Identifying the stability of a new wheat gliadin extract by protein analysis, skin tests and cell degranulation assay.

BACKGROUND: The commercial wheat extract for skin prick test (SPT) provides less sensitivity to predict wheat allergy, compared to in-house gliadin extracts. SPT is a preferred method to study extract stability as it is the aim of developing extract. The role of cell degranulation assay, a functional assay with the same mechanism as SPT, is not widely used to determine extract stability. OBJECTIVE: To study the stability of in-house gliadin extracts stored at different periods, by using protein analysis, SPT and degranulation assay of humanized rat basophilic-leukemia (RBL-SX38) cells. METHODS: Patients with a history of wheat allergy and positive SPT to wheat, were recruited. The gliadin extracts stored for 1, 6, 9, and 12 months at 2-8°C were used in SDS-PAGE, SPT and cell degranulation assay. The cell degranulation was determined by ß-hexosaminidase release. AR patients. RESULTS: Forty children were recruited. The gliadin extract stored for 9 and 12 months provided lighter protein bands than 1 and 6 months. However, the wheal diameters from SPT using extracts stored at different periods, were not significantly different (p = 0.09). There were also no significant differences of the ß-hexosaminidase released using 0.1 and 1 µg/mL of gliadin extracts stored at different periods (p > 0.05). The 10 µg/mL of gliadin extracts stored at longer periods, significantly stimulated higher ß-hexosaminidase release (p = 0.01). The extracts were sterile at all storage times. CONCLUSIONS: To determine the stability of in-house gliadin extracts, SPT or cell degranulation assay provided additional information to SDS-PAGE. The extracts were stable for up to 12 months.

Children with wheat anaphylaxis and with low wheat specific IgE have a different IgE immunoblot pattern than those with high wheat specific IgE.

BACKGROUND: Children with wheat anaphylaxis can present with a wide range of wheat-specific IgE (sIgE). OBJECTIVE: To identify differences in clinical features and predominant wheat allergens sensitized by these patients. METHODS: Children with history of wheat anaphylaxis were recruited. Skin prick test (SPT) to wheat, sIgE to wheat, omega-5 gliadin (ω5G), lipid transfer protein (LTP) were investigated. Profiles of IgE-bound wheat allergens were studied to identify predominant wheat allergens. RESULTS: Twenty-nine children (17 males) aged 1-18 years were enrolled. Sixteen patients (55.2%) had wheat-sIgE > 100 kUA/L (WAhi) and 13 patients (44.8%) had wheat-sIgE < 34 kUA/L (WAlo). The median of peak wheat-sIgE in WAhi and WAlo were 340.5 kUA/L (IQR 184.3, 564.5) and 12.2 kUA/L (IQR 1.4, 41.3), respectively. Oral food challenge test (OFC) was carried out in 12 of 13 patients in the WAlo group, all of which had positive results. Eight of these 12 patients developed anaphylaxis during OFC despite having wheat-sIgE less than 10 kUA/L. There were no differences in clinical characteristics and atopic history between WAhi vs. WAlo. Medium to low molecular weight gliadin (< 40 kDa) and glutenin (< 60 kDa) were commonly recognized by patients with WAhi. IgE immunoblot pattern among the WAlo group was more widely dispersed than those with WAhi. CONCLUSIONS: Wheat anaphylaxis can occur in patients with low wheat-sIgE. Predominant wheat allergens recognized by patients with WAlo were different than those with WAhi. Such difference could be responsible for anaphylaxis at even low levels of wheat-sIgE.

A potential role of gliadin extract skin prick test in IgE-mediated wheat allergy.

