Isolation and characterization of allergens from curvularia lunata 1

1.2 ALLERGENS 1.2.1 Definition and various allergen types The word “Allergen” is defined as an antigen that induces IgE antibody synthesis in atopic patients in response to the allerge

CHAPTER 1:

BACKGROUND AND INTRODUCTION

1.1 HYPERSENSITIVITY REACTIONS AND ALLERGY

1.1.1 Hypersensitivity Reactions

A normal immune system is beneficial to the human body in order to differentiate self from non-self and to neutralize potentially pathogenic organisms or substances Hypersensitivity refers to undesirable (damaging, discomfort-producing and sometimes fatal) reactions produced by the normal immune system In other words, hypersensitivity refers to a pre-sensitized state of an individual being abnormally sensitive to the foreign substances causing inflammation and cellular damage Hypersensitivity reactions were classified into four types: Type I, II, III and IV, based

on the mechanisms involved (Gel and Coombs, 1975) Later Type V and VI reactions were added (Rajan, 2003) to the above classification scheme Details of various hypersensitivity reactions are highlighted in Table 1.1

Table 1.1: Various types of hypersensitivity reactions (modified from Gel and Coombs, 1975)

I IgE-mediated immediate hypersensitivity

Systemic anaphylaxis, Asthma, Eczema, Hay fever

IV Cell-mediated delayed hypersensitivity

Contact dermatitis, Tubercular lesions

V

Stimulated antibody mediated

VI

Antibody dependent cell mediated

cytotoxic hypersensitivity Parasitic helminthes infections

1.1.2 Allergy

The term as well as the concept of “allergy” was first introduced by a Viennese pediatrician, von Pirquet in 1906 (Bendiner, 1981) Allergy is used to refer to a Type I hypersensitivity reaction Out of the four major hypersensitivity reactions, allergy has the most clearly defined and unambiguous immunological as well as pathological correlation

Allergy is characterized as a hyper response of IgE antibody to environmental substances like pollen, dust mites, animal dander, fungal spores, insect venom and food Allergic conditions include allergic rhinitis, conjunctivitis, asthma, etc., causing clinical symptoms like sneezing, coughing, wheezing and breathlessness with reversible airway obstruction, urticaria and anaphylaxis (Stewart and Thompson, 1996) The initiation of the process is brought up by presentation of processed environmental antigens to nạve Th precursor cells (ThP) by antigen presenting cells (APCs) bringing selective proliferation of Th2-polarised memory cells (Th2M), eventually causing production of antigen specific IgE by the B cells Re-exposure to this particular antigen elicits acute phase response brought by cross-linking of IgE receptors (FcεRI) on mast cells or basophils causing them to degranulate This results

in release of pro-inflammatory mediators like histamine, leukotrienes and prostaglandins These in turn cause symptoms of immediate allergic reactions as mentioned above The mast cells can also cause delayed type reactions, 4 -8 hours

after the immediate responses (Holt et al., Holgate, 1999) The mediators released by

mast cells induce release of cytokines and proteases causing tissue damage The late

phase or delayed type of reaction brings about nasal congestion in allergic rhinitis and bronchial obstruction in asthma which may lead to airway hyperresponsiveness (AHR)

in future The major cell types, molecules implicated in allergic reaction and the overall mechanism underlying allergic reaction is described in Figure 1.1

Allergy is often explained in terms of “atopy” The term atopy refers to a hereditary disorder marked by the tendency to develop immediate hypersensitivity reactions to specific antigens Hence it is also referred as “atopic allergy” As atopy is a hereditary disorder, the atopic individual shows a predisposition for a Th2-polarised response which is further enhanced by factors like lack of pathogens in environment, vaccination, industrialization, clean housing and bedding (Figure 1.2) The development of atopy is a two-step process As shown in Figure 1.3, phase 1 of atopic asthma involves antigen specific immunological memory This occurs normally in childhood and results in Th0/Th2-polarized memory, increasing risk for respiratory disease The first phase is not sufficient for the disease presentation The second phase

occurs only in the individuals with persistent inflammation (Holt et al., 1999)

1.2 ALLERGENS

1.2.1 Definition and various allergen types

The word “Allergen” is defined as an antigen that induces IgE antibody synthesis in atopic patients in response to the allergen, leading to release of histamine and other pharmacological mediators of immediate hypersensitivity from mast cells and basophils (Kurup and Banerjee, 2000) Commonly, the allergens are classified into two

Figure 1.1: Molecular and Cellular mechanism of allergy (Adapted from Holt et

al.,1999)

