205. Griffith IJ, Smith PM, Pollock J, Theerakulpisut P, Avjioglu A, Davies S, et al. Cloning and sequencing of Lol pI, the major allergenic protein of rye-grass pollen. FEBS letters. 1991;279(2):210-5.
206. Singh MB, Hough T, Theerakulpisut P, Avjioglu A, Davies S, Smith PM, et al. Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen: intracellular targeting to the amyloplast. Proc Natl Acad Sci U S A. 1991;88(4):1384-8.
207. Bhalla PL, Swoboda I, Singh MB. Antisense-mediated silencing of a gene encoding a major ryegrass pollen allergen. Proc Natl Acad Sci U S A. 1999;96(20):11676-80.
208. Suphioglu C, Singh MB, Taylor P, Bellomo R, Holmes P, Puy R, et al. Mechanism of grass-pollen-induced asthma. The Lancet. 1992;339(8793):569-72.
209. Schramm G, Bufe A, Petersen A, Haas H, Merget R, Schlaak M, et al. Discontinuous IgE-binding epitopes contain multiple continuous epitope regions: results of an epitope mapping on recombinant Hol l 5, a major allergen from velvet grass pollen. Clin Exp Allergy. 2001;31(2):331-41.
210. Esch RE, Klapper DG. Isolation and characterization of a major cross-reactive grass group I allergenic determinant. Mol Immunol. 1989;26(6):557-61.
211. Suphioglu C, Blaher B, Rolland JM, McCluskey J, Schappi G, Kenrick J, et al. Molecular basis of IgE-recognition of Lol p 5, a major allergen of rye-grass pollen. Mol Immunol. 1998;35(5):293-305.
212. Sigari N, Ghasri H. Correlation between hs-CRP and Asthma Control Indices. Tanaffos. 2013;12(3):44-8.
213. Pollart SM, Reid MJ, Fling JA, Chapman MD, Platts-Mills TA. Epidemiology of emergency room asthma in northern California: association with IgE antibody to ryegrass pollen. J Allergy Clin Immunol. 1988;82(2):224-30.
214. Bousquet J, Van Cauwenberge P, Khaltaev N. Allergic rhinitis and its impact on asthma. J Allergy Clin Immunol. 2001;108(5 Suppl):S147-334.
215. Bousquet J, Khaltaev N, Cruz AA, Denburg J, Fokkens WJ, Togias A, et al. Allergic Rhinitis and its Impact on Asthma (ARIA) 2008 update (in collaboration with the World Health Organization, GA(2)LEN and AllerGen). Allergy. 2008;63 Suppl 86:8-160.
PhD Thesis – References Nayyar Ahmed
196
216. Annesi-Maesano I, Rouve S, Desqueyroux H, Jankovski R, Klossek JM, Thibaudon M, et al. Grass pollen counts, air pollution levels and allergic rhinitis severity. Int Arch Allergy Immunol. 2012;158(4):397-404.
217. Kemp A, Ponsonby AL, Dwyer T, Cochrane J, Pezic A, Carmichael A, et al. The interaction between early life upper respiratory tract infection and birth during the pollen season on rye-sensitized hay fever and ryegrass sensitization--a birth cohort study. Pediatr Allergy Immunol. 2009;20(6):536-44.
218. Jacobs SJ, Pezza AB, Barras V, Bye J. A new 'bio-comfort' perspective for Melbourne based on heat stress, air pollution and pollen. Int J Biometeorol. 2014;58(2):263-75.
219. Glovsky MM, Jacobson K, Zakarian S, Jensen J, Moran J, Taylor PE. Meteorological Effects May Alter the Incidence of Rye-grass Pollen Related Asthma. J Allergy Clin Immunol. 2007;119(1, Supplement):S189.
220. Grote M, Vrtala S, Niederberger V, Valenta R, Reichelt R. Expulsion of allergen-containing materials from hydrated rye grass (Lolium perenne) pollen revealed by using immunogold field emission scanning and transmission electron microscopy. J Allergy Clin Immunol. 2000;105(6 Pt 1):1140-5.
221. Wark PA, Simpson J, Hensley MJ, Gibson PG. Airway inflammation in thunderstorm asthma. Clin Exp Allergy. 2002;32(12):1750-6.
222. Celenza A, Fothergill J, Kupek E, Shaw RJ. Thunderstorm associated asthma: a detailed analysis of environmental factors. BMJ 1996;312(7031):604-7.
223. Wardman AE, Stefani D, MacDonald JC. Thunderstorm-associated asthma or shortness of breath epidemic: a Canadian case report. Can Respir J. 2002;9(4):267-70.
224. Grundstein A, Sarnat SE, Klein M, Shepherd M, Naeher L, Mote T, et al. Thunderstorm associated asthma in Atlanta, Georgia. Thorax. 2008;63(7):659-60.
225. Jutel M, Jaeger L, Suck R, Meyer H, Fiebig H, Cromwell O. Allergen-specific immunotherapy with recombinant grass pollen allergens. J Allergy Clin Immunol. 2005;116(3):608-13.
226. Calderon M, Brandt T. Treatment of grass pollen allergy: focus on a standardized grass allergen extract - Grazax(R). Ther Clin Risk Manag. 2008;4(6):1255-60.
227. Winther L, Malling HJ, Mosbech H. Allergen-specific immunotherapy in birch- and grass-pollen-allergic rhinitis. II. Side-effects. Allergy. 2000;55(9):827-35.
228. Wallner M, Briza P, Thalhamer J, Ferreira F. Specific immunotherapy in pollen allergy. Curr Opin Mol Ther. 2007;9(2):160-7.
PhD Thesis – References Nayyar Ahmed
197
229. Pauli G, Malling HJ. Allergen-specific immunotherapy with recombinant allergens. Curr Top Microbiol Immunol. 2011;352:43-54.
230. Khinchi MS, Poulsen LK, Carat F, Andre C, Hansen AB, Malling HJ. Clinical efficacy of sublingual and subcutaneous birch pollen allergen-specific immunotherapy: a randomized, placebo-controlled, double-blind, double-dummy study. Allergy. 2004;59(1):45-53.
