Atsuo Urisu

Utility of ovomucoid-specific IgE concentrations in predicting symptomatic egg allergy

BACKGROUND Children with allergy to raw egg white might tolerate low amounts of heated egg. Ovomucoid-specific IgE antibodies have been suggested to be predictors of whether children could tolerate heat-treated egg. OBJECTIVE The aim was to evaluate the clinical usefulness and added diagnostic value of measurements of IgE antibodies to egg white, ovalbumin, and ovomucoid in children with egg allergy. METHODS One hundred eight patients (median age, 34.5 months) with suspected egg allergy underwent double-blind, placebo-controlled food challenges with raw and heated egg. The outcomes of the challenges were related to the serum concentration of specific IgE antibodies and total IgE by using ImmunoCAP. RESULTS Reactions to heated egg white were observed in 38 patients (considered allergic to raw and heated egg), 29 patients reacted to only raw egg white, and 41 patients were tolerant. Correlation was observed between the serologic parameters studied. Receiver operating characteristic analysis showed that egg white ImmunoCAP was useful in the diagnosis of allergy to raw egg white. The positive decision point, based on 95% clinical specificity, was 7.4 kU(A)/L, and the negative decision point, based on 95% clinical sensitivity, was 0.6 kU(A)/L. For reaction to heated egg white, ovomucoid ImmunoCAP was superior. The positive decision point was 10.8 kU(A)/L, and the negative decision point was 1.2 kU(A)/L. CONCLUSIONS Quantitative measurements of specific IgE antibodies to both egg white and ovomucoid and the evaluation against the suggested positive and negative decision points for specific IgE will be useful in the diagnosis of egg allergy.

Oral Allergy Syndrome

Oral allergy syndrome (OAS) is defined as the symptoms of IgE-mediated immediate allergy localized in the oral mucosa, and the characteristics depend on the lability of the antigen. Another term used for this syndrome is pollen-food allergy (PFS); the patient is sensitized with pollen via the airways and exhibits an allergic reaction to food antigen with a structural similarity to the pollen (class 2 food allergy). In addition to PFS, latex-fruit syndrome is also well-known as the disease exhibiting OAS. In treating the condition, it must be noted that most but not all symptoms of PFS are those of OAS. In many cases, antigens become edible by heating, but some are resistant to heating. Also, since the exacerbation of atopic dermatitis is occasionally observed after the intake of cooked antigens in asymptomatic individuals, careful inquiry of the history is important in designing the treatment. Immunotherapy against the cross-reacting pollen has also been attempted in PFS.

16-Kilodalton Rice Protein Is One of the Major Allergens in Rice Grain Extract and Responsible for Cross-Allergenicity between Cereal Grains in the Poaceae Family

Cross-allergenicity between five cereal grains including rice, wheat, corn, Japanese millet (Panicum crus-galli L. var. frumentaceum Trin.) and Italian millet (Setaria italica Beauv. var. germanica schrad.) was examined by radioallergosorbent test (RAST) and RAST inhibition assay. There were significant close correlations between every combinations of RAST values for the five cereal grain extracts. RAST inhibition assay of each extract against RAST discs coupled with other cereal grain extracts indicated marked cross-reactivity of IgE binding between these cereal grain extracts. Rice protein 16KD (RP16KD) was shown to be one of major allergens in rice grain extracts by immunoblotting analysis, histamine release assay from human leukocytes and RAST inhibition. Next, the involvement of RP16KD in the cross-allergenicity between these cereals was investigated. RAST values for RP16KD significantly correlated with that for Italian millet as well as rice but not with those for corn and wheat. There was a trend of positive correlation between RAST values for RP16KD and Japanese millet. In the RAST inhibition assay using sera with positive RAST for these five cereal grain extracts and RP16KD, RP16KD inhibited IgE binding to these all cereal discs in a dose-dependent manner. Similarly, all of the five cereal grain extracts showed an effective decrease in IgE binding to the RP16KD disc. These results indicated possible participation of IgE binding structure on RP16KD in cross-allergenicity between these cereal grain extracts in the Poaceae family.

