IgE antibodies to omega-5 gliadin associate with immediate symptoms on oral wheat challenge in Japanese children

Background: Gliadins have been implicated in immunoglobulin E (IgE)‐mediated allergy to ingested wheat and ω‐5‐gliadin is known to represent a major allergen in wheat‐dependent exercise‐induced anaphylaxis. Less known is whether ω‐5‐gliadin is a clinically relevant allergen in children with immediate allergy to ingested wheat. This study investigates whether specific IgE antibodies to ω‐5‐gliadin (sIgE‐ω‐5‐gliadin‐ab) could be used as a marker for oral wheat challenge outcome in wheat‐sensitized children. A secondary objective was to study whether the level of sIgE‐ω‐5‐gliadin was related to symptom severity in children with a positive challenge test. Methods: Serum samples from 88 children sensitized to wheat, of whom 35 underwent wheat challenge, were collected consecutively. sIgE‐ω‐5‐gliadin‐ab was related to a physician’s diagnosis of wheat allergy and challenge symptoms. Results: The mean concentration of sIgE‐ω‐5‐gliadin‐ab was 7.25 kU_A/l in patients with wheat allergy and 1.08 kU_A/l in patients with no wheat allergy (P < 0.01). sIgE‐ω‐5‐gliadin‐ab was only detected in 12 of the non‐wheat allergic children and 11 of them had a specific IgE to wheat below 1.30 kU_A/l. Children reacting with severe symptoms upon challenge (n = 8) had increased levels of sIgE‐ω‐5‐gliadin‐ab compared to children with moderate, mild or no symptoms (P < 0.001). Conclusions: The presence of sIgE‐ω‐5‐gliadin‐ab is related to the reaction level to wheat challenge outcome in wheat‐sensitized children. The sIgE‐ω‐5‐gliadin‐ab was found to be associated with a strong convincing history of wheat allergy also in those cases when oral food challenge was avoided. The sIgE‐ω‐5‐gliadin‐ab level may serve as a marker for clinical reactivity in wheat‐sensitized individuals.     

Sensitivity and specificity of recombinant omega-5 gliadin-specific IgE measurement for the diagnosis of wheat-dependent exercise-induced anaphylaxis

Background:  A recent study has shown that the measurement of specific IgE antibodies to B-cell epitope peptides of wheat ω-5 gliadin (Pep A) and high molecular weight glutenin subunit (Pep B) are useful to diagnose wheat-dependent exercise-induced anaphylaxis (WDEIA).
Aims of the study:  We sought to compare the sensitivity and specificity of the in vitro tests for measuring the specific IgE antibodies to recombinant ω-5 gliadin (rω-5 gliadin) with those for wheat, gluten, Pep A, and Pep B in identification of patients with WDEIA.
Methods:  Fifty patients with WDEIA, 25 healthy subjects and 25 patients with atopic dermatitis with specific IgE antibodies to wheat but without experience of allergic reactions after ingestion of wheat products were enrolled in this study. The concentrations of specific IgE antibodies were measured using ImmunoCAP^TM. The empirical receiver operating characteristics curves (ROC) for each test were prepared and the areas under the ROC curve ( AUC ) were compared.
Results:  In patients with WDEIA, the sensitivities of the allergen-specific IgE tests for wheat, gluten, Pep A, Pep B and rω-5 gliadin were 48%, 56%, 76%, 22%, and 80%, respectively. The seven of 10 WDEIA patients with no specific IgE antibodies to rω-5 gliadin had specific IgE antibodies to Pep B. The highest AUC (0.850) was observed in the test for rω-5 gliadin.
Conclusions:  Measuring the concentration of specific IgE antibodies to rω-5 gliadin is more useful than to wheat, gluten, or Pep A in the identification of patients with WDEIA.     

Exercise and aspirin increase levels of circulating gliadin peptides in patients with wheat-dependent exercise-induced anaphylaxis