Figure 1.2: Factors responsible for atopy (adapted from Umetsu et al., 2002)

Figure 1.3: Progression of allergic sensitization from early childhood to atopy in

adulthood (adapted from Holt et al., 1999)

types: Indoor and outdoor allergens (Boulet et al, 1997; Kerkhoff et al., 2003) Plant

pollens and fungi are the two major groups of outdoor allergens (Burge, 2000;

Kerkhoff et al., 2003) Indoor allergens are from house dust mites, dander of pets (cats and dogs), cockroaches and fungi (Burge, 2000; Kerkhoff et al., 2003) In

industrialized nations, atopic diseases affect up to 20% of the population (Kurup and Banerjee, 2000)

Biochemically, allergens are proteins, carbohydrates or glycoproteins which stimulate the immune system of the atopic individual and bind specifically to IgE produced in response to stimulation To date there are over 300 reported allergens which comprise molecules of various physiological and biochemical functions (Scheiner, 1995) Various allergens from pollens, house dust mites and cockroaches have been well studied, but the same is not true for fungal allergens (Scheiner, 1995) Although fungal allergens are important (as they are found both indoors and outdoors), very few fungal species and fungal allergens have been studied in detail for possible allergenicity

1.2.2 Recombinant allergens in allergy

Classically, allergologists used natural products such as total protein extracts for the diagnosis and treatment of allergies However, allergens prepared this way were highly heterogeneous in the mixture due to the varying amounts of allergenic and non-allergenic proteins Moreover, natural extracts had various drawbacks such as chances

of contamination from other allergen sources being prone to proteolysis, degradation and at times containing various lipopolysaccharides and endotoxins (Linhart and Valenta, 2005) With the development of molecular biology and recombinant DNA

technology, several recombinant allergens were cloned, expressed, purified and tested More than 300 allergen (nucleotide/protein) sequences are now available in Genbank (www.ncbi.nlm.nih.gov) and various databases These recombinant allergens would

soon be used in various diagnosis and treatments of allergy (Chapman et al., 2000)

These recombinant allergens may also be used to improve various forms of specific immunotherapy – SIT (Norman, 1993)

1.3 FUNGAL ALLERGY AND FUNGAL ALLERGENS

1.3.1 Fungi as environmental allergens

Fungi are eukaryotic, achlorophyllus, chitinous cell walled, unicellular/multicellular organisms which form a separate kingdom in classification (Whittaker, 1969) Fungi form a large group of organisms found in every ecological niche (Hawksworth, 2001)

Around 1.5 million species of fungi are present worldwide (Alexopoulos et al., 1996)

Based on the spore type produced, the life cycle of a typical fungus is divided into perfect (sexual) and imperfect (asexual) phases In modern terms, these states are

referred as the teleomorph and anamorph, respectively and the fungus showing both states, known as holomorph Conidium is a term used for asexual spores produced by

anamorphs of filamentous fungi Most fungi reproduce sexually by meiosis, producing spores on specialized structures such as basidia or in a specialized structure called the

ascus These types of fungi are referred as Fungi Perfecti Fungi liberate spores and

respirable mycelial fragments in large numbers Fungal species that produce airborne

spores are found under the phyla Dikaryomycota, Zygomycota and Oomycota (Horner

et al., 1995) Details of the classification of the fungal species under these phyla are shown in Table 1.2

Fungi cause a number of infectious diseases Many fungi produce toxins (Kendrick,

1985), some of which are potent carcinogens, e.g., Aflatoxins produced by Aspergillus

flavus Fungal spores have been identified as one of the sources of indoor and outdoor

allergies (Platts-Mills et al., 1996) Given their smaller size (>10µm), fungal spores can penetrate the lower respiratory tract causing allergies (Pepys, 1965; Dankaart et

al., 1991; Reponen et al., 2001) The immunological manifestations of fungal allergies

range from dermatitis, sinusitis and asthma, to bronchopulmonary mycoses,

pneumonitis and allergic alveolitis (Lehrer et al., 1983; Fink, 1998) The immune

responses in fungal allergies follow the same pattern as that of other inhalant allergens

such as pollens or house dust mites (Kauffman et al., 1995)

The most commonly found allergic fungi are Alternaria spp., Cladosporium spp.,

Epicoccum nigrum, Fusarium spp., Ganoderma spp., Penicillium spp., Aspergillus spp., etc., (Beaumont et al., 1985; Solomon and Matthews, 1988) Many yeasts and mushrooms capable of producing allergic reactions have also been reported (Horner et

al., 1995 and 1998) Generally, Aspergillus spp and Penicillium spp are considered as indoor fungi and are less commonly seen outdoors (Beaumont et al., 1985; Licorish et

al., 1985) Outdoor fungal spore counts are seen to be correlated with clinical symptoms (Malling, 1986) Most of the allergenic fungal genera belong to the class

