House dust mites (HDM) allergies, such as asthma and rhinitis, are well-known and widespread that have an impact on more than half of all hypersensitised patients globally. From the various groups of HDM, group 2 allergens are recognised influentially due to effecting over 80% of all HDM-allergic individuals. As individuals are unfamiliar of the levels of HDM in their households, the detection of mite allergens are of great importance. HDM reside in bedding, carpets and upholstery where they excrete allergenic compounds in their faeces that can cause allergic reactions in sensitive patients. Circumstances that direct the nature of an allergen-stimulated immunological response involve primarily the genetic makeup of an individual, but also include the allergen form and level of exposure.
A study of 19 participants of various age and ethnicity was conducted in Leicester, UK to research the local levels of exposure to HDM-allergens.
Through visits by representatives, participants had their mattress dust samples taken using the Ventiaâ„¢ Rapid Allergen test kit, which was used due to its ease of handling and speed of results. Dust samples were analysed for Group 2 allergens and additional data were collected including bedroom temperature and humidity levels, participant background information and sleeping characteristics, and also housing attributes.
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Group 2 allergens were only detected in 3 mattress dust samples, of which only 1 of the participants had an allergy disorder. No correlations could be made from the data available as results were not collected systematically, thereby providing unreliable data for analysis. However one conclusion that could be made from the results was that genetic susceptibility to hypersensitivity is the dominant factor for allergenicity.
Allergen-specific immunotherapy (SIT) is an alternative to general anti-inflammatory medication. Up to now SIT is the only therapeutic option available that can provide prolonged clinical efficacy, unlike conventional treatment options that suggest allergen evasion where possible and drugs for symptomatic relief. Even though present day SIT is clinically feasible, it is confined to treatment of particular HDM-allergens only and also restricted for use in mild to moderate allergic individuals as a result of side-effects. However increasing progressiveness in research endeavours has provided new approaches for increasing clinical efficacy. These include T-lymphocyte epitope mapping, regulatory T-cell inducement, allergoid synthesis and adjuvant preparations. These advancements in research can allow SIT to be the treatment of choice in allergic patients.
A clear affiliation exists between sensitisation to HDM and the occurrence of allergies (Platts-Mills et al., 1997; Custovic et al., 1995). Global protocols advise steering clear of mite allergens as the initial course of action to reduce clinical sensitivity (Colloff et al., 1992; Van Moerbeke, 1997).
HDM species, population statistics and allergen numbers alternate subject to geographic locality, climate conditions, moisture and property attributes. However, clear correlations cannot be formulated as epidemiological studies have not been able to provide enough evidence on patterns of allergen exposure relating to factors mentioned previously (Tsay et al., 2002).
HDM thrive in households due to a number of reasons. These include favourable temperatures for growth, comparatively high relative humidity levels, damp conditions and also number of inhabitants residing at address.
Type 1 hypersensitisation relates to about a quarter of populations in the industrialised world. The main development of sensitivity is the binding of IgE antibody molecules on effector cells by allergenic compounds. This induces secretion of inflammatory mediators that are responsible for various allergy-related symptoms (Ishizaka et al., 1966).
B cell antigenic determinants are generally structured but can also be irregular, where numerous isolated amino acid chains in the primary conformation come together on the exterior region as a result of molecular fragments folding the original protein (Laver et al., 1990; Sela, 1969). It is these regions on allergenic molecules that are identified by complementary binding sites on specific antibodies (Crameri, 2003).
Mimotopes can activate B cell signalling and stimulation without inducing allergen-specific T cell functioning (Scholl et al., 2002). Mimotopes can also provide conformational descriptions of B cell antigenic determinants and therefore allow production of allergen-specific blocking antibodies (Hantusch et al., 2004; Leitner et al., 1998) or cancerous gene products (Riemer et al., 2004; Spillner et al., 2003). As a result of wide-spread cross-reactions between HDM species, effective therapy directed towards Der p 1 and Der p2 could treat 80% of all HDM-allergic individuals worldwide (Meyer et al., 1994; van der Zee et al., 1988).
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The social and economic burden of allergenic disease within community settings is significant and includes health expenses, reduced work hours and disruption to education (Masoli et al., 2004).
Atopic people are the ones generally affected by allergies due to their genetic susceptibility to synthesise specific IgE antibodies to everyday environmental allergens. These include HDM, grass and tree pollen and food substances such as nuts.
Initially IgE antibodies are produced in sensitive people due to exposure to specific allergenic substances that results in binding of high-affinity IgE Fc receptors to basophils and mast cells. Re-exposure then leads to a biphasic-type response with first rapid hypersensitivity and then delayed inflammation (Rolland et al., 2009).
Rapid hypersensitivity occurs within minutes due to allergenic molecules binding to mast cell-attached specific IgE antibodies. This activates the release of inflammatory mediators such as histamine, prostaglandins and cytokines. A further surge of delayed inflammation, known as the late-stage response, then takes place as a result of mediator release from previously activated T-cells, eosinophils, basophils and macrophages (Kay, 2001).