Nucleotide sequence of a cDNA clone encoding a major allergenic protein in rice seeds Homology of the deduced amino acid sequence with members of α‐amylase/trypsin inhibitor family

A cDNA clone of rice major allergenic protein (RAP) was isolated from a cDNA library of maturing rice seeds. The cDNA had an open reading frame (486 nucleotides) which coded a 162 amino acid residue polypeptide comprising a 27‐residue signal peptide and a 135‐residue mature protein of Mr 14,764. The deduced amino acid sequence of RAP showed a considerable similarity to barley trypsin inhibitor [1983, J. Biol. Chem. 258, 7998–8003] and wheat α‐amylase inhibitor [1981, Phytochemistry 20, 1781–1784].

State of the art and new horizons in the diagnosis and management of egg allergy

To cite this article: Benhamou AH, Caubet J‐C, Eigenmann PA, Nowak‐We˛grzyn A, Marcos CP, Reche M, Urisu A. State of the art and new horizons in the diagnosis and management of egg allergy. Allergy 2010; 65: 283–289.

Molecular diagnosis of egg allergy.

Purpose of reviewAllergy to hens egg is common in infancy and childhood. Oral food challenges are often required to diagnose egg allergy, because of the limitation in the diagnostic accuracy of skin test and specific IgE to egg white. New molecular diagnostic technologies have been recently introduced into allergological research. In this article, we will review the recent literature regarding the potential value of these tests for the clinical management of egg-allergic patients. Recent findingsComponent-resolved diagnosis that can be combined with the microarray technology is promising as measurement of specific IgE antibodies to individual egg white components has been shown to predict different clinical patterns of egg allergy. Specific IgE to ovomucoid has been identified as a risk factor for persistent allergy and could indicate reactivity to heated egg. Ovomucoid and ovalbumin IgE and IgG4-binding epitope profiling could also help distinguish different clinical phenotypes of egg allergy. Particularly, egg-allergic patients with IgE antibodies reacting against sequential epitopes tend to have more persistent allergy. SummaryUsing recombinant allergens, IgE-binding epitopes, and microarrays, molecular-based technologies show promising results. However, none of these tests is ready to be used in clinical practice and oral food challenge remains the standard for the diagnosis of egg allergy.

Identification and characterization of the allergens in the tomato fruit by immunoblotting.

Background: Although the tomato fruit (Lycopersicon esculentum) has been widely investigated for breeding purposes, there have been few studies on tomato allergenicity. We attempted to identify the tomato fruit allergens and to compare the concentrations of IgE-binding proteins among the different growth stages with sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Methods: An immunoblot experiment on tomato fruit extracts was performed using sera from 11 patients with oral allergy syndrome (OAS) to tomatoes. Bands reacting with IgE from more than half of the OAS patients’ sera were excised and subjected to determination of N-terminal amino acid sequences using the automated Edman degradation method. Moreover, we compared the concentrations of these proteins at each growth stage of the tomato fruit with SDS-PAGE and immunoblotting. Results: Four proteins binding with IgE from more than half of the OAS patients’ sera were determined to be polygalacturonase 2A (PG2A), β-fructofuranosidase, superoxide dismutase (SOD) and pectinesterase (PE). The concentrations of PG2A, β-fructofuranosidase and PE were highest in the red ripening stage with both SDS-PAGE and immunoblotting. Conclusion: The concentrations of 3 of 4 tomato allergens increased during ripening.

Effects of Oral Administration of Lactobacillus acidophilus L-92 on the Symptoms and Serum Markers of Atopic Dermatitis in Children

Background: Few studies have investigated the complementary effects of long-term oral administration of Lactobacillus acidophilus on traditional medical therapy in the treatment of patients with atopic dermatitis (AD). Methods: The Atopic Dermatitis Area and Severity Index was used to evaluate AD severity. Symptom severity was assessed using the symptom score. The effect of medical therapy was evaluated by adding the medication score, calculated as the sum of each product of the amount of steroid ointment used for therapy and its designated strength graded on a 4-point scale, to the symptom score. The complementary effect of long-term oral administration of L. acidophilus strain L-92 (L-92) as a probiotic or biogenic strain in patients with AD was evaluated using the symptom-medication score, which was calculated as the sum of the symptom score and medication score. Both a preliminary casuistic study and a double-blinded, placebo-controlled study were performed to evaluate the effects of L-92 on the symptoms of AD in children. Results: Orally administered L-92 significantly ameliorated the symptoms of AD in Japanese children. L-92 also affected the serum concentrations of thymus and activation-regulated chemokine in a time-dependent manner.Conclusions: The results of the preliminary trial and the double-blinded, placebo-controlled study revealed a complementary effect of oral L-92 on the standard medical therapy (topical application of a steroid ointment) in patients with AD that was mediated, at least in part, by alterations in the Th1/Th2 balance.

