Nuclear magnetic resonance structure-based epitope mapping and modulation of dust mite group 13 allergen as a hypoallergen

IgE-mediated allergic response involves cross-linking of IgE bound on mast cells by specific surface epitopes of allergens. Structural studies on IgE epitopes of allergens are essential in understanding the characteristics of an allergen and for development of specific allergen immunotherapy. We have determined the structure of a group 13 dust mite allergen from Dermatophagoides farinae, Der f 13, using nuclear magnetic resonance. Sequence comparison of Der f 13 with homologous human fatty acid-binding proteins revealed unique surface charged residues on Der f 13 that may be involved in IgE binding and allergenicity. Site-directed mutagenesis and IgE binding assays have confirmed four surface charged residues on opposite sides of the protein that are involved in IgE binding. A triple mutant of Der f 13 (E41A_K63A_K91A) has been generated and found to have significantly reduced IgE binding and histamine release in skin prick tests on patients allergenic to group 13 dust mite allergens. The triple mutant is also able to induce PBMC proliferation in allergic patients with indices similar to those of wild-type Der f 13 and shift the secretion of cytokines from a Th2 to a Th1 pattern. Mouse IgG serum raised using the triple mutant is capable to block the binding of IgE from allergic patients to wild-type Der f 13, indicating potential for the triple mutant as a hypoallergen for specific immunotherapy. Findings in this study imply the importance of surface charged residues on IgE binding and allergenicity of an allergen, as was also demonstrated in other major allergens studied.     

Antigen-specific T cell tolerance down-regulates mast cell responses in vivo

Fel d I is the major cat allergen that induces asthma and allergic rhinitis in humans. To investigate the mechanism of allergic responses to this allergen, a mouse model was developed. Mice sensitized to chain 1 of Fel d I exhibited T cell responses, B cell responses, and mast cell responses when challenged with the protein. Subcutaneous injections of peptides containing the dominant T cell epitopes of the allergen induced T cell tolerance in presensitized mice. When challenged with the allergen intratracheally, these tolerized mice produced a decreased amount of histamine in vivo. The decrease in histamine release was not solely dependent on the reduction of allergen-specific IgE. These data show that mast cell activity in mice with an ongoing sensitivity to allergen can be regulated through peptide-induced T cell tolerance.     

Commensal bacteria-derived signals regulate basophil hematopoiesis and allergic inflammation

Commensal bacteria that colonize mammalian barrier surfaces are reported to influence T helper type 2 (T(H)2) cytokine-dependent inflammation and susceptibility to allergic disease, although the mechanisms that underlie these observations are poorly understood. In this report, we find that deliberate alteration of commensal bacterial populations via oral antibiotic treatment resulted in elevated serum IgE concentrations, increased steady-state circulating basophil populations and exaggerated basophil-mediated T(H)2 cell responses and allergic inflammation. Elevated serum IgE levels correlated with increased circulating basophil populations in mice and subjects with hyperimmunoglobulinemia E syndrome. Furthermore, B cell-intrinsic expression of myeloid differentiation factor 88 (MyD88) was required to limit serum IgE concentrations and circulating basophil populations in mice. Commensal-derived signals were found to influence basophil development by limiting proliferation of bone marrow-resident precursor populations. Collectively, these results identify a previously unrecognized pathway through which commensal-derived signals influence basophil hematopoiesis and susceptibility to T(H)2 cytokine-dependent inflammation and allergic disease.     

Characterization of Rhinitis According to the Asthma Status in Adults Using an Unsupervised Approach in the EGEA Study

Background

The classification of rhinitis in adults is missing in epidemiological studies.

Objective

To identify phenotypes of adult rhinitis using an unsupervised approach (data-driven) compared with a classical hypothesis-driven approach.

Methods

983 adults of the French Epidemiological Study on the Genetics and Environment of Asthma (EGEA) were studied. Self-reported symptoms related to rhinitis such as nasal symptoms, hay fever, sinusitis, conjunctivitis, and sensitivities to different triggers (dust, animals, hay/flowers, cold air…) were used. Allergic sensitization was defined by at least one positive skin prick test to 12 aeroallergens. Mixture model was used to cluster participants, independently in those without (Asthma-, n = 582) and with asthma (Asthma+, n = 401).

