Proliferation and release of IL-5 and IFN-gamma by peripheral blood mononuclear cells from cat-allergic asthmatics and rhinitics, non-cat-allergic asthmatics, and normal controls to peptides derived from Fel d 1 chain 1.

BACKGROUND: In general, T cells from normal, nonatopic individuals respond to aeroallergens with synthesis and release of IFN-gamma. In contrast, release of T(H)2-type cytokines by activated lymphocytes is a feature of allergic rhinitis and atopic asthma. OBJECTIVE: The purpose of this study was to determine differences in T-cell recognition of epitopes within allergenic sequences, in terms of proliferation and cytokine production, in subjects with atopic asthma compared with subjects with allergic rhinitis and normal controls. METHODS: Proliferative responses and IL-5/IFN-gamma release patterns from PBMCs from cat-allergic asthmatic, cat-allergic rhinitic, and non-cat-allergic asthmatic subjects and nonatopic normal controls were determined in primary cultures. Cells were challenged with 7 overlapping peptides spanning chain 1 of the major cat allergen, Fel d 1. RESULTS: The 4 groups did not differ with respect to the ability to mount proliferative responses to Fel d 1 peptides. In all groups, the IFN-gamma responses were predominantly to the amino terminus peptides. Cat-allergic and non-cat-allergic asthmatic subjects (and not cat-allergic rhinitic subjects and normal controls) made IL-5 responses to most of the Fel d 1 peptides, the result being a mixed (T(H)0) cytokine response at the N-terminus and a restricted (T(H)2) response at the C-terminus. CONCLUSION: Proliferative and IL-5/IFN-gamma responses of T cells from asthmatic and atopic rhinitic subjects and normal controls to allergen peptides can be dissociated. Furthermore, differing cytokine responses to peptides derived from a single antigen suggest that certain domains of the molecule might preferentially induce IL-5 rather than IFN-gamma and as a result could be more important in disease pathogenesis.     

High-dose allergen exposure leads to tolerance

Reports of decreased sensitization to cat allergen (Fel d 1) among individuals living with a cat or subjects exposed to high-dose cat allergen may be explained by the development of a form of high-dose tolerance resulting from natural exposure to an inhalant allergen. Although the epidemiological data regarding the relationship between exposure and sensitization to Fel d 1 are conflicting, the ability for high-dose Fel d 1 to induce a characteristic nonallergic immune response with a distinctive serum antibody profile has been established. Definition of this modified T-helper (Th)2 response to cat allergen, coupled with the renewed interest in regulatory T cells within the immunology field, has provided an avenue for exploring the mechanism by which IgE antibody-mediated responses are controlled. There is mounting evidence to suggest that the modified Th2 response is a variation of the allergic response and that the modified Th2-allergic axis is influenced by allergen dose and genetics. This article discusses putative immune mechanisms of tolerance within the context of an allergen-specific system. The relevance of high-dose allergen exposure and alternate factors such as endotoxin to the development of tolerance is considered. Fel d 1 exhibits unique molecular and immunological characteristics that may contribute to its tolerogenic properties. Major T-cell epitopes of Fel d 1 that preferentially induce regulatory factors have been defined. Furthermore, hightiter IgE antibody responses associated with atopic dermatitis are characterized by a defect in the T-cell repertoire that is specific to these epitopes. Identification of Fel d 1 epitopes that induce interleukin-10 may provide new targets for treatment.     

Non-allergenic antigen in allergic sensitization: responses to the mite ferritin heavy chain antigen by allergic and non-allergic subjects

BACKGROUND: The majority of house dust mite proteins are non-allergenic. There is, however, no information on the type of immune responses produced to these proteins and if the responses are affected by allergic sensitization. OBJECTIVE: To identify and produce a non-allergenic antigen of the house dust mite and compare antibody and T cell responses with the responses to allergens in sensitized and non-sensitized individuals. RESULTS: Ferritin heavy chain was cloned from a cDNA library as a candidate non-allergen of the house dust mite. It bound IgG but not IgE in the sera of allergic and non-allergic subjects and induced high T cell proliferative responses that correlated highly with the responses to the major allergen Der p 2. The cytokine response to the non-allergen was characterized by the release of high levels of both Th1 and Th2 cytokines from the PBMC of both allergic and non-allergic subjects. In contrast, the response to Der p 2 showed the expected high level of Th2 cytokine release from the PBMC of allergic subjects, while the Th2 cytokine production from PBMC of non-allergic subjects was low and even lower than that induced by ferritin heavy chain. The levels of IFN-gamma release were similar for all groups. Der p 2 induced significantly more IL-10 than ferritin in the non-allergic group. CONCLUSION: The T cell responses to a non-allergenic protein of the house dust mite were high and strongly correlated with the response to the major allergen. The non-allergenic protein induced high levels of Th1 and Th2 cytokine in both allergic and non-allergic subjects, while the allergen induced high levels of Th2 cytokine in allergic subjects and low levels in non-allergic subjects. The responses to the allergen were thus independently up- and down-regulated with no evidence of bystander regulation.     

