Ara h 8, a Bet v 1-homologous allergen from peanut, is a major allergen in patients with combined birch pollen and peanut allergy

BACKGROUND: We recently described patients with soybean allergy mainly mediated by cross-reactivity to birch pollen allergens. A majority of those patients were reported to have peanut allergy. OBJECTIVE: We sought to study the occurrence of peanut allergy in patients allergic to birch pollen and characterized the Bet v 1-homologous peanut allergen Ara h 8. METHODS: Recombinant Ara h 8 was cloned with degenerated primers and expressed in Escherichia coli. Nine Swiss and 11 Dutch patients with peanut and birch pollen allergy and a positive double-blind, placebo-controlled food challenge result to peanut were investigated for IgE reactivity to birch pollen and purified peanut allergens and cross-reactivity between birch and peanut. Ara h 8 stability against digestion and roasting was assessed by means of RAST inhibition. The IgE cross-linking potency of Ara h 8 was tested on the basis of basophil histamine release. RESULTS: During double-blind, placebo-controlled food challenge, all patients experienced symptoms in the oral cavity, progressing to more severe symptoms in 40% of patients. CAP-FEIA detected recombinant (r) Ara h 8-specific IgE in 85%. IgE binding to Ara h 8 was inhibited by Bet v 1 in peanut extract immunoblotting and in RAST inhibition. In EAST inhibition recombinant rAra h 8 inhibited IgE binding to peanut in 4 of 7 tested patient sera. Antipeanut response was dominated by Ara h 8 in 12 of 17 tested patients. Furthermore, our results demonstrate a low stability of Ara h 8 to roasting and no stability to gastric digestion. Basophil histamine release with rAra h 8 was more than 20% in 5 of 7 tested sera. CONCLUSIONS: Peanut allergy might be mediated in a subgroup of our patients by means of cross-reaction of Bet v 1 with the homologous peanut allergen Ara h 8.     

The non-specific lipid transfer protein, Ara h 9, is an important allergen in peanut

Background Plant food allergy in the Mediterranean area is mainly caused by non-specific lipid transfer proteins (nsLTP). The aim of this study was to characterize peanut nsLTP in comparison with peach nsLTP, Pru p 3, and assess its importance in peanut allergy.
Methods Peanut-allergic patients from Spain ( n =32) were included on the basis of a positive case history and either a positive skin prick test or specific IgE to peanut. For comparison, sera of 41 peanut-allergic subjects from outside the Mediterranean area were used. Natural Ara h 9 and two isoforms of recombinant Ara h 9, expressed in Pichia pastoris , were purified using a two-step chromatographic procedure. Allergen characterization was carried out by N-terminal sequencing, circular dichroism (CD) spectroscopy, immunoblotting, IgE inhibition tests and basophil histamine release assays.
Results Compared with natural peanut nsLTP, the recombinant proteins could be purified in high amounts from yeast supernatant (45 mg/L). The identity of the proteins was verified by N-terminal amino acid sequencing and with rabbit nsLTP-specific antibodies. CD spectroscopy revealed similar secondary structures for all preparations and Pru p 3. The Ara h 9 isoforms showed 62–68% amino acid sequence identity with Pru p 3. IgE antibody reactivity to rAra h 9 was present in 29/32 Spanish and 6/41 non-Mediterranean subjects. Recombinant Ara h 9 showed strong cross-reactivity to nPru p 3 and similar IgE-binding capacity as nAra h 9. The two Ara h 9 isoforms displayed similar IgE reactivity. In peanut-allergic patients with concomitant peach allergy, Ara h 9 showed a weaker allergenic potency than Pru p 3 in histamine release assays.
Conclusions Ara h 9 is a major allergen in peanut-allergic patients from the Mediterranean area. Ara h 9 is capable of inducing histamine release from basophils, but to a lesser extent than Pru p 3.     

