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IgE and T-cell responses to house dust mite allergen components
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IgE and T-cell responses to house dust mite allergen components Wayne R. Thomas Telethon Kids Institute, University of Western Australia, 100 Roberts Rd., Subiaco 6008, Western Australia, Australia
A R T I C LE I N FO
A B S T R A C T
Keywords: House dust mite Dermatophagoides Allergen Component IgE Tcell Antibody
Using the terminology for Dermatophagoides pteronyssinus, IgE responses to house dust mites have been shown to be mostly directed to the serodominant Der p 1, 2 and 23 allergen components with mid -tier responses to Der p 4, 5, 7 and 21 that are made by 30–50% of subjects with titers proportional to those of the serodominant specificities. This pattern can be seen to evolve in childhood and although responses to minor allergens appear to contribute little to the total IgE they are at least markers for a greater propensity to develop disease. While Der p 23 is a component that induces prevalent IgE responses, sometimes in the absence of responses to Der p 1and 2, not all studies have found high titers so further investigation is needed. From limited knowledge adult onset IgE responses might have a different pattern that is not so centered on Der p 1 and 2. Responses that induce under 3.5 IU/ml of IgE antibody are not usually associated with disease and should be examined for cross reactivity expected from IgE responses to other allergens and antigens of infectious agents. Scabies that has 40% endemicity in some regions and is spread by immigration can give rise to high-titer binding that can be recognized by component resolved diagnosis. Recent studies with synthetic peptides representing allergens and non-allergenic HDM proteins now offer new research avenues on HDM induced immune responses, including the ability to use peptides representing the serodominant allergens as defined reagents for long overdue reproducible T-cell investigations.
1. Introduction The recent progress towards compiling an exhaustive list of the IgE binding house dust mite (HDM) components is not only valuable for investigations in different geographic regions but as shown for Der p 21(Weghofer et al., 2008) and 23 (Weghofer et al., 2013) and Der f 35 (Fujimura et al., 2017) has added significantly to the knowledge of HDM allergy in well-studied populations. Responses to these relatively recently recognized components have been shown by gravimetric measurements and comparative titrations to contribute significantly to the total IgE binding and Der p 21 is a cross-reactive determinant for allergy to Blomia tropicalis (Tan et al., 2012). It is however important to appreciate that HDM sensitization is not constituted by random responses to different components (Thomas, 2015; Thomas, 2016) and that the size of IgE response is critical for the association with allergic disease. Titers of less than 3.5 IU/ml (10 times 0.35 IU/ml) of IgE antibody are strongly associated with a life untroubled by allergy (Simpson et al. 2005; Crane et al., 2012). Western immunoblotting has detected over 30 IgE-binding entities in HDM extracts (Tovey and Baldo,1987) but most of it was focused on seven important components. In keeping with this although extracts made from whole mite cultures and washed mite bodies had differences in binding to minor components they were quantitatively equally able to cross inhibit IgE
binding to each other. Studies using purified components and quantitative measurements have similarly identified 7–8 components that account for most of the known IgE binding to HDM extracts as well as a distinctive pattern of IgE binding (Thomas, 2015; Thomas, 2016) that is different for childhood and adult onset sensitization (O’Brien and Thomas, 1994) and from current determinations not always directly related to the specificity of T-cell responses (Oseroff et al., 2016). The term serodominant will be used here instead of a major allergen because the latter has become attached to a definition based on a 50% prevalence without a consideration of the amount of antibody or its contribution to the overall response. 2. Childhood sensitization The serodominance of the group 1 and 2 allergen components (Trombone et al., 2002) was evident from the first analyses of the IgE binding components of children. IgE antibodies to Der p 1and 2 contributed 50–60% of the IgE binding in 95% of children in Australia (Hales et al., 2006) and was the dominant combination in 78% of children in Singapore (Kidon et al., 2011) noting that some of this population were also sensitized to B. tropicalis. Further Hales et al. (2006) found that Der p 4, 5, and 7, that each bound IgE in 30–50% of subjects, were mid-tier components accounting for much of the residual
E-mail address: [email protected]. https://doi.org/10.1016/j.molimm.2018.03.016 Received 24 November 2017; Accepted 19 March 2018 0161-5890/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Thomas, W.R., Molecular Immunology (2018), https://doi.org/10.1016/j.molimm.2018.03.016
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IgE binding and binding with titers proportional to the combined titers to Der p 1 and 2. The contribution of the IgE binding to Der p 3, 8, 10 and 20 was insignificant and this was unsurprising from absorption assays of IgE binding to HDM extracts (Weghofer et al., 2005). Although with lower prevalences and an equivalent contribution from Der p 3, Kidon et al. found the same mid-tier components and the expected midtier response to the then recently discovered Der p 21 (Weghofer et al., 2008). Recent microarray studies have corroborated these observations and added the peritrophin-like allergen Der p 23 that bound IgE with similar prevalence to Der p 1and 2 and was the dominant IgE binding component in 5% of asthmatics (Resch et al., 2015). The pattern has been replicated across Europe and in Canada, USA and Japan and for the D. farinae allergens (Batard et al., 2016) but the titer of binding to the group 23 components was variable and mostly lower. The proportional relationship of the IgE binding of the mid-tier and serodominant components can be seen in the heat maps for individual patients shown by Resch et al. (2015) except for one low responder. The IgE binding of atopic but non-asthmatic subjects had the same pattern as that of asthmatics but with lower titers and fewer detectable responses ((Resch et al., 2015). A result that could have been emphasized was that, as inferred from binding to extracts, children with high binding to serodominant and mid-tier components did not necessarily develop asthma. Although the clinical history of subjects in the allergic rhinitis sample examined for IgE binding to components by Mueller et al. (2016) was not reported in detail, and the IgE titers to all allergens were very low, the higher-titer responses showed the Der p 1, 2 and 23 dominance described for asthma. There was a heterogeneous response in the very low responders that might have been from the cat-allergic subjects included in the panel and since a high proportion bound the glutathione-S-transferase, Der p 8, could include cockroach cross reactivity (Mueller et al., 2015). Providing a new perspective, Posa et al. (2017) showed that the evolution of responses to the components paralleled their IgE binding hierarchy. Children first showed antibodies to Der p 1, 2 and 23 and then progressed to anti-mid-tier responses binding Der p 4, 5, 7 and 21 with responses to the minor components Der p 11, 14, 15, 18 and clone 16 not becoming apparent until 5–6 years. Responses to the mid-tier components did not affect the propensity of the children to develop asthma but it was increased two-fold for children who had responses to serodominant, mid-tier and minor components. It might be that responses to the dominant and mid-tier are part of a single pathway of sensitization as shown by the frequent correlation of their responses (Hales et al., 2006; Hales et al., 2013) while the minor allergens sensitize via a different pathway that interacts for disease. It has already been reported that the IgE titers to the chitinase- and chitin binding-like components Der p 15 and 18 correlate well with themselves but not with those to the serodominant and mid-tier components (Hales et al., 2013). Alternatively subjects that respond to poorly stimulating allergens might just have a greater propensity to develop disease. Although interesting it needs to be appreciated that the subjects that bound all three groups of components (Posa et al., 2017) only constituted 18.5% of the children that developed asthma. Hales et al. (2006) examining antibodies to Der p 1, 2, 3, 4, 5, 7, 8, 10 and 20, found children admitted to the emergency department for asthma exacerbations did not have a broader recognition of allergens than children with stable asthma indicating that only some minor components might contribute to disease. The properties and denomination of the allergens examined during the evolution of disease in children are shown in Table 1.