231. Asher I, Mahlab-Guri K, Sthoeger Z. The immunological mechanisms contributing to the clinical efficacy of allergen specific immunotherapy (SIT) in allergic diseases. Harefuah. 2013;152(9):529-33, 64, 63.
232. Passalacqua G, Canonica GW. Long-lasting clinical efficacy of allergen specific immunotherapy. Allergy. 2002;57(4):275-6.
233. Ohashi Y, Nakai Y, Tanaka A, Kakinoki Y, Washio Y, Nakai Y. Allergen-specific immunotherapy for allergic rhinitis: a new insight into its clinical efficacy and mechanism. Acta oto-laryngologica Supplementum. 1998;538:178-90.
234. Demoly P, Calderon MA. Dosing and efficacy in specific immunotherapy. Allergy. 2011;66 Suppl 95:38-40.
235. Duksal F, Akcay A, Becerir T, Ergin A, Becerir C, Guler N. Rising trend of allergic rhinitis prevalence among Turkish schoolchildren. Int J Pediatr Otorhinolaryngol. 2013;77(9):1434-9.
236. Banac S, Rozmanic V, Manestar K, Korotaj-Rozmanic Z, Lah-Tomulic K, Vidovic I, et al. Rising trends in the prevalence of asthma and allergic diseases among school children in the north-west coastal part of Croatia. J Asthma. 2013;50(8):810-4.
237. Wong GW, Leung TF, Ko FW. Changing prevalence of allergic diseases in the Asia-pacific region. Allergy Asthma Immunol Res. 2013;5(5):251-7.
238. Koplin J, Allen K, Gurrin L, Peters R, Lowe A, Tang M, et al. The Impact of Family History of Allergy on Risk of Food Allergy: A Population-Based Study of Infants. Int J Environ Res Publ Health. 2013;10(11):5364-77.
239. Mallen CD, Mottram S, Wynne-Jones G, Thomas E. Birth-related exposures and asthma and allergy in adulthood: a population-based cross-sectional study of young adults in North Staffordshire. J Asthma. 2008;45(4):309-12.
240. Kelly-Welch AE, Hanson EM, Boothby MR, Keegan AD. Interleukin-4 and interleukin-13 signaling connections maps. Science (New York, NY). 2003;300(5625):1527-8.
241. Venkayya R, Lam M, Willkom M, Grunig G, Corry DB, Erle DJ. The Th2 lymphocyte products IL-4 and IL-13 rapidly induce airway hyperresponsiveness
PhD Thesis – References Nayyar Ahmed
198
through direct effects on resident airway cells. Am J Respir Cell Mol Biol. 2002;26(2):202-8.
242. Chomarat P, Banchereau J. Interleukin-4 and interleukin-13: their similarities and discrepancies. Int Rev Immunol. 1998;17(1-4):1-52.
243. Schnyder B, Lugli S, Feng N, Etter H, Lutz RA, Ryffel B, et al. Interleukin-4 (IL-4) and IL-13 bind to a shared heterodimeric complex on endothelial cells mediating vascular cell adhesion molecule-1 induction in the absence of the common gamma chain. Blood. 1996;87(10):4286-95.
244. Deo SS, Mistry KJ, Kakade AM, Niphadkar PV. Role played by Th2 type cytokines in IgE mediated allergy and asthma. Lung India. 2010;27(2):66-71.
245. Williams CM, Rahman S, Hubeau C, Ma HL. Cytokine pathways in allergic disease. Toxicol Pathol. 2012;40(2):205-15.
246. Georas SN, Guo J, De Fanis U, Casolaro V. T-helper cell type-2 regulation in allergic disease. Eur Respir J. 2005;26(6):1119-37.
247. Zurawski SM, Vega F, Jr., Huyghe B, Zurawski G. Receptors for interleukin-13 and interleukin-4 are complex and share a novel component that functions in signal transduction. The EMBO journal. 1993;12(7):2663-70.
248. Murata T, Taguchi J, Puri RK, Mohri H. Sharing of receptor subunits and signal transduction pathway between the IL-4 and IL-13 receptor system. Int J Hematol. 1999;69(1):13-20.
249. Mueller TD, Zhang JL, Sebald W, Duschl A. Structure, binding, and antagonists in the IL-4/IL-13 receptor system. Biochimica et biophysica acta. 2002;1592(3):237-50.
250. Parks KW, Casale TB. Anti-immunoglobulin E monoclonal antibody administered with immunotherapy. Allergy Asthma Proc. 2006;27(2 Suppl 1):S33-6.
251. Schwartz HJ. Problems with skin testing in patients with allergic bronchopulmonary aspergillosis. Ann Allergy Asthma Immunol. 1995;75(6 Pt 1):533-5.
252. Akkerdaas JH, Wensing M, Knulst AC, Krebitz M, Breiteneder H, de Vries S, et al. How accurate and safe is the diagnosis of hazelnut allergy by means of commercial skin prick test reagents? Int Arch Allergy Immunol. 2003;132(2):132-40.
253. Focke M, Marth K, Valenta R. Molecular composition and biological activity of commercial birch pollen allergen extracts. Eur J Clin Invest. 2009;39(5):429-36.
PhD Thesis – References Nayyar Ahmed
199
254. Rosewich M, Schulze J, Eickmeier O, Telles T, Rose MA, Schubert R, et al. Tolerance induction after specific immunotherapy with pollen allergoids adjuvanted by monophosphoryl lipid A in children. Clin Exp Immunol. 2010;160(3):403-10.
255. Kopp HG, Hooper AT, Shmelkov SV, Rafii S. Beta-galactosidase staining on bone marrow. The osteoclast pitfall. Histol Histopathol. 2007;22(9):971-6.
256. Smith J, Kontermann RE, Embleton J, Kumar S. Antibody phage display technologies with special reference to angiogenesis. FASEB J. 2005;19(3):331-41.