The effects of roasting on the allergenic properties of peanut proteins

BACKGROUND: Because of the widespread use of peanut products, peanut allergenicity is a major health concern in the United States. The effect or effects of thermal processing (roasting) on the allergenic properties of peanut proteins have rarely been addressed. OBJECTIVE: We sought to assess the biochemical effects of roasting on the allergenic properties of peanut proteins. METHODS: Competitive inhibition ELISA was used to compare the IgE-binding properties of roasted and raw peanut extracts. A well-characterized in vitro model was used to test whether the Maillard reaction contributes to the allergenic properties of peanut proteins. The allergic properties were measured by using ELISA, digestion by gastric secretions, and stability of the proteins to heat and degradation. RESULTS: Here we report that roasted peanuts from two different sources bound IgE from patients with peanut allergy at approximately 90-fold higher levels than the raw peanuts from the same peanut cultivars. The purified major allergens Ara h 1 and Ara h 2 were subjected to the Maillard reaction in vitro and compared with corresponding unreacted samples for allergenic properties. Ara h 1 and Ara h 2 bound higher levels of IgE and were more resistant to heat and digestion by gastrointestinal enzymes once they had undergone the Maillard reaction. CONCLUSIONS: The data presented here indicate that thermal processing may play an important role in enhancing the allergenic properties of peanuts and that the protein modifications made by the Maillard reaction contribute to this effect.     

Relevance of Ara h1, Ara h2 and Ara h3 in peanut-allergic patients, as determined by immunoglobulin E Western blotting, basophil–histamine release and intracutaneous testing: Ara h2 is the most important peanut allergen

BACKGROUND: A number of allergenic proteins in peanut has been described and the relative importance of these allergens is yet to be determined. OBJECTIVES: We have investigated the relevance of previously identified peanut allergens in well-characterized peanut-allergic patients by in vitro, ex vivo and in vivo assays. METHODS: Thirty-two adult peanut-allergic patients were included based on careful and standardized patient history and the presence of peanut-specific IgE. The diagnosis peanut allergy was confirmed using double-blind placebo-controlled food challenges in 23 patients. Major peanut allergens Ara h1, Ara h2 and Ara h3 were purified from peanuts using ion-exchange chromatography. IgE immunoblotting was performed and IgE-cross-linking capacity was examined by measuring histamine release (HR) after incubating patient basophils as well as passively sensitized basophils with several dilutions of the allergens. Intracutaneous tests (ICTs) using 10-fold dilution steps of the purified allergens and crude peanut extract were performed. RESULTS: Ara h2 was recognized most frequently (26 out of 32) in all tests and induced both positive skin tests and basophil degranulation at low concentrations, whereas Ara h1 and Ara h3 were recognized less frequently and reacted only at 100-fold higher concentrations as analysed with HR and intracutaneous testing (ICT). Next to the three tested allergens, proteins with molecular weights of somewhat smaller than 15 kDa were identified as a IgE-binding proteins on immunoblot in the majority of the patients (20 out of 32). CONCLUSION: We conclude that Ara h2 is, for our patient group, the most important peanut allergen, and that previously unidentified peanut proteins with molecular weights of somewhat smaller than 15 kDa may be important allergens as well. ICT in combination with basophil-HR and IgE immunoblotting provides insight in the patient specificity towards the individual peanut allergens.     

Mixed antibody and T cell responses to peanut and the peanut allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6 in an oral sensitization model

BACKGROUND: Peanut allergy is known for its severity and persistence through life. Several peanut proteins have been identified as allergenic and are indicated as Ara h 1-7. Very little is known about the mechanisms that underlie sensitization to peanut proteins. OBJECTIVE: The purpose of the present study was to reveal the immune responses that are induced against peanut and the peanut allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6 during sensitization, including the very early responses. METHODS: Humoral and T cell responses against peanut and the peanut allergens were examined in an early and later stage of sensitization in an established murine model of peanut anaphylaxis. Therefore C3H/HeJ mice were orally exposed to two different doses of peanut extract plus cholera toxin. RESULTS: Oral sensitization to peanut was characterized by an antigen-induced mixed cytokine response in the spleen (IL-4, IL-5, IL-10 and IFN-gamma), which could already be observed 7 days after the onset of exposure. Additionally, polyisotypic humoral responses (IgE, IgG1 and IgG2a) against peanut were found in the serum. Moreover, we demonstrated that these T helper (Th)1/Th2 cytokine and antibody responses were also directed specifically against the major peanut allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6. CONCLUSIONS: This study implicates that both Th1 and Th2 phenomena are involved in the development of peanut allergy in the C3H/HeJ murine model. Furthermore, we show that the present oral model is suitable to examine immune responses to food allergens during different stages of sensitization upon treatment with a whole food extract.     