IgE antibody-specific activity in human allergic disease

IgE antibody concentration, affinity, clonality and specific activity (also known as the allergen-specific IgE to total IgE ratio) influence the translation of IgE responses into clinically evident allergic symptoms following allergen exposure. Reported IgE-specific activity levels >3–4% place allergic individuals undergoing anti-IgE (Omalizumab^®) therapy at a disadvantage for poor resolution of their allergy symptoms following manufacturer’s recommended dosing schemes. We investigated the hypothesis that the specific activity of the IgE antibody response is highly variable with respect to age, allergen specificity and an individual’s total serum IgE level. Second, we investigated whether the IgE-specific activity level influences the extent and rate of loss of effector cell mediator release. IgE-specific activity distributions were plotted against age, allergen specificity and total serum IgE using 18,950 paired total IgE and allergen-specific IgE antibody data obtained from the analysis of sera from 3,614 allergic subjects and covering 182 allergen specificities. The fraction of specific IgE antibody of the total serum IgE was dependent on age of the individual, epitope specificity (clonality) and total serum IgE. The youngest group of allergic individuals with the lowest total serum IgE levels tended to have the highest allergen-specific IgE to total IgE ratios. Hymenoptera venom (54%), peanut (33%) and milk (27%) were the three allergen specificities that elicited the highest frequency of IgE-specific activities >4% among sensitized individuals. A prospective double-blind, placebo-controlled clinical study involving anti-IgE treatment of cat-allergic subjects showed IgE-specific activity was remarkably constant over the 16-week course of treatment, despite the up to 8-fold rise in total serum IgE following repetitive Omalizumab^® administration. Changes in specific and total IgE levels paralleled each other in patients receiving anti-IgE therapy. The fastest rate of reduction in cat allergen-induced basophil histamine release following anti-IgE therapy was observed when the cat-specific IgE to total IgE ratio was <2.5%. This reflected the more rapid loss of surface cat-specific IgE antibody with anti-IgE therapy in allergic individuals who displayed a more diverse IgE antibody repertoire. We conclude that IgE-specific activity is an age-, IgE heterogeneity- and total serum IgE-dependent variable that influences the magnitude of effector cell mediator release, and by inference, ultimate allergic symptom induction.     

Fel d 4, a cat lipocalin allergen

BACKGROUND: Cat allergy is unique among allergy to mammals in that the major allergen Fel d 1 is a uteroglobin-like protein and not a lipocalin. The biochemical spectrum of the cat allergens is thus uncertain, particularly with regard to the role that a cat lipocalin protein may play in sensitization to cats in allergic individuals. OBJECTIVE: To analyse cDNA encoding a lipocalin allergen and the corresponding recombinant allergen at both the molecular and immunological levels. METHODS: A submandibular salivary gland cDNA expression library was constructed and screened for clones producing IgE-binding polypeptides. cDNA encoding a lipocalin allergen and its corresponding recombinant allergen were analysed. RESULTS: An IgE binding molecule with high sequence identity to the boar salivary lipocalin and the horse lipocalin Equ c 1 allergen was isolated and designated, Fel d 4. Serum from 62.96% of cat-allergic subjects examined had measurable IgE antibody to Fel d 4 but typically at low levels. Despite this in 47% of sera the anti-Fel d 4 IgE titres were higher than the anti-Fel d 1 titres. IgE binding to the lipocalin allergen could be blocked by an allergen extract from cow and to a lesser degree by extracts from horse and dog. CONCLUSION: Fel d 4 is a lipocalin allergen produced by the cat, which binds IgE at relatively high frequency in cat-sensitive individuals. The allergen provides not only a means for investigating differences in the immune response to lipocalin allergens from that found for other mammalian species but also an important reagent for the diagnosis of cat allergy.     

