Expressed sequence tags analysis of major allergens producing dust mites and molecular characterization of their allergens

farinae as an importance source of house dust mite allergens 15 1.6.3 Cross-reactivity- a common feature especially among taxonomic 1.7.2 Mite allergens- important source of indoor alle

EXPRESSED SEQUENCE TAGS ANALYSIS OF MAJOR ALLERGENS PRODUCING DUST MITES AND MOLECULAR CHARACTERIZATION

OF THEIR ALLERGENS

ONG SEOW THENG

(B Sc (Hons.), UPM)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF PAEDIATRICS

Acknowledgements

My gratitude goes to my supervisor Dr Chew Fook Tim and co-supervisor Dr Lim Saw Hoon for always being encouraging, supportive, and optimistic I also appreciate that you always put trust in my abilities and given me a lots of flexibilities

in pursuing the research work I would also like to specially thank Dr Chew for the invaluable recommendations, ideas, guidance and perseverance that you have provided that shaped this project

I am greatly indebted to Dr Shang Huishen, for his tremendous support and generous sharing of his expertise in molecular biology, for always being optimistic and calm, especially in times when things are difficult I owe a big thank-you to Dr Bi Xuezhi, for sharing his excellent experience in protein work and his prompt assistance whenever the computers are not working properly

I wish to thank everyone in Dr Chew’s and Dr Lim’s lab, past and present, for making my stay in the laboratory a pleasant one Thanks especially to Aaron, for always being there whenever we need your help and amazing us by offering better solutions than we can expect Xiaoshan, I owe you for your tremendous help in the statistical analyses Fei Ling, Tan Ching, Yang Rui, Wun Long, Kuee Theng, Sook Mei, Teng Nging and Yun Feng, I really appreciate your concern, encouragements and fruitful discussions Hai Sim, Winnie, Chye Fong, Madam Toh, Cheng Hui, Wee Kee and Xiao Le I am grateful your warm support and guidance when I first started my work Dr Adrian Loo, Dr Li Bin, Puay Ann, Kavita, Bee Leng, Gek Huey, Resma,

Lubna, Shirley, Qingqing, Hema, Saurabh, Su Yin and Kelly, thanks for your assistance and moral supports There are just too many people that I would like to express my gratitudes for their concern and dedications to the completion of this work

I may not have listed you here but you know who you are Thank you!

Last but not least, I cannot thank my family enough for their immeasurable love, support, patience, encouragement and every persistent effort to guide me to where I am now All my friends outside the lab, I thank you for reminding me of the more important aspects of life, for friendship and for lots of fun

1.4 Immunoglobulin E 9

1.4.2 Roles of IgE in laboratory diagnosis 10

1.6 House dust mites as an important cause of allergy 13

1.6.2 D farinae as an importance source of house dust mite allergens 15 1.6.3 Cross-reactivity- a common feature especially among taxonomic

1.7.2 Mite allergens- important source of indoor allergens 20 1.7.3 Isoallergens- many allergens in nature exist in several isoforms 26

1.7.4 Cross-reactivity- common feature among proteins derived from taxonomically related organisms and/or evolutionary conserved

1.8 Molecular biology for allergen research, diagnosis and treatment 30

1.8.1 Expression systems for recombinant allergens production 30 1.8.2 Recombinant allergens essential in for research and diagnosis 35 1.8.3 Molecular modification of recombinant proteins- for allergen

Chapter 2: Materials and methods

2.1 Expressed Sequence Tagging (EST) approach 50

2.1.3 Plasmid DNA isolation and sequencing 51

2.1.5 DNA sequence analysis using software package 52

2.1.7 Cataloging of ESTs into functional categories 53

2.2 Isolation and sequencing of full length putative D farinae allergens 54

2.2.1 Bacterial host strains for transformation 54 2.2.2 Computer-based characterization and analysis 56 2.2.3 Sequencing of full length putative allergen EST clones 56 2.2.4 Isolation of full length putative allergen clones 56

2.2.4.2 RT-PCR to isolate full length clones of known mite

allergens not being identified from EST collection 57 2.2.4.3 RACE for truncated EST clones and DNA fragments

2.2.4.4 Full-length cloning and sequence analysis 58

2.3 Sub-cloning of putative allergens into expression vectors 59

2.3.1 Ligation cloning of putative allergens into PET32a (+)

2.3.2 Ligation Independent Cloning (LIC) of putative allergens into

2.4 Expression of putative allergen genes in E coli 63

2.5.4 Measurement and calculation of the dot blot and inhibition results 66

2.5.5.1 Sodium Dodecyl Sulphate-Polyacrylamide Gel

2.6 Generation of mite FABP homologues deletion mutants 69

2.6.1 PCR amplification of deletion mutants for epitope localizations 69

2.6.3 Immuno-dot blots and Western blots on deletion mutants 71

2.7 Statistical analyses 72

2.9.2 Digestion and electrophoresis of genomic DNA 74

2.9.5 Hybridization and immunological detection 74

3.1 EST database generate information on dust mite genome 76

3.1.1 D farinae cDNA library construction 76

3.1.4 Contig Assembly and unigenes identification 81

3.1.8 Biological function assignments of ESTs based on

3.1.9 EST as tool for rapid identification and characterization

3.2 EST databases aid in dust mite allergy research 94

3.2.1 Identification of allergen homologues in D farinae EST collection 94

3.2.2 ESTs matching to allergen homologues from various sources 98 3.2.3

3.2.5 Identification of Non-Allergen Homologues from EST database 101 3.2.6 EST catalogue can be utilized for expression profiling 102 3.2.7 ESTs as a means of facilitating proteomic studies and