T cell responses to major peanut allergens in children with and without peanut allergy

Background T cell responses involved in peanut allergy are poorly understood. Objective To investigate T cell responses towards major peanut allergens in peanut‐allergic (PA) subjects compared with peanut‐sensitized (PS) non‐allergic children and non‐atopic (NA) controls. Methods Eighteen PA children, seven non‐allergic PS children and 11 NA adults were included. Peripheral blood mononuclear cells were stimulated with a crude peanut extract (CPE). Short‐term T cell lines were generated and subsequently stimulated with CPE and purified Ara h 1, Ara h 2, Ara h 3 and Ara h 6. The proliferation and production of IL‐13, IFN‐γ, IL‐10 and TNF‐α were analysed. Results Proliferation to CPE and major allergens was enhanced in PA subjects. The primary response to CPE was comparable with PS subjects, with increased production of IL‐13 and IFN‐γ compared with NA. Production of IL‐10 was not observed. In short‐term T cell lines, the response to CPE was stronger in PA than in PS and NA subjects. Only PA children had a detectable response to major peanut allergens, characterized by IL‐13 production. The response was the highest after Ara h 3 stimulation, and the lowest after Ara h 2 stimulation. No significant correlation was observed between peanut‐specific IgE levels and T cell responses to CPE. Conclusion T cell responses to CPE in PA and PS children were characterized by Th1 and Th2 cytokines. Only PA children showed enhanced Th2 responses to Ara h 1, Ara h 3 and Ara h 6. Cite this as: A. E. Flinterman, S. G. M. A. Pasmans, C. F. den Hartog Jager, M. O. Hoekstra, C. A. F. M. Bruijnzeel‐Koomen, E. F. Knol and E. van Hoffen, Clinical & Experimental Allergy, 2010 (40) 590–597.     

Digested Ara h 1 has sensitizing capacity in Brown Norway rats

Background Food allergies are a public health issue of growing concern, with peanuts in particular being associated with severe reactions. The peanut allergen, Ara h 1, belongs to the cupin plant food allergen family, which, unlike other structural families, appears to be broken down rapidly following gastrointestinal digestion.
Objective Using Ara h 1 as a model allergen, the ability of digested protein to sensitize has been investigated.
Methods Ara h 1 was purified from whole roasted peanuts. Intact Ara h 1 was digested in an in vitro model, simulating the human gastrointestinal digestion process. Digestion products were analysed for peptide sizes and their ability to aggregate. Brown Norway (BN) rats, used as an animal model, were immunized with purified intact Ara h 1 or the gastrointestinal digestion products thereof. The sensitizing capacity was evaluated by analyses of specific antibody (IgG1, IgG2a and IgE) responses and ability to trigger mediator release of rat basophilic leukaemia (RBL)-2H3 cells.
Results The present study showed that Ara h 1 was broken down, resulting in peptide fragments of sizes <2.0 kDa, of which approximately 50% was in aggregated complexes of M _r up to 20 kDa. Ara h 1 digesta were shown to have sensitizing capacity in BN rats, being capable of inducing specific IgG and IgE antibodies. The IgE response was functional, having the capacity to induce specific degranulation of RBL cells.
Conclusion From this study, it can be concluded that lability of a food allergen to gastrointestinal digestion does not necessarily abrogate its allergenic sensitizing potential.     