257. Gnanasekar M, Rao KVN, He Y-X, Mishra PK, Nutman TB, Kaliraj P, et al. Novel Phage Display-Based Subtractive Screening To Identify Vaccine Candidates of Brugia malayi. Infect Immun. 2004;72(8):4707-15.
258. Tikunova NV, Morozova VV. Phage display on the base of filamentous bacteriophages: application for recombinant antibodies selection. Acta naturae. 2009;1(3):20-8.
259. Devlin JJ, Panganiban LC, Devlin PE. Random peptide libraries: a source of specific protein binding molecules. Science. 1990;249(4967):404-6.
260. Gazarian T, Selisko B, Herion P, Gazarian K. Isolation and structure-functional characterization of phage display library-derived mimotopes of noxiustoxin, a neurotoxin of the scorpion Centruroides noxius Hoffmann. Mol Immunol. 2000;37(12-13):755-66.
261. Bloemen K, Verstraelen S, Van Den Heuvel R, Witters H, Nelissen I, Schoeters G. The allergic cascade: review of the most important molecules in the asthmatic lung. Immunol Lett. 2007;113(1):6-18.
262. Valenta R, Campana R, Marth K, van Hage M. Allergen-specific immunotherapy: from therapeutic vaccines to prophylactic approaches. J Intern Med. 2012;272(2):144-57.
263. Moverare R, Elfman L, Vesterinen E, Metso T, Haahtela T. Development of new IgE specificities to allergenic components in birch pollen extract during specific immunotherapy studied with immunoblotting and Pharmacia CAP System. Allergy. 2002;57(5):423-30.
264. Keegan AD, Beckmann MP, Park LS, Paul WE. The IL-4 receptor: biochemical characterization of IL-4-binding molecules in a T cell line expressing large numbers of receptors. J Immunol. 1991;146(7):2272-9.
265. Pande J, Szewczyk MM, Grover AK. Phage display: concept, innovations, applications and future. Biotechnol Adv. 2010;28(6):849-58.
PhD Thesis – References Nayyar Ahmed
200
266. Paschke M. Phage display systems and their applications. Appl Microbiol Biotechnol. 2006;70(1):2-11.
267. Sidhu SS. Phage display in pharmaceutical biotechnology. Current Opinion in Biotechnology. 2000;11(6):610-6.
268. Newton J, Deutscher S. Phage Peptide Display. In: Semmler W, Schwaiger M, editors. Molecular Imaging II. Handbook of Experimental Pharmacology. 185/2: Springer Berlin Heidelberg; 2008. p. 145-63.
269. Smith GP, Petrenko VA. Phage Display. Chem Rev. 1997;97(2):391-410.
270. Oldenburg KR, Loganathan D, Goldstein IJ, Schultz PG, Gallop MA. Peptide ligands for a sugar-binding protein isolated from a random peptide library. Proc Natl Acad Sci U S A. 1992;89(12):5393-7.
271. Zhao P, Grabinski T, Gao C, Skinner RS, Giambernardi T, Su Y, et al. Identification of a met-binding peptide from a phage display library. Clin Cancer Res. 2007;13(20):6049-55.
272. Kelso A. Cytokines: Principles and prospects. Immunol Cell Biol. 1998;76(4):300-17.
273. Ihle JN. Cytokine receptor signalling. Nature. 1995;377(6550):591-4.
274. Starr R, Willson TA, Viney EM, Murray LJ, Rayner JR, Jenkins BJ, et al. A family of cytokine-inducible inhibitors of signalling. Nature. 1997;387(6636):917-21.
275. Luster AD. Chemokines--chemotactic cytokines that mediate inflammation. N Engl J Med. 1998;338(7):436-45.
276. Romagnani S. Cytokines and chemoattractants in allergic inflammation. Mol Immunol. 2002;38(12-13):881-5.
277. O'Shea JJ. Jaks, STATs, cytokine signal transduction, and immunoregulation: are we there yet? Immunity. 1997;7(1):1-11.
278. Pousset F, Cremona S, Dantzer R, Kelley KW, Parnet P. IL-10 and IL-4 regulate type-I and type-II IL-1 receptors expression on IL-1 beta-activated mouse primary astrocytes. J Neurochem. 2001;79(4):726-36.
279. Gadina M, Hilton D, Johnston JA, Morinobu A, Lighvani A, Zhou YJ, et al. Signaling by type I and II cytokine receptors: ten years after. Curr Opin Immunol. 2001;13(3):363-73.
PhD Thesis – References Nayyar Ahmed
201
280. David M, Ford D, Bertoglio J, Maizel AL, Pierre J. Induction of the IL-13 receptor alpha2-chain by IL-4 and IL-13 in human keratinocytes: involvement of STAT6, ERK and p38 MAPK pathways. Oncogene. 2001;20(46):6660-8.
281. Zurawski G, de Vries JE. Interleukin 13, an interleukin 4-like cytokine that acts on monocytes and B cells, but not on T cells. Immunol Today. 1994;15(1):19-26.
282. May RD, Monk PD, Cohen ES, Manuel D, Dempsey F, Davis NHE, et al. Preclinical development of CAT-354, an IL-13 neutralizing antibody, for the treatment of severe uncontrolled asthma. Brit J Pharmacol. 2012;166(1):177-93.
283. Oettgen HC, Geha RS. IgE regulation and roles in asthma pathogenesis. J Allergy Clin Immunol. 2001;107(3):429-40.
284. Lorentz A, Wilke M, Sellge G, Worthmann H, Klempnauer J, Manns MP, et al. IL-4-Induced Priming of Human Intestinal Mast Cells for Enhanced Survival and Th2 Cytokine Generation Is Reversible and Associated with Increased Activity of ERK1/2 and c-Fos. J Immunol. 2005;174(11):6751-6.
285. Hershey GK. IL-13 receptors and signaling pathways: an evolving web. J Allergy Clin Immunol. 2003;111(4):677-90; quiz 91.
286. D'Amato G, Liccardi G, Noschese P, Salzillo A, D'Amato M, Cazzola M. Anti-IgE monoclonal antibody (omalizumab) in the treatment of atopic asthma and allergic respiratory diseases. Curr Drug Targets Inflamm Allergy. 2004;3(3):227-9.