BACKGROUND: Food-dependent exercise-induced anaphylaxis (FDEIA) is an allergic reaction characteristically induced by intense exercise combined with the ingestion of causative food. Recent reports have shown that aspirin intake is a contributing factor in some patients with FDEIA. Wheat is known to be the most frequent causative food, and the IgE-binding epitopes of a major wheat allergen (omega-5 gliadin) in wheat-dependent exercise induced anaphylaxis (WDEIA) have already been clarified. However, the mechanism of eliciting the symptom in WDEIA remains not fully understood. OBJECTIVES: The aim of this study was to examine the relationship of serum gliadin levels and allergic symptoms induced by exercise or aspirin in patients with WDEIA. METHODS: Six patients with a history of recurrent anaphylaxis associated with wheat ingestion were diagnosed as having WDEIA by the provocation test, which included wheat ingestion, exercise, aspirin intake and a combination of these challenges. During the tests, serum levels of gliadins were monitored by gliadin-specific sandwich ELISA. The effects of exercise and aspirin on serum gliadin levels were also investigated in four healthy subjects. RESULTS: Immunoreactive gliadins appeared in the sera of patients during the provocation test with both wheat-exercise and wheat-aspirin challenges in parallel with allergic symptoms. Serum gliadin levels also increased under the two same challenge conditions in the healthy subjects, although they exhibited no allergic symptoms. However, low levels of gliadin were detected in the sera of both patients and healthy subjects when challenged with wheat alone. CONCLUSION: We demonstrated for the first time that blood gliadin levels correlate with clinical symptoms induced by exercise and aspirin in patients with WDEIA. These findings suggest that exercise and aspirin facilitate allergen absorption from the gastrointestinal tract.     

Life-threatening, recurrent anaphylaxis caused by allergy to gliadin and exercise

Background Exercise-induced urticaria or anaphylaxis is regarded as a distinct form of physical allergy. In some patients the symptoms occur only after ingestion of various food products in connection with exercise. We have come across patients with cereal dependent exercise-induced anaphylaxis. Objectives The purpose of the present study was to analyse the allergens in cereals responsible for the severe anaphylactic symptoms and to verify the test methods suitable for screening the patients with cereal dependent exercise-induced anaphylaxis. Methods The patients underwent skin-prick tests (SPT) with common inhalant and food allergens as well as with various cereal extracts. IgE-immunoblotting was used to identify the allergenic fractions. Results Five patients found positive in SPT with NaC1 wheat suspension had IgE antibodies to wheat, rye, barley and oats, especially directed against the ethanolsoluble protein fractions in immunoblotting. No IgE antibodies were detected against other cereals. The patients had been unaware of any cereal allergy since anaphylaxis occurred only in association with exercise postprandially. The patients were directed to follow a gluten-free diet and have been free from symptoms, being able to continue their outdoor physical activities. Conclusion Wheat gliadin and the corresponding ethanol-soluble proteins of taxonomically closely related cereals were found to be the allergens in cereal-dependent exercise-induced anaphylaxis. Skin-prick testing with NaC1 wheat suspension was a simple and practical test to screen patients with this kind of occult, possibly life-threatening, allergy to cereals.     

IgE detection to α/β/γ‐gliadin and its clinical relevance in wheat‐dependent exercise‐induced anaphylaxis

Wheat‐dependent exercise‐induced anaphylaxis (WDEIA) is characterized by anaphylactic reactions after wheat ingestion and physical exercise. IgE antibodies to recombinant ω₅‐gliadin are detectable in a majority of WDEIA patients, but other wheat allergens may also play a role in elicitation of WDEIA. Here, we performed a comprehensive analysis of IgE reactivity to different wheat proteins in 17 patients with confirmed WDEIA by ImmunoCAP research prototypes and a semi‐quantitative microarray immunoassay with α/β/γ‐gliadin, high‐molecular‐weight (HMW) glutenin, alpha‐amylase inhibitor (AAI) dimer, and wheat lipid transfer protein (LTP). By ImmunoCAP, IgE to recombinant ω₅‐gliadin was detectable in 14/17 patients (82%), to α/β/γ‐gliadin in 82% including the three patients lacking IgE to ω₅‐gliadin, and to HMW glutenin in 59%. The microarray revealed specifically γ‐gliadin as the second most important allergen. These results demonstrate the additional diagnostic value of α/β‐ and γ‐gliadin in particular in ω₅‐gliadin‐negative patients in the diagnosis of WDEIA.     

Humoral and cellular responses to gliadin in wheat-dependent, exercise-induced anaphylaxis