Peanut protein allergens: gastric digestion is carried out exclusively by pepsin

BACKGROUND: A major characteristic of many food allergens, including Ara h 1, a major peanut allergen, is their resistance to gastric digestion. One estimate of the allergenic potential of a possible protein allergen is its stability under simulated gastric conditions. OBJECTIVE: Because the rate and extent of digestion of allergenic proteins will affect the severity of any subsequent allergic response, it is important to correlate protein allergen digestion in simulated gastric fluid with that in actual gastric fluid. METHODS: A major peanut allergen, Ara h 1, was digested in vitro by using both pepsin and porcine gastric fluid. Several comparisons between the 2 sets of proteolytic conditions were assessed including pH optima and the effect of temperature, denaturants, and specific enzyme inhibitors. RESULTS: In vitro digestion of Ara h 1 with pepsin and porcine gastric fluid resulted in virtually identical hydrolysis patterns as observed on SDS-PAGE. The protease activity of both pepsin and gastric fluid were inhibited at high pH and in the presence of pepstatin. However, both remained active in 4 mol/L urea and at 60 degrees C. CONCLUSIONS: Protein digestion in the porcine stomach is carried out by pepsin. In vivo gastric digestion is modeled accurately by peptic hydrolysis. Digestion conditions in vivo are comparable to experimental conditions in vitro provided that the acidic nature of the stomach contents is optimal for characterization of the allergen under standard pepsin digestion conditions. Additional experimentation using crude food extracts, both in the presence and absence of a complete meal, is needed to elucidate the complete physiologic nature of food allergen digestion.     

The effects of gastric digestion on codfish allergenicity

BACKGROUND: In a recent murine study, we showed that impaired gastric digestion supports the induction of fish allergy by protecting the digestion-sensitive major allergen parvalbumin and thus enhancing its sensitizing properties. OBJECTIVE: The aim of the present study was to investigate whether impairment of peptic degradation might also play a role in the effector phase of codfish allergy. METHODS: The resistance of cod proteins to digestion by simulated gastric fluid was assessed in vitro . Gastric solutions with pH values ranging from 1.25 to 5.0 were prepared, and the influence of the pH on protein degradation was evaluated by means of SDS-PAGE and IgE immunoblotting. The allergenic potency of digested and undigested cod extract was further characterized in RAST inhibition and basophil histamine release experiments. RESULTS: The digestion experiments revealed that codfish proteins were degraded within 1 minute under physiologic gastric conditions. An only marginal pH shift from 2.5 to 2.75 abrogated completely the digestion of cod allergens. In RAST inhibition experiments digested cod extracts showed a reduced IgE-binding capability that was dependent on the digestion time. Moreover, peptic fragments expressed a 10,000 times reduced allergenic potency, as evaluated on the basis of histamine release from human basophils. CONCLUSION: Codfish allergens have a grossly reduced ability to trigger an intestinal allergic reaction when they are physiologically degraded. Impairment of the physiologic digestion might thus lower the threshold levels of a food allergen in sensitized patients.     

Peanut allergen Ara h 2-specific T cells are activated via Ras-Erk MAP kinase pathway signalling and identified by CD154 expression

Background. Peanut allergy is driven by peanut-specific Th2 cells. Ara h 2, a peanut 2S albumin, is one of the major peanut allergens. Methods. An Ara h 2-specific T cell line was generated from peanut sensitised C3H/HeJ mice and used to analyse allergen-induced cytokine secretion and activation of signalling molecules. Results. Peanut stimulated splenocytes from peanut sensitised mice had strong proliferation responses and highly elevated IL-4 secretion. The Ara h 2-specific T cells generated from sensitised mice were mainly CD4+ T helper cells with a Th2 cytokine profile. The expression of CD154 on the surface of these T cell clones was detected following Ara h 2 stimulation. Ara h 2 stimulation induced phosphorylation of ZAP-70 and Erk1/2 in the T cells. Conclusions. CD154 is a useful marker for identifying activated, allergen-specific T cells. The Ras-Erk mitogen-activated protein kinase pathway is involved in the activation of Ara h 2-specific Th2 cells.     