Table 1 House dust mite allergen components in the development of childhood asthmaa. Serodominant Group 1 Cysteine protease (that requires the addition of reducing agents to extracts for activity) Group 2 ML domain protein with characteristic lipopolysaccharide binding Group 23 Non-chitin binding peritrophin-like protein Mid-tier Group 4 Group 5 Group 7 Group 21 Minor Der p 11 Group 14
Group 15 Group 18 Clone 16
a
Alpha amylase Coiled-coil bundle of unknown function Bacterial permeability increasing/lipid binding protein homologue Homologous to group 5 Paramyosin is an unstable allergen. Amongst minor IgE binding associated with enhanced development of asthma Large lipid transport proteins susceptible to degradation producing IgE binding peptides. amongst minor IgE binding associated with enhanced development of asthma Chitinase-like proteinamongst minor IgE binding associated with enhanced development of asthma Chitin binding domain containing amongst minor IgE binding associated with enhanced development of asthma Unspecified recombinant protein amongst minor IgE binding associated with enhanced development of asthma
Developmental evolution as described by Posa et al. (2017).
production is part of normal immune responses, even those of healthy children to bacterial antigens (Hales et al., 2012), and responses to allergens can provide collateral help for IgE production to bystander antigens (Eisenbarth et al., 2004), it would not be surprising to find low IgE responses to many mite proteins. Accordingly newly denominated IgE-binding proteins need to be assessed with quantitative measurements and comparisons with other components for their contribution to allergic responses. As detailed elsewhere (Thomas, 2016) such assessments have yet to materialize for many of the newly-described D. farinae components and not only do many discrepancies need to be resolved but a recent proteomic analysis failed to detect IgE or IgG binding to most of the allergens designated Der f 24–30 (Oseroff et al., 2016). This might have been due to the particular extract however the accompanying analysis of T-cell responses to peptides representing these components also showed little reactivity indicating a need to identify the extent of the binding or regional differences. A newly described IgE binding protein that warrants corroboration is Der f 35 found to have quantitatively similar IgE binding as Der f 2 and like the group 2 components is a ML domain protein (Fujimura et al., 2017). Further analysis of the group 11 paramyosin allergens are indicated from the finding of a high frequency in atopic dermatitis but not asthma patients (Banerjee et al., 2015) and its recently described binding by sera from clinically undefined adolescent and adult patients (Conti et al., 2017). The latter study also found from immunoblotting that Der p 14 was frequently reactive. Both these allergens are members of the minor allergen panel used by Posa et al. (2017) but both need the production of authentically structured recombinant proteins or the isolation of stable natural components since IgE antibodies to other allergens are highly dependent on conformation (Greene and Thomas, 1992). The high IgE binding titers measured to recombinant fragments of Der f 14 in Japanese patients continue to point the importance of this component (ElRamlawy et al., 2018), which was also one of the strongest inducers of T-cell responses (Oseroff et al., 2016).
4. Cross reactivity 3. Newly reported IgE binding components The cross reactivity of the group 10 and 20 components with their tropomyosin (Fernandes et al., 2003) and arginine kinase (Binder et al. 2001) homologues in other arthropods is especially implicated in shellfish allergy. Tropomyosin however only induces IgE in a small percentage of HDM-sensitized subjects (Hales et al., 2006; Resch et al.,
The International Union of Immunological Societies/World Health Organization (IUIS/WHO) allergen nomenclature subcommittee assigns a name to a protein if it is tendered that it binds IgE antibodies from sera of five subjects allergic to HDM regardless of titer. Since IgE 2
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Table 2 Allergen cross reactivities. Group Group Group Group
4 8 10 20
Group 21
High titer IgE anti-alpha amylase binding antibodies associated with scabies infection or previous exposure Glutathione-S- transferase shows weak cross reactions with cockroach helminth Tropomyosin with highly conserved primary structure that can mediate cross reaction with shellfish and helminths Arginine kinase is major body protein with a highly conserved primary structure that can mediate cross reaction with shellfish and insects and high-titer IgE antibody is associated with active scabies infection Coiled coil structure of unknown function homologous group 5 component shows cross reactivity with Blomia tropicalis.
indigenous populations of Northern Australia and the Pacific and Indian Ocean Islands, 10% in South America and 5% across Africa (Romani et al., 2015). Increases in infestation due to immigration have been reported across Europe as documented by the 14% incidence for immigrants in Italy compared to 1.4% for natives (Limina et al., 2015). Even incidences of 0.27% and 0.07% reported for the UK and USA would, with the persistence of antibodies, result in 1–2% of the population having anti-scabies antibodies over a 10-year period. The problem with immigration has been amplified by the spread of scabies in refugee centers, as shown in Germany where between 2004 and 2014, the number of outbreaks rose 10 fold (Thomas et al., 2017). It could be particularly significant in low-income regions where the causation of asthma is uncertain and allergic asthma is treated as infection (Ostergaard et al., 2012). A conundrum that has arisen from the whole genome sequences of scabies (Rider et al., 2015; Mofiz et al., 2016) is the absence of a gene for the alpha amylase or a related molecule. The induction of antiamylase antibodies has been corroborated in experimentally infected pigs (Sandeman et al., 2001) and the high-titers make cross reactivity with a distantly related protein unlikely. Possibilities are that the binding could be to carbohydrate or to an amylase of a symbiotic microorganism. Since the reactivity in pigs could be detected with bacterial and blow fly amylases it might also be due to interactions with one of organisms with skin lesions. Another possibility is as a specificity of the autoantibodies found after scabies infestations (Toet et al., 2014). The denomination and properties of cross reacting HDM allergen components are shown in Table 2.