BACKGROUND: Wheat extracts containing both water/salt and alcohol soluble proteins may increase extract's accuracy for diagnosing IgE-mediated wheat allergy. OBJECTIVE: This study aimed to determine the performance of new invented in-house prepared wheat extracts for skin prick test (SPT). METHODS: Children aged 1-18 years with history of immediate wheat allergy were recruited. Four in-house prepared wheat extracts (wheat-Coca-10%EtOH, and 3 new invented extracts, wheat-salt, gliadin, and glutenin) and a commercial wheat extract were used for SPT. Serum specific IgE (sIgE) to wheat and omega-5 (ω-5) gliadin were also determined. Oral food challenge (OFC) with wheat flours was performed in all patients except those with history of wheat-induced anaphylaxis or with recent symptoms within the past 6 months. RESULTS: Thirty-one children were recruited. Of those, 14 were excluded from OFC (12 with history of anaphylaxis and 2 with recent symptom). OFC was positive in 8 of 17 children. Of the 5 extracts and sIgE to wheat and ω-5 gliadin, gliadin extract provided the best SPT performance with 84.2% sensitivity, 88.9% specificity, 94.1% positive predictive value (PPV), 72.7% negative predictive value (NPV), 7.59 positive likelihood ratio (LR), 0.18 negative LR, and 85.7% accuracy. CONCLUSIONS: Compared to other in-house and commercial wheat extracts and sIgE to wheat and ω-5 gliadin, SPT with an in-house gliadin extract yielded the highest performance for the diagnosis IgE-mediated wheat allergy.

Comprehending the allergen repertoire of shrimp for precision molecular diagnosis of shrimp allergy.

BACKGROUND: Clinical management of shrimp allergy is hampered by the lack of accurate tests. Molecular diagnosis has been shown to more accurately reflect the clinical reactivity but the full spectrum of shrimp allergens and their clinical relevance are yet to be established. We therefore sought to comprehend the allergen repertoire of shrimp, investigate and compare the sensitization pattern and diagnostic value of the allergens in allergic subjects of two distinct populations. METHODS: Sera were collected from 85 subjects with challenge-proven or doctor-diagnosed shrimp allergy in Hong Kong and Thailand. The IgE-binding proteins of Penaeus monodon were probed by Western blotting and identified by mass spectrometry. Recombinant shrimp allergens were synthesized and analyzed for IgE sensitization by ELISA. RESULTS: Ten IgE-binding proteins were identified, and a comprehensive panel of 11 recombinant shrimp allergens was generated. The major shrimp allergens among Hong Kong subjects were troponin C (Pen m 6) and glycogen phosphorylase (Pen m 14, 47.1%), tropomyosin (Pen m 1, 41.2%) and sarcoplasmic-calcium binding protein (Pen m 4, 35.3%), while those among Thai subjects were Pen m 1 (68.8%), Pen m 6 (50.0%) and fatty acid-binding protein (Pen m 13, 37.5%). Component-based tests yielded significantly higher area under curve values (0.77-0.96) than shrimp extract-IgE test (0.70-0.75). Yet the best component test differed between populations; Pen m 1-IgE test added diagnostic value only in the Thai cohort, whereas sensitizations to other components were better predictors of shrimp allergy in Hong Kong patients. CONCLUSION: Pen m 14 was identified as a novel shrimp allergen predictive of challenge outcome. Molecular diagnosis better predicts shrimp allergy than conventional tests, but the relevant component is population dependent.

Defining Biomarkers to Predict Natural Resolution in Shrimp Allergy.

PURPOSE: Tolerance to shrimp has been reported in some patients with a history of shrimp allergy. The predictors of the natural resolution of shrimp allergy have not been widely explored. This study aimed to investigate the role of specific IgE (sIgE) and specific IgG4 (sIgG4) to shrimp extracts and the cross-reactive shrimp allergens tropomyosin (TM), arginine kinase (AK) and myosin light-chain (MLC), as markers of persistent or resolved shrimp allergy (PSA or RSA). METHODS: Seventeen patients with a 10-year history of allergy to Penaeus monodon (Pm) and/or Macrobachium rosenbergii (Mr) were recruited. Oral shrimp challenges identified 10 patients with PSA and 7 patients with RSA. Sera from these patients were evaluated for sIgE and sIgG4 to Mr and Pm extracts as well as to TM, AK and MLC. RESULTS: The levels of sIgE to Mr and Pm extracts were lower in the RSA than in the PSA groups (P = 0.05 and P = 0.008, respectively), but sIgG4 or sIgG4:sIgE ratio did not show statistical significance. The sIgE to AK and MLC, but not TM, were lower in the RSA group than in the PSA group (P = 0.009 and P = 0.0008, respectively). There was no difference in sIgG4 to TM, AK and MLC between both groups. The ratio of sIgG4:sIgE to MLC, but not TM or AK, was higher in the RSA than in the PSA group (P = 0.02). A higher diversity of sIgE to shrimp components was found in the PSA group than in the RSA group (P = 0.006). CONCLUSIONS: Specific bioassays can be used to identify patients with RSA. Oral shrimp challenges in these patients may provide a higher rate of passing the challenges and finally reintroducing shrimp in their diet.