Influence of bronchial allergen challenge on histamine release by human basophils

BACKGROUND: Basophils can be primed by cytokines such as interleukin (IL) -3, IL-5 or granulocyte macrophage-colony stimulating factor (GM-CSF). It has been described that the concentrations of these cytokines are enhanced at sites of allergic inflammation as well as systemic in allergic asthma. OBJECTIVE: To investigate the priming status of basophils as detected by thapsigargin-induced histamine release during bronchial allergen challenge. METHODS: Ten subjects allergic to house dust mite were challenged via an aerosol delivery system. Spontaneous leucocyte histamine release as well as histamine release induced by various stimuli was measured in vitro at several time points. In addition, lung function parameters, serum IL-5 and blood eosinophil counts were evaluated. RESULTS: We found no effect of bronchial allergen challenge upon spontaneous leucocyte histamine release, nor upon histamine release induced by anti-immunoglobulin (Ig) E, house dust mite extract, C5a, fMLP, IL-3, PMA+ thapsigargin or IL-3+ thapsigargin. However, the priming status of basophils as measured by thapsigargin-induced histamine release was enhanced at 24 h after bronchial allergen challenge. Analysis of the individual data showed a heterogeneous initial response (30 min, 6 h) followed by a predominant increase at 24 h after allergen challenge. This increase in the thapsigargin-induced histamine release correlated with the increase in serum IL-5 levels at 24 h after allergen challenge. CONCLUSION: The priming status of human basophils as measured by thapsigargin-induced histamine release is enhanced 24 h after allergen challenge.     

Does skin prick test reactivity to purified allergens correlate with clinical severity of peanut allergy?

BACKGROUND: Recognition of specific peanut allergens or the diversity of IgE binding to peanut allergens may play a role in the elicitation of severe allergic reactions. OBJECTIVE: To investigate whether sensitization to individual allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6 is correlated with clinical severity. METHODS: The reactivity of purified peanut allergens was measured by skin prick test (SPT) and by IgE immunoblot in 30 patients. The results were related to the clinical reactivity by history, and in 25 of them to the eliciting dose (ED). RESULTS: The majority of patients recognized Ara h 2 and Ara h 6. Patients with severe symptoms had a higher SPT response to Ara h 2 and Ara h 6 at low concentrations (0.1 micro g/mL) and to Ara h 1 and Ara h 3 at higher concentrations (100 micro g/mL), compared with patients with mild symptoms. They also recognized a greater number of allergens and showed a higher cumulative SPT response compared with patients with mild symptoms. No significant differences were observed between patients with a low or high ED. CONCLUSIONS: Ara h 2 and Ara h 6 appeared to be more potent than Ara h 1 and Ara h 3. Both SPT reactivity to low concentrations of Ara h 2 and Ara h 6 and to higher concentrations of Ara h 1 and Ara h 3 were shown to be indicative of severe symptoms.     

Children with peanut allergy recognize predominantly Ara h2 and Ara h6, which remains stable over time

BACKGROUND: In peanut-allergic adults, IgE is mainly directed to Ara h1 and Ara h2. More recently, a role for Ara h6 has been suggested. In contrast to adults, IgE in children can fluctuate over time. Therefore, children may have a more dynamic reactivity to peanut. OBJECTIVE: To examine the IgE reactivity to major peanut allergens in peanut-allergic children at two subsequent time-points. METHODS: Twenty children (3-15 years old) with peanut allergy, confirmed by a double-blind placebo-controlled food challenge (DBPCFC), were included. Just before and 20 months after DBPCFC, IgE reactivity to purified Ara h1, Ara h2, Ara h3 and Ara h6 was studied by immunoblots and skin prick tests (SPTs). RESULTS: Before DBPCFC, all peanut-allergic children showed IgE reactivity to Ara h2; Ara h6 was recognized by 16 children, and Ara h1 and Ara h3 by 10 children. After 20 months, peanut-specific IgE levels (median 23 kU/L) and the individual recognition of major allergens were comparable with the levels and recognition before challenge (median 28.2 kU/L). SPT with Ara h2 and Ara h6 was positive in most children, whereas SPT with Ara h1 and Ara h3 was positive in approximately half of the children. Ara h6 induced the largest weals. None of the parameters were related to the severity of peanut allergy. CONCLUSION: Ara h2 and Ara h6 are the most frequently recognized major peanut allergens in children. The individual reactivity to the major peanut allergens remained stable over time, despite DBPCFC.     