2011; Kidon et al., 2011) even in Singapore (Kidon et al., 2011) where shellfish allergy is common. Several studies have shown that a high percentage of shellfish allergic subjects show HDM IgE reactivity (Giuffrida et al., 2014) but tropomyosin and arginine kinase are only occasionally involved (Becker et al., 2012; Giuffrida et al., 2014). The other proteins could include ubiquitin (Gamez et al., 2014), hemocyanin (Kamath et al., 2017) and enolase (Kamath et al., 2017) but the reactivity with the HDM counterparts has not been shown. Subjects with Der p 10-reactive IgE, thought to be HDM-allergic by reactivity to extracts, have even been found to have little IgE binding to the serodominant HDM components indicating a different source of sensitization (Resch et al., 2011: Mohamad Yadzir et al., 2014) that could from serological investigations be shrimp, cockroach and anisakis (Mohamad Yadzir et al., 2014). IgE binding to Der p 10 in sera from respiratory patients in Zimbabwe however is associated with IgE binding Der p 2 so there might be interactions. Besides Anisakis sp. cross reactivity with tropomyosins of other helminths has been frequently reported including Ascaris lumbricoides (Buendia et al., 2015) and Onchocerca volvulus (Santiago et al., 2011). Glutathione-S-transferases have been implicated in cross reactivities with cockroaches and parasites but sera from cockroach allergic patients bind to Der p 8 with 100 fold less activity than that to the homologous Bla g 5 consistent with the low conservation of surface amino acids (Mueller et al., 2015). There was also little cross reactivity to Ascaris glutathione-S-transferase in keeping with an absence of notable IgE binding to glutathione-S-transferase of Schistosoma japonicum from the widely used pGEX expression vectors. Paramyosin cross reactivity has been a subject of speculation since it is a common antigen in parasite infections but its amino acid sequence is not well conserved. Assays of IgE binding to Blo t 11 paramyosin of B. tropicalis by sera from subjects infected with Ascaris lumbricoides have however provided evidence for this possibility (Valmonte et al., 2012). Enhanced IgE responses to Der p 1 and 2 by filarial infection has also been reported but the proposal that it might be due to homologous proteins is unlikely given the 23 and 33% sequence identities and that responses to Der p 7, for which homology was not identified, were also increased (Santiago et al., 2015). Der p 4 and 20 bind IgE from the sera of scabies infected subjects at high titer in the absence of binding to Der p 1, 2, 5, 7 and 10 and peptides representing Der p 14 (Walton et al., 2015). While cross reactivity of HDM and scabies extracts has been long recognized (Arlian et al., 1991; Taskapan and Harmanyeri, 2005) the high titers have been revealed by the component analysis. Antibodies to Der p 4 and 20 were associated with active infection while antibodies only to Der p 4 were associated with previous exposure. Circumstantial evidence indicates that a previously reported predominance of IgE binding to Der p 4 and not the serodominant allergens in a population of Australian aboriginals (Hales et al., 2007) had its genesis in scabies infection. Their high IgE binding and skin test reactions to HDM extracts, in association with a low prevalence of asthma skewed to adult disease, erroneously fostered speculation on environmental effects on asthma (Bremner et al., 1998). Asthma in northern Australia where scabies infection affects 40% of some communities is a serious problem with an uncertain etiology that can now and only be investigated for a contribution by HDM allergy by component resolved diagnosis. Current scabies infestations have an astonishing prevalence 40% in
5. Adult onset asthma About 60% of childhood asthma, mainly mild intermittent asthma, disappears with age but about 0.4% of the adults develop asthma each year (Thomsen et al., 2005) and this persists for half the subjects. Thus in a 20 year period in Switzerland 5.1% of men and 7.5% of women developed doctor-diagnosed asthma (Hansen et al., 2015) so later in life childhood-onset and adult-onset asthma become equally prevalent (Tan et al., 2016). Remission rates are low and about half develop a severe decline in lung function (Tuomisto et al., 2015). About half have nonatopic disease but atopy is also a risk factor for asthma (Thomsen et al., 2005; Bodner et al., 1998) and for resistance to remission (Ronmark et al., 2007). About 30% is attributed to HDM and fungal allergy (Jaakkola et al., 2006). Treatment with the anti-IL-5 monoclonal antibody reslizumab has been far more effective for adult-onset than childhood-onset asthma (Brusselle et al., 2017) and anti-IgE treatment with omalizumab less effective (Sposato et al., 2016). The one study of IgE binding to HDM components showed that only 20% of asthmatic and rhinitis patients diagnosed as adults had IgE binding to Der p 1 and/or Der p 2 compared to 80% of children (O’Brien and Thomas, 1994) and a heterogeneous pattern of binding by western blotting. In companion studies (Hales et al., 2006) adult volunteers sensitized from childhood showed responses focused on Der p 1 and 2 (Hales et al., 2006). Accordingly distinct pathways of HDM sensitization that differ with the age of the onset need to be considered and the testing of formulations for immunotherapy in trials with adult onset disease using components associated with early onset disease questioned. The average very low titer of 1.6 IU/ml of IgE anti- Der p 1, in one of the 3
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chymotrypsin Der p 6 (Yasueda et al., 1993; King et al., 1996) and the collagenolytic Der p 9 (King et al., 1996) was first characterized as only binding IgE from 16% of HDM-allergic subjects (Heymann et al., 1989) with similar low binding found in the component analyses of Hales et al. 2006 and Kidon et al. 2011. Although Yasueda et al. (1993) found 40% IgE positivity to Der p 3 the titers were low by Phadebas calibration. Similar IgE binding to the group 1 and 2 allergens has however been reported in some studies (Ando et al., 1993; King et al., 1996). Most titers were low in the small sample of Ando et al. (1993) and the detail of King et al. (1996) reveals that the Der p 3 results were from a different study that found that twice the amount of Der p 3 was required to inhibit RAST responses compared to Der p 1 and explicitly used an impure Der p 3 preparation (Stewart et al., 1992). A recent re-examination showed that the use of an enzymatically inactive mutant of Der p 3 could detect more IgE binding than wild type Der p 3 (Bouaziz et al., 2015) but the titers were still much lower than those to Der p 1.