3.3.2 Expression vector pET 32a (+) as choice for recombinant

3.3.3 Protein profiles of recombinant allergens 107

3.4 Immuno-characterizations of allergenic homologues cloned from

3.4.1 Prevalence of dust mites in Singapore and Italy 111 3.4.2 Overview of IgE binding reactivity of the allergen

3.4.4 Isoallergen homologues for group 7 and group 13 allergen

identified in D farinae EST collection 151

3.4.4.1 Isoallergens in dust mite allergy 151

3.4.4.2 Group 7 allergen and its isoallergen homologue showed

significant difference in IgE binding reactivity 152 3.4.4.3 Fatty acid binding protein and its isoallergen homologues

showed significant difference in IgE binding reactivity 163 3.4.5 Allergen homologues other than mite origin- potential pan

3.4.5.1 Mal f 6 homologue in D farinae EST collection 194

3.4.5.2 Gal d 2 homologue in D farinae EST collection 197

3.4.5.4 Alt a 10 homologue in D farinae EST collection 206

3.5 IgE binding reactivity to allergen homologues can be classified into 5 categories based on IgE binding frequency and magnitude 209

Chapter 4: Conclusion and future prospects

4.1 EST catalogue generates information on dust mite genome 212 4.2 Different IgE binding patterns detected from various mite allergens 212 4.3 IgE cross-reactivity profiles of Group 7, serine proteases, FABP

4.4 Epitope analysis of FABP homologues by deletion mutants indicating the presence of a conserved immuno-dominant epitope 214

Summary

The prevalence of asthma and allergy is increasing and negatively impact the quality of life More than 90% of the allergic individuals are sensitized to dust mites, especially

the Dermatophagoides pteronyssinus and D farinae in the temperate regions, and

Blomia tropicalis in the tropics Expressed Sequence Tagging (EST) is a useful

approach for genome level transcript profiling EST database of D farinae was generated and the database generated was compared to a parallel study of B tropicalis

More than 50% of the ESTs matched a known protein in the GenBank, in which most

of them belong to functional groups related to the general house keeping proteins, such

as metabolism, gene expression and protein synthesis

About 4% (B tropicalis) and 6% (D farinae) of the ESTs encode for more

than 20 distinct allergen homologues identified from mites and other sources A total

of 20 allergen homologues including their isoforms were cloned and expressed in

Escherichia coli The IgE binding reactivity of the recombinant proteins were tested

using 55 Singaporean and 42 Italian sera which have reacted to D farinae crude

extracts Majority of the known dust mite allergens reacted to the similar patterns as their homologues in various dust mite species reported previously No significant difference was detected between the Singaporean and Italian populations for majority

of the recombinant proteins in terms of IgE binding frequency Group 2, 3, 5, 6, 7, 10,

13 and Mag 1 allergens are important allergenic proteins since they either bind IgE at high frequency and/or bind IgE at high magnitude

The rest of the proteins could also be unique and important to a specific individual Allergenic proteins with house keeping functions such as tropomyosin, proteases, heat shock proteins, arginine kinase are normally abundant and can be easily identified from various organisms crossing taxonomic kingdoms, justifying its potential to be important pan allergens Moreover, the newly identified allergen homologues from fungus, yeast and food also show 30-42% of IgE binding reactivity

in dust mite allergic sera

IgE binding and cross-reactivity of group 7 and 13 isoallergens have been studied IgE binding reactivity of group 7 isoform, DF167 is postulated to be largely attributed to the shared epitopes that most probably derived from sequence and/or structural similarity to Der f 7 (DF47) and other group 7 allergens Southern analyses indicate that the isoforms of group 13 allergen could be members of the multigenic family of fatty acid binding protein (FABP) Different IgE binding rates were observed for all the 5 FABP isoallergens, ranging from 6%-55% and these isoallergens also demonstrate some degree of cross-reactivity Deletion studies and molecular modeling

of the group 13 isoallergens suggest the presence of immuno-dominant epitope(s) within the FABP N-terminal region and/or this region is important for maintaining the proper protein folding

Understanding the allergenic components in mites is essential for effective diagnosis and immunotherapy EST approach is indeed a rapid and efficient way of