Targeting the MHC class II pathway of antigen presentation enhances immunogenicity and safety of allergen immunotherapy

Background:  Current s.c. allergen-specific immunotherapy (SIT) leads to amelioration of IgE-mediated allergy, but it requires numerous allergen injections over several years and is frequently associated with severe side-effects. The aim of this study was to test whether modified recombinant allergens can improve therapeutic efficacy in SIT while reducing allergic side-effects.
Methods:  The major cat allergen Fel d 1 was fused to a TAT-derived protein translocation domain and to a truncated invariant chain for targeting the MHC class II pathway (MAT-Fel d 1). The immunogenicity was evaluated in mice, while potential safety issues were assessed by cellular antigen stimulation test (CAST) using basophils from cat-dander-allergic patients.
Results:  MAT-Fel d 1 enhanced induction of Fel d 1-specific IgG2a antibody responses as well as the secretion of IFN-γ and IL-2 from T cells. Subcutaneous allergen-specific immunotherapy of mice using the modified Fel d 1 provided stronger protection against anaphylaxis than SIT with unmodified Fel d 1, and MAT-Fel d 1 caused less degranulation of human basophils than native Fel d 1.
Conclusion:  MAT-Fel d 1 allergen enhanced protective antibody and Th1 responses in mice, while reducing human basophil degranulation. Immunotherapy using MAT-Fel d 1 allergen therefore has the potential to enhance SIT efficacy and safety, thus, shortening SIT. This should increase patient compliance and lower treatment costs.     

Cat allergen-induced cytokine secretion and Fel d 1–immunoglobulin G immune complexes in cord blood

BACKGROUND: We have recently obtained evidence for the presence of immune complexes (IC) in cord blood from allergic and non-allergic mothers. Such complexes could conceivably provide the fetus with the initial trigger for the priming of the T cell system already in utero. OBJECTIVE: To relate the presence of Fel d 1-IgG IC to T cell cytokine production in cord blood mononuclear cells (CBMCs) after stimulation with cat allergen. METHODS: CBMC obtained from babies of 15 allergic and 22 non-allergic mothers were cultured in the presence of cat allergen. The production of IFN-gamma, IL-5, IL-10 and IL-13 was determined by ELISA. Furthermore, IgG1 and IgG4 antibodies to cat allergen in cord blood samples were measured by ELISA. A more sensitive ELISA was used to measure Fel d 1-IgG IC. RESULTS: The prevalence and levels of IC were similar in cord blood from children of allergic and non-allergic mothers. The production of IL-5, IL-10. IL-13 and IFN-gamma by CBMC was not influenced by maternal atopy, but IFN-gamma was less commonly detected in samples with IC. There was no association between the presence of IC and any other cytokines. The levels of IgG1 and IgG4 antibodies were similar in both groups, and tended to be associated with the presence of IC. CONCLUSION: Immune complexes in cord blood may represent a normal mechanism for inducing primary immune responses, as the responses in babies from allergic and non-allergic mothers were largely similar. Low levels of IFN-gamma seems to be related with the presence of IC in cord blood.     

Characterization of the human T cell response to antigen 5 from Vespula vulgaris (Ves v 5)

BACKGROUND: The T cell reactivity to the major allergen of bee venom, phospholipase A2, has been thoroughly characterized. In contrast, only little is known about the human cellular response to major allergens from wasp venom. OBJECTIVE: To characterize the human T cell response to antigen 5 from Vespula vulgaris, Ves v 5. METHODS: Recombinant Ves v 5 was used to establish allergen-specific T cell lines (TCL) and T cell clones (TCC) from the peripheral blood of vespid-allergic and non-allergic individuals. Ves v 5-specific TCL were mapped for T cell epitopes using overlapping synthetic peptides representing the complete amino acid sequence of Ves v 5. Ves v 5-specific TCC were analysed for antigen-induced secretion of IL-4, IFN-gamma and IL-10. RESULTS: Seventeen distinct T cell epitopes were recognized by allergic individuals among which Ves v 5(181-192) was identified as a dominant T cell epitope. Partially different epitopes were observed in TCL from non-allergic subjects and the dominant epitope Ves v 5(181-192) was not prevalent in these cultures. Ves v 5-specific TCC isolated from allergic individuals did not show the typical T helper type 2 (Th2)-like cytokine profile in response to specific stimulation, i.e. high amounts of IL-4 and low IFN-gamma. TCC from non-allergic individuals showed a Th1-like cytokine pattern. CONCLUSIONS: Our findings provide evidence that the allergic T cell response to Ves v 5 is not Th2-dominated and that different immunogenic sites on this major wasp venom allergen are recognized by allergic and non-allergic individuals.     