Results

Three clusters were identified in both groups: 1) Cluster A (55% in Asthma-, and 22% in Asthma+) mainly characterized by the absence of nasal symptoms, 2) Cluster B (23% in Asthma-, 36% in Asthma+) mainly characterized by nasal symptoms all over the year, sinusitis and a low prevalence of positive skin prick tests, and 3) Cluster C (22% in Asthma-, 42% in Asthma+) mainly characterized by a peak of nasal symptoms during spring, a high prevalence of positive skin prick tests and a high report of hay fever, allergic rhinitis and conjunctivitis. The highest rate of polysensitization (80%) was found in participants with comorbid asthma and allergic rhinitis.

Conclusion

This cluster analysis highlighted three clusters of rhinitis with similar characteristics than those known by clinicians but differing according to allergic sensitization, and this whatever the asthma status. These clusters could be easily rebuilt using a small number of variables.     

New In-vitro Test for IgE-mediated Hypersensitivity in Man

A new simple and sensitive in-vitro method for the diagnosis of type 1 (IgE-mediated) hypersensitivity in man is described. Sliced human skin is passively sensitized by reaginic serum from allergic patients and the presence of antigen-specific IgE on the sensitized slices is detected by assay of antigen-evoked histamine release. Serum from 12 out of 14 patients with clinical respiratory allergy and positive skin tests gave significant antigen-specific histamine release. This method, which is essentially an in-vitro model of the Prausnitz-Küstner reaction, should prove of value in the diagnosis of human reaginic hypersensitivity in man.     

Another smoking hazard: raised serum IgE concentration and increased risk of occupational allergy.

Individual smoking histories of a general population sample and of two groups of workers exposed to occupational allergens were related to serum IgE concentrations and results of radioallergosorbent and prick tests in the workers. The geometric mean IgE concentration was higher in smokers than in non-smokers. The distribution of serum IgE values in the two groups showed an apparent difference, with a bimodal appearance in the smokers. Evidence of sensitisation against occupational allergens was more common in workers who smoked. The adjuvant effect of smoking on IgE antibody production might be due to damage to airways mucosa and supports the mucosal theory of atopy.     

Potentiation of histamine release by sodium cromoglycate.

Human lung slices passively sensitized with allergic serum released histamine when incubated with specific antigen and anti-IgE but anti-IgG had no effect. Sodium cromoglycate (SCG) inhibited antigen induced histamine release but the dose-response curve was bell-shaped. Inhibition of anti-IgE induced release was linearly related to dose, whereas that induced by anti-IgG was potentiated by increasing doses of SCG. After sensitization with allergic serum in which IgE had been inactivated by heating, specific antigen released little or no histamine but this was potentiated by SCG. It is concluded that SCG inhibits IgE mediated but potentiates IgG mediated allregic reactions thus explaining its characteristic dose-response curve $\text{in vitro}$.     

New Type of Allergic Asthma due to IgG “Reaginic” Antibody

This study was undertaken to examine the possibility that IgE is not the only immunoglobulin responsible for immediate allergic reactions. A group of asthmatics were investigated in whom immediate allergic reactivity of the bronchi to common inhalant allergens had been confirmed by provocation tests. Their sera were fractionated and the reaginic activity of the immunoglobulin classes was studied by passive cutaneous anaphylaxis testing in monkeys. The results showed that the immediate allergic reactions were due to IgE antibodies in most patients, but there was a group with reactions due to short-term anaphylactic IgG antibodies. It was not possible to inhibit the IgG-mediated responses with disodium cromoglycate. As these two groups had clearly different serum IgE levels the estimation of IgE provided an important guide to the management of these patients.     