List of figures

Figure 1: Allergy mechanism: sensitization; immediate reaction; and late

Figure 2: TR and NKT cells regulate the differentiation and function of TH1

Figure 3: Taxonomic distribution of common dust mites 16

Figure 4: An outline of the generation of EST collection 44

Figure 5: Redundancy rate of ESTs generated for D farinae and B tropicalis 85

Figure 6: Determination of E-value as cut-off point using first 1000 ESTs

Figure 7: Classification of 1722 and 1432 ESTs from the D farinae and B

tropicalis cDNA libraries, respectively 91

Figure 8: Comparison of the highly redundant genes between the two dust

Figure 9: Coomassie brilliant blue-stained 15% SDS-PAGE of purified

recombinant allergens incorporated with fusion tags 110 Figure 10: Representative IgE dot blots of 43 positive sera and 7 negative sera

of 2 geographical distinct populations on the recombinant allergens 116

Figure 11: Percentage of IgE binding frequency and IgE binding strength

to D farinae, D pteronyssinus and B tropicalis recombinant allergens of Singaporean patients allergic to D farinae 117

Figure 12: Percentage of IgE binding frequency and IgE binding strength

to D farinae, D pteronyssinus and B tropicalis recombinant allergens of Italian patients allergic to D farinae 118

Figure 13: Comparison of the sensitization to D farinae, D pteronyssinus

and B tropicalis recombinant allergens between the Singaporean and Italian populations allergic to D farinae 119

Figure 14: Western blots of 4 individual sera on D farinae recombinant

Figure 15: Multiple sequences alignment of group 2 mite allergens 125

Figure 16: Multiple sequences alignment of Der f 5, Der p 5, Blo t 5 and

Figure 17: Multiple sequences alignment of Der f 8 and Der p 8 131

Figure 18: Nucleotide and deduced amino acid sequence of the DF1894 132

Figure 19: Multiple sequences alignment of the mite group 14 and Mag 1

Figure 20: A magnified version of multiple sequences alignment of the mite

Figure 21: Multiple sequences alignment of the D farinae Mag 29 allergens 139

Figure 22: Multiple sequences alignment of 18 N-terminal amino acid residues

of the mature protein of Der p 9, Der f 9 and Blo t 9 allergens 142

Figure 23: Multiple sequences alignment of Der f 9, Der p 9 and Blo t 9

Figure 24: IgE binding reactivity of Der f 9, Der p 9 and Blo t 9 tested by

55 Singaporean sera and 42 Italian sera that are sensitized to

Figure 25: Multiple sequences alignment of Der f 3 and the newly cloned

Figure 26: Multiple sequences alignment of Der f 3, Der f 6 and Der f 9 149

Figure 27: Venn diagram showing IgE binding of a total of 97 D farinae

sensitized sera to Der f 3, Der f 6 and Der f 9 allergens 150

Figure 28: Amino acid sequence alignment between DF47 and Der f 7 154

Figure 29: Amino acid sequence alignment between DF47 and DF167 155

Figure 30: Multiple sequences alignment of the group 7 allergens, DF47,

DF167, Der f 7, Der p 7, Blo t 7 and Lep d 7 156

Figure 31: Phylogenetic tree was created based on distance matrix of DF47,

DF167, Der f 7, Der p 7, Blo t 7 and Lep d 7 157

Figure 33: Dose dependent inhibition curve of IgE immunoblot for group 7

Figure 34: Inhibition of IgE binding to group 7 allergens 162

Figure 35: Multiple sequences alignment of mites FABP homologues,

Figure 36: Phylogenetic tree of all the group 13 allergens (Aca s 13, Blo t 13

and Lep d 13), mites FABP homologues (DF414, DF1096, BT796, BT1313 and BT1694) and FABPs identified from various sources including the nematode Sm14, Fh15, adipose FABP, brain FABP, heart FABP, retinoic FABP (crustacean) and liver FABP 168

Figure 37: Genomic DNA of D farinae and B tropicalis analyzed by

Figure 38: Restriction enzyme analysis of the cDNA sequence of DF414;

Figure 39: Southern blot analysis of mites FABP homologues 173

Figure 40: IgE binding frequency of the Singaporean and Italian sera towards

Figure 41: Venn diagram showing IgE binding to DF414 and DF1096;

DF414 and BT796; and BT796, BT1313 and BT1694 176

Figure 42: IgE binding reactivity of mites FABP homologues tested by 55

Singaporean sera and 42 Italian sera that are sensitized to

Figure 43: Dose dependent inhibition curve of IgE immunoblots for mites

Figure 46: IgE binding to FABP homologues and their deletion mutants 187

Figure 47: Secondary structure of DF414 based on Chemical Shift Index

Figure 48: Homology models of DF414, DF1096, BT796, BT1313 and

Figure 49: Box plots drawn based on densitometric index scored in IgE

binding assay on deletion mutants of DF414, DF1096, BT796,

Figure 52: Venn diagram showing IgE binding of a total of 120 allergic

sera to DF Gal d 2 homologue and egg white; and

Figure 53: Evaluation of cross-reactivity between DF Gal d 2 homologue,

Figure 54: A space-filled model of 10VA with IgE binding fragments 41-172

and 301-305 colored in green, with the rest of the atoms in grey 204 Figure 55: Predictive homology model of DF Gal d 2 homologue was created