Dendritic cells (DCs) are antigen-presenting cells, which take up allergens that are classed as foreign to the immune system. Allergen uptake may be facilitated by IgE-specific antibodies bound to CD23 receptors on DCs (van der Heijden et al., 1993). These are then transported to local lymph nodes where they are drained and also where they present processed antigenic material on their surface as peptides in conjunction with MHC class II molecules to T-cells who have complementary allergen receptors. Binding of specific allergenic epitopes to T-cell receptors, in conjunction with relevant cytokine stimulation, promotes antigen-specific B-cell differentiation and proliferation to antibody-secreting plasma cells.
CD4+ T-cells play an essential role in controlling the allergenic immune response and this differs in atopic and non-atopic humans. In atopic people high levels of interleukins (IL) 4, 5, 10 and 13 are produced by activated allergen-specific Th2 cells that then trigger B-cell antibody switching to IgE, along with mediator release and mucus secretions from goblet cells. However, in non-atopic individuals, stimulated allergen-specific T-cells are Th1-induced leading to increased secretions of interferon-gamma and IL-2, which in turn results in non-allergenic complementary IgG antibodies being produced (Kapsenberg et al., 1992).
The circumstances that direct the nature of an immunological response involve the genetic makeup of a person, the allergen form and level of exposure, the characteristic antigen-presenting cell and its stage of development, and finally the array of cytokines present or attracted (Gardner et al., 2003).
Dermatophagoide pteronyssinus (D. pteronyssinus) contains high concentrations of Group 1 and 2 allergens in its faeces of which Der p 1 and Der p 2 are clinically the most immunogenic (Stewart et al., 1994). Around 80 percent of sera tested from hypersensitive individuals had IgE antibodies to Der p 1 and Der p 2, compared to 20 percent who did not. There are other allergens also present in reduced amounts in extracts that can also induce elevated levels of antibody production. Furthermore, non-allergenic polypeptides may also be highly allergenic, stimulating intracellular Th1/Th2 cytokine responses, such as ferritin heavy chain molecules (Epton et al., 2002).
Der p 2 is made up of 129 amino acids subunits and has a molecular weight of 14 kDa (Chua et al., 1991). IgE-directed antibody production is amplified by cytokines IL-4 or IL-13 by binding at the C-terminal portion of Der p 2 at positions 41-80, 64-105 and 81-129 (Punnonen et al., 1993; Kobayashi et al., 1996). D. pteronyssinus and D. farinae are the leading inducers of hypersensitivity in over 80% of young adults with bronchial asthma (Heymann et al., 1989; Lin et al., 1991). Dominant allergens of these dust mites have been placed into three distinct groups. Group 1 contains Der p 1 and Der f 1, while Group 2 contains Der p 2 and Der f 2. Likewise Group 3 contains the Der p 3 and Der f 3 allergens. The genetic code of the Group 1 and 2 allergens has already been solved (Thomas et al., 1988; Dilworth et al., 1991). Interestingly it has been found that both Der p 2 and Der f 2 possess significant genetic similarities with each other (Trudinger et al., 1991; Yuuki et al., 1991).
Even though Der p 1 and Der p 2 are composed of singular genes, they are extremely polymorphic, existing in a variety of isoforms (Thomas et al., 2004). Studies report thirteen of the twenty genetic combinations of Der p 1 as being unique (Smith et al., 2001) and containing mixed cysteine and serine enzymatic activity (Takai et al., 2005). They are also potent stimulators of nitric oxide release from bronchiolar macrophages (Peake et al., 2003).
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As allergens from dust mites have both common and species-specific determinants, these can be shared with other mite families resulting in high or variable cross-reactivity (Spieksma, 1970; Thomas and Smith, 1998). It has been shown that the homology of Group 2 allergens is higher compared to those of Group 1 (Ferrandiz et al., 1998).
In 1964, D. pteronyssinus and D. farinae were first described as being the cause of asthma-like reactions (Voorhorst et al., 1969; Spieksma, 1970; Wharton, 1976). Evidence supported exposure to their allergenic properties as being an important factor for sensitisation, disease progression and aggravator of symptoms (Arlian, 1991; Colloff et al., 1992; Pope et al., 1993).
Studies on mite-allergic patients globally confirmed that allergens were the cause of symptoms of asthma, rhinitis and conjunctivitis that often exacerbated after awakening from sleep (Platts-Mills et al., 2000). However, other HDM extract substances do contribute substantially to symptoms such as the allergen-induced late-asthmatic phase or bronchial hypersensitivity (Van der Veen et al., 2001). A study conducted in Croatia reported patients with severe asthma as having elevated serum levels of both total and allergen-specific IgE to D. pteronyssinus (Kovac et al., 2007).
D. pteronyssinus also plays an essential part in triggering elevated levels of IgE antibodies, increasing the reactivity and also exposure in patients with atopic dermatitis (Katoh et al., 2004; Kuljanac, 2006).
D. pteronyssinus can also cause allergic conjunctivitis by increasing IgE antibody levels, particularly during the autumn season when house dust mite exposure is high.