Effect of cobalamin on the allergic response in mice

Cyanocobalamin, deoxyadenosylcobalamin and methylcobalamin were separately administered to mice sensitized with the ovalbumin antigen to investigate the allergic response. The serum IgE and pulmonary histamine concentrations were significantly lower in all cobalamin (Cbl)-administered groups. The production of interleukin (IL)-2 and IL-4 in splenocytes was also lower in all Cbl-administered groups. These results show that Cbls were effective in mitigating allergic reactions and IL-2 production. CD3+ CD28+ (CD28 is an accessory molecule related to IL-2 production) and CD4+ CD28+ in splenocytes were higher in all the Cbl-administered groups. However, CD3+ CD28-, CD4+ CD28- and CD5+ CD25- (CD25: IL-2 R alpha/p55) were lower in the Cbl-administered groups. In addition, Cbl specifically inhibited the cellular phosphorylation of tyrosine induced by ovalbumin sensitization. These results indicate that the signal in a cell by CD 28 was restrained by Cbl. We infer that Cbl administration significantly reduced the IL-2 concentration, and secondarily the IL-4, IgE and histamine concentrations.     

Safety evaluation of genetically modified mustard (V4) seeds in terms of allergenicity: Comparison with native crop

Genetically modified (GM) mustard line (V4) with increased carotenoid content was compared with native mustard to find the difference in allergenic potential, if any. Simulated gastric fluid (SGF) digestibility of crude protein extract from GM as well as its native counterpart mustard crop was envisaged to understand the intended or unintended changes in GM crop along with IgE immunoblotting. BALB/c mice were used as model for allergenicity studies for monitoring total and specific IgE, specific IgG1, histamine level, histopathology, and systemic anaphylaxis score. Allergenicity of mustard was checked in humans by clinical history, skin prick test and IgE levels. Similar results were evident by significant increase in total IgE, specific IgE, IgG1, histamine levels, in GM and native mustard in comparison to control group. Prominent anaphylactic symptoms (score 2: 60%; score 3: 20%; score 4: 20% in native mustard and score 2: 40%; score 3: 40%; score 4: 20% in GM mustard) and eruptive histopathological changes were observed in both GM and native mustard when compared with controls. One protein of approximately 16 kDa was found stable up to 1 h in both GM as well as non GM mustard. IgE immunoblotting detected three protein components of approximately 29, 24 and 16 kDa in both GM and non GM varieties. Collectively, our data demonstrate substantially equivalent allergic responses against GM as well as its native counterpart. Therefore, the GM mustard may be as safe as its native counterpart with reference to allergenic responses.     

Seasonal variation in food allergy to apple

The aim of the study was to investigate the possibility of a seasonal variation in reactivity to apples in 27 birch pollen allergic patients. Before and during the birch pollen season 1998, the patients were subjected to double-blind, placebo-controlled food challenges (DBPCFCs) with grated fresh Golden Delicious apple followed by an open food challenge with whole fresh apple. The clinical reactions elicited during the challenges were evaluated both by the patients and the investigators. Moreover, the skin reactivity and the in vitro reactivity to apple were evaluated by skin prick test (SPT), leukocyte histamine release (HR), measurement of specific IgE, and immunoblotting experiments. The sensitivity of the DBPCFC, when compared with the result of the open challenge, was 0.74 (14/19) before the season and 0.80 (16/20) during the season. None of the patients reacted to the blinded challenge without a subsequent reaction to the open challenge. One placebo reaction was registered both before and in season, but not in the same patient. The patient scores of the first positive challenges, and the maximal scores of each combined blinded and open challenge session, were significantly increased during the pollen season (P<0.05). The scores of the open challenge were significantly higher than the scores of the DBPCFC both before the season and during the in-season challenges (P<0.05). Specific IgE against Golden Delicious increased during season (P<0.05), while neither SPT, HR, nor immunoblotting experiments could confirm an increase in reactivity. In conclusion, the results of the oral challenge tests indicated an increase in clinical reactivity to apples during the birch pollen season in birch pollen allergic individuals.     

Incidence of allergic rhinitis in general practice, 1981-92.