Mechanisms of T cell peptide epitope-dependent late asthmatic reactions

Short peptide sequences corresponding to T cell epitopes have been identified in the major cat allergen Fel d 1. In order to directly activate allergen-specific T cells in cat-allergic asthmatic individuals, peptides were administered by intradermal injection. Subsequently, a proportion of subjects experienced a delayed reduction of airway calibre manifested as a decrease in FEV(1). Changes in lung function occurred approximately 3 h after peptide injection, peaked at 6 h and resembled an isolated late asthmatic reaction (LAR). Using molecular tissue typing techniques, it was determined that many of the individuals experiencing isolated LAR expressed particular HLA-DR molecules. These molecules were shown in subsequent experiments to bind individual peptides within the preparation and thus to activate T cells in a major histocompatibility complex (MHC)-restricted fashion. The precise mechanisms whereby MHC-restricted activation of allergen-specific T cells gives rise to bronchoconstriction are currently under investigation.     

Chimeric human fcgamma-allergen fusion proteins in the prevention of allergy

Allergic responses are strongly associated with Th2-type immune responses, and modulation of the skewed Th2 response toward a more balanced response is the major goal of allergen immunotherapy (IT) in allergic disorders. To achieve this goal, several approaches have been tested. The authors previously showed that a human immunoglobulin (Ig) Fcgamma-Fcvarepsilon fusion protein (GE2) that directly cross-links FcvarepsilonRI and FcgammaRIIb on human mast cells and basophils was able to inhibit degranulation, and they reasoned that human gamma-allergen fusion protein would achieve a similar inhibitory effect in an allergen-specific fashion while preserving the immunogenicity of the allergen component. Therefore, the authors constructed and developed a human-cat chimeric fusion protein composed of the human Fcgamma1 and the cat allergen Fel d1 (Felis domesticus) for cat allergen-specific IT. This article summarizes the therapeutic features and potential of this novel fusion protein for allergic IT.     

Detection of an allergen in dog dander that cross-reacts with the major cat allergen, Fel d 1

BACKGROUND: A considerable proportion of animal-allergic patients are sensitized to both cat and dog allergens but knowledge about cross-reactive allergens in cat and dog dander is limited. OBJECTIVE: To investigate whether dog dander contains an allergen that cross-reacts with the major cat allergen, Fel d 1. METHODS: Recombinant Fel d 1 with the same immunological properties as natural Fel d 1 was used for quantitative (CAP) IgE competition experiments performed with sera obtained from cat-allergic patients (n=36). A Fel d 1 cross-reactive dog allergen was characterized by one- and two-dimensional immunoblotting using rFel d 1 for IgE inhibition experiments and with monospecific, polyclonal rabbit anti-recombinant Fel d 1 antibodies. RESULTS: In 25% of Fel d 1-reactive cat-allergic patients, more than 50% inhibition of IgE reactivity to dog allergens was achieved with recombinant Fel d 1. An Fel d 1 cross-reactive 20 kDa allergen with a pI of approximately 3.4 was detected in dander extracts of several different dog breeds. CONCLUSION: This is the first report demonstrating the presence of an Fel d 1-like allergen in dog dander extracts, which may be responsible for double positivity to cat and dog in serology. However, the clinical relevance of this cross-sensitization needs to be confirmed. These results are important for the diagnostic and therapeutic use of dog dander allergen extracts.     

Modified T-cell activation pattern during specific immunotherapy (SIT) in cat-allergic patients