using the crystal structure of ovalbumin (10VA) as template 205

Figure 56: Multiple sequences alignment of DF Alt a 10 homologue,

List of Tables

Table 1: IgE binding rates of the cloned dust mite allergens and their

Table 2: Overview of the productions of recombinant mite allergens:

from the aspects of the production yield, bioactivities and types

Table 3: Identity and percentage of homology of EST clones corresponding

Table 4: List of primers used in RACE protocol as well as obtaining full

Table 5: Synthetic oligonucleotides used in PCR to amplify the putative

allergens for pET32a (+) vector cloning 61

Table 6: Concentrations of inhibitors used in dot blot inhibition assay 65

Table 7: Synthetic oligonucleotides used as PCR primers to amplify the

deletion mutants of the mites FABP homologues 70

Table 8: Overview of the characteristics and statistical data of the

Table 9: Summary and comparisons of gene expression levels in D farinae

Table 10: Number of D farinae and B tropicalis EST copy number matching

to allergen homologues from dust mites and other sources 96

Table 11: Estimated molecular weight of the expressed allergen with the

Table 12: Profile of allergen collections of D farinae and B tropicalis 114

Table 13: Percentage of amino acid sequence homology between the

Table 14: IgE reactivity profiles of the sera selected for inhibition studies 181

Table 15: Classification of allergen homologues based on their IgE binding

rate and IgE binding strength according to the populations tested 211

List of abbreviations

Chemical and reagents

BCIP 5-bromo-4-chloro-3-indolyl phosphate

dH20 distilled water EDTA ethylenediaminetetraacetic acid IPTG isopropyl-β-thiogalactopyranoside NBT nitroblue tetrazolium

PBS phosphate-buffered saline PVDF polyvinyldiflouride

Units and Measurements

IU international unit

pH abbreviation of "potential of hydrogen"

dbEST GenBank database of Expressed Sequence Tags dsDNA double stranded deoxyribonucleic acid

EMBL European Molecular Biology Laboratory

EST expressed sequence tag FABP fatty acid binding protein

MHC major histocompatibility complex

NCBI National Center for Biotechnology Information NIH National Institution of Health

PCR polymerase chain reaction

RACE Rapid amplification cDNA ends RAST radioallergosorbent Test RNA ribodeoxyribonucleic acid RT-PCR reverse transcription- polymerase chain reaction

Chapter 1: Introduction

1.1 The immune system as a defense system

One of the most easily understood functions of the immune system is its role in protecting against foreign antigens The immune responses fall into 2 broad categories, the innate and adaptive immunities The innate immune responses act as first line of defense and are mediated by the phagocytic cells such as the monocytes, macrophages and neutrophils They engulf, present and initiate inflammatory responses using non-specific recognition systems The important difference between innate and adaptive immunity is that the latter is highly specific for a particular antigen The adaptive immune system also “remembers” the antigen and will respond more vigorously on second exposure

Lymphocytes are central to all adaptive immune responses and these cells fall into 2 basic categories, the T cells and B cells The antigen specificity of the immune system is due to both the B cell receptor and the T cell receptor (TCR) for antigens The antigen receptor for B cell is a membrane bound IgM molecule Having recognized its specific antigen, B cells proliferate and differentiate into plasma cells, which produce large amounts of antibodies that activate other parts of the immune system T cells come in several different types and they have a variety of functions T helper (TH) cells carry the CD4 marker and recognize their specific antigens in association with major histocompatibility complex (MHC) class II molecules TH cells

interferon (IFN)-γ; while TH2 cells on the other hand produce cytokines such as IL-4, IL-5, IL-13 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and are more effective at stimulating B cells to proliferate and produce antibodies (Romagnani, 1991) Another type of T cells, the cytotoxic T (TC) cells carry CD8 marker and recognize antigens in association with MHC class I molecules, and are responsible for destruction of host cells which have been infected

The B cell receptor binds antigen directly whereas the TCR recognizes antigen- derived processed peptides in the context of MHC molecules that are present on antigen presenting cells (APC) The professional APC includes B cells, dendritic cells and monocyte/macrophages

1.2 Allergy- Type I immediate hypersensitivity

When an individual has been immunologically primed, further contact with antigen will lead to secondary boosting of the immune response However, the reaction may be excessive and lead to gross tissue changes (hypersensitivity) if the antigen is present in relatively large amounts or if the humoral and cellular immune state is at a heightened level It should be emphasized that the mechanisms underlying these inappropriate reactions are those normally employed by the body in combating infections (Roitt, 1997) Coombs and Gell (1975) described 4 types of hypersensitivity reaction, to which a fifth, viz “stimulatory” was added later Types I, II, III and V hypersensitivity are antibody-mediated; the fourth is primarily mediated by T-cells and macrophages

Allergy is commonly regarded as type I or immediate hypersensitivity, in which IgE antibody responses to the allergen