HDM allergens such as Der p 1 have also been reported to have a prenatal influence on atopic dermatitis activity. A Korean study found that HDM-positive asthmatics were more likely to have been born in the autumn season (Yoo et al., 2005). Increased exposure to HDM during pregnancy could also influence the clinical outcomes of the foetus, resulting in dermatitis during the first year of life, due to a higher rate of sensitisation to house mites (Hagendorens et al., 2004).
Other studies have detailed how the degree of hypersensitivity is greater in those with allergies and that elevated HDM levels increase this degree of hypersensitivity (Proceedings of an international scientific workshop, 1998).
Bronchial asthma is a long-standing, damaging condition noticeable by significant saturation of the mucosa by lymphocytes and eosinophils. There is also an abnormal multiplication of goblet cells and elevated serum IgE levels (Robinson et al., 1992). The symptoms of asthma are brought about through the introduction of background dust mite allergens leading to hypersensitivity and persistent manifestation (Illi et al., 2006). Th2 CD4+ T cells have been connected with hypersensitivity and asthma through the generation of Th2-driven cytokine release (Walker et al., 1992; Gavett et al., 1994). However activation through this immunogenic route is still relatively unclear. It is possible that dendritic cells (DCs) that have an important intermediary role in connecting the innate and adaptive responses of the immune system play a part. They set in motion and direct which route the immune system pathway should follow (Hart et al., 1997).
T-cell characterisation and proliferation through cytokine generation and intracellular signalling pathways are brought about by specific co-stimulatory fragments displayed on their surface. Swift entry of DCs into the respiratory mucous membranes is known to result in hypersensitivity (Jahnsen et al., 2001).
CCL20 is the only known chemokine molecule that binds to the cell surface CCR6 site recognisable on undeveloped DCs (Power et al., 1997). Binding to CCR6 activates CD1a antigen-positive DCs to migrate to the bronchial mucosa where they play an important role in hypersensitivity that results in asthmatic inflammation (Dieu-Nosjean et al., 2000).
Even though antigen presentation plays a crucial role in promoting the maturation of DCs leading to induction of the immune response, other chemical substances or signal pathways may also be involved. An example of this is activation of intracellular cytokine signalling pathways via pathogen recognition receptor binding, initiating an innate immune response (Gallucci et al., 2001). However, the involvement of HDM in the process of triggering and releasing chemokine-activated DCs is unfamiliar.
Der p 1 and Der p 2 are digestive enzymes released as waste matter from D. pteronyssinus (Tovey et al., 1981). They play an important role in the adaptive response phase, establishing T-cell activity and IgE antibody production (Rawle et al., 1984; O'Brien et al., 1992). Some other incidents of HDM presentation on mite-sensitive individuals involve the protease activity of their allergens that results in abrasion of the mucosal epithelial barrier. This in turn leads to alterations in the structure and function of the membrane allowing entry of antigenic substances into the interstitial space (Herbert et al., 1995).
It has also been deduced that the cysteine proteases of Der p 1 are involved in complement activation within the epithelia, thereby triggering innate immune signals (Maruo et al., 1997). Another study mentions allergenic signal transduction mechanisms involved within the bronchiolar epithelium associated with protease activity of the allergens (Kondo et al., 2004).
HDM are also involved in production of other peptide molecules that could possibly act as ligands for pathogen recognition receptor sites. In particular for Toll-like receptors abundant on mucosal epithelial surfaces or non Toll-like receptors such as C-type lectin and Dectin-1 receptor sites.
A study conducted last year made progress in providing detailed information on how dust mite allergens are able to activate innate immunity (Rolland et al., 2009). They showed that protease activity of allergenic peptides was in fact not part of the sequence of events that led to commencement of the CCL20 signalling pathway. They suggested an exclusive pathway leading to epithelial surface activation by dust mites that did not involve Toll-like receptors. Their reason being that binding to those types of ligands was particularly insensitive in causing hypersensitisation.
It is non-Toll like Î²-glucan receptor binding by dust mite allergens that triggers stimulation of the bronchoepithelium. They showed how HDM-achieved CCL20 formation was abolished through the use of Î²-glucanase therapy and also enzymatic inhibition through the use of similar Î²-glucans compounds.
Binding of Dectin-1 by other types of antigenic substances resulted in stimulation of Spleen tyrosine kinase that is an inducer of immunological signalling processes linked to CCL20 production (Brown et al., 2003). Inhibition of this tyrosine kinase prevented HDM-initiated CCL20 formation, confirming association of a Dectin-type receptor.
The structural composition of Î²-glucan linkage was also important in the degree of inhibition and 1-3, 1-6- Î²-glucan linkage-type compounds were more potent than just the 1, 3- Î²-glucan type, putting forward a theory that structural linkage is of importance (Nathan et al., 2009).
The stage at which immunological activation by HDM is initiated has long being thought to be at the adaptive stage but is still an ongoing debate that needs to be solved to help further the understanding of hypersensitivity.