BACKGROUND: Wheat-dependent, exercise-induced anaphylaxis (WDEIA) is a severe allergy where wheat ingestion together with physical exercise induces anaphylaxis. We have previously shown that patients with WDEIA have IgE antibodies against gliadin proteins and identified omega-5 gliadin (Tri a 19) as a major allergen. OBJECTIVE: The aim of this study was to examine gliadin-specific IgG subclass, IgA and IgE antibodies, basophil histamine release and cell-mediated responses in WDEIA. METHODS: Sera and peripheral blood mononuclear cells (PBMC) were obtained from patients with WDEIA and from controls without wheat allergy. Serum antibodies to crude gliadin extract (CGE) and purified omega-5 gliadin were measured by ELISA and basophil reactivity by histamine-release test. Gliadin-induced cell-mediated responses were assessed by lymphocyte proliferation assay, and cytokine mRNA expression with real-time quantitative PCR. RESULTS: All patients with WDEIA, but none of the controls, had IgE antibodies to CGE and omega-5 gliadin. Both allergens released high levels of histamine from the basophils of patients with WDEIA. Levels of IgA antibodies to CGE and omega-5 gliadin were significantly elevated in the patients, but the distribution of IgG subclass antibodies showed no statistically significant differences between the two groups. Proliferative responses of PBMC to CGE were increased in patients with WDEIA, and stimulation of PBMC with CGE caused, both in patients and in controls, a clear induction of IL-10 mRNA. Compared with the controls, induction of IL-10 mRNA expression in patients with WDEIA was significantly (P < 0.01) suppressed. CONCLUSION: These results suggest that, in addition to IgE antibodies against omega-5 gliadin, specific IgA antibodies may be involved in the pathogenesis of WDEIA. Decreased expression of IL-10 mRNA in PBMC during gliadin stimulation may facilitate the development of gliadin-specific T cell responses.     

Demonstration of IgE and IgG antibodies against venoms in the blood of victims of fatal sting anaphylaxis

Nine people died from insect sting anaphylaxis in North Carolina from 1979 to 1981. Postmortem blood specimens from eight of these subjects were analyzed for IgE and IgG antibodies against venoms. All eight were RAST positive to at least one of the venoms. IgE and IgG anti-venom levels were comparable to those of a group of untreated sting-allergic individuals. RAST to venoms was also performed on several control groups of sera and from 3% to 50% positive RAST results were found. The highest incidence was in rural North Carolina outdoor workers. The incidence of positive RAST results in the sting-death group was significantly different from that in the control groups. This is the first demonstration of IgE antibodies against venoms in the sera of victims of fatal anaphylaxis from stings and adds further evidence for the role of IgE in sting anaphylaxis.     

Transglutaminase-mediated cross-linking of a peptic fraction of omega-5 gliadin enhances IgE reactivity in wheat-dependent,exercise-induced anaphylaxis

BACKGROUND: Patients with wheat-dependent, exercise-induced anaphylaxis (WDEIA) experience recurrent anaphylactic reactions when exercising after ingestion of wheat products. We have identified omega-5 gliadin (Tri a 19) as a major allergen in WDEIA, but the role of exercise in eliciting the symptoms remains obscure. OBJECTIVE: The aim was to examine whether tissue transglutaminase (tTG)-mediated cross-linking could be involved in modulating the IgE-binding ability and in vivo reactivity of digested omega-5 gliadin peptides in WDEIA. METHODS: Purified omega-5 gliadin was digested with pepsin or with pepsin and trypsin and treated with tTG. The binding of IgE antibodies in pooled sera from 10 patients with WDEIA was studied by means of immunoblotting before and after tTG treatment of the digested peptides. The peptides derived from pepsin digestion were separated by means of gel-filtration chromatography, and IgE reactivity of 4 different peptide fractions was studied by immunoblotting before and after tTG treatment. The fraction showing the greatest degree of cross-linking by tTG was further studied by means of IgE ELISA, ELISA inhibition, and skin prick testing. RESULTS: The IgE-binding ability of omega-5 gliadin was retained after pepsin and pepsin-trypsin digestion. tTG treatment of the whole peptic digest formed large peptide complexes, with molecular weights ranging from 40 to greater than 200 kd. These cross-linked aggregates bound IgE antibodies in immunoblotting more intensely than untreated, pepsin-digested, or pepsin-trypsin-digested omega-5 gliadin. A gel-filtration fraction of the whole peptic digest corresponding to the highest peak of the chromatogram and showing the greatest degree of tTG-mediated cross-linking showed an increase in serum IgE reactivity in ELISA after tTG treatment, as well as a shift of reactivity to cross-linked complexes. In the 20 patients with WDEIA, the mean skin prick test wheal elicited by this tTG-treated peptic fraction was 77% larger (P <.001) than that elicited by the untreated peptic fraction and 56% larger (P <.01) than that elicited by intact omega-5 gliadin. CONCLUSIONS: Omega-5 gliadin-derived peptides are cross-linked by tTG, which causes a marked increase in IgE binding both in vitro and in vivo. Activation of tTG during exercise in the intestinal mucosa of patients with WDEIA could lead to the formation of large allergen complexes capable of eliciting anaphylactic reactions.     