287. van Odijk J, Ahlstedt S, Bengtsson U, Hulthen L, Borres MP. Specific IgE antibodies to peanut in western Sweden--has the occurrence of peanut allergy increased without an increase in consumption? Allergy. 2001;56(6):573-7.
288. van Odijk J, Bengtsson U, Borres MP, Hulthen L, Ahlstedt S. Specific immunoglobulin E antibodies to peanut over time in relation to peanut intake, symptoms and age. Pediatr Allergy Immunol. 2004;15(5):442-8.
289. Shields RL, Whether WR, Zioncheck K, O'Connell L, Fendly B, Presta LG, et al. Inhibition of allergic reactions with antibodies to IgE. Int Arch Allergy Immunol. 1995;107(1-3):308-12.
290. Gounni AS, Lamkhioued B, Ochiai K, Tanaka Y, Delaporte E, Capron A, et al. High-affinity IgE receptor on eosinophils is involved in defence against parasites. Nature. 1994;367(6459):183-6.
291. Joseph M, Auriault C, Capron A, Vorng H, Viens P. A new function for platelets: IgE-dependent killing of schistosomes. Nature. 1983;303(5920):810-2.
292. Capron M, Capron A. Immunoglobulin E and effector cells in schistosomiasis. Science. 1994;264(5167):1876-7.
PhD Thesis – References Nayyar Ahmed
202
293. Dunne DW, Butterworth AE, Fulford AJ, Ouma JH, Sturrock RF. Human IgE responses to Schistosoma mansoni and resistance to reinfection. Mem Inst Oswaldo Cruz. 1992;87 Suppl 4:99-103.
294. Arshad SH. Primary prevention of asthma and allergy. J Allergy Clin Immunol. 2005;116(1):3-14; quiz 5.
295. Gergen PJ. Understanding the economic burden of asthma. J Allergy Clin Immunol. 2001;107(5 Suppl):S445-8.
296. Lepski S, Brockmeyer J. Impact of dietary factors and food processing on food allergy. Mol Nutr Food Res. 2013;57(1):145-52.
297. Tedeschi A, Barcella M, Bo GA, Miadonna A. Onset of allergy and asthma symptoms in extra-European immigrants to Milan, Italy: possible role of environmental factors. Clin Exp Allergy. 2003;33(4):449-54.
298. Wang DY. Risk factors of allergic rhinitis: genetic or environmental? Ther Clin Risk Manag. 2005;1(2):115-23.
299. Blumer N, Renz H. Consumption of omega3-fatty acids during perinatal life: role in immuno-modulation and allergy prevention. J Perinat Med. 2007;35 Suppl 1:S12-8.
300. Simopoulos AP. Omega-3 fatty acids in inflammation and autoimmune diseases. J Am Coll Nutr. 2002;21(6):495-505.
301. Dunstan JA, Mori TA, Barden A, Beilin LJ, Taylor AL, Holt PG, et al. Fish oil supplementation in pregnancy modifies neonatal allergen-specific immune responses and clinical outcomes in infants at high risk of atopy: a randomized, controlled trial. J Allergy Clin Immunol. 2003;112(6):1178-84.
302. Furuhjelm C, Warstedt K, Larsson J, Fredriksson M, Bottcher MF, Falth-Magnusson K, et al. Fish oil supplementation in pregnancy and lactation may decrease the risk of infant allergy. Acta paediatrica (Oslo, Norway : 1992). 2009;98(9):1461-7.
303. Lauritzen L, Kjaer TM, Fruekilde MB, Michaelsen KF, Frokiaer H. Fish oil supplementation of lactating mothers affects cytokine production in 2 1/2-year-old children. Lipids. 2005;40(7):669-76.
304. Gottrand F. Long-chain polyunsaturated fatty acids influence the immune system of infants. J Nutr. 2008;138(9):1807s-12s.
305. Burks AW, Tang M, Sicherer S, Muraro A, Eigenmann PA, Ebisawa M, et al. ICON: food allergy. J Allergy Clin Immunol. 2012;129(4):906-20.
306. West CE, Videky DJ, Prescott SL. Role of diet in the development of immune tolerance in the context of allergic disease. Curr Opin Pediatr. 2010;22(5):635-41.
PhD Thesis – References Nayyar Ahmed
203
307. Kremmyda LS, Vlachava M, Noakes PS, Diaper ND, Miles EA, Calder PC. Atopy risk in infants and children in relation to early exposure to fish, oily fish, or long-chain omega-3 fatty acids: a systematic review. Clin Rev Allergy Immunol. 2011;41(1):36-66.
308. Shek LP, Chong MF-F, Lim JY, Soh S-E, Chong Y-S. Role of Dietary Long-Chain Polyunsaturated Fatty Acids in Infant Allergies and Respiratory Diseases. Clin Dev Immunol. 2012;2012:8.
309. van den Elsen L, Garssen J, Willemsen L. Long chain N-3 polyunsaturated fatty acids in the prevention of allergic and cardiovascular disease. Curr Pharm Des. 2012;18(16):2375-92.
310. Rosell MS, Lloyd-Wright Z, Appleby PN, Sanders TA, Allen NE, Key TJ. Long-chain n–3 polyunsaturated fatty acids in plasma in British meat-eating, vegetarian, and vegan men. Am J Clin Nutr. 2005;82(2):327-34.
311. Zhou L, Nilsson A. Sources of eicosanoid precursor fatty acid pools in tissues. J Lipid Res. 2001;42(10):1521-42.
312. Simopoulos AP. Essential fatty acids in health and chronic disease. Am J Clin Nutr. 1999;70(3 Suppl):560s-9s.
313. Kull I, Bergstrom A, Lilja G, Pershagen G, Wickman M. Fish consumption during the first year of life and development of allergic diseases during childhood. Allergy. 2006;61(8):1009-15.
314. Alm B, Aberg N, Erdes L, Mollborg P, Pettersson R, Norvenius SG, et al. Early introduction of fish decreases the risk of eczema in infants. Arch Dis Child. 2009;94(1):11-5.