A past study used ragweed pollen substances containing inherent NADPH oxidases which they used to investigate immunogenicity at the respiratory airways. The results they attained showed capability of the allergens to activate neutrophil movement to the bronchiolar tissues as part of the innate response but however did not result in any adaptive immune processes. A major allergenic pollen compound Amb a 1 was also used that did not contain NADPH enzymatic activity and so was not able to activate even an initial immune response (Boldogh et al., 2005).
From this it can be postulated that HDM must be capable of engaging both arms of the immune response purely because innate immunity must precede activation of the adaptive phase. However the process of immunological initiation is still unknown.
The advancement of atopic disease is thought to be aggravated by surrounding and genetic elements resulting in divergence of Th1 and Th2 intracellular signalling pathways. Substantial research has led us to believe that it is the stimulation of the Th2 lymphocytic pathway induced by background allergenic substances that results in exacerbating the immune response. Specific production of IL-4, IL-5 and IL-13 cytokines is triggered by allergens that amplify Th2 signalling pathways. IL-5 is particularly effective at inducing eosinophil production and recruitment at the bronchiolar epithelium (Zimmermann et al., 2003) whereas IL-4 and IL-13 function to stimulate generation of IgE antibodies (Ngoc et al., 2005) within the bronchioles and bloodstream. However the individual roles of these cytokines in inducing signalling pathways leading to hypersensitivity and disease are not known.
HDM-specialised stimulation of the Th2 signalling pathway and the involvement of Der p 1 in leading to allergic disease states were investigated not long ago in infants between the ages of birth and 5 years (Weber-Chrysochoou et al., 2005). The results they obtained showed IL-5 influence between ages 3-5 as being correlated to hypersensitivity and asthma at year 5, but not eczema. In comparison HDM-specific IL-10, IL-13 and interferon-Î³ influences were unrelated at year 5. Also during the first 5 years after birth the levels of HDM-linked IL-5, IL-10 and IL-13 increased whereas levels of interferon-Î³ were irregular during that period.
The infants that took part in the clinical trial were chosen due to having a marked possibility of asthma and allergic disease. Children from families with a history of allergies were considered, as their background environment constituted a greater risk of HDM-associated hypersensitivity, asthma or wheezing symptoms. Also, the degree of HDM-related T-cellular stimulation in these infants was proposed to be of greater sensitivity (Murphy and Reiner, 2002).
This study showed how HDM-associated cytokine-specific signalling effects varied in infants during the ages of 18 months and 3 years and then at ages 3 to 5. Cytokine affects at 18 months did not lead to predisposition to allergic disease influences at age 5. However, IL-5 and IL-13 HDM-specific influences at age 3 were slightly correlated to levels at age 5, but not of IL-10 and interferon-Î³. Therefore allergen-related cytokine responses, particularly of interferon-Î³ need to be studied in more depth to enhance our understanding of early-onset hypersensitivity. A further point to note from the study was that HDM-associated T cell effects were related to asthma but not eczema at age 5, leading to believe that HDM-specific T cell responses may perhaps have a higher bearing on clinical expression of asthmatic symptoms than eczema.
HDM-related allergies are well-known and wide-spread that have an impact on more than half of all hypersensitised patients (Boulet et al., 1997). Group 2 HDM allergens hold considerable IgE-binding ability and activity (van der Zee et al., 1988). Der p 2 and Der f 2 do not possess N-glycosylation sites (Thomas et al., 1989) but do share genetic sequence similarity of 87% (Chua et al., 1996). Three disulphide bonds hold the allergenic structures together that are made up of two anti-parallel beta-sheets (Johannessen et al., 2005; Mueller et al., 1997). The T-cell binding sites on Der p 2 are distributed all over the surface (O'Brien et al., 1995) whereas IgE binding sites are related to Der p 2 arrangement, apart from a few IgE-specific recombinant peptide pieces (Chua et al., 1991; Ichikawa et al., 2005).
Asthmatic disease is classed into four types ranging from mild-occasional to severe-permanent (NIH Publication, 2002). Clinically confirming the disease can be accomplished by the use of spirometry in those of whom physical examinations provide a negative prognosis.
A previous study has shown that grown-up individuals with elevated levels of asthma severity have a serious type of the disease in adolescence (Limb et al., 2005).
Measurement of blood levels of total and specific-IgE has a significant part to play in diagnosing pre-teen asthma. IgE antibodies circulate inside and outside of blood vessels, where extravascularily they are found on mastocytes and basophils (Platts-Mills et al., 2001).
High serum IgE levels is known to be the case in individuals with parasitic disease (Heinzel et al., 1995). Studies have also shown that IgE concentrations differ in patients depending on intensity of immunological inducement as a result of factors such as prevalence and dose of allergen presentation (Nelde et al., 2001). Not many asthmatic patients have low IgE antibody concentrations (Burrows et al., 1989) and the likelihood of wheezing symptoms are relatively lower in infants with low IgE to nasal allergens (Simpson et al., 2005).