Rye γ-70 and γ-35 secalins and barley γ-3 hordein cross-react with ω-5 gliadin, a major allergen in wheat-dependent, exercise-induced anaphylaxis

Patients with wheat-dependent, exercise-induced anaphylaxis experience severe allergic reactions when exercising after ingestion of wheat. The major wheat allergen associated with these reactions is a omega-5 gliadin, and patients following a gluten-free diet have remained free of symptoms. The aim of this study was to examine whether allergens cross-reacting with wheat omega-5 gliadin are present in rye, barley and oats. Sera from 23 adult patients with wheat-dependent, exercise-induced anaphylaxis were examined. Cereal allergens cross-reacting with wheat omega-5 gliadin were identified by immunoblot inhibition. The cross-reactive allergens were purified by gel filtration and reversed-phase chromatography and submitted to amino acid sequencing. Cross-reactivity was further studied by IgE ELISA and ELISA inhibition, and in vivo reactivity by skin prick testing. In immunoblotting rabbit anti-omega-5 gliadin antibodies bound to 70 kDa and 32 kDa proteins in rye and a 34-kDa protein in barley, but not to proteins in oats. N-terminal sequencing identified these proteins as rye gamma-70 secalin, rye gamma- 35 secalin and barley gamma-3 hordein, correspondingly. In ELISA 21/23 (91%) patients with wheat-dependent, exercise-induced anaphylaxis showed IgE antibodies to purified gamma-70 secalin, 19/23 (83%) to gamma-35 secalin and 21/23 (91%) to gamma-3 hordein. In ELISA inhibition omega-5 gliadin inhibited over 90% of the IgE binding of pooled patient sera to solid-phase gamma-secalins and gamma-3 hordein. Skin prick testing gave positive reactions to gamma-70 secalin in 10/15 (67%) patients, to gamma-35 secalin in 3/15 (20%) patients and to gamma-3 hordein in 7/15 (47%) patients. The results of this study show that gamma-70 and gamma-35 secalins in rye and gamma-3 hordein in barley cross-react with omega-5 gliadin, a major allergen in wheat-dependent, exercise-induced anaphylaxis. These findings suggest that also rye and barley may elicit symptoms in patients with wheat-dependent, exercise-induced anaphylaxis.     

抗赭曲霉毒素A单克隆杂交瘤细胞系的建立及特性

用杂交瘤技术制备了4株稳定产生抗赭曲霉毒素A单克隆抗体的杂交瘤细胞株,命名为1A9、5F2、5G5和6G11。1A9、5G5和6G11的Ig亚类为IgG1,5F2的亚类为IgG2a。抗体腹水效价为500000～1000000。6G11检测纯毒素的线性范围为2～500ng/ml,最低检出量为1ng/ml。交叉反应的结果还表明,该单抗与共试的其他结构类似物无反应,具有较高的特异性。     

Detection of anti-delta antibodies among acute hepatitis B virus-infected patients

A total of 130 acute HBsAg-positive subjects were tested for the presence of delta hepatitis antibodies (anti-HD). Of the 130 subjects, 56 were females and 74 were males. All patients were individuals attending the two teaching hospitals, i.e., Parirenyatwa and Harare Central hospitals. All the sera were examined by the enzyme-linked immunosorbent assay (ELISA), and results were read spectrophotometrically at a wavelength of 492 nm. Of 130, 11 (19.64%) males had anti-HD present in the serum with an overall prevalence of 21 (16.15%). The positivity rates of HBeAg and anti-HBc were 64 (49.23%) and 116 (89.23%), respectively. There was no significant difference between the positive rate in males as compared to females.     

Rapid enzyme-linked immunosorbent assay of whole blood for detection of antibodies to japanese encephalitis virus

The application of the rapid system of enzyme-linked immunosorbent assay (ELISA) was studied to quantify antibodies to Japanese encephalitis virus in large-scale epidemiological surveys, especially by testing under field conditions. The assay system, with 15 min for the first reaction and 30 min each for the second and the third reactions, was highly reproducible (coefficients of variation with swine positive sera were less than 5.8%) and was significantly correlated with the routine assay system with 1 h for each reaction (correlation coefficient was 0.960). Compared with the haemagglutination inhibition test, the rapid system gave a correlation coefficient of 0.916 and qualitative agreeement of 96.1%. The substitution of whole blood for serum in the first reaction was also examined not only to avoid serum separation but also to apply this system to antibody quantification in animals from which sufficient amounts of sera cannot be easily obtained: only 2 μl were needed for the test. The results obtained with 51-fold diluted whole blood had a linear relationship to those obtained with 100-fold diluted sera in swine and humans.     