315. Atkins PC, Zweiman B, Littman B, Presti C, von Allmena C, Moskovitza A, et al. Products of arachidonic acid metabolism and the effects of cyclooxygenase inhibition on ongoing cutaneous allergic reactions in human beings. J Allergy Clin Immunol.95(3):742-7.
316. Talbot SF, Atkins PC, Goetzl EJ, Zweiman B. Accumulation of leukotriene C4 and histamine in human allergic skin reactions. J Clin Invest. 1985;76(2):650-6.
317. Massey WA, Hubbard WC, Liu MC, Kagey-Sobotka A, Cooper P, Lichtenstein LM. Profile of prostanoid release following antigen challenge in vivo in the skin of man. Brit J Dermatol. 1991;125(6):529-34.
318. Pla AF, Grange C, Antoniotti S, Tomatis C, Merlino A, Bussolati B, et al. Arachidonic Acid–Induced Ca2+ Entry Is Involved in Early Steps of Tumor Angiogenesis. Mol Cancer Res. 2008;6(4):535-45.
PhD Thesis – References Nayyar Ahmed
204
319. Inagaki N, Nagai H, Koda A. Effect of vitamin E on IgE antibody formation in mice. J Pharmacobiodyn. 1984;7(1):70-4.
320. Stubbs CD, Smith AD. The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim Biophys Acta. 1984;779(1):89-137.
321. Graber R, Sumida C, Nunez EA. Fatty acids and cell signal transduction. J Lipid Mediat Cell Signal. 1994;9(2):91-116.
322. Holgate ST. The epidemic of allergy and asthma. Nature 97(3): 103–110. 2004.
323. Kukkonen K, Savilahti E, Haahtela T, Juntunen-Backman K, Korpela R, Poussa T, et al. Probiotics and prebiotic galacto-oligosaccharides in the prevention of allergic diseases: a randomized, double-blind, placebo-controlled trial. J Allergy Clin Immunol. 2007;119(1):192-8.
324. Murdoch JR, Lloyd CM. Chronic inflammation and asthma. Mutat Res. 2010;690(1-2):24-39.
325. Suijkerbuijk AW, de Wit GA, Wijga AH, Heijmans M, Hoogendoorn M, Rutten-van Molken M, et al. Societal costs of asthma, COPD and respiratory allergy. Nederlands tijdschrift voor geneeskunde. 2013;157(46):A6562.
326. Singh AM, Busse WW. Asthma exacerbations · 2: Aetiology. Thorax. 2006;61(9):809-16.
327. Kim JM, Lin SY, Suarez-Cuervo C, Chelladurai Y, Ramanathan M, Segal JB, et al. Allergen-specific immunotherapy for pediatric asthma and rhinoconjunctivitis: a systematic review. Pediatrics. 2013;131(6):1155-67.
328. Rao CV, Janakiram NB, Mohammed A. Lipoxygenase and Cyclooxygenase Pathways and Colorectal Cancer Prevention. Curr Colorectal Cancer Rep. 2012;8(4):316-24.
329. Miles MV, Putnam PE, Miles L, Tang PH, DeGrauw AJ, Wong BL, et al. Acquired coenzyme Q10 deficiency in children with recurrent food intolerance and allergies. Mitochondrion. 2011;11(1):127-35.
330. Lee BJ, Tseng YF, Yen CH, Lin PT. Effects of coenzyme Q10 supplementation (300 mg/day) on antioxidation and anti-inflammation in coronary artery disease patients during statins therapy: a randomized, placebo-controlled trial. Nutr J. 2013;12(1):142.
331. Schmelzer C, Lindner I, Rimbach G, Niklowitz P, Menke T, Doring F. Functions of coenzyme Q10 in inflammation and gene expression. Biofactors. 2008;32(1-4):179-83.
PhD Thesis – References Nayyar Ahmed
205
332. Levy BD. Resolvin D1 and Resolvin E1 Promote the Resolution of Allergic Airway Inflammation via Shared and Distinct Molecular Counter-Regulatory Pathways. Front Immunol. 2012;3:390.
333. Aoki H, Hisada T, Ishizuka T, Utsugi M, Kawata T, Shimizu Y, et al. Resolvin E1 dampens airway inflammation and hyperresponsiveness in a murine model of asthma. Biochem Biophys Res Commun. 2008;367(2):509-15.
334. Li L, Wu Y, Wang Y, Wu J, Song L, Xian W, et al. Resolvin D1 promotes the interleukin-4-induced alternative activation in BV-2 microglial cells. J Neuroinflammation. 2014;11:72.
335. Spite M, Norling LV, Summers L, Yang R, Cooper D, Petasis NA, et al. Resolvin D2 is a potent regulator of leukocytes and controls microbial sepsis. Nature. 2009;461(7268):1287-91.
336. Lynch NR, Hagel IA, Palenque ME, Di Prisco MC, Escudero JE, Corao LA, et al. Relationship between helminthic infection and IgE response in atopic and nonatopic children in a tropical environment. J Allergy Clin Immunol. 1998;101(2 Pt 1):217-21.
337. D'Amato G, Spieksma FT, Liccardi G, Jager S, Russo M, Kontou-Fili K, et al. Pollen-related allergy in Europe. Allergy. 1998;53(6):567-78.
338. Schappi GF, Taylor PE, Pain MC, Cameron PA, Dent AW, Staff IA, et al. Concentrations of major grass group 5 allergens in pollen grains and atmospheric particles: implications for hay fever and allergic asthma sufferers sensitized to grass pollen allergens. Clin Exp Allergy. 1999;29(5):633-41.
339. Erbas B, Akram M, Dharmage SC, Tham R, Dennekamp M, Newbigin E, et al. The role of seasonal grass pollen on childhood asthma emergency department presentations. Clin Exp Allergy. 2012;42(5):799-805.