In this study we will investigate the levels of group 2 HDM-allergens in a small local population and then to use the understanding of the prevalence and mechanisms of action of HDM-allergens to explore the immunotherapeutic options available for long-term clinical effectiveness of allergy disorders.
Materials and Methods
5 representatives were chosen, after gaining consent from their household habitants, to go to their respective addresses and test bedding for group 2 HDM allergens using the Ventiaâ„¢ Rapid Allergen Test kit. Prior to this they were told to leave a temperature/hydrometer in the participant's bedroom, preferably overnight, before testing bedding for dust mite allergens. Also a results sheet was provided for the representatives for each participant that was to be filled in (shown in appendix).
The process for collecting and testing dust mite samples was as follows:
A plastic filter tube was inserted into the Duststreamâ„¢ collector, which was then itself securely attached to the end of a standard vacuum cleaner tube. The area of bedding to be tested was planned using A4 paper as a template. Two different areas were chosen to be tested for 60 seconds each, giving a total sampling time of 120 seconds (2 minutes). After 2 minutes of hoovering, the collector was removed for the end of the vacuum cleaner nozzle and then a bottom cap firmly attached onto the base. The testing solution vial was then snapped open and the contents poured into the open end of the collector. The top cap was then used to close the open end of the collector resulting in both ends being placed shut. The collector was then gently shaken for a minute before being left to stand for an additional 4 minutes. The Ventiaâ„¢ Rapid Allergen test cassette was then removed from its foiled wrapping and laid on a flat surface. The top cap was then taken off the collector and a dropper used to draw the dust solution. 5 drops of sample solution were then released into the sample well of the test cassette and the dropper discarded. After 10 minutes of allowing the test line to develop, the intensity of the indicator line at the "T" mark was then viewed. A dark red test line indicated high levels of HDM allergens detected. A pink/red test line indicated medium levels of HDM allergens detected. A light pink test line indicated low levels of dust mite allergens detected No test line indicated a negative result, hence no dust mite allergens detected.
Insight of the techniques involved in HDM survival can allow more appropriate control measures to be implemented. From past studies relative humidity levels have been established as being the main influence in allowing HDM to thrive (Arlian, 1992; Arlian et al., 1992). However HDM do have distinctive biological systems that can allow them to endure cold temperatures and reduced relative humidity levels for long periods of time, such as the newly hatched forms.
Increasing levels of humidity within buildings can alter the quantity of HDM and therefore removing sources of indoor moisture is advantageous in lowering exposure to them. Examples include reducing moisture from bathrooms, in kitchens where food is prepared and also limiting the amount of inhabitants residing (Colloff et al., 1992). Age of homes, dampness, thickness of glass and condensation levels in windows all need to be considered as they can affect HDM exposure (Pauli et al., 2000).
Adjustable risk influences linked to lowering of HDM allergen levels include sleeping on a new/near-new mattress, increased bedroom floor level and constant air conditioning.
In a recent European Community Respiratory Health Survey (Zock et al., 2006), HDM allergen levels were analysed and results concluded detection of Der p 1 and Der f 1 in mattress samples from 50% of homes.
Both types of HDM are common in European countries, even though most areas have either one or the other. It has also been found that D. farinae is more widespread than previously thought, and that temperature does affect the type of species that survives or exists.
The two HDM species are more prevalent in the south of Europe than in the north, however D. pteronyssinus levels depreciated more than D. farinae during the cold winter months.
HDM thrive in households due to a number of reasons that include sustenance from skin scales, favourable room temperatures for growth and high moisture levels (Arbes et al., 2003). Elevated relative humidity levels are affiliated with increased scores of Der p 1 but have no influence on Der f 1 levels as D. farinae is more capable of persevering through dry spells (Arlian and Morgan, 2003).
A connection also exists between periods of winter where temperatures are decreased and the use of central heating. In these situations D. pteronyssinus levels fall sharply compared to D. farinae (Arlian and Morgan, 2003). Another situation also exists where reasonably cold weather in countries with moderate climate accounts for a 2-fold reduction in Der p 1 numbers.
Altitude also plays a part in the presence of HDM, where high altitude diminishes allergen numbers significantly.
The analysis of household contents in relation to numbers of HDM allergens is also essential in reducing susceptibility to asthma and hypersensitivity. Nonetheless there is a degree of ambiguity and doubtfulness relating to efficacy of individual interference methods and the control of inflammatory disease (Recer, 2004).
A study revealed how 25 hypersensitive patients below the age of 11 in England were examined for IgE-specific skin reactions to 4 different HDM species. All patients were sensitive to D. pteronyssinus, 20 of them to D. farinae and also 20 to D. microceras. Significantly all HDM samples from several areas of each of the patient's households exhibited detectable levels of D. pteronyssinus, but not for the other species. Another selection of 20 hypersensitive patients of resembling ages were not tested but had coinciding susceptibility to the 4 HDM species. The results therefore indicate that circumstances adjunct to HDM exposure are influential in the progression of allergen-specific IgE reactions to HDM (Young et al., 1990).