Omega-5 and Gamma Gliadin are the Major Allergens in Adult-Onset IgE-Mediated Wheat Allergy: Results from Thai Cohort with Oral Food Challenge.

BACKGROUND: Various clinical patterns based on routes of sensitization and sensitized allergens are reported in adult-onset IgE-mediated wheat allergy. There is still a paucity of data on IgE-bound wheat allergen profiles in wheat challenge-proven adult-onset wheat allergic cases. Therefore, we aim to identify the major sensitized allergens in Thai adult-onset wheat allergic patients whose first symptom occurred after the age of 18 years despite previous tolerance. METHODS: This cross-sectional pilot study recruited patients from the Thai Adult-onset IgE-mediated Wheat Allergy Cohort (TAWAC). The sera of patients with mostly challenge-proven cases were selected for allergen study, including ImmunoCAP and IgE-bound gliadins along with glutenins profiles. The IgE-bound proteins were identified by liquid chromatography-tandem mass spectrophotometry (LC-MS/MS). Direct binding of IgE to recombinant gliadin and glutenin was performed to confirm the results of immunoblot and LC-MS/MS. RESULTS: Eleven wheat-dependent exercise-induced anaphylaxis (WDEIA) and 4 typical wheat allergy (WA) patients were enrolled. Serum IgE from >50% of bound proteins had a molecular weight ranging from 35 to 55 kDa in both gliadin and glutenin extracts. Further, ELISA demonstrated that γ-gliadin and ω5-gliadin were the most important major allergens. Other major allergens include α/ß-gliadin, HMW glutenin, and possibly α-amylase inhibitor or LWM glutenin. Gamma-gliadin sensitization was found in all WA patients (4/4), while ω-5 gliadin was found in all WDEIA patients (11/11) from ELISA. CONCLUSION: Wheat γ-gliadin and ω-5 gliadin are major wheat allergens among adult-onset wheat allergy patients in Thailand. Component-resolved diagnosis using γ-gliadin might be helpful in high suspicion of wheat allergy.

Manila grass (Zoysia matrella) Zoy m 1 allergen may contribute to allergic sensitization in tropical/subtropical regions due to extensive cross-reactivity with other group-1 grass pollen allergens.

BACKGROUND: Pollen of grasses in Chloridoideae and Panicoideae subfamilies is a major source of grass group-1 allergens in tropical/subtropical areas. Previously, most studies of subtropical grass pollen allergens have focused on Cynodon dactylon (Bermuda grass-Chloridoideae) and Sorghum halepense (Johnson grass-Panicoideae). However, little information is available about allergenicity of pollen from Zoysia matrella (Manila grass or Zoysia grass-Chloridoideae), which is among the most popular turfgrasses in tropical/subtropical areas. OBJECTIVE: This study aimed to investigate the IgE reactivity and cross-reactivity of grass group-1 allergen from Z. matrella. In addition, the clinical relevance of Z. matrella in comparison with other species was assessed. METHODS: IgE reactivity and cross-reactivity between recombinant proteins of group-1 allergen from Z. matrella (Zoy m 1) and C. dactylon (Cyn d 1) were determined by ELISA and immunoblot assays. Clinical relevance of Z. matrella pollen in Thai atopic patients was assessed using its pollen crude extract for skin-prick test, in comparison with extracts from four other pollen species. RESULTS: The Zoy m 1 had high IgE binding and could interfere with binding to C. dactylon crude extract. In addition, Z. matrella pollen extract elicited positive skin-prick test results comparable to previously reported allergenic species. Group-1 grass pollen allergen was confirmed to be a major allergen from Z. matrella among Thai atopic patients and was officially designated Zoy m 1.0101. CONCLUSIONS: Zoy m 1 allergen is a major allergen from Z. matrella that cross-reacts with other group-1 grass pollen allergens in the tropical/subtropical region.