1.3 Mechanism of allergy

As illustrated in figure 1, when an innocuous environmental antigen is taken up by a local APC, the antigen is processed and presented as antigenic peptides to a T helper cell Non-atopic populations will mount a low grade immunological response and produce allergen specific IgG1 and IgG4 antibodies (Kemeny et al., 1989) Their T

cells respond to the antigen with a modest degree of proliferation and production of IFN-γ which is typical of TH1 cells (Romagnani, 1991; Ebner et al., 1995; Till et al.,

1997) It is well known that IFN-γ inhibits the effects of IL-4 (Pène et al., 1988) thus

inhibits the development of TH2 cells Atopic individuals, by contrast, build up exaggerated allergen specific IgE response, a typical type I hypersensitivity response

to an antigen TH2 cells seem to be preferentially stimulated by the APCs, resulting in the release of IL-4 and IL-13 which promote the isotype switch of B cells to IgE

production (Pène et al., 1988; Finkelman et al., 1988; Punnonen et al., 1993; Emson et

al., 1998; Till et al., 1997) and IL-5 which is required for eosinophil growth and

enhances basophil histamine release (Weller, 1992; Hogan et al., 1998)

Secreted IgE is then bound to the high-affinity receptors, FcεRI on mast cells and/or basophils Upon re-exposure to allergen, cross-linking of allergen specific IgE

mediators (leukotrienes, and cytokines IL-4, IL-5 and IL-13) (Kinet, 1999) Immediate hypersensitivity reactions are then developed and the symptoms include weal and flare eruptions on the skin, itching, coughing, wheezing, sneezing, blocked nose, watery eyes, and more serious conditions such as asthma and anaphylaxis (The Allergy Report, 2000)

Late reactions begin 2 to 4 hours after exposure to allergen and can last for 24 hours before subsiding This is mainly caused by the presentation of allergens to TH2 cells, which become activated, proliferate and release proinflammatory cytokines that promote IgE production, eosinophil chemoattraction and increased numbers of mucosal mast cells Inflammatory leukocytes (e.g., neutrophils, basophils, eosinophils) are involved but the late response is primarily mediated by eosinophils and mast cells

in atopic individuals Eosinophils produce mediators (e.g major basic protein, eosinophil cationic protein and leukotrienes) that promote tissue damage associated with chronic allergic reactions (Dombrowicz and Capron, 2001; The Allergy Report, 2000)

Figure 1: Allergy mechanism: (a) sensitization; (b) immediate reaction; and (c) late

reaction (adapted from Valenta, 1999)

Since 1986, the TH1/TH2 paradigm has dominated our understanding of the pathophysiology of asthma and allergic disease However, it has become apparent in recent years that other immunologic principles are important in explaining the regulation of asthma and allergy and those new paradigms encompassing a more diverse set of cell types must be developed to explain the immunologic events that regulate these disorders

Over the past 5 years, a great deal of interest has focused on regulatory T cells that appear to control the development of autoimmune disease and transplant rejection and that might also play a critical role in controlling the expression of asthma and allergy The term “regulatory T cell (TR)” refers to cells that actively control or

suppress the function of other cells, generally in an inhibitory fashion The specific mechanisms by which the regulatory cells function or the specific characteristics of these cells are still being investigated However, the 5 most promising recent candidates of CD4 regulatory/suppressor T cells are TH3, TR1, TR, CD4+CD25+, and

natural killer (NK) T cells (Umetsu et al., 2003)

Studies have shown that T cells engineered to secrete TGF-β, in contrast to IFN-γ–secreting TH1 cells, could very effectively reduce airway inflammation and

airway hyper-reactivity (Hansen et al., 2000).Inflammation in asthma can be inhibited

not only by TGF-β–secreting cells but also by IL-10–secreting cells (Nakao et al.,

2000) As illustrated in figure 2, dendritic cells (DC) producing IL-12 and IL-18 enhance TH1 cell development, whereas DCs expressing ICOS ligand and producing IL-10 enhance the development of TH2 and TR cells TR cells and TH2 cells develop from a common precursor cell producing IL-4, but as T cells differentiate, they lose

the capacity to produce IL-4 but retain the capacity to produce IL-10 TR cells inhibit the function of both TH1 and TH2 cells NKT cells, which produce large quantities of IFN-γ and IL-4, have critical effects on TH1/TH2 cell differentiation and are required for the development of allergen-induced airway hyper-reactivity Therapies that enhance the development of regulatory T cells and inhibit the function of NKT cells in patients with allergic disease and asthma might be useful in the prevention and treatment of these disorders

Figure 2: TR and NKT cells regulate the differentiation (blue arrows) and function of

TH1 cells and TH2 cells (adapted from Umetsu et al., 2003) Green arrows indicate

promotion effect while red arrows indicate inhibitory effect

TH0 CellNKT Cell

TH3 Cell

TH2 Cell

TRCell

IL-4 producing Cell IL-4

IL-10

Inhibition

Inhibition

IL-12IL-18

Mature Dendritic Cell

Mature Dendritic Cell

IL-10ICOS-L

TH0 CellNKT Cell

TH3 Cell

TH2 Cell

TRCell

IL-4 producing Cell IL-4

IL-10

Inhibition

Inhibition

IL-12IL-18

Mature Dendritic Cell

Mature Dendritic Cell

IL-10ICOS-L

1.4 Immunoglobulin E

1.4.1 IgE and allergy

IgE was identified in 1967 as the reagin in serum that mediates the immediate-type wheal and flare reaction (Ishizaka and Ishizaka, 1967; Johansson and Benich, 1967) In