Past studies conclude that conventionally enforced interventions like water-proof bedding coverings have no convincing bearings on reducing allergen numbers (Van Strien et al., 2003), whereas removal of carpeting has shown to be an efficient approach (Wickens et al., 2004).
Unfortunately though capable adjustments to mattresses is more challenging but essential as this is a prominent mite environment where HDM allergen numbers are found in abundance compared to other home areas of living.
Allergen-specific immunotherapy (SIT) is an alternative to anti-inflammatory medication in patients who may be prone to suffering from serious side-effects. It is available for those in whom there are obvious presentations of symptoms when in contact with specific allergenic substances along with IgE elevations.
Initially it was deduced that corrective dosage of an antigenic substance could result in a situation where therapeutic and immunological resistance to a specific allergen could predominate. Since then subcutaneous SIT (SSIT) has progressed as a form of generalised treatment and as a result guidelines have been designated (Bousquet et al., 1998).
Routine SSIT entails injecting accumulating doses of a specific allergen over a period of time proceeded by repetitive injections at a peak dosage level during a 3-5 duration (Rolland et al., 2000). Clinical trials have confirmed effectiveness in patients with rhinitis (Calderon et al., 2007) and mild asthmatic symptoms (Abramson et al., 2003). Significantly SSIT is capable of restricting development of additional sensitive reactions in mono-sensitive individuals (Purello-D'Ambrosio et al., 2001).
Unfortunately even with progression and regulation of treatment practices, SSIT is not advised for individuals with modest or serious asthma. Adjustment of SSIT, particularly allergen preparation is essential to achieve global alertness of this method of clinical treatment.
Clinical sublingual immunotherapy (SLIT) differs with traditional types of treatment options available for allergic diseases in that, not only does it alleviate symptoms; it also adjusts the characteristic advancement of inflammation (Bousquet et al., 1998; Frew, 2003).
Several clinical trials have shown SLIT to be reliable and efficient at treating hypersensitive individuals. However it can take up to two years before noticeable clinical effectiveness (Bousquet et al., 2001; Passalacqua et al., 2004).
The mechanism of action of SLIT involves inhibiting IgE-specific (allergen-activated) pathways, in addition to goblet cell proliferation, airways inflammation and also hyper-responsiveness. However the exact course of action is still unknown. It is believed that suppression of IgE results in an increase in allergen-induced IgG levels, mostly IgG4 (Akdis et al., 2006). This process is then thought to equilibrate the Th1/Th2 balance that in allergen-stimulated individuals is more towards the Th2 response (Brimnes et al., 2006).
Another study mentions IL-10 production by regulatory T-cells as being involved in suppressing allergen-induced IgE levels, while at the same time elevating IgG4 levels. Another product of regulatory T cell action, TGF-Î², is also thought to be involved. It stimulates IgA formation that is considered to down-regulate IgE levels (Jutel et al., 2003).
Further studies suggest SLIT works by altering the array of cytokines secreted by helper T cells. A number of Th2 cytokines are involved in the production and development of inflammatory disease. The disproportions between IL-4 (a Th2 cytokine) and IFN-Î³ (a Th1 cytokine) are considered to elevate IgE levels in hypersensitive humans. As a result, therapeutic effectiveness is correlated with decreases in IL-4 and IgE in those undergoing SIT (Benjaponpitak et al., 1999). It has been found that immunotherapy reduces the Der p 2-specific IFN-Î³/IL-4 ratio (O'Brien et al., 1997).
New proposals for SIT
Allergy-related conditions are an exclusive type of immunological disorders, with regards to SIT, that can presently be treated routinely and successfully. However, as mentioned previously, SIT is prone to causing unwanted side-effects that for particular allergens and chronic asthmatics, prevents periodic use. Developments in knowledge of biological mechanisms that determine the pathway and degree of immunological responses to antigen stimulation lead the way for improvements in effectiveness and therapy. The formulation type and dosage of allergenic products are especially necessary as well as simultaneous delivery of adjuvant particles and medication (Rolland et al., 2009). Antigen-presenting cell subsets diversify in relation to pattern recognition receptors and as a result can be pinpointed primarily through selective allergenic compositions and adjuvants. The differences in subset populations and activity at various bodily sites could also help in finding the most efficacious therapeutic applications. In this way particular divisions of the immunological system may also be marked with advanced SIT products.
HDM structure and activity
Cloning and sequencing of influential HDM allergenic determinants has laid an essential framework to aid conformational remodelling for peak immune regulation. Allergenic compounds are classified into groups corresponding to their architecture and function. However, mutations or isoforms including cross-reactivities of inter-related species need to be considered when producing vaccines. In recent times, the influence of conformational arrangement and function of antigenic determinants is known to be crucial in stimulating immunological responses. Crystallised images have been modelled exposing IgE epitopes on exterior surfaces (Verdino et al., 2008).