OBJECTIVE: The aim of the study was to analyse early effects of specific immunotherapy (SIT) on immune functions in cat-allergic patients. METHODS: Immunological responses of peripheral blood mononuclear cells from eight cat-allergic patients were analysed before and after SIT in comparison with 11 nonallergic controls. Cells were stimulated in vitro with either bacterial superantigen, mitogen, or cat allergen. Production of IL-12 and TH1/TH2 cytokines was analysed by ELISA and lymphocyte subset distribution was assessed by flow-cytometry. RESULTS: We found a significantly reduced secretion of IL-12 (P < 0.05) from cells of allergic individuals compared with the controls. This finding was associated with significantly lower IFN-gamma production after stimulation with allergen (P < 0.05) that did not increase during SIT. However, no significant differences were seen after stimulation with mitogen indicating an allergen specific IFN-gamma secretion response in allergic individuals. Prior to SIT IL-5 production was significantly higher in cells of allergic donors stimulated with allergen < 0.005 or mitogen (< 0.05). After reaching the maintenance dose for SIT, allergen-induced IL-5 production returned to normal levels, whereas it remained elevated after stimulation with mitogen. These changes were associated with a reduced frequency of CD45 RO T cells following SIT. CONCLUSION: These results suggest that SIT exerts early effects on allergen-specific T-cell responses with selective inhibition of the up-regulated TH2 immune response.     

Molecular determinants of allergen-induced effector cell degranulation

BACKGROUND: Allergen-induced effector cell degranulation is a key event in allergic inflammation and leads to early-phase symptoms, such as allergic rhinitis, conjunctivitis, urticaria, or bronchial asthma. OBJECTIVE: We sought to study molecular determinants of effector cell degranulation using a monoclonal IgE antibody specific for a peptide epitope of one of the most important respiratory allergens, the major grass pollen allergen Phl p 1, as a model system. METHODS: A hybridoma cell line producing a monoclonal IgE antibody against a Phl p 1-derived peptide, P1, was established by means of immunization of mice and used to sensitize rat basophil leukemia cells, which were exposed to P1 monomer, P1 dimer, and P1 polymer. RESULTS: It is demonstrated that the number of IgE epitopes on an allergen molecule and the concentration of allergen-specific IgE antibodies determine the extent of degranulation. The P1 monomer did not cause mediator release and prevented degranulation induced by polymeric P1. CONCLUSION: Our results suggest that the number of IgE epitopes on an allergen molecule determines its allergenic activity and explains why increases of allergen-specific IgE levels make patients more sensitive to allergens. Allergen-derived monomeric structures isolated by means of combinatorial chemistry might be used to develop new therapeutic strategies for allergy. CLINICAL IMPLICATIONS: Our study reveals molecular factors that determine the immediate allergenic activity of allergens and hence influence clinical sensitivity to these allergens.     

The performance of a component-based allergen-microarray in clinical practice

BACKGROUND: Currently, the diagnosis of IgE-mediated allergy is based on allergen-specific history and diagnostic procedures using natural allergen extracts for in vivo and in vitro tests. OBJECTIVE: The aim of the study was to comparatively analyse a new component-based allergen-microarray and the 'quasi-standard' ImmunoCAP for their clinical relevance in patients with allergic rhinoconjunctivitis to five aeroallergens [house dust mite (HDM), cat dander, birch, grass and mugwort pollen] in a prospective, double-centre study. METHODS: We enrolled 120 subjects at the two study centres. Allergic patients were defined as having an allergen-specific history plus a concomitant positive skin-prick test (SPT) to natural allergen extracts and specific serum IgE was measured by both methods. Each allergen was analysed separately. RESULTS: The microarray performed equally well in receiver-operating characteristic curve (ROC) analyses when compared with the CAP in cat (23 allergic vs 97 non-allergic, ROC area under the curve microarray 0.950 vs CAP 0.894, P = 0.211), birch (31/89, 0.908 vs 0.878, P = 0.483) and grass pollen (47/73, 0.923 vs 0.915, P = 0.770). It was slightly less sensitive in HDM-allergic subjects (26 allergic vs 94 non-allergic, ROC area microarray 0.808 vs CAP 0.911, P = 0.053) and displayed a reduced sensitivity in the mugwort pollen-allergic patients (17/103, 0.723 vs 0.879, P = 0.032). CONCLUSIONS: Component-based testing and the whole-allergen CAP are equally relevant in the diagnosis of grass-, birch- and cat-allergic patients. Although slightly less sensitive, the microarray is sufficient for the diagnosis of HDM-allergic patients, but needs alternative and/or additional components for detecting mugwort allergy.     