OBJECTIVES--To determine the epidemiology of hay fever and to consider the role of pollution. DESIGN--Examination of data on weekly incidence of allergic rhinitis and hay fever by age, sex, region, and location. SETTING--Royal College of General Practitioners Weekly Returns Service. Practice data were based on registered populations of 220,000 in 1981, rising to 700,000 in 1992 from England and Wales. MAIN OUTCOME MEASURES--Numbers of new cases of hay fever and allergic rhinitis. Data on pollen counts for Darlington, Derby, and London. RESULTS--The incidence of allergic rhinitis fluctuated greatly from year to year but showed no trend. Peaks in hay fever coincided with peak pollen counts. No important differences were found between urban and rural locations or different parts of the country with respect to both size and timing of the peaks. Incidence was highest in children (5-14 years). CONCLUSIONS--The similarity of the results throughout England and Wales does not support an important role for local pollutants in hay fever. However, the possibility that levels of pollutants are high enough to act as an adjuvant in hay fever across the whole study area has not been excluded.     

Does IgE bind to and activate eosinophils from patients with allergy?

Human eosinophils have been reported to express both the mRNA and protein for the high affinity IgE receptor (FcepsilonRI); it is speculated that this receptor plays a role in eosinophil mediator release in allergic diseases. However, questions still remain. How much of the FcepsilonRI protein is actually expressed on the cell surface of the eosinophil? If they are present, are these IgE receptors associated with effector functions of eosinophils? To address these issues, we studied blood eosinophils from patients with ragweed hay fever. A high level of low affinity IgG receptor (FcgammaRII, CD32), but no expression of FcepsilonRI, was detectable on the eosinophil surface by standard FACS analysis. However, after in vitro sensitization with biotinylated chimeric IgE (cIgE), cell-bound cIgE was detected by PE-conjugated streptavidin. This cIgE binding was partially inhibited by anti-FcepsilonRI mAb, suggesting that eosinophils do express minimal amounts of FcepsilonRI detectable only by a sensitive method. Indeed, FACS analysis of whole blood showed that eosinophils express approximately 0.5% of the FcepsilonRI that basophils express. When stimulated with human IgE or anti-human IgE, these eosinophils did not exert effector functions; there was neither production of leukotriene C4 or superoxide anion nor any detectable degranulation response. In contrast, eosinophils possessed membrane-bound human IgG and showed functional responses when stimulated with human IgG or anti-human IgG. Thus, IgG and/or cytokines, such as IL-5, appear to be more important for eosinophil activation in allergic diseases than IgE.     

Measurement of IgE on human basophils: relation to serum IgE and anti-IgE-induced histamine release.

The number of IgE molecules bound to human basophils was calculated from direct measurements of the IgE dissociated after exposing leukocytes to pH 3.7 acetate buffer in the cold. In 18 donors studied, cell-bound IgE ranged from 4000 to 500,000 molecules/basophil and correlated with the serum IgE concentration (r = 0.89, p less than 0.001) which ranged from 5 to 3,000 ng/ml. Sensitivity of these cells to anti-IgE was tested to explore the relationship between cell-bound IgE and the concentration of anti-IgE required for histamine release. Cells from some nonatopic donors (4000 to 100,000 IgE molecules/basophil) were as sensitive as cells from allergic donors (100,00 to 500,000 IgE molecules/basophil). Moreover, cells from donors having approximately the same cell-bound IgE concentration varied widely in their sensitivity to anti-IgE. We conclude that an intrinsic property of human basophils ("releasability") is an important parameter in determing mediator release.     

The Tec family kinase, IL-2-inducible T cell kinase, differentially controls mast cell responses

The Tec family tyrosine kinase, IL-2-inducible T cell kinase (Itk), is expressed in T cells and mast cells. Mice lacking Itk exhibit impaired Th2 cytokine secretion; however, they have increased circulating serum IgE, but exhibit few immunological symptoms of allergic airway responses. We have examined the role of Itk in mast cell function and FcepsilonRI signaling. We report in this study that Itk null mice have reduced allergen/IgE-induced histamine release, as well as early airway hyperresponsiveness in vivo. This is due to the increased levels of IgE in the serum of these mice, because the transfer of Itk null bone marrow-derived cultured mast cells into mast cell-deficient W/W(v) animals is able to fully rescue histamine release in the W/W(v) mice. Further analysis of Itk null bone marrow-derived cultured mast cells in vitro revealed that whereas they have normal degranulation responses, they secrete elevated levels of cytokines, including IL-13 and TNF-alpha, particularly in response to unliganded IgE. Analysis of biochemical events downstream of the FcepsilonRI revealed little difference in overall tyrosine phosphorylation of specific substrates or calcium responses; however, these cells express elevated levels of NFAT, which was largely nuclear. Our results suggest that the reduced mast cell response in vivo in Itk null mice is due to elevated levels of IgE in these mice. Our results also suggest that Itk differentially modulates mast cell degranulation and cytokine production in part by regulating expression and activation of NFAT proteins in these cells.     