Natural resolution of non-anaphylactic shrimp allergy in patients diagnosed 10 years earlier by oral food challenge.

BACKGROUND: Shrimp allergy is considered a lifelong condition. The natural resolution of shrimp allergy is not well studied. OBJECTIVE: To investigate the natural resolution of shrimp allergy among a cohort of patients diagnosed with shrimp allergy 10 years earlier by oral shrimp challenge. METHODS: A prospective study recruited patients diagnosed with shrimp allergy to Penaeus monodon (Pm), Macrobrachium rosenbergii (Mr), or both from a study conducted during 2005-2006. The current oral shrimp challenges were conducted during 2015-2016. The negative oral shrimp challenge was designated 'resolved shrimp allergy' (RSA), with a positive challenge designated 'persistent shrimp allergy' (PSA). Skin prick and prick-to-prick testing to shrimp were used to determine sensitization. RESULTS: Sixty patients who had positive shrimp challenge from the previous cohort were contacted. Patients who had previous anaphylactic reaction (8 subjects) or allergic reaction after shrimp ingestion within 6 months (6 subjects), were not included. Nine patients refused to participate and 20 patients could not be contacted. Seventeen patients were included. Three were previously diagnosed with allergy to Pm, 3 to Mr, and 11 to both species. RSA was observed in 1 patient with isolated Pm allergy, and in 3 patients with isolated Mr allergy. Three of 9 patients with dual allergy had RSA to both species. RSA patients had significantly smaller size of shrimp skin test than PSA patients at both diagnosis and follow-up. CONCLUSIONS: At ten years after diagnosis, 46% of patients had RSA. These patients had significantly smaller size of shrimp skin test than PSA patients.

Clinical Characteristics and Proposed Wheat-Cofactor Challenge Protocol with a High Diagnostic Yield in Adult-Onset IgE-Mediated Wheat Allergy.

BACKGROUND: IgE-mediated wheat allergy in adults can be childhood or adulthood onset. Adult-onset wheat allergy has been reported, but data on clinical characteristics and practical food challenge protocols are scarce. OBJECTIVE: We aimed to describe the clinical characteristics of adult-onset wheat allergy, laboratory results, and outcomes of a modified 3-day challenge protocol using a combination of aspirin, wheat, and exercise. PATIENTS AND METHODS: Patients with histories compatible with adult-onset wheat allergy were recruited from Siriraj Hospital, Thailand. Clinical history, skin prick tests (SPTs), and specific IgE (sIgE) levels were ascertained. Patients with no food challenge contraindications were asked to volunteer for wheat challenge. A modified 3-day protocol using 300 mg of acetylsalicylic acid, 60-75 g of wheat flour, and exercise was used for confirmatory diagnosis of conventional wheat allergy (WA) and wheat-dependent exercise-induced anaphylaxis (WDEIA). RESULTS: Thirty-three patients were recruited. The mean age of onset was 29.7 years (SD 10.5). SPTs yielded positivity rates of 9.1%, 84.8%, and 81.8% in commercial wheat, in-house gliadin, and in-house glutenin extracts, respectively. sIgE yielded a positivity rate of 61% and 88% in wheat and ω5-gliadin, respectively. Eighteen patients underwent oral wheat challenges. Of these, 17 patients (94.4%) had positive challenges leading to definite diagnoses of WA (35%), and WDEIA (65%). One WDEIA patient developed hypotensive anaphylaxis in the protocol. CONCLUSION: WDEIA was the most common phenotype. Our modified 3-day protocol could differentiate WA and WDEIA and yielded a high positivity rate (94.4%). It should be used cautiously as severe reactions can occur.

Clinical and in vitro cross-reactivity of cereal grains in children with IgE-mediated wheat allergy.