Tomato profilin Lyc e 1: IgE cross-reactivity and allergenic potency

BACKGROUND: To date, very little data are available about the nature of tomato allergens. Immunoglobulin E (IgE) cross-reactive profilins have been suggested to account for allergic symptoms in patients suffering from tomato allergy. METHODS: The cDNA of tomato profilin was amplified by reversely transcribed polymerase chain reaction (RT-PCR) from total RNA extracted from ripe tomato fruit. The gene was cloned into the pET101D expression plasmid and the protein was produced in Escherichia coli BL21. Purification was performed via poly-l-proline (PLP) affinity chromatography. IgE reactivity of recombinant tomato profilin was investigated by immunoblot and enzyme-linked immunosorbent assay. IgE-inhibition studies were performed to analyse cross-reactivity with other profilins. To determine the allergenic activity of the recombinant protein, basophil histamine release assays using sera of patients with adverse reactions to tomato were performed. RESULTS: Profilin was identified as a new minor allergen in tomato fruits. The recombinant tomato profilin comprises 131 amino acids and high sequence identity to other allergenic food and pollen profilins. It was shown to be IgE-reactive with a prevalence of 22% (11/50) in tomato-allergic patients. In patients with tomato allergy and multiple sensitization to other foods and birch pollen, IgE directed against tomato profilin showed a strong cross-reactivity with profilins from plant food sources and birch pollen. The tomato profilin was able to induce mediator release from human basophils. CONCLUSION: The tomato profilin is a minor allergen in tomato fruit. Thus, it shows biological activity, as confirmed by in vitro histamine release assays with human basophils and thereby has the potential to account for clinical symptoms in tomato-allergic patients.     

The major peanut allergen,Ara h 2, functions as a trypsin inhibitor,and roasting enhances this function

BACKGROUND: The widespread use of peanut products, the severity of the symptoms, and its persistence in afflicted individuals has made peanut allergy a major health concern in western countries such as the United States, United Kingdom, and Canada. In a previous study, the authors showed that the allergenic properties of peanut proteins are enhanced as a result of thermal processing. OBJECTIVE: The purpose of this investigation was to determine whether any specific functions are associated with the major peanut allergen, Ara h 2, and whether the functionality of this protein is influenced by processing. An assay was developed and used to assess structure/function changes in Ara h 2 induced by roasting and the effect of these alterations on the allergenic properties of this major peanut allergen. METHODS: A protein domain homology search was used to determine possible functions for Ara h 2. One of the putative functions (protease inhibition) was tested by means of appropriate enzyme assays and protein gel electrophoresis. Circular dichroism was used to compare the structural properties of Ara h 2 purified from raw and roasted peanuts. RESULTS: Ara h 2 purified from peanuts is homologous to and functions as a trypsin inhibitor. Roasting caused a 3.6-fold increase in trypsin inhibitory activity. Functional and structural comparison of the Ara h 2 purified from roasted peanuts to native and reduced Ara h 2 from raw peanuts revealed that the roasted Ara h 2 mimics the behavior of native Ara h 2 in a partially reduced form. CONCLUSIONS: The data indicate that thermal processing might play an important role in enhancing the allergenic properties of peanuts. Not only has it previously been shown to affect the structural and allergic properties of peanut proteins but also, for the first time, the functional characteristics of an allergen. These structural and functional alterations are likely to influence the allergenicity of peanuts.     

Comparison of physiological and in vitro porcine gastric fluid digestion

BACKGROUND: In previous studies, the major peanut allergen Ara h 1 was digested in vitro using pepsin and porcine gastric fluid. The results suggested that in vivo gastric digestion of allergen protein can be modeled accurately by peptic hydrolysis in vitro. In the current investigation, studies were designed to follow the gastrointestinal (GI) digestion of peanut allergens under true physiological conditions. In vitro digestion with porcine gastric fluid was compared with actual physiological digestion of peanut allergens in the porcine digestive tract in vivo. METHODS: Analysis of physiologic digestion was performed in piglets administered a 20-gram bolus of peanut meal followed by periodic sampling and analysis of GI contents. The pH was monitored, and digesta were analyzed by SDS-PAGE and immunoblot analysis. RESULTS: Peanut meal initially neutralized stomach contents to a pH of approximately 7, which was subsequently acidified by HCl secretion within 30 min. Acidification to pH 2-4 resulted in active pepsin digestion of soluble protein in the stomach. Soluble intact protein/allergens were rapidly degraded to pepsin-resistant peptides in the stomach followed by hydrolysis of these fragments in the small intestine. Particulate material was evident in both the stomach and small intestine that could contribute to continued release of peanut allergens Ara h 1, 2 and 3. CONCLUSIONS: Porcine gastric digestion of peanut proteins resembles true physiological digestion only under optimal physiologic conditions. Soluble proteins are rapidly digested and insoluble material continues to release IgE-reactive proteins throughout the GI tract. GI digestion of food allergens can play a prominent role when assessing allergens within the context of a food matrix or meal and during the sensitization phase of IgE-mediated allergy.     