Diagnosis of <Emphasis Type="Italic">Chenopodium album</Emphasis> allergy with a cocktail of recombinant allergens as a tool for component-resolved diagnosis

Chenopodium album pollen is one of the main sources of pollen allergy in desert and semi-desert areas and contains three identified allergens, so the aim of this study is comparison of the diagnostic potential of C. album recombinant allergens in an allergenic cocktail and C. album pollen extract. Diagnostic potential of the allergenic cocktail was investigated in 32 individuals using skin prick test and obtained results were compared with the acquired results from C. album pollen extract. Specific IgE reactivity against the pollen extract and allergenic cocktail was determined by ELISA and western blotting tests. Inhibition assays were performed for the allergenic cocktail characterization. The exact sensitization profile of all patients was identified which showed that 72, 81 and 46% of allergic patients had IgE reactivity to rChe a 1, rChe a 2 and rChe a 3, respectively. Almost all of C. album allergic patients (30/32) had specific IgE against the allergenic cocktail. In addition, there was a high correlation between IgE levels against the allergenic cocktail and IgE levels against the pollen extract. The allergenic cocktail was able to completely inhibit IgE binding to natural Che a 1, Che a 2 and Che a 3 in C. album extract. In addition, positive skin test reactions were seen in allergic patients that tested by the allergenic cocktail. The reliable results obtained from this study confirmed that the allergenic cocktail with high diagnostic potential could be replaced with natural C. album allergen extracts in skin prick test and serologic tests.     

Allergenic pollen and pollinosis in Warsaw

Summary Our study of fifty two hay fever patients included twenty six solely allergic to grass pollen and twenty six exhibiting allergy to various pollen species, such as hazel, birch, oak, poplar, andArtemisia. Their total and specific IgE response was evalutated by the immunoenzymatic method, while clinical reactivity was assessed by recording nasal and bronchial symptom scores between mid-March and mid-July. Simultaneously pollen counts were made. Polysensitized patients showed significantly higher levels of both total and specific IgE, which testifies to the enhanced quantitative and qualitative IgE. Multisensitized patients reacted earlier than patients sensitized to grass pollen only, which confirms that non-grass plants flowering only in the spring cause the priming effect on the nasal and bronchial mucosa. The early symptoms may be attributable to tree pollen sensitivity or may refletct higher grass pollen IgE levels in the polysensitized group. Characteristically, nasal symptoms preceded bronchial symptoms of several weeks.On comparing nasal washing from the polysensitized patients to washing from patients with grass pollen, we found much cytological material with the predominance of eosinophils.     

Establishment of an allergic rhinitis model in mice for the evaluation of nasal symptoms

The purpose of our study was to establish a new model of allergic rhinitis in mice, eliciting symptoms such as sneezing, infiltration of eosinophils into the nasal mucosa, and antigen-specific IgE production. One of the major human T-cell epitopes in Cry j 1, an allergen of Japanese cedar pollen, is also a major murine T-cell epitope in B10.S mice. Thus we tried to establish an allergic rhinitis model in B10.S mice with Cry j 1 as the antigen. We sensitized B10.S mice subcutaneously with Cry j 1/alum three times at intervals of 1 week. Five weeks after the final sensitization, we challenged the mice by instilling Cry j 1 intranasally from the day after intranasal histamine pretreatment. Soon after, we counted the number of sneezes. We then evaluated the infiltration of eosinophils into the nasal tissues and also measured the serum levels of antigen-specific IgE antibody. In addition, we confirmed the effects of ketotifen fumarate and dexamethasone hydrochloride on these animals. In Cry j 1-sensitized B10.S mice, sneezes, eosinophil peroxidase (EPO) activity in nasal tissues, and Cry j 1-specific IgE clearly increased after intranasal histamine pretreatment and 5 days of continuous intranasal Cry j 1 challenge. Both ketotifen and dexamethasone inhibited the increase in sneezing, and dexamethasone also inhibited EPO activity and Cry j 1-specific IgE. Thus we succeeded in establishing a new model of allergic rhinitis in Cry j 1-sensitized B10.S mice, which exhibited sneezing, eosinophil infiltration into the nasal mucosa, and Cry j 1-specific IgE production.     