INTRODUCTION AND OBJECTIVES: Wheat and cereal grains have a broad range of cross-reactivity, but the clinical relevance of this cross-reactivity is uncertain. This study aimed to evaluate clinical and in vitro cross-reactivity with barley, oat, and Job's tears among wheat-allergic patients. MATERIALS AND METHODS: Patients aged 5 to 15 years with IgE-mediated wheat allergy were enrolled. Skin prick test (SPT) and specific IgE (sIgE) to wheat, barley, and oat, and SPT to Job's tears were performed. Oral food challenge (OFC) was conducted if the SPT was ≤5 mm in size and there was no history of anaphylaxis to each grain. Profiles of sIgE bound allergens of wheat, barley, and oat, and inhibition ELISA of IgE binding to barley and oat with wheat were performed. RESULTS: Ten patients with a median age of 8 years were enrolled. Nine of those patients had a history of wheat anaphylaxis. The median SPT size and sIgE level to wheat was 7.3 mm and 146.5 kUA/l, respectively. The cross-reactivity rate for barley, oat, and Job's tears was 60.0%, 33.3%, and 20.0%, respectively. Significantly larger SPT size and higher sIgE level were observed in patients with positive cross-reactivity to barley and oat when compared to patients without cross-reactivity. Barley and oat extracts inhibited 59% and 16% of sIgE bound to wheat gliadins and glutenins, respectively. CONCLUSION: The cross-reactivity rate was quite low for oat and Job's tears compared to that of barley; therefore, avoidance of all cereal grains may be unnecessary in patients with severe wheat allergy.

Accuracy of in-house alcohol-dissolved wheat extract for diagnosing IgE-mediated wheat allergy.

BACKGROUND: The standard method for diagnosing immediate wheat allergy is oral food challenge test (OFC). However, OFC can provoke anaphylaxis during the challenge process. Skin prick test (SPT) using commercial wheat extract yielded unsatisfactory result for diagnosis of wheat allergy. As a result, an in-house, alcohol-dissolved (Coca-10% EtOH) wheat extract was developed to improve accuracy of the SPT. OBJECTIVE: To determine the accuracy of in-house, alcohol-dissolved wheat extract in children with immediate wheat allergy. METHODS: This prospective cross-sectional study included children with history of immediate reaction after wheat ingestion. SPTs with commercial and in-house Coca-10% EtOH wheat extract were performed and wheat and omega-5 (ω-5) gliadin specific IgE (sIgE) were measured. Patients with no history of recent anaphylaxis after wheat ingestion underwent OFC with 31 grams of wheat flour. RESULTS: Thirty children were recruited. Thirteen of those had history of anaphylaxis after wheat ingestion. Eleven of the remaining 17 children (64.7%) had a positive result for wheat challenge test. Wheal size of 3 mm for both in-house and commercial wheat extract yielded the best accuracy for the test. Using these cutoff parameters, in-house Coca-10% EtOH wheat extract yielded 91.7% sensitivity, 66.7% specificity, and 86.7% accuracy. Comparatively, the commercial extract yielded 70.8% sensitivity, 100% specificity, and 76.6% accuracy. CONCLUSIONS: SPT using in-house Coca-10% EtOH wheat extract yielded better accuracy than commercial extract for diagnosing immediate type wheat allergy in children.

Reduced Renal Colonization and Enhanced Protection by Leptospiral Factor H Binding Proteins as a Multisubunit Vaccine Against Leptospirosis in Hamsters.

Subunit vaccines conferring complete protection against leptospirosis are not currently available. The interactions of factor H binding proteins (FHBPs) on pathogenic leptospires and host factor H are crucial for immune evasion by inhibition of complement-mediated killing. The inhibition of these interactions may be a potential strategy to clear leptospires in the host. This study aimed to evaluate a multisubunit vaccine composed of four known leptospiral FHBPs: LigA domain 7-13 (LigAc), LenA, LcpA, and Lsa23, for its protective efficacy in hamsters. The mono and multisubunit vaccines formulated with LMQ adjuvant, a combination of neutral liposome, monophosphoryl lipid A, and Quillaja saponaria fraction 21, induced high and comparable specific antibody (IgG) production against individual antigens. Hamsters immunized with the multisubunit vaccine showed 60% survival following the challenge by 20 LD50 of Leptospira interrogans serovar Pomona. No significant difference in survival rate and pathological findings of target organs was observed after vaccinations with multisubunit or mono-LigAc vaccines. However, the multisubunit vaccine significantly reduced leptospiral burden in surviving hamsters in comparison with the monosubunit vaccines. Therefore, the multisubunit vaccine conferred partial protection and reduced renal colonization against virulence Leptospira infection in hamsters. Our multisubunit formulation could represent a promising vaccine against leptospirosis.