340. D'Amato G, Liccardi G, Frenguelli G. Thunderstorm-asthma and pollen allergy. Allergy. 2007;62(1):11-6.
341. Bellomo R, Gigliotti P, Treloar A, Holmes P, Suphioglu C, Singh MB, et al. Two consecutive thunderstorm associated epidemics of asthma in the city of Melbourne. The possible role of rye grass pollen. Med J Aust. 1992;156(12):834-7.
342. Frenz DA. Interpreting atmospheric pollen counts for use in clinical allergy: allergic symptomology. Ann Allergy Asthma Immunol. 2001;86(2):150-7, quiz 8.
343. Caillaud D, Thibaudon M, Martin S, Segala C, Besancenot JP, Clot B, et al. Short-term effects of airborne ragweed pollen on clinical symptoms of hay fever in a panel of 30 patients. J Investig Allergol Clin Immunol. 2014;24(4):249-56.
PhD Thesis – References Nayyar Ahmed
206
344. Smart I, Tuddenham W, Knox R. Aerobiology of Grass Pollen in the City Atmosphere of Melbourne: Effects of Weather Parameters and Pollen Sources. Aust J Bot. 1979;27(3):333-42.
345. Haberle SG, Bowman DMJS, Newnham RM, Johnston FH, Beggs PJ, Buters J, et al. The Macroecology of Airborne Pollen in Australian and New Zealand Urban Areas. PLoS ONE. 2014;9(5):e97925.
346. Snider JL, Oosterhuis DM. How does timing, duration and severity of heat stress influence pollen-pistil interactions in angiosperms? Plant Signal Behav. 2011;6(7):930-3.
347. Norris-Hill J, Emberlin J. Diurnal variation of pollen concentration in the air of north-central london. Grana. 1991;30(1):229-34.
348. Howden ML, McDonald CF, Sutherland MF. Thunderstorm asthma--a timely reminder. Med J Aust. 2011;195(9):512-3.
349. Taylor PE, Flagan RC, Valenta R, Glovsky MM. Release of allergens as respirable aerosols: A link between grass pollen and asthma. J Allergy Clin Immunol. 2002;109(1):51-6.
350. Baxi SN, Phipatanakul W. The role of allergen exposure and avoidance in asthma. Adolesc Med State Art Rev. 2010;21(1):57-71, viii-ix.
351. Burton MD, Papalia L, Eusebius NP, O'Hehir RE, Rolland JM. Characterization of the human T cell response to rye grass pollen allergens Lol p 1 and Lol p 5. Allergy. 2002;57(12):1136-44.
352. Schäppi GF, Suphioglu C, Taylor PE, Knox RB. Concentrations of the major birch tree allergen Bet v 1 in pollen and respirable fine particles in the atmosphere. J Allergy Clin Immunol. 1997;100(5):656-61.
353. Hopkin J. Immune and genetic aspects of asthma, allergy and parasitic worm infections: evolutionary links. Parasite Immunol. 2009;31(5):267-73.
354. Keegan K, Johnson DE, Williams LT, Hayman MJ. Isolation of an additional member of the fibroblast growth factor receptor family, FGFR-3. Proc Natl Acad Sci U S A. 1991;88(4):1095-9.
355. Irving MB, Pan O, Scott JK. Random-peptide libraries and antigen-fragment libraries for epitope mapping and the development of vaccines and diagnostics. Curr Opin Chem Biol. 2001;5(3):314-24.
356. Bottger V, Bottger A. Epitope mapping using phage display peptide libraries. Methods Mol Biol. 2009;524:181-201.
PhD Thesis – References Nayyar Ahmed
207
357. Wang LF, Yu M. Epitope mapping using phage-display random fragment libraries. Methods Mol Biol. 2009;524:315-32.
358. Huang J, Honda W. CED: a conformational epitope database. BMC Immunol. 2006;7(1):7.
359. Pacios LF, Tordesillas L, Cuesta-Herranz J, Compes E, Sanchez-Monge R, Palacin A, et al. Mimotope mapping as a complementary strategy to define allergen IgE-epitopes: peach Pru p 3 allergen as a model. Mol Immunol. 2008;45(8):2269-76.
360. Luzzago A, Felici F, Tramontano A, Pessi A, Cortese R. Mimicking of discontinuous epitopes by phage-displayed peptides, I. Epitope mapping of human H ferritin using a phage library of constrained peptides. Gene. 1993;128(1):51-7.
361. Ganglberger E, Grunberger K, Sponer B, Radauer C, Breiteneder H, Boltz-Nitulescu G, et al. Allergen mimotopes for 3-dimensional epitope search and induction of antibodies inhibiting human IgE. FASEB J. 2000;14(14):2177-84.
362. Jensen-Jarolim E, Ganglberger E, Leitner A, Radauer C, Scheiner O, Breiteneder H. Nonapeptides selected by phage display mimic the binding sites of monoclonal antibodies BIP1 and BIP4 on Bet v 1, the major birch pollen allergen. Int Arch Allergy Immunol. 1999;118(2-4):224-5.
363. Riemer A, Scheiner O, Jensen-Jarolim E. Allergen mimotopes. Methods (San Diego, Calif). 2004;32(3):321-7.
364. Furmonaviciene R, Tighe PJ, Clark MR, Sewell HF, Shakib F. The use of phage-peptide libraries to define the epitope specificity of a mouse monoclonal anti-Der p 1 antibody representative of a major component of the human immunoglobulin E anti-Der p 1 response. Clin Exp Allergy. 1999;29(11):1563-71.
365. Kraich M, Klein M, Patino E, Harrer H, Nickel J, Sebald W, et al. A modular interface of IL-4 allows for scalable affinity without affecting specificity for the IL-4 receptor. Biomed Central Biology. 2006;4(1):13.
366. Minton K. Allergy and Asthma: What 'drives' IL-4 versus IL-13 signalling? Nature Rev Immunol. 2008;8(3):166-7.
367. Hantusch B, Krieger S, Untersmayr E, Scholl I, Knittelfelder R, Flicker S, et al. Mapping of conformational IgE epitopes on Phl p 5a by using mimotopes from a phage display library. J Allergy Clin Immunol. 2004;114(6):1294-300.