Clinical Significance of IgE–Binding Activity to Enzymatic Digests of Ovomucoid in the Diagnosis and the Prediction of the Outgrowing of Egg White Hypersensitivity

Background: We frequently encounter subjects without overt symptoms despite high IgE antibodies to egg white and its components. The measurements of these antibodies are not necessarily efficient for the diagnosis or the prediction of the outcome of egg allergy in children. Methods: Specific IgE antibodies to egg white and its components, including ovomucoid, ovalbumin, ovotransferrin and lysozyme, were measured by direct RAST assays. IgE–binding activity to ovomucoid degraded by pepsin, trypsin and chymotrypsin was examined by RAST inhibition. Thirty subjects were divided into two groups with positive (n=18; mean age ± SD = 42 ±25 months) and negative (n=12; mean age ± SD = 48 ±31 months) oral challenge tests with egg white antigens. The individuals with positive results to the first challenge tests were given the second provocation tests at mean intervals of 32 months. IgE–binding activity of the sera collected on the first challenge to these ovomucoid fragments was compared between subjects with positive and negative reactions to the follow–up challenge tests. Results: There were no significant differences in IgE antibody titers to egg white and its components between the positive and negative groups at the first and the second challenge tests. IgE–binding activity to ovomucoid digests after treatments with pepsin (p = 0.000008) and trypsin (p=0.037), except chymotrypsin (p=0.062), were significantly higher in subjects with positive challenge tests than in those with negative results. The difference was most remarkable in the IgE–binding to pepsin digests; the average concentrations (mean – SD and mean + SD) needed for 50% RAST inhibition in the positive group and in the negative group were 2.6 μg/ml (0.3 and 25) and 94.2 μg/ml (24.7 and 358.7), respectively. A significant difference was still observed in the inhibition tests using filtrates of pepsin digests with a membrane with MW 10,000 (p=0.014) and 3,000 (p=0.042) of cutoff. The concentration (mean= 0.8, mean – SD=0.2, mean + SD=3.4; μg/ml) of pepsin–treated ovomucoid required for 50% RAST inhibition in the subjects with positive second challenge results was significantly (p=0.033) lower than that (mean=6.8, mean–SD=0.6, mean + SD=73.9) of the negative group. Conclusion: IgE–binding activity to pepsin–digested ovomucoid was of diagnostic value to distinguish the challenge–positive subjects from the negative subjects. Subjects with high IgE–binding activity to pepsin–treated ovomucoid are unlikely to outgrow egg white allergy.

Primary structure and allergenic activity of trypsin inhibitors from the seeds of buckwheat (Fagopyrum esculentum Moench).

The complete amino acid sequences of two trypsin inhibitors BWI‐2a and BWI‐2b from the seeds of buckwheat (Fagopyrum esculentum Moench) were determined. BWI‐2b consists of 51 amino acid residues containing two disulfide bonds. BWI‐2a shares all amino acids with BWI‐2b except for the C‐terminal tripeptide: BWI‐2a lacks Glu‐Gly‐Asn and ends with the Asp residue, making a total of 48 residues in the chain. The two disulfide bonds connect Cys11 to Cys32 and Cys15 to Cys28. BWI‐2b shows no relatedness to the other buckwheat trypsin inhibitor reported [Belozersky et al. (1995) FEBS Lett. 371, 264–266]. Sequence comparison of BWI‐2b with those of the other proteins included in PIR showed that BWI‐2b is significantly homologous to the N‐terminal region of storage proteins classified in the vicilin family. Furthermore, the allergenic activity of BWI‐2b and the other buckwheat trypsin inhibitor BWI‐1 was examined using the radioallergosorbent test. The result indicated that both inhibitors BWI‐2b and BWI‐1 have IgE binding activity, albeit to a low extent, suggesting that they might be minor allergenic proteins in buckwheat seeds.