Beta-Expansin of Bermuda, Johnson, and Para grass pollens, is a major cross-reactive allergen for Allergic Rhinitis patients in subtropical climate.

BACKGROUND: Subtropical grass pollens of Bermuda (BGP), Johnson (JGP), and Para or buffalo grass (PGP), are common causes of pollen allergies in warm climate area. Allergic rhinitis (AR) patients had positive skin prick test (SPT) to extract of these 3 grass pollens. However, no allergenic proteins of 3 grass pollens have never been studied. OBJECTIVE: To identify major allergens of BGP, JGP, and PGP in Thai grass pollen-allergic patients and to examine their sIgE cross-reactivity. METHODS: Serum of nine AR patients with positive SPT to at least 2 of 3 studied pollens were collected. Based on availability, only ImmunoCAP of BGP and JGP were available to determine a level of sIgE. Profiles of sIgE bound proteins from BGP, JGP, and PGP, were obtained by immunoblot. Major IgE bound protein was identified by liquid chromatography-tandem mass spectrophotometry (LC-MS/MS). Cross-reactivity of purified major allergen of the 3 grass pollens was determined by inhibition of sIgE in both ELISA and immunoblot. RESULTS: AR patients who have positive SPT to extract of BGP, JGP, and PGP, were 9, 8, and 6, respectively. Positive sIgE (> 0.35 kUA/L) to BGP and JGP were found in 9 and 8 patients, respectively. Eight profiles of IgE bound proteins of the 3 grass pollens showed 29-30 kDa pollen protein as major allergen and was identified as beta-expansin (ExpB). Moreover, purified ExpB of the 3 grass pollens cross-inhibited serum sIgE. CONCLUSION: ~30 kDa ExpB of BGP, JGP, and PGP, is major cross-reactive allergen for AR Thai patients.

Effects of Ser47-Point Mutation on Conformation Structure and Allergenicity of the Allergen of Der p 2, a Major House Dust Mite Allergen.

PURPOSE: Hypoallergenic recombinant Der p 2 has been produced by various genetic manipulations, but mutation of a naturally polymorphic amino acid residue known to affect IgE binding has not been studied. This study aimed to determine the effect of a point mutation (S47W) of residue 47 of Der p 2 on its structure and immunoglobulin (Ig) E binding. Its ability to induce pro-inflammatory responses and to induce blocking IgG antibody was also determined. METHODS: S47 of recombinant Der p 2.0110, one of the predominant variants in Bangkok, was mutated to W (S47W). S47W secreted from Pichia pastoris was examined for secondary structure and for the formation of a hydrophobic cavity by 8-Anilino-1-naphthalenesulfonic acid (ANS) staining. Monoclonal and human IgE-antibody binding was determined by enzyme-linked immunosorbent assay. Allergen-induced degranulation by human epsilon receptor expressed-rat basophil was determined. Stimulation of the pro-inflammatory cytokine interleukin (IL)-8 release from human bronchial epithelial (BEAS2B) cells and inhibition of IgE binding to the wild type allergen by S47W-induced IgG were determined. RESULTS: S47W reduced secondary structure and failed to bind the hydrophobic ANS ligand as well as a monoclonal antibody known to be dependent on the nature of the side chain of residue 114 in an adjacent loop. It could also not stimulate IL-8 release from BEAS2B cells. IgE from house dust mite (HDM)-allergic Thais bound S47W with 100-fold weaker avidity, whereas IgE of HDM-allergic Australians did not. S47W still induced basophil degranulation, although requiring higher concentrations for some subjects. Anti-S47W antiserum-immunized mice blocked the binding of human IgE to wild type Der p 2. CONCLUSIONS: The mutant S47W had altered structure and reduced ability to stimulate pro-inflammatory responses and to bind IgE, but retained its ability to induce blocking antibodies. It thus represents a hypoallergen produced by a single mutation of a non-solvent-accessible amino acid.

Identification of wheat sensitization using an in-house wheat extract in Coca-10% alcohol solution in children with wheat anaphylaxis.