368. Ma M, Ma J, Shi Y, Wu H, Zhao W, Huang W, et al. Prokaryotic expression, purification, and production of polyclonal antibody against novel human serum inhibited related protein I (SI1). Protein J. 2010;29(2):75-80.
PhD Thesis – References Nayyar Ahmed
208
369. Tan WF, Wang LL, Li Q, Luo Y, Na DX, Ma Z, et al. Prokaryotic expression and polyclonal antibody preparation of human novel gene CTRP4. Chinese journal of cellular and molecular immunology. 2012;28(6):614-7.
370. Saper CB. A guide to the perplexed on the specificity of antibodies. J Histochem Cytochem. 2009;57(1):1-5.
371. Kobayashi M, Yanagihara E, Hayashi T. Specificity problem of polyclonal rabbit antibody. J Clin Pathol. 1988;41(6):705-6.
372. Vira S, Mekhedov E, Humphrey G, Blank PS. Fluorescent-labeled antibodies: Balancing functionality and degree of labeling. Anal Biochem. 2010;402(2):146-50.
373. Takai H, Kato A, Nakamura T, Tachibana T, Sakurai T, Nanami M, et al. The importance of characterization of FITC-labeled antibodies used in tissue cross-reactivity studies. Acta histochemica. 2011;113(4):472-6.
374. Tungtrakanpoung R, Pitaksajjakul P, Na-Ngarm N, Chaicumpa W, Ekpo P, Saengjaruk P, et al. Mimotope of Leptospira from phage-displayed random peptide library is reactive with both monoclonal antibodies and patients' sera. Vet Microbiol. 2006;115(1-3):54-63.
375. Mullaney PB, Marks DJ, Pallavicini GM. Mimotopes and proteome analyses using human genomic and cDNA epitope phage display. Comp Funct Genomics. 2002;3(3):254-63.
376. Fehrsen J, du Plessis DH. Cross-reactive epitope mimics in a fragmented-genome phage display library derived from the rickettsia, Cowdria ruminantium. Immunotechnology. 1999;4(3-4):175-84.
377. Shang Y, Singh PR, Chisti MM, Mernaugh R, Zeng X. Immobilization of a Human Epidermal Growth Factor Receptor 2 Mimotope-Derived Synthetic Peptide on Au and Its Potential Application for Detection of Herceptin in Human Serum by Quartz Crystal Microbalance. Anal Chem. 2011;83(23):8928-36.
378. Pavan S, Berti F. Short peptides as biosensor transducers. Anal Bioanal Chem. 2012;402(10):3055-70.
379. Goldman ER, Pazirandeh MP, Mauro JM, King KD, Frey JC, Anderson GP. Phage-displayed peptides as biosensor reagents. J Mol Recognit. 2000;13(6):382-7.
380. Magliani W, Conti S, Salati A, Arseni S, Ravanetti L, Frazzi R, et al. Engineered killer mimotopes: new synthetic peptides for antimicrobial therapy. Curr Med Chem. 2004;11(13):1793-800.
381. Saphire EO, Montero M, Menendez A, van Houten NE, Irving MB, Pantophlet R, et al. Structure of a high-affinity "mimotope" peptide bound to HIV-1-neutralizing
PhD Thesis – References Nayyar Ahmed
209
antibody b12 explains its inability to elicit gp120 cross-reactive antibodies. J Mol Biol. 2007;369(3):696-709.
382. Gill K, Singh AK, Kapoor V, Nigam L, Kumar R, Holla P, et al. Development of peptide inhibitor as a therapeutic agent against head and neck squamous cell carcinoma (HNSCC) targeting p38α MAP kinase. Biochim Biophys Acta. 2013;1830(3):2763-9.
383. Lee E, Lee SJ, Koskimaki JE, Han Z, Pandey NB, Popel AS. Inhibition of breast cancer growth and metastasis by a biomimetic peptide. Sci Rep. 2014;20(4):7139.
384. Pietrzkowski Z, Mulholland G, Gomella L, Jameson BA, Wernicke D, Baserga R. Inhibition of growth of prostatic cancer cell lines by peptide analogues of insulin-like growth factor 1. Cancer research. 1993;53(5):1102-6.
385. Sillerud LO, Larson RS. Design and Structure of Peptide and Peptidomimetic Antagonists of Protein- Protein Interaction. Curr Protein Pept Sci. 2005;6(2):151-69.
386. Molek P, Strukelj B, Bratkovic T. Peptide Phage Display as a Tool for Drug Discovery: Targeting Membrane Receptors. Molecules. 2011;16(1):857-87.
387. Huang Z. Impact of impurities on IC50 values of P450 inhibitors. Drug Metab Lett. 2011;5(3):156-62.
388. Dragan CA, Hartmann RW, Bureik M. A fission yeast-based test system for the determination of IC50 values of anti-prostate tumor drugs acting on CYP21. J Enzyme Inhib Med Chem. 2006;21(5):547-56.
389. Jozwiak K, Moaddel R, Yamaguchi R, Ravichandran S, Collins JR, Wainer IW. Qualitative assessment of IC50 values of inhibitors of the neuronal nicotinic acetylcholine receptor using a single chromatographic experiment and multivariate cluster analysis. J Chromatogr B Analyt Technol Biomed Life Sci. 2005;819(1):169-74.
390. Gholivand K, Shariatinia Z, Ghadimi S, Mojahed F, Alizadegan AM, Khajeh K, et al. Acetylcholinesterase inhibition by diaza- and dioxophosphole compounds: synthesis and determination of IC50 values. J Enzyme Inhib Med Chem. 2004;19(5):403-7.
391. Caldwell GW, Yan Z, Lang W, Masucci JA. The IC(50) concept revisited. Curr Top Med Chem. 2012;12(11):1282-90.
392. Burton OT, Oettgen HC. Beyond immediate hypersensitivity: evolving roles for IgE antibodies in immune homeostasis and allergic diseases. Immunol Rev. 2011;242(1):128-43.