Peanut- and cow's milk-specific IgE, Th2 cells and local anaphylactic reaction are induced in Balb/c mice orally sensitized with cholera toxin

BACKGROUND: The development of animal models developing specific immunoglobulin (Ig)E presenting the same specificity as human IgE and similar clinical symptoms as those observed in allergic patients are of great interest for the understanding of mechanisms involved in the induction and regulation of food allergy. METHODS: Balb/c female mice were sensitized with whole peanut protein extract (WPPE) by means of intraperitoneal (i.p.) injections with alum or gavages with cholera toxin (CT). The WPPE specific IgE, IgG1 and IgG2a were monitored. Th2 cells activation was analysed assaying interleukin (IL)-4 and IL-5 vs IFNgamma on reactivated splenocytes. Local anaphylactic reaction was evaluated by assaying histamine in faecal samples. The oral sensitization protocol was further extended to cow's milk proteins (CMP). RESULTS: Balb/c mice developed high peanut-specific IgE and IgG1 responses either after i.p. or oral sensitizations. In both cases, antibodies were specific to polymer of glycinin fragments, containing polypeptides from Ara h3/4, and to a lesser extent to Ara h1 and Ara h2. Interleukin-4 and IL-5 production were evidenced. Balb/c mice could also be sensitized to CMP, as demonstrated by CMP-specific IL-4 and IL-5 secretions and induction of IgE specific for whole caseins, beta-lactoglobulin, serum bovine albumin and lactoferrin. Of interest was the occurrence of a local anaphylactic reaction in the peanut and CM models. CONCLUSIONS: In contrast with previous authors, Balb/c mice were sensitized and evidenced an allergic reaction after oral administrations of peanut or CMP plus CT, providing an interesting model for further studies on immunopathogenic mechanisms.     

Secretion of cytokines,histamine and leukotrienes in chronic urticaria

BACKGROUND: Approximately 35-40% of patients with chronic urticaria have an IgG autoantibody to the IgE receptor which can activate basophils and mast cells so that they release histamine. In this study we assessed the cytokine profile present in chronic urticaria sera, and then measured cytokine and leukotriene release from basophils and mast cells upon incubation with chronic urticaria sera. Finally we assessed cytokine expression at the single-cell level and characterized the T cell subpopulations involved in their production. We chose IL-4 as representative of Th2 lymphocytes and IFN-gamma for Th1 lymphocytes. METHODS: We analyzed IL-4, IL-5 and IFN-gamma in 60 chronic urticaria sera versus 51 controls. Sera were incubated with purified human basophils and cutaneous mast cells and the release of histamine, IL-4 and leukotrienes (C(4), D(4), E(4)) was quantitated. Immunoblotting was performed to identify IgG antibody to FcepsilonRIalpha, alpha subunit. We measured intracellular cytokine production in peripheral blood mononuclear cells of 17 chronic urticaria patients compared to 50 healthy controls. RESULTS: We found higher IL-4 levels (p = 0.028) in the sera of chronic urticaria patients (1.03 pg/ml) versus healthy donors (0.20 pg/ml) but no difference between urticaria sera and atopic control sera (0.52 pg/ml). We did not detect IFN-gamma or IL-5 in any serum. However, sera that activated basophils so that they released histamine also produced leukotriene and IL-4, and leukotriene production by cutaneous mast cells and basophils was closely correlated. However, there was no correlation between immunoblotting and the functional ability to induce either histamine or IL-4. After stimulating with PMA-ionomycin we found significant differences in CD4+ lymphocyte production of IL-4 and IFN-gamma with no differences in CD8+ lymphocyte production of either cytokine. CONCLUSION: Our data support the presence of basophil and mast cell activators in the sera of patients with chronic urticaria which can lead to the production of leukotrienes and IL-4 in addition to the histamine. IL-4 levels are similar to those seen in atopic subjects. We found that CD4+ T cells from patients with chronic urticaria are activated and tend to produce higher cytokine levels than CD4+ T cells from healthy controls. There were no differences when cytokine production by CD8+ lymphocytes was similarly assessed. These results are consistent with the histology found in biopsies of chronic urticaria lesions, where a CD4+-predominant infiltrate is found with cytokine production suggesting either a Th0 response or a mixture of Th1 and Th2 lymphocytes.     