1968, the WHO international Reference Center for Immunoglobulins decided that enough critical data were available to announce the presence of a fifth immunoglobulin

isotype, IgE (Benich et al., 1968) Human IgE is an immunoglobulin of approximately

190 kDa that circulates in the blood as a monomer The concentration of IgE in serum

is the lowest of the five human immunoglobulin isotypes (0-0.0001 g/L, constituting 0.004% of total serum immunoglobulin) and is highly age dependant Serum IgE concentration is low in cord serum (<2 kIU/L) and rises with age until a person is 10-

15 years old Those with an allergic predisposition show an earlier and steeper rise Total serum IgE levels decline from the second to the eighth decades of life (Prussin and Metcalfe, 2003)

It has long been considered that IgE has evolved to play a major role in the defense against parasitic worms Many epidemiological studies from regions with high prevalence of parasitic diseases have shown that B cells producing IgE are abundant in

the sites that are prone to parasitic invasion such as skin, lungs and guts (McGarvey et

al., 1992; Santiago et al., 1998; Roitt, 1997) Joseph et al (1983) also reported the

involvement of IgE in the cell-mediated killing of schistosomes

Over the last 30 years, overproduction of IgE antibody has become closely linked to the pathogenesis of allergic disease IgE levels are often raised in allergic disease and grossly elevated in parasitic infestations When assessing children or adults for the presence of atopic disease, a raised level of IgE aids the diagnosis although a normal IgE level does not exclude atopy In children, normal IgE values vary with age, reaching adult levels in early adolescence IgE levels remain constant from the age of

20, with a slight decrease after the age of 65 to 70 (Sears et al., 1980)

Healthy, non-atopic individuals have very low levels of IgE in their serum, while patients with atopic manifestations usually have significantly increased values Population studies have shown that 70-80% of atopic patients have IgE levels higher than one standard deviation above the geometric mean for age, while only about 5% of

these patients have levels below the geometric mean (Wittig et al., 1980)

1.4.2 Roles of IgE in laboratory diagnosis

The presence of allergen specific IgE antibody supports the diagnosis of allergic disease Allergen specific IgE antibody is the most important analyte in the diagnosis

of Type I hypersensitivity reactions (Hamilton and Adkinson, 2003) Of the individuals with a clinical history consistent with allergic disease, a subset will have a positive IgE antibody test When considering the predictive value of laboratory tests, these allergen-specific IgE antibody tests can be considered true positives since specific IgE to inhalant allergens are usually negative in patients who do not experience a clinically relevant allergy (Hamilton and Adinson, 2003) Identification of pathologically

increased levels of specific IgE antibodies to various allergens in the serum often reveals the specific allergen(s) responsible for the patient’s symptoms This is important, not only for the diagnosis but also, most importantly, for determining the optimal therapy for the patient

Allergen specific IgE levels in a given individual depend on the degree and duration of exposure to both the allergen in question and to cross-reactive allergens The level of total IgE can be clinically applied to prediction of atopic allergy in small children with hereditary predisposition and bronchiolitis, to differentiation between atopic and non-atopic eczema, allergic and non-allergic asthma In a typical case of atopy, the dose of inhalant allergen necessary to induce sensitization and symptoms is

extremely low (Wahn et al., 1997) However, it should be remembered that many

individuals have positive responses to tests for allergens but show no clinical reactivity

to these allergens (Prussin and Metcalfe, 2003)

The presence of IgE antibodies does not necessarily mean clinically active disease, although such antibodies represent a risk in the appropriate circumstances However, if a patient does have a very high IgE and no evidence of a worm infection,

allergy does become increasingly likely (Roitt, 1997) On the other hand, a low or

normal IgE level does not necessarily exclude an allergic etiology Some patients may

be uniquely sensitized to only one relatively weak allergen, leading to a very limited IgE production that has no effect on the total IgE concentration (Zetterstrom and

1.5 Increasing prevalence of allergy

The prevalence of asthma and allergy is increasing over the past 30 years It is estimated that over 40% of the world population is atopic Asthma occurs in around 10-15% in the pediatric population and is estimated to affect between 100-150 million people worldwide (Johansson, 2000) The results of the first phase of the International Study of Asthma and Allergies in Childhood (ISAAC) program (1998) have revealed that the highest asthma prevalence rates were found in Australia, New Zealand, the United Kingdom, the USA and some cities in Latin America

In Singapore, 1 in 5 school children (Goh et al., 1996) and 4% among the adult population (Ng et al., 1994) were reported to have asthma In addition, more than 90%

of the patients with asthma and/or allergic rhinitis to dust mites and other inhalant

allergens are sensitized to Blomia tropicalis, Dermatophagoides pteronyssinus and D

farinae (Chew et al., 1999c)