rare immunotherapy trials to provide information on component binding, shows this might indeed be a major problem. Most trials recruit subjects with 0.35 or 0.7 IU/ml of anti-HDM extract antibody both of which are markers for a low likelihood HDM-allergy associated disease (Simpson et al., 2005; Crane et al., 2012). 6. Component response to T-cells Peripheral blood mononuclear cell (PBMC) responses to Der p 1 and 2 have shown the Th2 cytokine release expected from potent allergens (Hales et al., 2000; Hales et al., 2002). Increased IFN-γ release from PBMC of allergic subjects has also been detected (O’Brien et al., 2000; Hales et al., 2002), a finding corroborated with T-cell lines showing the induction of a more mixed cytokine profile with Der p 2 than that induced by a pollen allergen (Wambre et al., 2011) and by similar results for PBMC stimulated by representative peptides of the serodominant allergens (Hinz et al., 2015). Der p 23 has also been shown to induce IL5, IL-13 and IFN-γ release from the PBMC of allergic subjects (Banerjee et al., 2014). Hales et al., (2000) comparing responses to Der p 1 and 7 found no difference in Th2 cytokines or correlation with IgE titers but did show that more IFN-γ was induced by Der p 7 than Der p 1 especially in non-allergic subjects. The largely non-allergenic ferritin induced mixed Th1/Th2 cytokines from PBMC of both allergic and nonallergic subjects but interestingly the anti-Der p 2 Th2 cytokine response was higher than that to ferritin in allergic subjects but less in the non-allergic with more IL-10 (Epton et al., 2002) suggesting active regulatory responses to curb sensitization (Hinz et al., 2016; Sette and Schulten, 2017) long been known to be mediated by several T and Bcell compartments (Asherson et al., 1977). Epitope mapping with synthetic peptides has been used to define the T-cell reactivity to serodominant group 1, 2 and 23 allergens of D. pteronyssinus and D. farinae (Hinz et al., 2015). Many epitopes for the group 1 and 2 were identified and only a few for the smaller group 23. Peptides recognizing 11–13 regions per allergen could account for 90% of the reactivity to the group 1 components and peptides recognizing 6 regions similarly accounted for most of responses to the group 2 components. In contrast most responses to group 23 components were to single epitopes. A pool from peptides representing the groups 1, 2 and 23 components induced ex-vivo T-cell responses from PBMC with Th2 cytokine titers correlating with the IgE titers of the donors and, like the aforementioned studies, higher IL-10, IL-17 and IFN-γ induction from allergic donors. This pool can now be used to replace extracts and be free from contaminating innate immune stimulators. Caveats for these studies are that HDM extracts were used to expand the PBMC before epitope mapping and that important epitopes can be missed by their juxtaposition to inhibitory sequences in peptides (Jarnicki and Thomas, 2002). The study was recently extended to peptides representing all the then WHO/IUIS denominated components although only those with MHC binding motifs. With the exception of the group 21 there were readily detected epitopes for the well-characterized IgE binding components but few for the group 24–30 identified from proteomic immunoblotting. As before however the mapping depended on expanding the PBMC with HDM extracts so responses to components poorly represented by the extract used would be under-represented. There were also T-cell responses to previously unknown IgE-binding components identified in an accompanying IgE immunoblotting analysis, especially one that showed high T-cell responses in PBMC of 20% of the donors. High T-cell reactivity was also identified for a number of proteins without IgE binding that should be invaluable for comparing allergic and non-allergic responses. The donors had an average age of 49 so stratification with the age of onset of senstization would be of interest. The most highly stimulatory IgE binding components were intriguing. They included the amylases and the arginine kinases considered above for their cross reactivity but were topped by the serine proteases Der p 3, 6 and 6 that are of uncertain allergenicity. The group 3 trypsin, which has been reported to show more IgE binding than the
7. Conclusions The measurement of IgE antibodies has shown that HDM allergy developing in childhood produces serodominant responses to group 1, 2 and 23 components and mid-tier responses to the group 4, 5, 7 and 21 components which, when present, are proportional to the size of those to the combined group 1 and 2. Despite the low titers to other components there is evidence from the evolution of sensitization during childhood that the production of IgE antibodies to some of the minor IgE binding components is associated with enhanced clinical allergy. The ability of physicians to attribute the cause to allergic responses to a particular source of allergens on clinical history is often very poor showing frequent over-attribution and uncertainty (Williams et al., 2003; Szeinbach et al., 2004) so there is a case for component resolved diagnosis with HDM components and components of other sources of allergen and components that mediate cross reactivity. Identifying IgE cross reactivity induced by scabies might now be a priority for allergy diagnosis in regions with endemic scabies and in regions of scabies resurgence due to immigration. It is probable but not sufficiently proven that HDM allergy in adult onset asthma could involve responses to different components which is not only important for the assessment of allergy but for trials of immunotherapy that might be using medicaments formulated from studies of childhood onset asthma (O’Hehir et al., 2009). There has been little interest shown in investigating T-cell responses to the HDM components and thus investigating immune responses to known allergens in known doses in the absence of variable levels of extraneous antigens and innate immune stimuli. The information recently made available on peptides pools that can be bought and used to stimulate ex-vivo T-cell responses correlated with IgE titers has the potential for widespread utility and as does the information on the T-cell epitopes from other allergenic and non-allergenic components. References Ando, T., Homma, R., Ino, Y., Ito, G., Miyahara, A., Yanagihara, T., Kimura, H., Ikeda, S., Yamakawa, H., Iwaki, M., Okumura, Y., Suko, M., Haida, M., Okudaira, H., 1993. Trypsin-like protease of mites: purification and characterization of trypsin-like protease from mite faecal extract Dermatophagoides farinae. Relationship between trypsin-like protease and Der f III. Clin. Exp. Allergy 23, 777–784. Arlian, L.G., Vyszenski-Moher, D.L., Ahmed, S.G., Estes, S.A., 1991. Cross-antigenicity between the scabies mite, Sarcoptes scabiei, and the house dust mite, Dermatophagoides pteronyssinus. J. Invest. Dermatol. 96, 349–354. Asherson, G.L., Zembala, M., Perera, M.A., Mayhew, B., Thomas, W.R., 1977. Production of immunity and unresponsiveness in the mouse by feeding contact sensitizing agents and the role of suppressor cells in the peyer’s patches, mesenteric lymph nodes and other lymphoid tissues. Cell. Immunol. 33, 145–155. Banerjee, S., Resch, Y., Chen, K.-W., Swoboda, I., Focke-Tejkl, M., Blatt, K., Novak, N., Wickman, M., van Hage, M., Ferrara, R., Mari, A., Purohit, A., Pauli, G., Sibanda, E.N., Ndlovu, P., Thomas, W.R., Krzyzanek, V., Tacke, S., Malkus, U., Valent, P., Valenta, R., Vrtala, S., 2015. Der p 11 is a major allergen for house dust mite-allergic patients suffering from atopic dermatitis. J. Invest. Dermatol. 135, 102–109. Banerjee, S., Weber, M., Blatt, K., Swoboda, I., Focke-Tejkl, M., Valent, P., Valenta, R.,
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IgE and T-cell responses to house dust mite allergen components Wayne R. Thomas Telethon Kids Institute, University of Western Australia, 100 Roberts Rd., Subiaco 6008, Western Australia, Australia
A R T I C LE I N FO
A B S T R A C T
Keywords: House dust mite Dermatophagoides Allergen Component IgE Tcell Antibody
Using the terminology for Dermatophagoides pteronyssinus, IgE responses to house dust mites have been shown to be mostly directed to the serodominant Der p 1, 2 and 23 allergen components with mid -tier responses to Der p 4, 5, 7 and 21 that are made by 30–50% of subjects with titers proportional to those of the serodominant specificities. This pattern can be seen to evolve in childhood and although responses to minor allergens appear to contribute little to the total IgE they are at least markers for a greater propensity to develop disease. While Der p 23 is a component that induces prevalent IgE responses, sometimes in the absence of responses to Der p 1and 2, not all studies have found high titers so further investigation is needed. From limited knowledge adult onset IgE responses might have a different pattern that is not so centered on Der p 1 and 2. Responses that induce under 3.5 IU/ml of IgE antibody are not usually associated with disease and should be examined for cross reactivity expected from IgE responses to other allergens and antigens of infectious agents. Scabies that has 40% endemicity in some regions and is spread by immigration can give rise to high-titer binding that can be recognized by component resolved diagnosis. Recent studies with synthetic peptides representing allergens and non-allergenic HDM proteins now offer new research avenues on HDM induced immune responses, including the ability to use peptides representing the serodominant allergens as defined reagents for long overdue reproducible T-cell investigations.