IFN-gamma-enhanced allergen penetration across respiratory epithelium augments allergic inflammation

BACKGROUND: Respiratory allergen contact is the critical event in the elicitation and boosting of allergen-specific immune responses, as well as in the induction of immediate and late inflammatory reactions. OBJECTIVE: We sought to investigate the influence of various factors of allergic inflammation on the integrity and barrier function of respiratory epithelium for allergens. METHODS: We cultured the human bronchial epithelial cell line 16HBE14o- in a transwell culture system as a surrogate of intact respiratory epithelium and used purified iodine 125-labeled recombinant major birch pollen allergen (rBet v 1) to study the extent, kinetics, and factors influencing transepithelial allergen penetration. RESULTS: Culture supernatants from activated allergen-specific T H 1 clones decreased transepithelial resistance. A screening of various factors (histamine, IFN-gamma, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-8, IL-12, and TNF-alpha) identified IFN-gamma as a potent factor capable of reducing epithelial barrier properties and enhancing transepithelial allergen penetration. Increased submucosal allergen concentrations caused by IFN-gamma-mediated reduction of epithelial barrier function provoked a more than 7-fold augmentation of histamine release from sensitized basophils. CONCLUSION: These results demonstrate that the T H 1 cell-derived cytokine IFN-gamma facilitates allergen penetration through the respiratory epithelium and thereby can aggravate allergic inflammation.     

Lactobacillus buchneri S-layer as carrier for an Ara h 2-derived peptide for peanut allergen-specific immunotherapy

Peanut allergy is an IgE-mediated severe hypersensitivity disorder. The lack of a treatment of this potentially fatal allergy has led to intensive research on vaccine development. Here, we describe the design and initial characterization of a carrier-bound peptide derived from the most potent peanut allergen, Ara h 2, as a candidate vaccine. Based on the adjuvant capability of bacterial surface (S-) layers, a fusion protein of the S-layer protein SlpB from Lactobacillus buchneri CD034 and the Ara h 2-derived peptide AH3a42 was produced. This peptide comprised immunodominant B-cell epitopes as well as one T cell epitope. The fusion protein SlpB-AH3a42 was expressed in E. coli, purified, and tested for its IgE binding capacity as well as for its ability to activate sensitized rat basophil leukemia (RBL) cells. The capacity of Ara h 2-specific IgG rabbit-antibodies raised against SlpB-AH3a42 or Ara h 2 to inhibit IgE-binding was determined by ELISA inhibition assays using sera of peanut allergic patients sensitized to Ara h 2. IgE specific to the SlpB-AH3a42 fusion protein was detected in 69% (25 of 36) of the sera. Despite the recognition by IgE, the SlpB-AH3a42 fusion protein was unable to induce β-hexosaminidase release from sensitized RBL cells at concentrations up to 100 ng per ml. The inhibition of IgE-binding to the natural allergen observed after pre-incubation of the 20 sera with rabbit anti-SlpB-AH3a42 IgG was more than 30% for four sera, more than 20% for eight sera, and below 10% for eight sera. In comparison, anti-Ara h 2 rabbit IgG antibodies inhibited binding to Ara h 2 by 48% ±13.5%. Our data provide evidence for the feasibility of this novel approach towards the development of a peanut allergen peptide-based carrier-bound vaccine. Our experiments further indicate that more than one allergen-peptide will be needed to induce a broader protection of patients allergic to Ara h 2.