A newly-forming discipline is the identification of allergenic derivatives with consideration to determinants that bind to various pattern recognition receptors of antigen presenting cells or regulatory T cells. Contaminated allergenic products are composed of various molecular constituents. Investigation of these constituents and their immunological effect can establish applicable strategies for formulating filtered allergenic particulates for SIT. For instance it has been found recently that lecithins from pollen extracts can be expressed by CD1, inducing expansion and generation of Th1 and Th2 cells and also possibly natural killer T cells (Ageaet et al., 2005). Preserving these adjuvant-like constituents in allergenic formulations to provide successful immunological treatment warrants additional study.
In numerous situations the biochemical action, such as catalytic activity of an allergen, may direct an immunological response (Chapman et al., 2007). For instance, enzymatic activity of Der p 1 causes structural alterations of superficial B and T-cell binding sites inducing Th2-like immune reactions (Furmonaviciene et al., 2007). NAD(P)+ oxidase of pollen extracts likewise holds similar immunological effect (Dharajiya et al., 2008). Therefore, preserving catalytic activity of allergenic compounds in certain situations may not be beneficial for therapy as they may have Th2-stimulating hallmarks. Furthermore, multiple allergen-containing formulations that have intracellular enzymatic activity may cause degradation of allergenic material (Nelson, 2007).
It is largely acknowledged that efficacious therapy is affiliated with adjusted T-cell stimulation to allergenic determinants, with abnormal effects of therapy being mainly IgE-dependent. Significantly increased therapeutic allergen levels may bolster T-cell-specific anergy, elimination and also immunological alterations from Th2 to Th1-like responses (Rolland et al., 2009). As a result, production of hypoallergenic mixtures that preserve T-cell activity and tolerate high dosage administration will allow , in addition to reliability, more potent utilisation of SIT.
Chemical alterations of allergenic fragments
An original concept to reduce therapeutic side-effects involves manufacture of allergenic derivatives known as allergoids. Research of formaldehyde treatment of allergens presented signs of increased immune tolerance whilst preserving therapeutic effectiveness (Negro et al., 1999).
A separate technique for reducing IgE responsiveness to allergenic determinants is cyanide utilisation, which at intermediate pH levels, results in carbamylation of amino acid residua (Ariano et al., 2005). Such allergenic formulations have been researched for therapeutic efficacy with positive results, as reduced IgE responsiveness and increase in Th1-type cytokine levels were observable (Burastero et al., 2008). Thus immunotherapy utilising these types of hypoallergenic derivatives may increase efficacy. However, customary practice of preparing these compounds is not well systemised and differentiation of allergoids is problematic.
Recombinant allergen therapy
Enhanced modification of allergen-specific IgE is achievable through the use of recombinant technology. Genetic sequences for majority of the significant allergens have been unravelled allowing production of hypoallergenic recombinant particles determined by appropriate features of specific allergenic molecules.
For allergenic substances with potent IgE reactivities linked to their carbohydrate moieties, the use of bacteria for production of non-glycosylated allergenic compounds cater for first-line strategies, however for a number of dust mite substances, site-directed mutagenesis of crucial peptide regions for structural IgE antigenic determinants is the prudent approach. An example is breakage of molecular disulphide bonds via substitution of amino acid residues decreasing or weakening IgE reactivity of HDM-allergens such as Der p 2 and Der f 2 (Olsson et al., 1998; Bonura et al., 2001).
Further concepts entail the formation of recombinant deletion mutants, monomers, particles and also artificially created allergens using understanding of allergen conformation (Okada et al., 1998).
Complete immunological identification of recombinant allergenic molecules with reduced reactivity has not been fully achieved. Also preserved T cell activity is important for SIT effectiveness, therefore proper washing of recombinant antigens and removal of bacterial impurities, particularly endotoxins, is critical for therapy.
The recombinant allergen concept is advantageous as particles will preserve various T-cell epitopes and therefore regulate the various lineages of allergen-specific T-cells. Successions of clinical vaccination trials based on recombinant allergen therapy have been run with encouraging developments (Valenta and Niederberger, 2007).
Not long ago the marked deliverance of recombinant allergen molecules for MHC class II display on antigen presenting cells has been analysed for optimising dust mite presentation to T-cells. This technique may be able to elevate T-cell reactivity without the need for issuing further allergen dosages.
Random T-cell epitope identification
A different, untried technique for discovering allergen-specific T-cell determinants, is identification through random peptide collections (Sparbier and Walden, 1999). Epitopes identified in this way may possibly be mimotopes or conformational substitutes rather than naturally existing. Also both inhibitory and non-inhibitory mimotopes may be chosen determined by the characteristic immunological pathways wanted.
Despite epitope mapping commonly displaying several T-cell binding regions on allergenic particles (O'Hehir et al., 2007), influential domains are evident. Crucially, the excessive range of indiscrimination recognised in displaying of T-cell binding molecules in population research requires independent modification of peptides for appropriate group 2 major histocompatibility complex haploid genotypes.
In a unique vaccination plan, several T-cell binding peptides were integrated to comprise numerous active sites. However it is unclear if multi-allergen-specific T-cell cultures can be biologically effective in this way or if combinations of specifc peptides are more efficacious. Essentially nominal formulations need to be trialled for reduced IgE response and side-effects beforehand. If positive clinical results can be established, then this technique has the upper-hand of ease of mass-production.