1. Introduction The recent progress towards compiling an exhaustive list of the IgE binding house dust mite (HDM) components is not only valuable for investigations in different geographic regions but as shown for Der p 21(Weghofer et al., 2008) and 23 (Weghofer et al., 2013) and Der f 35 (Fujimura et al., 2017) has added significantly to the knowledge of HDM allergy in well-studied populations. Responses to these relatively recently recognized components have been shown by gravimetric measurements and comparative titrations to contribute significantly to the total IgE binding and Der p 21 is a cross-reactive determinant for allergy to Blomia tropicalis (Tan et al., 2012). It is however important to appreciate that HDM sensitization is not constituted by random responses to different components (Thomas, 2015; Thomas, 2016) and that the size of IgE response is critical for the association with allergic disease. Titers of less than 3.5 IU/ml (10 times 0.35 IU/ml) of IgE antibody are strongly associated with a life untroubled by allergy (Simpson et al. 2005; Crane et al., 2012). Western immunoblotting has detected over 30 IgE-binding entities in HDM extracts (Tovey and Baldo,1987) but most of it was focused on seven important components. In keeping with this although extracts made from whole mite cultures and washed mite bodies had differences in binding to minor components they were quantitatively equally able to cross inhibit IgE
binding to each other. Studies using purified components and quantitative measurements have similarly identified 7–8 components that account for most of the known IgE binding to HDM extracts as well as a distinctive pattern of IgE binding (Thomas, 2015; Thomas, 2016) that is different for childhood and adult onset sensitization (O’Brien and Thomas, 1994) and from current determinations not always directly related to the specificity of T-cell responses (Oseroff et al., 2016). The term serodominant will be used here instead of a major allergen because the latter has become attached to a definition based on a 50% prevalence without a consideration of the amount of antibody or its contribution to the overall response. 2. Childhood sensitization The serodominance of the group 1 and 2 allergen components (Trombone et al., 2002) was evident from the first analyses of the IgE binding components of children. IgE antibodies to Der p 1and 2 contributed 50–60% of the IgE binding in 95% of children in Australia (Hales et al., 2006) and was the dominant combination in 78% of children in Singapore (Kidon et al., 2011) noting that some of this population were also sensitized to B. tropicalis. Further Hales et al. (2006) found that Der p 4, 5, and 7, that each bound IgE in 30–50% of subjects, were mid-tier components accounting for much of the residual
E-mail address: [email protected]. https://doi.org/10.1016/j.molimm.2018.03.016 Received 24 November 2017; Accepted 19 March 2018 0161-5890/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Thomas, W.R., Molecular Immunology (2018), https://doi.org/10.1016/j.molimm.2018.03.016
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IgE binding and binding with titers proportional to the combined titers to Der p 1 and 2. The contribution of the IgE binding to Der p 3, 8, 10 and 20 was insignificant and this was unsurprising from absorption assays of IgE binding to HDM extracts (Weghofer et al., 2005). Although with lower prevalences and an equivalent contribution from Der p 3, Kidon et al. found the same mid-tier components and the expected midtier response to the then recently discovered Der p 21 (Weghofer et al., 2008). Recent microarray studies have corroborated these observations and added the peritrophin-like allergen Der p 23 that bound IgE with similar prevalence to Der p 1and 2 and was the dominant IgE binding component in 5% of asthmatics (Resch et al., 2015). The pattern has been replicated across Europe and in Canada, USA and Japan and for the D. farinae allergens (Batard et al., 2016) but the titer of binding to the group 23 components was variable and mostly lower. The proportional relationship of the IgE binding of the mid-tier and serodominant components can be seen in the heat maps for individual patients shown by Resch et al. (2015) except for one low responder. The IgE binding of atopic but non-asthmatic subjects had the same pattern as that of asthmatics but with lower titers and fewer detectable responses ((Resch et al., 2015). A result that could have been emphasized was that, as inferred from binding to extracts, children with high binding to serodominant and mid-tier components did not necessarily develop asthma. Although the clinical history of subjects in the allergic rhinitis sample examined for IgE binding to components by Mueller et al. (2016) was not reported in detail, and the IgE titers to all allergens were very low, the higher-titer responses showed the Der p 1, 2 and 23 dominance described for asthma. There was a heterogeneous response in the very low responders that might have been from the cat-allergic subjects included in the panel and since a high proportion bound the glutathione-S-transferase, Der p 8, could include cockroach cross reactivity (Mueller et al., 2015). Providing a new perspective, Posa et al. (2017) showed that the evolution of responses to the components paralleled their IgE binding hierarchy. Children first showed antibodies to Der p 1, 2 and 23 and then progressed to anti-mid-tier responses binding Der p 4, 5, 7 and 21 with responses to the minor components Der p 11, 14, 15, 18 and clone 16 not becoming apparent until 5–6 years. Responses to the mid-tier components did not affect the propensity of the children to develop asthma but it was increased two-fold for children who had responses to serodominant, mid-tier and minor components. It might be that responses to the dominant and mid-tier are part of a single pathway of sensitization as shown by the frequent correlation of their responses (Hales et al., 2006; Hales et al., 2013) while the minor allergens sensitize via a different pathway that interacts for disease. It has already been reported that the IgE titers to the chitinase- and chitin binding-like components Der p 15 and 18 correlate well with themselves but not with those to the serodominant and mid-tier components (Hales et al., 2013). Alternatively subjects that respond to poorly stimulating allergens might just have a greater propensity to develop disease. Although interesting it needs to be appreciated that the subjects that bound all three groups of components (Posa et al., 2017) only constituted 18.5% of the children that developed asthma. Hales et al. (2006) examining antibodies to Der p 1, 2, 3, 4, 5, 7, 8, 10 and 20, found children admitted to the emergency department for asthma exacerbations did not have a broader recognition of allergens than children with stable asthma indicating that only some minor components might contribute to disease. The properties and denomination of the allergens examined during the evolution of disease in children are shown in Table 1.