Various hypothetical concepts have been proposed to explain the increase in prevalence of atopic diseases, including increased awareness of the diseases, improved diagnostics, genetic susceptibility (Ruffilli and Bonini, 1997), increased allergen

exposure due to atmospheric and environmental pollutions (Muranaka et al., 1986; Behrendt et al., 1992; Martinez et al., 1998) and decreased stimulation of the immune system because of improved hygiene (Strachan, 1989; Yazdanbakhsh et al., 2002)

1.6 House dust mites as an important cause of allergy

1.6.1 General aspects of mites

Mites are normal inhabitants in our environment and are abundant in house dust as well as barns and grain stores Mites are small organisms, 0.1–1 mm in size with four pairs of legs and belong to the subclass of Acari (mites and ticks), forming an important division of arthropodan class Arachnida About 40 000 different mite species are thought to exist (Nathansson, 1969) and the taxonomic positions of some common dust mites are illustrated in Figure 3

The life cycle of mites is generally consist of five stages: egg, larva, protonymph, tritonymph and adult It takes about one month to develop from egg to adult and the adult mites may grow for another one to three months depends on the environmental temperature, humidity, food and space available The most favorable conditions for dust mites are a temperature of 23-30o C and 80-90% relative humidity The house dust mites primarily feed on skin scales and dander while the storage mites feed on plants and micro-organisms (Ho and Nadchatram, 1984; Andersen 1988; Hart,

1998; Arlian et al., 1999)

Any mite species present in the human environment in more than 100/g of dust could be considered as risk factors for sensitization and asthma (Platts-Mills and De Weck, 1989) The common allergen producing mites can broadly be divided into two

exist in stored agricultural products and in farming environments However, storage mites are now also being recognized as important contributors to the allergen content

in house dust from indoor urban dwellings (Wraith et al., 1979; Iversen and Dahl,

1990) Therefore, the term dust mites have been commonly used for all free-living, non-parasitic mites that are found regularly in house dust in indoor environment

The occurrence and relative importance of different dust mites varies in different geographical regions 13 mite species have been found in house dust, 3 of which are very common in homes worldwide and are the major source of mite

allergen The most common of these species are D farinae, D pteronyssinus, and

Euroglyphus maynei, which are found in temperate climates (Arlian et al., 1992) In

tropical climates, the storage mite B tropicalis (Family Echymyopodidae) can be a prevalent mite in dwellings, along with other Pyroglyphid mites (Fernandez-Caldas et

al., 1990; Arlian et al., 1992; Chew et al., 1999) In addition, other astigmatid mites

(storage mites) can be found in homes and are a potent source of allergens Most

notable are species in the families Glycyphagidae (Glycyphagus domesticus and

Lepidoglyphus destructor), Acaridae (Tyrophagus putrescentiae and Acarus siro), and

Chortoglyphidae (Chortoglyphus ancutatus) Predaceous mites (e.g Cheyletus) and

parasitic mites of plants (Tetranychidae, spider mites and Tarsonemidae) can also be present in homes However, the significance of these as sources of indoor allergens is yet to be determined (Arlian and Platts-Mills, 2001)

1.6.2 D farinae as an importance source of house dust mite allergens

The role of mites of the genus Dermatophagoides as the single most important source

of house dust allergens was established 30 years ago (Voorhorst et al., 1964) Since

then, multiple epidemiological studies have shown strong casual links between sensitization to its allergens and the development of asthma (Arlian and Platts-Mills, 2001)

D farinae is highly prevalent in the temperate regions such as Southern and Central parts of America (Arlian et al, 1992),Poland (Solarz, 2001), Italy (Verini et

al., 2001; Bigliocchi et al., 1996), Germany (Lau et al., 1989; Kuher et al., 1994), Sweden (Wickman et al., 1991), France (Dornelas de Andrade et al., 1995), United

Kingdom (Doull et al., 1997), Portugal (Placido et al., 1996), Denmark (Mosbech et

al., 1991), Korea (Ree et al., 1997), Thailand (Malainual et al., 1995), Indonesia (Woolcock et al., 1984), Hong Kong (Leung et al., 1997) and Taiwan (Chang and Hsieh, 1989) In Singapore, B tropicalis is the most prevalent mite species identified (62% of total mites) however, D farinae is not frequently found in Singapore, only being part of a 1% of mites identified (Chew et al., 1999)

Figure 3: Taxonomic distribution of common dust mites (adapted from Olsson and

van Hage-Hamsten, 2000)

The examination of house dust mite extracts has indicated that about 4-32 different proteins with molecular weights ranging from 11 to over 100 kDa, can induce

IgE antibody in allergic patients (Arlian et al., 1987a; Arlian et al., 1987b; Tovey and Baldo, 1987; Baldo et al., 1989) Several studies subsequently revealed that most of

the sera recognized a unique combination of 1 to 7 bands, although some did have a

broader activity (Ford et al., 1989; Lin et al., 1991; Nakanishi and Shimokata, 1990)