BACKGROUND: Identification of wheat sensitization by a skin prick test (SPT) is essential for children with wheat-induced anaphylaxis, since oral food challenge can cause serious adverse effects. Wheat allergens are both water/salt and alcohol soluble. The preparation of wheat extract for SPT containing both water/salt and alcohol soluble allergen is needed. OBJECTIVE: To determine if a wheat extract using Coca's solution containing 10% alcohol (Coca-10% EtOH), prepared in-house, contians both water/salt and alcohol soluble allergens. METHODS: Serum of children with a history of anaphylaxis after wheat ingestion was used. Wheat flour was extracted in Coca-10% alcohol solution. An SPT with both commercial and in-house wheat extracts was performed as well as specific IgE (sIgE) for wheat and omega-5 gliadin. Direct and IgE inhibition immunoblots were performed to determine serum sIgE levels against water/salt as well as alcohol soluble (gliadins and glutenins) allergens in the extracts. RESULTS: Six children with history of wheat anaphylaxis had positive SPT to both commercial and in-house extracts. They also had different levels of sIgE against wheat and omega-5 gliadin allergens. The results of direct immunoblotting showed all tested sera had sIgE bound to ~35 kDa wheat protein. Further IgE inhibition immunoblotting identified the ~35 kDa wheat protein as gliadin but not gluten allergen. CONCLUSION: The in-house prepared Coca-10% EtOH solution could extract both water/salt and alcohol soluble allergens. The ~35 kDa gliadin appears to be a major wheat allergen among tested individuals.

Cloning and characterization of recombinant tropomyosin of giant freshwater shrimp M. rosenbergii to determine major allergens causing allergic reactions among shrimp-allergic children.

BACKGROUND: Seawater and freshwater shrimp are some of the most common causes of food allergy among children in Thailand. Tropomyosin has been reported as a major allergen for shrimp allergic populations around the world. Despite a high number of shrimp-allergic Thai children, however, it is unknown whether shrimp tropomyosin is a major cause of allergic reactions. OBJECTIVES: To clone and characterize tropomyosin of giant freshwater shrimp Macrobrachium rosenbergii (Mr) and determine whether this tropomyosin is a major cross-reactive allergen for Thai children with shrimp allergy. METHODS: Recombinant shrimp Mr tropomyosin (Mac r1.0101) was expressed in yeast Pichia pastoris. Secondary structure composition of purified recombinant Mac r1.0101 was determined by Circular Dichroism. IgE reactivity was examined by immunoblot, direct binding ELISA and inhibition of IgE ELISA using serum from shrimp-allergic children. RESULTS AND CONCLUSION: The amino acid sequence of Mac r1.0101 showed 2 polymorphic amino acids (F44 and S45) indicating a variant of tropomyosin. Purified recombinant Mac r1.0101 obtained a nature-like α-helix structure which can be bound by serum-specific IgE. The binding affinity of serum-specific IgE to Mac r1.0101 based on the IC50 value was ~1.8 ng/ml. Ten of 13 shrimp-allergic Thai children had serum-specific IgE against Mac r1.0101, but at different levels. Results of the inhibition of specific IgE using Mac r1.0101 showed that 7 of the tested serum samples also had specific IgE against other shrimp allergens in addition to IgE against Mac r1.0101. Tropomyosin therefore appears to be a major cross-reactive allergen for Thai children who are allergic to both seawater and giant freshwater shrimp.

Effect of Amino Acid Polymorphisms of House Dust Mite Der p 2 Variants on Allergic Sensitization.