Table 1 House dust mite allergen components in the development of childhood asthmaa. Serodominant Group 1 Cysteine protease (that requires the addition of reducing agents to extracts for activity) Group 2 ML domain protein with characteristic lipopolysaccharide binding Group 23 Non-chitin binding peritrophin-like protein Mid-tier Group 4 Group 5 Group 7 Group 21 Minor Der p 11 Group 14
Group 15 Group 18 Clone 16
a
Alpha amylase Coiled-coil bundle of unknown function Bacterial permeability increasing/lipid binding protein homologue Homologous to group 5 Paramyosin is an unstable allergen. Amongst minor IgE binding associated with enhanced development of asthma Large lipid transport proteins susceptible to degradation producing IgE binding peptides. amongst minor IgE binding associated with enhanced development of asthma Chitinase-like proteinamongst minor IgE binding associated with enhanced development of asthma Chitin binding domain containing amongst minor IgE binding associated with enhanced development of asthma Unspecified recombinant protein amongst minor IgE binding associated with enhanced development of asthma
Developmental evolution as described by Posa et al. (2017).
production is part of normal immune responses, even those of healthy children to bacterial antigens (Hales et al., 2012), and responses to allergens can provide collateral help for IgE production to bystander antigens (Eisenbarth et al., 2004), it would not be surprising to find low IgE responses to many mite proteins. Accordingly newly denominated IgE-binding proteins need to be assessed with quantitative measurements and comparisons with other components for their contribution to allergic responses. As detailed elsewhere (Thomas, 2016) such assessments have yet to materialize for many of the newly-described D. farinae components and not only do many discrepancies need to be resolved but a recent proteomic analysis failed to detect IgE or IgG binding to most of the allergens designated Der f 24–30 (Oseroff et al., 2016). This might have been due to the particular extract however the accompanying analysis of T-cell responses to peptides representing these components also showed little reactivity indicating a need to identify the extent of the binding or regional differences. A newly described IgE binding protein that warrants corroboration is Der f 35 found to have quantitatively similar IgE binding as Der f 2 and like the group 2 components is a ML domain protein (Fujimura et al., 2017). Further analysis of the group 11 paramyosin allergens are indicated from the finding of a high frequency in atopic dermatitis but not asthma patients (Banerjee et al., 2015) and its recently described binding by sera from clinically undefined adolescent and adult patients (Conti et al., 2017). The latter study also found from immunoblotting that Der p 14 was frequently reactive. Both these allergens are members of the minor allergen panel used by Posa et al. (2017) but both need the production of authentically structured recombinant proteins or the isolation of stable natural components since IgE antibodies to other allergens are highly dependent on conformation (Greene and Thomas, 1992). The high IgE binding titers measured to recombinant fragments of Der f 14 in Japanese patients continue to point the importance of this component (ElRamlawy et al., 2018), which was also one of the strongest inducers of T-cell responses (Oseroff et al., 2016).
4. Cross reactivity 3. Newly reported IgE binding components The cross reactivity of the group 10 and 20 components with their tropomyosin (Fernandes et al., 2003) and arginine kinase (Binder et al. 2001) homologues in other arthropods is especially implicated in shellfish allergy. Tropomyosin however only induces IgE in a small percentage of HDM-sensitized subjects (Hales et al., 2006; Resch et al.,
The International Union of Immunological Societies/World Health Organization (IUIS/WHO) allergen nomenclature subcommittee assigns a name to a protein if it is tendered that it binds IgE antibodies from sera of five subjects allergic to HDM regardless of titer. Since IgE 2
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Table 2 Allergen cross reactivities. Group Group Group Group
4 8 10 20
Group 21
High titer IgE anti-alpha amylase binding antibodies associated with scabies infection or previous exposure Glutathione-S- transferase shows weak cross reactions with cockroach helminth Tropomyosin with highly conserved primary structure that can mediate cross reaction with shellfish and helminths Arginine kinase is major body protein with a highly conserved primary structure that can mediate cross reaction with shellfish and insects and high-titer IgE antibody is associated with active scabies infection Coiled coil structure of unknown function homologous group 5 component shows cross reactivity with Blomia tropicalis.
indigenous populations of Northern Australia and the Pacific and Indian Ocean Islands, 10% in South America and 5% across Africa (Romani et al., 2015). Increases in infestation due to immigration have been reported across Europe as documented by the 14% incidence for immigrants in Italy compared to 1.4% for natives (Limina et al., 2015). Even incidences of 0.27% and 0.07% reported for the UK and USA would, with the persistence of antibodies, result in 1–2% of the population having anti-scabies antibodies over a 10-year period. The problem with immigration has been amplified by the spread of scabies in refugee centers, as shown in Germany where between 2004 and 2014, the number of outbreaks rose 10 fold (Thomas et al., 2017). It could be particularly significant in low-income regions where the causation of asthma is uncertain and allergic asthma is treated as infection (Ostergaard et al., 2012). A conundrum that has arisen from the whole genome sequences of scabies (Rider et al., 2015; Mofiz et al., 2016) is the absence of a gene for the alpha amylase or a related molecule. The induction of antiamylase antibodies has been corroborated in experimentally infected pigs (Sandeman et al., 2001) and the high-titers make cross reactivity with a distantly related protein unlikely. Possibilities are that the binding could be to carbohydrate or to an amylase of a symbiotic microorganism. Since the reactivity in pigs could be detected with bacterial and blow fly amylases it might also be due to interactions with one of organisms with skin lesions. Another possibility is as a specificity of the autoantibodies found after scabies infestations (Toet et al., 2014). The denomination and properties of cross reacting HDM allergen components are shown in Table 2.