To date, 20 different groups of allergen have been reported from dust mites, in which

17 of them are from the genus of Dermatophagoides, while the rest of the 3 allergen groups (group 12, 13 and 19) have only been reported from storage mites and/or B

tropicalis (Allergen Nomenclature, 2003; Peurta et al., 1996; Caraballo et al., 1997;

Eriksson et al., 1999; Eriksson et al., 2001)

1.6.3 Cross-reactivity- a common feature especially among taxonomic related mites

The majority of house dust mite allergic patients are co-sensitized to D pteronyssinus and D farinae (Biliotti et al., 1972) High degree of cross-reactivity among the

Dermatophagoides spp (D pteronyssinus, D farinae, D siboney and D microceras)

was also reported (Ferrandiz et al., 1997) A significant relationship between the house dust mites and E maynei has also been found where all of the suspected allergic patients showed positive skin prick test to both E maynei and the house dust mites (Kemp et al., 1997)

Some investigators have found little or no cross-reactivity among

Dermatophagoides spp and storage mites (Chew et al., 1999b; van Hage-Hamsten et

al., 1987) Griffin et al (1989) concluded that there is limited cross-reactivity among

A siro, G destructor and D pteronyssinus and these mites have common and

species-specific allergenic determinants On the other hand, Lucznska et al (1990) found that house dust mites seem to cross-react more strongly to A siro and T putrescentiae than

to L destructor In another study, Puerta et al (1991) reported that the cross-reactivity

between D farinae and B tropicalis appears to be greater than that between D farinae and L destructor A slight cross-reactivity was reported to exist between house dust mites and the mange mites, Psoroptes cuniculi and P ovis (Stewart et al., 1986)

Simultaneous sensitization to two or more allergens may originate from reaction or be caused by co-sensitization Cross-reaction occurs when an antibody originally raised against one allergen binds to a similar allergen from another source; co-sensitization takes place when different IgE antibodies bind to different allergens at the same time The distinction between cross-reaction and co-sensitization can be

cross-determined through in vitro inhibition experiments in which blocking of IgE binding

activity occur if the mite proteins are cross-reactive However, it is important to note that immunoblotting recognized two types of cross-reactivity; one due to proteins and the other due to sugars (glycans on glycoproteins or called cross-reacting carbohydrate

determinant) (Aalberse et al., 1981; Aalberse et al., 2001)

1.7 Allergen

1.7.1 General features of allergens

Allergens are antigens that stimulate the production of, and combine specifically with IgE antibodies (Blumenthal and Rosenberg, 1999) Most allergens reacting with IgE

and IgG antibodies are proteins, often with carbohydrate side chains (Aalberse et al.,

1997), but in certain circumstances pure carbohydrates have been considered to be

allergens (Aalberse et al., 2001; Fotisch et al., 1999) Nevertheless, allergens are often

derived from common and usually harmless substances such as pollens, mold spores, animal dander, dust, foods, insect venoms, and drugs (The Allergy Report, 2000)

There appear to be three restrictions for a molecule to become an allergen: (1)

it has to possess a surface to which the antibody can form a complementary surface; (2) it has to have an amino acid sequence in its backbone that is able to bind the MHC

II alleles of the responding individual; and (3) the free energy of interaction of the allergen with the antibody should be adequate to ensure binding at low concentrations (Blumenthal and Rosenberg, 1999) However, there is no unifying theory for why some proteins are allergenic and others not There are also no characteristic features of the allergens other than to be able to reach and stimulate immune cells and mast cells Any protein, therefore, may be allergenic, especially if it avoids the activation of TH2 suppressive mechanisms (Aalberse, 2000)

weeds, grasses, trees, animal dander, molds, house dust mites, parasites, insect venoms, occupational allergens, drugs, and foods Today, more than 2000 allergen (including variants and isoforms) protein or nucleotide sequences have been deposited

in the GenBank and other databases Allergenic proteins could be identified using serum containing high level of IgE antibody in combination with a number of immunochemical assays that separate proteins based on their net charge (isoelectric focusing), size (Western blot analysis), and ability to bind to IgE antibody (competitive inhibition immunoassay)

1.7.2 Mite allergens- important source of indoor allergens

Dust mites are common sources of indoor allergens (Thomas et al., 1999) besides cockroaches (Arruda et al., 2001), cats and dogs (Chapman and Wood, 2001) Mite

allergens are divided into specific groups on the basis of their biochemical composition, sequence homology and molecular weight A protein can be included in the Allergen Nomenclature if the prevalence of IgE reactivity is >5% and if it elicits IgE responses in as few as 5 patients The nomenclature of the allergens which is recommended by International Union of Immunologic Societies Subcommittee/ World Health Organization (IUIS/WHO), include the first 3 letters of the genus, the first letter

of the species and an Arabic numeral to indicate the chronological order of

purification For example, Der p 1 is the first isolate from D pteronyssinus Allergens

from different species of the same or different genus which share the common biochemical properties are considered to belong to the same group (WHO/IUS, 1994)