Human serum IgE-mediated mast cell degranulation shows poor correlation to allergen-specific IgE content

BACKGROUND: Although allergen-specific IgE content in serum can be determined immunochemically, little is known about the relationship between this parameter and the strength of the degranulation response upon allergen triggering. OBJECTIVES: Analyse the degranulation capacity of immunochemically defined purified and serum IgE after challenge with anti-IgE or allergen using a rat mast cell line (RBL) transfected with the alpha-chain of the human high-affinity IgE receptor (FcepsilonRI). METHODS: Purified IgE specific for 4-hydroxy-3nitrophenylacetyl, purified IgE of unknown specificity, and sera from allergic patients sensitive to Dermatophagoides pteronyssinus and Dactylis glomerata were assessed. Degranulation was measured by a beta-hexosaminidase release assay after anti-IgE or allergen-specific challenge. RESULTS: For purified monoclonal IgE a significant correlation (r = 0.97) was found between the proportion of bound allergen-specific IgE and the strength of the degranulation response. In contrast, no correlation (r = 0.27) was detected after sensitization with serum IgE. CONCLUSION: Our studies demonstrate that mast cell activation mediated through IgE from allergic patients is a result of complex relationships that are not only dependent on allergen-specific IgE content but also relate to the capacity to efficiently sensitize and trigger the signalling responses that lead to degranulation.     

"Accentuate the negative, eliminate the positive": engineering allergy therapeutics to block allergic reactivity through negative signaling

By targeting the dominant-negative signaling receptor FcgammaRIIb expressed on proallergic cells, we have developed 2 novel platforms for the treatment of IgE-mediated allergic disease. First is a genetically engineered bifunctional human fusion protein GE2, which is comprised of the Fc portions of human IgE and IgG1 with an interposed flexible linker designed as a long-term parenteral allergen-nonspecific therapy. GE2 blocks the effector phase of the IgE response in vitro in mice and human subjects and in vivo in the skin and airway and systemically in mice and monkeys. Whether reactivity against human GE2 in human subjects will limit its applicability remains to be determined. The second platform is designed to provide a safer form of allergen-specific immunotherapy and consists of genetically engineered chimeric human Fcgamma-allergen proteins, with Fcgamma-Fel d 1 as the prototype. The allergen portion binds to specific IgE on FcepsilonRs, whereas the Fcgamma portion coaggregates inhibitory FcgammaRIIb and drives inhibition of allergic reactivity. Fcgamma-Fel d 1 blocked human mast cell Fel d 1-induced allergic reactivity in vitro and in vivo in murine models while functioning as an immunogen but not as an allergen.     

A chimeric human-cat Fcgamma-Fel d1 fusion protein inhibits systemic, pulmonary, and cutaneous allergic reactivity to intratracheal challenge in mice sensitized to Fel d1, the major cat allergen

Co-aggregation of FcepsilonRI with FcgammaRIIb can block FcepsilonRI-mediated reactivity and Fc gamma:allergen chimeric proteins, by co-crosslinking FcgammaRIIb to allergen-specific IgE bound to the FcepsilonRI can block allergen-specific reactivity. We evaluated whether a human cat chimeric fusion protein (GFD) composed of part of the human Ig G1 Fc fused to the major cat allergen (Fel d1) would function as allergen immunotherapy while not inducing acute allergic reactivity in mice sensitized to Fel d1. Injection of GFD 6 h prior to Fel d1 challenge acutely blocked systemic and skin reactivity to Fel d1 challenge while mice given subcutaneous immunotherapy with GFD at days 37, 38, and 39 showed inhibition of systemic, lung, and cutaneous reactivity to Fel d1 2 weeks later. GFD immunotherapy did not induce systemic reactivity. Overall, the Fcgamma-Fel d1 chimeric fusion protein blocked Fel d1-induced IgE-mediated reactivity but did not induce in vivo mediator release on its own; suggesting that this approach using allergen combined with Fc gamma1 so as to achieve inhibitory signaling may provide an enhanced form of allergen immunotherapy.     

T cell responses to recombinant isoforms, synthetic peptides and a mutant variant of Lep d 2, a major allergen from the dust mite Lepidoglyphus destructor