PURPOSE: The sequence variations of the Der p 2 allergen of Dermatophagoides pteronyssinus diverge along 2 pathways with particular amino acid substitutions at positions 40,47,111, and 114. The environmental prevalence and IgE binding to Der p 2 variants differ among regions. To compare IgE binding to Der p 2 variants between sera from Bangkok, Thailand and Perth, Western Australia with different variants and to determine the variant-specificity of antibodies induced by vaccination with recombinant variants. METHODS: The structures of recombinant variants produced in yeast were compared by circular dichroism and 1-anilinonaphthalene 8-sulfonic acid staining of their lipid-binding cavity. Sera from subjects in Bangkok and Perth where different variants are found were compared by the affinity (IC50) of IgE cross-reactivity to different variants and by direct IgE binding. Mice were immunized with the variants Der p 2.0101 and Der p 2.0110, and their IgG binding to Der p 2.0103, 2.0104, and 2.0109 was measured. RESULTS: The secondary structures of the recombinant variants resembled the natural allergen but with differences in ANS binding. The IC50 of Der p 2.0101 required 7-fold higher concentrations to inhibit IgE binding to the high-IgE-binding Der p 2.0104 than for homologous inhibition in sera from Bangkok where it is absent, while in sera from Perth that have both variants the IC50 was the same and low. Reciprocal results were obtained for Der p 2.0110 not found in Perth. Direct binding revealed that Der p 2.0104 was best for detecting IgE in both regions, followed by Der p 2.0101 with binding to other variants showing larger differences. Mouse anti-Der p 2.0101 antibodies had a high affinity of cross-reactivity but bound poorly to other variants. CONCLUSIONS: The affinity of IgE antibody cross-reactivity, the direct IgE binding, and the specificities of antibodies induced by vaccination show that measures of allergic sensitization and therapeutic strategies could be optimized with knowledge of Der p 2 variants.

Amaranthus species around Bangkok, Thailand and the release of allergenic proteins from their pollens.

BACKGROUND: Pollen of Amaranthus L., commonly known as careless weed or Phak-khom in Thai, has become one of the major causes of airway allergy in many countries including Thailand. Despite its recognized importance, there is no available information about which Amaranthus species are producing allergenic pollen more likely to affect Thai patients. Furthermore, only allergenic proteins released from pollen can cause allergy. OBJECTIVE: This study aimed to survey species of Amaranthus found in Bangkok and to investigate the impact of water on pollen damage and protein release from Amaranthus pollens. METHODS: Amaranthus inflorescences were sampled and identified using the identification key provided in "Flora of Thailand". Shed pollens were collected on day 1, 3 and 7 after shedding. Ten mg of pollens in distilled water including damaged pollens were counted under a light microscope. In addition, supernatant was analyzed for concentration of proteins released from pollens using Bradford's assay. Profiles of released proteins and IgE binding proteins were analyzed by SDS-PAGE and Western blot. RESULTS: Three species of Amaranthus-A. hybridus, A. spinosus, and A. viridis were identified. SDS-PAGE analysis revealed at least twelve protein bands with MW ranging from 10 to 80 kDa. Water caused more damage to pollens and higher amount of proteins were recovered from pollens 1 day after shedding than from 3- and 7-days old pollens. The results of Western blot showed IgE-bound proteins with MW ranging from 30 to 50 kDa. CONCLUSIONS: Water could damage pollens and time after shedding and significantly affected the amount of allergenic proteins released from pollen.

Stability and potency of raw and boiled shrimp extracts for skin prick test.

BACKGROUND: The difference of stability between raw and boiled shrimp extracts used in prick tests has never been investigated despite its potential consequences in tests development. The aim of this study was to compare the raw and boiled shrimp extracts of two species; Macrobrachium rosenbergii (freshwater shrimp) and Penaeus monodon (seawater shrimp) held at 4 ?C for different periods of time for their stability and potency in vivo by using the skin prick test (SPT) method. METHODS: Raw and boiled M. rosenbergii and P. monodon extracts were prepared and stored at 4 ?C for 1, 7, 14 and 30 days. Thirty patients were pricked with raw and boiled shrimp extracts at all storage times, as well as prick to prick skin test (PTP) to fresh raw and boiled shrimps of both species. The mean wheal diameter (MWD) resulting from prick tests for all shrimp extracts was measured and compared. RESULTS: The shrimp extracts of all storage times yielded positive skin test results in the range of 90% - 100%. Raw P. monodon extracts induced larger wheals than boiled extracts at all storage times. There was no significant difference of MWD between raw and boiled M. rosenbergii extracts on day 1, 7, and 14. Significant correlations between MWD of PTP to fresh shrimps and SPT to all shrimp extracts were observed. All shrimp extracts were sterile at all storage times. CONCLUSIONS: Raw and boiled M. rosenbergii and P. monodon extracts were stable and sterile at 4 ?C for at most 30 days. SPT with these extracts induced more than 10 mm in shrimp allergy patients and the results were comparable with PTP to fresh shrimps.