PhD Thesis – References Nayyar Ahmed
210
393. Gurish MF, Bryce PJ, Tao H, Kisselgof AB, Thornton EM, Miller HR, et al. IgE enhances parasite clearance and regulates mast cell responses in mice infected with Trichinella spiralis. J Immunol. 2004;172(2):1139-45.
394. Kis LL, Gerasimčik N, Salamon D, Persson EK, Nagy N, Klein G, et al. STAT6 signaling pathway activated by the cytokines IL-4 and IL-13 induces expression of the Epstein-Barr virus–encoded protein LMP-1 in absence of EBNA-2: implications for the type II EBV latent gene expression in Hodgkin lymphoma. Blood. 2011;117(1):165-74.
395. VanMeter AR, Ehringer WD, Stillwell W, Blumenthal EJ, Jenski LJ. Aged lymphocyte proliferation following incorporation and retention of dietary omega-3 fatty acids. Mech Ageing Dev. 1994;75(2):95-114.
396. Koch C, Dolle S, Metzger M, Rasche C, Jungclas H, Ruhl R, et al. Docosahexaenoic acid (DHA) supplementation in atopic eczema: a randomized, double-blind, controlled trial. Br J Dermatol. 2008;158(4):786-92.
397. Monick MM, Robeff PK, Butler NS, Flaherty DM, Carter AB, Peterson MW, et al. Phosphatidylinositol 3-kinase activity negatively regulates stability of cyclooxygenase 2 mRNA. J Biol Chem. 2002;277(36):32992-3000.
398. Miles EA, Aston L, Calder PC. In vitro effects of eicosanoids derived from different 20-carbon fatty acids on T helper type 1 and T helper type 2 cytokine production in human whole-blood cultures. Clin Exp Allergy. 2003;33(5):624-32.
399. Bagga D, Wang L, Farias-Eisner R, Glaspy JA, Reddy ST. Differential effects of prostaglandin derived from ω-6 and ω-3 polyunsaturated fatty acids on COX-2 expression and IL-6 secretion. Proc Natl Acad Sci U S A. 2003;100(4):1751-6.
400. Adegboyega PA, Ololade O. Immunohistochemical expression of cyclooxygenase-2 in normal kidneys. Appl Immunohistochem Mol Morphol. 2004;12(1):71-4.
401. HARRIS RC. Cyclooxygenase-2 in the Kidney. J Am Soc Nephrol. 2000;11(12):2387-94.
402. Fedyk ER, Phipps RP. Prostaglandin E2 receptors of the EP2 and EP4 subtypes regulate activation and differentiation of mouse B lymphocytes to IgE-secreting cells. Proc Natl Acad Sci U S A. 1996;93(20):10978-83.
403. Miles EA, Calder PC. Omega-6 and omega-3 polyunsaturated fatty acids and allergic diseases in infancy and childhood. Curr Pharm Des. 2014;20(6):946-53.
404. Serhan CN, Gotlinger K, Hong S, Arita M. Resolvins, docosatrienes, and neuroprotectins, novel omega-3-derived mediators, and their aspirin-triggered
PhD Thesis – References Nayyar Ahmed
211
endogenous epimers: an overview of their protective roles in catabasis. Prostaglandins Other Lipid Mediat. 2004;73(3-4):155-72.
405. Puc M, Puc MI. Allergenic airborne grass pollen in Szczecin, Poland. Ann Agr Env Med. 2004;11(2):237-44.
406. van Zijll de Jong E, Guthridge KM, Spangenberg GC, Forster JW. Sequence Analysis of SSR-Flanking Regions Identifies Genome Affinities between Pasture Grass Fungal Endophyte Taxa. Int J Evol Biol. 2011;2011:921312.
407. Chauhan SVS, Goyal R. Pollen calendar of Agra city with special reference to allergenic significance. Journal of Environmental Biology. 2006;27(2):p275.
408. Jackson SL, Bayliss KL. Spore traps need improvement to fulfil plant biosecurity requirements. Plant Pathol. 2011;60(5):801-10.
409. Chen J, Saunders SC, Crow TR, Naiman RJ, Brosofske KD, Mroz GD, et al. Microclimate in Forest Ecosystem and Landscape Ecology: Variations in local climate can be used to monitor and compare the effects of different management regimes. BioScience. 1999;49(4):288-97.
410. Schäppi G, Taylor P, Kenrick J, Staff I, Suphioglu C. Predicting the grass pollen count from meteorological data with regard to estimating the severity of hayfever symptoms in Melbourne (Australia). Aerobiologia. 1998;14(1):29-37.
411. Hayden TJ, Muscatello DJ. Increased presentations to emergency departments for asthma associated with rye grass pollen season in inland NSW. N S W Public Health Bull. 2011;22(7-8):154-8.
412. Tominaga M, Barbosa SR, Poletti EF, Zukerman-Schpector J, Marchetto R, Schreier S, et al. Fmoc-POAC: [(9-fluorenylmethyloxycarbonyl)-2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-c arboxylic acid]: a novel protected spin labeled beta-amino acid for peptide and protein chemistry. Chem Pharm Bull (Tokyo). 2001;49(8):1027-9.
413. Ottinger EA, Xu Q, Barany G. Intramolecular pyrophosphate formation during N alpha-9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis of peptides containing adjacent phosphotyrosine residues. Pept Res. 1996;9(5):223-8.
414. Dong N, Ma QQ, Shan AS, Lv YF, Hu W, Gu Y, et al. Novel design of short antimicrobial peptides derived from the bactericidal domain of avian beta-defensin-4. Protein Pept Lett. 2012;8:8.
415. Kitz R, Rose MA, Schubert R, Beermann C, Kaufmann A, Böhles HJ, et al. Omega-3 polyunsaturated fatty acids and bronchial inflammation in grass pollen allergy after allergen challenge. Respir Med. 2010;104(12):1793-8.
PhD Thesis – References Nayyar Ahmed
212
416. Weise C, Heunemann C, Loddenkemper C, Herz U, van Tol EA, Worm M. Dietary docosahexaenoic acid in combination with arachidonic acid ameliorates allergen-induced dermatitis in mice. Pediatr Allergy Immunol. 2011;22(5):497-504.