BACKGROUND: The dust mite Lepidoglyphus destructor is an important cause of allergic reactions to dust, especially in farming environments. Two isoforms, recombinant (r)Lep d 2.01 and rLep d 2.02, of the major allergen Lep d 2, have previously been expressed as recombinant proteins. These isoforms differ 10.4% at the amino acid level. Furthermore, a mutant form of Lep d 2.01 (rLep d 2.6Cys) with a highly reduced IgE reactivity, has also been produced. OBJECTIVE: To investigate the T cell responses to the recombinant isoforms of Lep d 2, the Lep d 2.6Cys mutant and peptides of Lep d 2, in allergic and non-allergic individuals. METHODS: Peripheral blood mononuclear cells from 18 allergic and 16 non-allergic individuals were stimulated with the different antigens and the proliferative responses were measured. The cytokine production (interleukin (IL)-4, IL-5 and interferon (IFN)-gamma) were measured by ELISA. RESULTS: Higher T cell proliferation was measured to isoform 01 than to 02 in 28/34 subjects. The responses to rLep d 2.6Cys were lower than to isoform 01 in most subjects, but higher than to Lep d 2.02. Two immuno-dominant peptides, corresponding to amino acid residue 11-25 and 61-75 were identified. The atopic subjects produced significantly lower IFN-gamma in response to Lep d 2.01 as compared to the non-atopics. CONCLUSIONS: There was a significant difference in T cell response between the two isoforms of rLep d 2. The hypoallergenic mutant rLep d 2.6Cys was able to evoke a T cell response with a magnitude which is between the two isoforms. Amino acid residue 11-25 and 61-75 are the most frequently recognized parts of Lep d 2 and are likely to contain the immuno-dominant T cell epitopes.     

''Entopy'': localized mucosal allergic disease in the absence of systemic responses for atopy

BACKGROUND: The Th2 immune response in the nasal mucosa of subjects with allergic rhinitis is mediated by allergen-specific IgE. Moreover, these subjects show positive responses for markers of systemic atopy, including allergen-specific skin sensitivity and raised serum IgE titres. In contrast, idiopathic rhinitis (IR) subjects with similar histological nasal mucosal features differ in being defined as non-allergic because they have negative atopic responses. OBJECTIVE: We hypothesized that it is possible to have an allergic Th2 disease pathway localized in the nasal mucosa of 'non-allergic' rhinitis subjects despite an absence of atopic responses. METHODS: The presence of house dust mite and grass pollen-specific IgE antibodies was investigated in non-atopic (n=10) and atopic (n=11) subjects with persistent rhinitis and compared to normal (n=12) control subjects. Biotin-labelled allergen was used to localize specific allergen-binding antibodies in situ in sections of nasal mucosa. RESULTS: Grass pollen allergen binding was detected in the nasal mucosa of 3/10 non-atopic IR subjects but, in contrast, dust mite-specific antibodies were not detected. Specific antibodies were present in a total of 8/11 mucosal samples from the allergic group, but none was detected in normal control tissues. CONCLUSION: These findings support the concept of localized nasal allergy in 'non-atopic' rhinitis subjects. We propose the term 'entopy' to define this phenomenon and believe that this concept has a wider implication for localized allergic responses in other mucosal sites.     

Diesel exhaust particles directly induce activated mast cells to degranulate and increase histamine levels and symptom severity

BACKGROUND: The ability of combustion products, such as diesel exhaust particles (DEPs), to modulate the immune system has now been firmly established. DEPs can synergize with allergen at the human upper respiratory mucosa to enhance allergen-specific IgE production, initiate a T(H)2 cytokine environment, and even promote primary allergic sensitization. Experiments suggest that these effects result from the initial activation of mast cells to produce IL-4. OBJECTIVE: We sought to demonstrate that in vivo mast cell activation by DEPs plus allergen will also affect the release of classic mast cell mediators and consequently enhance the immediate-phase response. METHODS: Dust mite-sensitive subjects were challenged intranasally with allergen, and symptom scores and histamine levels in nasal wash samples were compared after prechallenge with 0.3 mg of DEPs. RESULTS: If the subjects were first sprayed with DEPs, mean symptom scores rose from 3.7 to 9.9; additionally, only one fifth of the amount of intranasal dust mite allergen was required to induce clinical symptoms. DEPs alone had no effect. The changes in symptoms correlated with histamine levels measured in nasal lavage specimens from these subjects. Although challenge with DEPs alone did not induce histamine release, challenge with both DEPs and allergen resulted in 3-fold higher histamine concentrations than those seen with allergen alone. In contrast, carbon black particles (elemental carbon devoid of chemicals) had no effect. The role of chemicals was confirmed because degranulation of a murine mast cell line by FcepsilonRI cross-linking was increased significantly (by 72%) by the soluble organic chemicals extracted from DEPs. CONCLUSIONS: Overall, these results suggest that exposure to DEPs can enhance the severity of clinical symptoms to allergen by enhancing mast cell degranulation.