2011; Kidon et al., 2011) even in Singapore (Kidon et al., 2011) where shellfish allergy is common. Several studies have shown that a high percentage of shellfish allergic subjects show HDM IgE reactivity (Giuffrida et al., 2014) but tropomyosin and arginine kinase are only occasionally involved (Becker et al., 2012; Giuffrida et al., 2014). The other proteins could include ubiquitin (Gamez et al., 2014), hemocyanin (Kamath et al., 2017) and enolase (Kamath et al., 2017) but the reactivity with the HDM counterparts has not been shown. Subjects with Der p 10-reactive IgE, thought to be HDM-allergic by reactivity to extracts, have even been found to have little IgE binding to the serodominant HDM components indicating a different source of sensitization (Resch et al., 2011: Mohamad Yadzir et al., 2014) that could from serological investigations be shrimp, cockroach and anisakis (Mohamad Yadzir et al., 2014). IgE binding to Der p 10 in sera from respiratory patients in Zimbabwe however is associated with IgE binding Der p 2 so there might be interactions. Besides Anisakis sp. cross reactivity with tropomyosins of other helminths has been frequently reported including Ascaris lumbricoides (Buendia et al., 2015) and Onchocerca volvulus (Santiago et al., 2011). Glutathione-S-transferases have been implicated in cross reactivities with cockroaches and parasites but sera from cockroach allergic patients bind to Der p 8 with 100 fold less activity than that to the homologous Bla g 5 consistent with the low conservation of surface amino acids (Mueller et al., 2015). There was also little cross reactivity to Ascaris glutathione-S-transferase in keeping with an absence of notable IgE binding to glutathione-S-transferase of Schistosoma japonicum from the widely used pGEX expression vectors. Paramyosin cross reactivity has been a subject of speculation since it is a common antigen in parasite infections but its amino acid sequence is not well conserved. Assays of IgE binding to Blo t 11 paramyosin of B. tropicalis by sera from subjects infected with Ascaris lumbricoides have however provided evidence for this possibility (Valmonte et al., 2012). Enhanced IgE responses to Der p 1 and 2 by filarial infection has also been reported but the proposal that it might be due to homologous proteins is unlikely given the 23 and 33% sequence identities and that responses to Der p 7, for which homology was not identified, were also increased (Santiago et al., 2015). Der p 4 and 20 bind IgE from the sera of scabies infected subjects at high titer in the absence of binding to Der p 1, 2, 5, 7 and 10 and peptides representing Der p 14 (Walton et al., 2015). While cross reactivity of HDM and scabies extracts has been long recognized (Arlian et al., 1991; Taskapan and Harmanyeri, 2005) the high titers have been revealed by the component analysis. Antibodies to Der p 4 and 20 were associated with active infection while antibodies only to Der p 4 were associated with previous exposure. Circumstantial evidence indicates that a previously reported predominance of IgE binding to Der p 4 and not the serodominant allergens in a population of Australian aboriginals (Hales et al., 2007) had its genesis in scabies infection. Their high IgE binding and skin test reactions to HDM extracts, in association with a low prevalence of asthma skewed to adult disease, erroneously fostered speculation on environmental effects on asthma (Bremner et al., 1998). Asthma in northern Australia where scabies infection affects 40% of some communities is a serious problem with an uncertain etiology that can now and only be investigated for a contribution by HDM allergy by component resolved diagnosis. Current scabies infestations have an astonishing prevalence 40% in
5. Adult onset asthma About 60% of childhood asthma, mainly mild intermittent asthma, disappears with age but about 0.4% of the adults develop asthma each year (Thomsen et al., 2005) and this persists for half the subjects. Thus in a 20 year period in Switzerland 5.1% of men and 7.5% of women developed doctor-diagnosed asthma (Hansen et al., 2015) so later in life childhood-onset and adult-onset asthma become equally prevalent (Tan et al., 2016). Remission rates are low and about half develop a severe decline in lung function (Tuomisto et al., 2015). About half have nonatopic disease but atopy is also a risk factor for asthma (Thomsen et al., 2005; Bodner et al., 1998) and for resistance to remission (Ronmark et al., 2007). About 30% is attributed to HDM and fungal allergy (Jaakkola et al., 2006). Treatment with the anti-IL-5 monoclonal antibody reslizumab has been far more effective for adult-onset than childhood-onset asthma (Brusselle et al., 2017) and anti-IgE treatment with omalizumab less effective (Sposato et al., 2016). The one study of IgE binding to HDM components showed that only 20% of asthmatic and rhinitis patients diagnosed as adults had IgE binding to Der p 1 and/or Der p 2 compared to 80% of children (O’Brien and Thomas, 1994) and a heterogeneous pattern of binding by western blotting. In companion studies (Hales et al., 2006) adult volunteers sensitized from childhood showed responses focused on Der p 1 and 2 (Hales et al., 2006). Accordingly distinct pathways of HDM sensitization that differ with the age of the onset need to be considered and the testing of formulations for immunotherapy in trials with adult onset disease using components associated with early onset disease questioned. The average very low titer of 1.6 IU/ml of IgE anti- Der p 1, in one of the 3
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chymotrypsin Der p 6 (Yasueda et al., 1993; King et al., 1996) and the collagenolytic Der p 9 (King et al., 1996) was first characterized as only binding IgE from 16% of HDM-allergic subjects (Heymann et al., 1989) with similar low binding found in the component analyses of Hales et al. 2006 and Kidon et al. 2011. Although Yasueda et al. (1993) found 40% IgE positivity to Der p 3 the titers were low by Phadebas calibration. Similar IgE binding to the group 1 and 2 allergens has however been reported in some studies (Ando et al., 1993; King et al., 1996). Most titers were low in the small sample of Ando et al. (1993) and the detail of King et al. (1996) reveals that the Der p 3 results were from a different study that found that twice the amount of Der p 3 was required to inhibit RAST responses compared to Der p 1 and explicitly used an impure Der p 3 preparation (Stewart et al., 1992). A recent re-examination showed that the use of an enzymatically inactive mutant of Der p 3 could detect more IgE binding than wild type Der p 3 (Bouaziz et al., 2015) but the titers were still much lower than those to Der p 1.