Standardization of food allergen extracts for skin prick test

The aim of the study was to standardize and evaluate technically optimized food allergen extracts for use in skin prick test (SPT). The standardization procedure comprised 36 allergic histories in 32 food allergic patients with 21 healthy, non-atopic individuals serving as controls. The patients had a history of allergic symptoms upon ingestion of either cow’s milk (n=3), hen’s egg (n=9), wheat (n=4), hazelnut (n=14) or cod (n=6). They also had specific IgE in serum to the food in question and a positive SPT with a fresh preparation of the food. The diagnosis had been confirmed by a double-blind, placebo-controlled food challenge, except for the hazelnut-allergic patients. The controls were subjected to an open food challenge with all the foods to ensure tolerance. The standardization was performed by means of titrated SPT in accordance with the guidelines on biological standardization from the Nordic Council on Medicine. Regression analysis of the skin wheal areas was performed for each patient and the median protein concentration of allergen preparation (median C_h10) eliciting a wheal area of the same size as histamine 10 mg/ml was calculated. The median C_h10 was 0.56 mg/ml for milk, 0.88 mg/ml for egg, 5.4 mg/ml for wheat, 2.1 mg/ml for hazelnut and 0.017 mg/ml for the cod extract. The sensitivity of the median C_h10 estimated from the SPT data was 1 for milk, 0.98 for egg, 1 for wheat, 1 for hazelnut and 0.87 for the cod extract. The allergenic activity of the hazelnut extract was further investigated by leukocyte histamine release (HR) and immunoblotting experiments using sera from 27 hazelnut allergic patients. The clinical sensitivity of the optimized hazelnut extract evaluated by HR was 0.78 compared to 0.30 for a commercially available hazelnut extract (Soluprick). Immunoblotting results showed a stronger IgE binding capacity and additional IgE-binding bands of the optimized hazelnut extract compared with the Soluprick extract.     

Soybean 𝛃-conglycinin-induced gut hypersensitivity reaction in a piglet model

Soybean is a major protein source in human and animal nutrition, but has also been described as a source of allergenic reactions. The objective of this study was to investigate anaphylactic reactions induced by purified soybean β-conglycinin in 10-day old piglets, and to elaborate the molecular and cellular mechanisms of intestinal injury resulting from soybean hypersensitivity. We investigated the oral allergy syndrome and anaphylactic reactions in piglets caused by soybean β-conglycinin with an intragastric feeding protocol without using an adjuvant. Physical symptoms, including lethargy, diarrhoea and respiratory distress were monitored to determine the anaphylactic reactions. Immunological assessment was conducted through measurement of immunoglobulin E (IgE) antibody concentration. Jejunal tissue was assessed for morphologic changes after the oral challenge, and histamine release, mRNA expression and endogenous production of cytokines were analysed. The results showed that β-conglycinin reduced growth of piglets, and after oral challenge, sensitised piglets displayed signs of allergic hypersensitivity. Furthermore, the levels of both total IgE and antigen-specific IgE in the sera were increased. Histological examination of jejunum revealed intestinal morphology damage, higher levels of interleukin-4, interferon-γ and significant levels of histamine were detected in the tissues. Our results indicate that purified β-conglycinin possesses intrinsic immune-stimulating capacity and can induce an allergic reaction, which was IgE mediated. Both T helper2 and T helper1 responses may play important roles in the intestinal injury resulting from soybean hypersensitivity.