IgA subclass antibodies to gliadin in serum and intestinal juice of patients with coeliac disease

Serum IgA anti-gliadin antibodies (AGA) were positive in 25 (68%) of 37 untreated adults with coeliac disease belonging mostly to IgA1 subclass (88%) and only in a few cases to IgA2 (12%). Antisecretory component IgA AGA were present in serum of seven patients (28%) by immunofluorescence and in nine (36%) by ELISA. The search for IgA AGA in jejunal juice of eight untreated children with coeliac disease was positive in seven cases (88%), with consistent finding of antisecretory component IgA AGA. These antibodies belonged with equal proportion to IgA1 and IgA2 subclasses. This study shows that in intestinal secretions of untreated coeliac disease cases the IgA immune response to gliadin is confined to polymeric anti-secretory component IgA with the same prevalence of IgA1 and IgA2 subclasses, while in serum IgA AGA are largely monomeric, more frequently of IgA1 than of IgA2 subclass, and with a lower proportion of polymeric anti-secretory component IgA (20-36%). The finding of secretory IgA AGA in serum of patients with coeliac disease could result from a spill-over from the intestinal mucosal synthesis into the circulation.     

Serum IgA, IgG, and IgM responses to different enteroviruses as measured by a coxsackie B5-based indirect ELISA.

An enterovirus-specific indirect ELISA, based on a single local isolate of coxsackie B5 as antigen, was used to study the IgA, IgG, and IgM responses in 19 patients with a recent or current enterovirus infection. Twelve different enterovirus serotypes were isolated from 15 patients. Paired serum samples were available from 10 and a single serum from 5 of these 15 patients. In addition, 4 patients diagnosed by a significant titer rise of complement fixing antibodies to enterovirus were included. A serological diagnosis, defined as an increase in titer of enterovirus IgG and/or presence of enterovirus IgM, were established in all 14 patients with paired sera. Enterovirus IgM was present in either a single serum or in both sera in 13 of them. Out of 5 patients with a single serum sample only, enterovirus IgA or enterovirus IgM was found in 4. Specific IgA was present in either a single serum or in both sera in 14 of the 19 patients. Seven of the 10 enterovirus isolate patients with paired sera had a significant titer rise of complement fixing antibodies; however, all 10 were diagnosed by ELISA. Among 64 healthy controls 2 had enterovirus IgA and none had enterovirus IgM. In conclusion, the use of a single antigen-based ELISA was found to be reliable for the diagnosis of recent and current enterovirus infections.     

Wheat omega-5 gliadin is a major allergen in children with immediate allergy to ingested wheat

BACKGROUND: Sensitization to wheat by ingestion can lead to food allergy symptoms and wheat-dependent, exercise-induced anaphylaxis. Sensitization by inhalation causes bakers' asthma and rhinitis. Wheat allergens have been characterized at the molecular level in bakers' asthma and in wheat-dependent, exercise-induced anaphylaxis, in which omega-5 gliadin (Tri a 19) is a major allergen. However, little information is available regarding allergens responsible for hypersensitivity reactions to ingested wheat in children. OBJECTIVE: The aim of this study was to examine whether children with allergy to ingested wheat have IgE antibodies to omega-5 gliadin. METHODS: Sera were obtained from 40 children (mean age, 2.5 years; range, 0.7-8.2 years) with suspected wheat allergy who presented with atopic dermatitis and/or gastrointestinal and/or respiratory symptoms. Wheat allergy was diagnosed with open or double-blinded, placebo-controlled oral wheat challenge. Wheat omega-5 gliadin was purified by reversed-phase chromatography, and serum IgE antibodies to omega-5 gliadin were measured by means of ELISA. In vivo reactivity was studied by skin prick testing. Control sera were obtained from 22 children with no evidence of food allergies. RESULTS: In oral wheat challenge, 19 children (48%) reacted with immediate and 8 children (20%) with delayed hypersensitivity symptoms. Sixteen (84%) of the children with immediate symptoms had IgE antibodies to purified omega-5 gliadin in ELISA. In contrast, IgE antibodies to omega-5 gliadin were not detected in any of the children with delayed or negative challenge test results or in the control children. The diagnostic specificity and positive predictive value of omega-5 gliadin ELISA were each 100% for immediate challenge reactions. Skin prick testing with omega-5 gliadin was positive in 6 of 7 children with immediate challenge symptoms and negative in 2 children with delayed challenge symptoms. CONCLUSION: The results of this study show that omega-5 gliadin is a significant allergen in young children with immediate allergic reactions to ingested wheat. IgE testing with omega-5 gliadin could be used to reduce the need for oral wheat challenges in children.