rare immunotherapy trials to provide information on component binding, shows this might indeed be a major problem. Most trials recruit subjects with 0.35 or 0.7 IU/ml of anti-HDM extract antibody both of which are markers for a low likelihood HDM-allergy associated disease (Simpson et al., 2005; Crane et al., 2012). 6. Component response to T-cells Peripheral blood mononuclear cell (PBMC) responses to Der p 1 and 2 have shown the Th2 cytokine release expected from potent allergens (Hales et al., 2000; Hales et al., 2002). Increased IFN-γ release from PBMC of allergic subjects has also been detected (O’Brien et al., 2000; Hales et al., 2002), a finding corroborated with T-cell lines showing the induction of a more mixed cytokine profile with Der p 2 than that induced by a pollen allergen (Wambre et al., 2011) and by similar results for PBMC stimulated by representative peptides of the serodominant allergens (Hinz et al., 2015). Der p 23 has also been shown to induce IL5, IL-13 and IFN-γ release from the PBMC of allergic subjects (Banerjee et al., 2014). Hales et al., (2000) comparing responses to Der p 1 and 7 found no difference in Th2 cytokines or correlation with IgE titers but did show that more IFN-γ was induced by Der p 7 than Der p 1 especially in non-allergic subjects. The largely non-allergenic ferritin induced mixed Th1/Th2 cytokines from PBMC of both allergic and nonallergic subjects but interestingly the anti-Der p 2 Th2 cytokine response was higher than that to ferritin in allergic subjects but less in the non-allergic with more IL-10 (Epton et al., 2002) suggesting active regulatory responses to curb sensitization (Hinz et al., 2016; Sette and Schulten, 2017) long been known to be mediated by several T and Bcell compartments (Asherson et al., 1977). Epitope mapping with synthetic peptides has been used to define the T-cell reactivity to serodominant group 1, 2 and 23 allergens of D. pteronyssinus and D. farinae (Hinz et al., 2015). Many epitopes for the group 1 and 2 were identified and only a few for the smaller group 23. Peptides recognizing 11–13 regions per allergen could account for 90% of the reactivity to the group 1 components and peptides recognizing 6 regions similarly accounted for most of responses to the group 2 components. In contrast most responses to group 23 components were to single epitopes. A pool from peptides representing the groups 1, 2 and 23 components induced ex-vivo T-cell responses from PBMC with Th2 cytokine titers correlating with the IgE titers of the donors and, like the aforementioned studies, higher IL-10, IL-17 and IFN-γ induction from allergic donors. This pool can now be used to replace extracts and be free from contaminating innate immune stimulators. Caveats for these studies are that HDM extracts were used to expand the PBMC before epitope mapping and that important epitopes can be missed by their juxtaposition to inhibitory sequences in peptides (Jarnicki and Thomas, 2002). The study was recently extended to peptides representing all the then WHO/IUIS denominated components although only those with MHC binding motifs. With the exception of the group 21 there were readily detected epitopes for the well-characterized IgE binding components but few for the group 24–30 identified from proteomic immunoblotting. As before however the mapping depended on expanding the PBMC with HDM extracts so responses to components poorly represented by the extract used would be under-represented. There were also T-cell responses to previously unknown IgE-binding components identified in an accompanying IgE immunoblotting analysis, especially one that showed high T-cell responses in PBMC of 20% of the donors. High T-cell reactivity was also identified for a number of proteins without IgE binding that should be invaluable for comparing allergic and non-allergic responses. The donors had an average age of 49 so stratification with the age of onset of senstization would be of interest. The most highly stimulatory IgE binding components were intriguing. They included the amylases and the arginine kinases considered above for their cross reactivity but were topped by the serine proteases Der p 3, 6 and 6 that are of uncertain allergenicity. The group 3 trypsin, which has been reported to show more IgE binding than the
7. Conclusions The measurement of IgE antibodies has shown that HDM allergy developing in childhood produces serodominant responses to group 1, 2 and 23 components and mid-tier responses to the group 4, 5, 7 and 21 components which, when present, are proportional to the size of those to the combined group 1 and 2. Despite the low titers to other components there is evidence from the evolution of sensitization during childhood that the production of IgE antibodies to some of the minor IgE binding components is associated with enhanced clinical allergy. The ability of physicians to attribute the cause to allergic responses to a particular source of allergens on clinical history is often very poor showing frequent over-attribution and uncertainty (Williams et al., 2003; Szeinbach et al., 2004) so there is a case for component resolved diagnosis with HDM components and components of other sources of allergen and components that mediate cross reactivity. Identifying IgE cross reactivity induced by scabies might now be a priority for allergy diagnosis in regions with endemic scabies and in regions of scabies resurgence due to immigration. It is probable but not sufficiently proven that HDM allergy in adult onset asthma could involve responses to different components which is not only important for the assessment of allergy but for trials of immunotherapy that might be using medicaments formulated from studies of childhood onset asthma (O’Hehir et al., 2009). There has been little interest shown in investigating T-cell responses to the HDM components and thus investigating immune responses to known allergens in known doses in the absence of variable levels of extraneous antigens and innate immune stimuli. The information recently made available on peptides pools that can be bought and used to stimulate ex-vivo T-cell responses correlated with IgE titers has the potential for widespread utility and as does the information on the T-cell epitopes from other allergenic and non-allergenic components. References Ando, T., Homma, R., Ino, Y., Ito, G., Miyahara, A., Yanagihara, T., Kimura, H., Ikeda, S., Yamakawa, H., Iwaki, M., Okumura, Y., Suko, M., Haida, M., Okudaira, H., 1993. Trypsin-like protease of mites: purification and characterization of trypsin-like protease from mite faecal extract Dermatophagoides farinae. Relationship between trypsin-like protease and Der f III. Clin. Exp. Allergy 23, 777–784. Arlian, L.G., Vyszenski-Moher, D.L., Ahmed, S.G., Estes, S.A., 1991. Cross-antigenicity between the scabies mite, Sarcoptes scabiei, and the house dust mite, Dermatophagoides pteronyssinus. J. Invest. Dermatol. 96, 349–354. Asherson, G.L., Zembala, M., Perera, M.A., Mayhew, B., Thomas, W.R., 1977. Production of immunity and unresponsiveness in the mouse by feeding contact sensitizing agents and the role of suppressor cells in the peyer’s patches, mesenteric lymph nodes and other lymphoid tissues. Cell. Immunol. 33, 145–155. Banerjee, S., Resch, Y., Chen, K.-W., Swoboda, I., Focke-Tejkl, M., Blatt, K., Novak, N., Wickman, M., van Hage, M., Ferrara, R., Mari, A., Purohit, A., Pauli, G., Sibanda, E.N., Ndlovu, P., Thomas, W.R., Krzyzanek, V., Tacke, S., Malkus, U., Valent, P., Valenta, R., Vrtala, S., 2015. Der p 11 is a major allergen for house dust mite-allergic patients suffering from atopic dermatitis. J. Invest. Dermatol. 135, 102–109. Banerjee, S., Weber, M., Blatt, K., Swoboda, I., Focke-Tejkl, M., Valent, P., Valenta, R.,
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