Even with advances in design of allergenic preparations, there is a specific requirement to pinpoint antigen-presenting cells (APCs) to accomplish maximum inhibition of negative immunological responses. Standard SIT products are conventionally aqueous, yet tablet dosage forms are obtaining significance due to enhanced safety and efficacy of delivery (Frew, 2008).
Better understanding of cellular processes of allergen and adjuvant detection by APCs and later immunological responses lead the way to production of improved preparations. Like all allergen and adjuvant particulates, the distinct pattern recognition receptors used for engagement may direct the allergen-induced cytokine lineage. An interesting challenge is if these receptor binding sites exhibited on regulatory T cells can be marked with allergen immunotherapy.
Previous studies centered on ideas to selectively initiate Th1-type signals so that undesirable Th2 signals could be suppressed. Live or denatured M. tuberculosis have previously been used in atopic Japanese youth to suppress th2 effects of allergenic substances (Shirakawa et al., 1997). Although encouraging anti-inflammatory data were derived in murine prototypes using M. vaccae (Yazi et al., 2008), therapeutic trials have been ineffective (Renz, 2004). Fascinatingly though, research revealed that the therapeutic efficacy of Mycobacterial use was preferentially associated with stimulation of regulatory T-cell activity rather than Th1 (Zuany-Amorim et al., 1992).
As a result further investigations need to be carried out to derive the definitive processes required for switching of selective T-cell responses to bacterial fragments including the use of constituents as adjuvant products in allergenic formulations.
Microbial polysaccharides such as monophosphoryl lipid A (MPL) are also being researched for their immuno-regulatory abilities. MPL extracted from S. minnesota is capable of binding to TLR4 along with stimulation of dendritic cells, leading to generation of IFN-Î³ by peripheral blood mononuclear cells and Th1/Th2 cytokines by monocytic cells. MPL has been used as an adjuvant in recent times in vaccine formulations for seasonal rhinitis manifestations with encouraging results (Puggioni et al., 2005).
A further application involved production of a protein admixture comprised of the surface layer from a mutated from of G. stearothermophilus and Bet v1. This fusion protein researched in vitro lead to generation of IL-10 and IL-12 cytokines from dendritic cells in hypersensitised patients. The role of TLR2 was believed to be influential, leading to the use of a manufactured TLR2 ligand that successfully stimulated Th1 and regulatory T-cell responses (Gerstmayr et al., 2007).
Superficial microbial PAMPs can also stimulate IL-10 or TGF-Î² secretion, thereby encouraging regulatory T cell expansion (Mills, 2004). As a result research into their use as adjuvants may be feasible.
Substantial awareness has been raised regarding applicability of microbial DNA for regulating immune responses. C-phosphate-G-rich (CpGr) DNA of bacteria are able to stimulate IFN-Î³ secretion by natural killer cells and also IL-12 and interferon secretion by dendritic cells through binding with TLR9 (Kline, 2007).
Research has also shown bacterial DNA to be involved in modulating the action of IDO1 that leads to production of kynurenines, which are influential compounds regulating T-cell activity. Manufactured bacterial oligodeoxynucleotide analogues (ODNs) exhibited profound adjuvant capabilities for reducing hypersensitivity in murine designs (Broide et al., 1998). Distinct varieties of CpGr ODNs have been characterised, diversifying in configuration, strength and immunological effect (Klinman et al., 2004). CpGr ODNs may be administered separately in formulations for hypersensitive therapy as fusion proteins, or supplied in conjunction with specific allergenic substances.
In a separate investigation, a vaccine composed of Amb a1 interfused to immuno-stimulating bacterial DNA was administered to 25 rhinitis sufferers. Manifestations of disease were considerably reduced in comparison to a placebo group and also no signs of systemic side-effects were apparent (Creticos et al., 2006).
CpGr ODNs can be transported in virus-type particles (VLPs) to TLR9, thus avoiding enzymatic breakdown. This mechanism of delivery prevents autoimmune reactions or side-effects from occurring (Storni et al., 2004).
In conclusion allergic disorders are serious global health dilemmas that have a debilitating effect on individuals and to societies as a whole, with SIT being the only therapeutic option available at this moment in time that has the ability to switch the characteristic nature of hypersensitivity. Even though SIT is efficacious, it is confined to particular HDM allergens and restricted to use in certain individuals because of its susceptibility to side-effects. Standard SSIT is the treatment of choice, however further research has warranted SLIT approval as a result of its better reliability, efficiency of use and also application. Insight into allergen conformation and activity and the biochemistry of successful SIT demands research and progress of novel immunotherapeutic options. Notable developments in research endeavours include progress in T-lymphocytic epitope mapping, allergoids synthesis, and regulatory T cell stimulation mechanisms to name but a few. Collectively these advancements allow increased prospects of standardisation of SIT with the ultimate vision of successfully treating allergic disease.