Ascomycetes

Table 1.2: Taxonomic distribution of various airborne spores-producing fungal

genera (adapted from Horner et al., 1995)

Class Ascomycetes (including imperfect forms)

Order Dothidiales Alternaria, Cladosporium, Epicoccum Order Eurotiales……….……Aspergillus, Penicillium

Order Agaricales………Coprinus, Pleurotus, Psilocybe

Order Aphyllophorales……… Ganoderma, Merulius

Order Lycoperdales……… Calvatia, Geaster

1.3.2 Recombinant fungal allergens

As explained earlier, recombinant allergens are thought to offer towards allergy diagnosis as well as therapeutics Although, the breakthrough in recombinant allergens

is promising, compared to other allergens (like dust mites, pollen and foods), recombinant fungal allergens are less documented and are less studied To date, there are only around 90 recombinant fungal allergens submitted to the International Union

of Immunological Societies (IUIS): Allergen nomenclature sub-committee which maintains the list of available recombinant allergens (Table 1.3) Taking into account the importance of fungi as environmental allergens and the uniqueness of fungal airspora, it is of great importance to identify and study these recombinant fungal allergens in detail

1.3.3 Global prevalence of fungal allergy

Fungal spores are present worldwide and many species can be observed at most times

of the year (Horner et al., 1995; Chou et al., 2003) Worldwide, more than 80 genera

of the major fungal groups have been associated with symptoms of respiratory tract

allergy (Horner et al., 1995) Fungal spores are usually present in outdoor air

throughout the year in high numbers and frequently exceed pollen concentrations by

100 to 1,000-fold (Lehrer et al., 1983) Globally, fungal allergy is prevalent at 20 to 30% among atopic individuals and up to 6% in the general population (Portnoy et al.,

1987) Epidemiological study on 16,204 civilians in the U.S.A showed that 3.6% of

the population was sensitized to the fungus Alternaria alternata (Gergen et al., 1987)

Generally, the fungal allergic subjects are seen to have IgEs to various fungal species

Table 1.3: Fungal allergens as approved by the allergen nomenclature

committee (adapted from www.allergen.org/List.htm)

Fungal allergen name Biochemical type

X78222, U87806

X78227, P42041

Cladosporium herbarum

Asp f 11 peptidyl-prolyl isomeras 24

Aspergillus niger

Penicillium brevicompactum

Penicillium chrysogenum

(formerly P.notatum)

Penicillium citrinum

Penicillium oxalicum

Fusarium culmorum

Trichophyton rubrum

Tri r 2

Mala s 12 glucose-methanol-choline oxidoreductase 67 AJ871960

1.3.4 Prevalence of fungal spores in Singapore environment

In line with the global prevalence of fungi, an aerobiology survey conducted in

Singapore showed abundant presence of fungal spores (Tan et al., 1992) Fungal

spores were found to occur perennially in the Singapore air Numerically, the fungal spores dominated around 86-89% of the total airspora, exceeding fern and pollen spore

counts (Lim et al., 1998) Cladosporium (48%) was the most abundant spore type, followed by Didymosphaeria (31%) and Pithomyces (12%), Curvularia (5%) and

Drechslera (2%) (Lim et al., 1998) The abundance of Cladosporium and Curvularia

was consistent with that of the surveys carried out in different parts of the world but

the presence of Didymosphaeria and Pithomyces was unique as it had not been

reported elsewhere (Lim et al., 1998) The abundance of Pithomyces was different

from the fungal profile reported in the neighboring country and some other parts of the

world where it constituted less than 1% (Lim et al., 1998) This suggests that the

fungal airflora in Singapore was distinct and different on some aspects

A five year survey (June 1990-June95) was also conducted to study the indoor as well

as to follow the sporulation patterns of various fungal spores It was observed that

spores of Didymosphaeria, Pithomyces and Curvularia were present in the

environment for more than 80% of the days sampled (Figure 1.4) This data suggests that the climatic conditions of Singapore favor growth of these fungi almost all year round Distinct seasonal variations in the spore densities were observed despite the

absence of climatic seasons in Singapore (Lim et al., 1998) An average spore count of

1688 spores m-3day-1 was found while the maximum spore load was found around 19,000 spores m-3day-1 (Lim et al., 1998)