sIgE Binding Research Articles

Thermostable allergens in canned fish: Evaluating risks for fish allergy.

Major fish allergens, including parvalbumin (PV), are heat stable and can withstand extensive cooking processes. Thus, the management of fish allergy generally relies on complete avoidance. Fish-allergic patients may be advised to consume canned fish, as some fish-allergic individuals have reported tolerance to canned fish. However, the safety of consuming canned fish has not been evaluated with comprehensive immunological and molecular analysis of canned fish products. We characterized the in vitro immunoreactivity of serum obtained from fish-allergic subjects to canned fish. Seventeen canned fish products (salmon n = 8; tuna n = 7; sardine n = 2) were assessed for the content and integrity of PV using allergen-specific antibodies. Subsequently, the sIgE binding of five selected products was evaluated for individual fish-allergic patients (n = 53). Finally, sIgE-binding proteins were identified by mass spectrometry. The canned fish showed a markedly reduced PV content and binding to PV-specific antibodies compared with conventionally cooked fish. However, PV and other heat-stable fish allergens, including tropomyosin and collagen, still maintained their sIgE-binding capacity. Of 53 patients, 66% showed sIgE binding to canned fish proteins. The canned sardine contained proteins bound to sIgE from 51% of patients, followed by canned salmon (43%-45%) and tuna (8%-17%). PV was the major allergen in canned salmon and sardine. Tropomyosin and/or collagen also showed sIgE binding. We showed that canned fish products may not be safe for all fish-allergic patients. Canned fish products should only be considered into the diet of individuals with fish allergy, after detailed evaluation which may include in vitro diagnostics to various heat-stable fish allergens and food challenge conducted in suitable environments.

The effect of soy processing on its allergenicity: Discrepancy between IgE binding and basophil stimulation tests

Presently, many studies assess allergenicity via IgE immunoblotting and IgE binding tests; however, IgE detection does not always signal the manifestation of a clinical allergy. However, the capacity of food allergens to trigger basophils makes it possible to use the in vitro functional basophil activation test (BAT) to assess allergenicity. The effect of the Maillard reaction (MR) on the allergenic potential of processed soy proteins was evaluated by two IgE binding tests (Competitive ELISA and Inhibition ImmunoCAP), a Western Blot and a functional BAT; with the aim to analyze whether the sIgE binding results correspond to the functional assay results. The results between the IgE binding tests and the functional assay were in-line for 2 of the 6 studied patients. For one patient there was no correlation between any of the results. For the raw soy protein extract heated with glucose for 10 and 30 min (SH SPE + Glu and LH SPE + Glu, respectively), the results were in-line for only 3 out of the 6 patients. Thus, the present study shows a discrepancy between IgE binding tests and basophil stimulation when assessing the effect of soy processing on its allergenicity. Since IgE-binding capacity does not always correlate to IgE cross-linking capacity, the conclusions of the allergenic potential based on the IgE binding tests alone should be drawn with care and further studies on this matter would benefit from the inclusion of a functional assay such as the BAT.

Evaluation of Allergenicity on a ω-5 Gliadin-Deficient Cultivar in Wheat-Dependent Exercise-Induced Anaphylaxis

Purposeω-5 gliadin is the major allergen that causes wheat-dependent exercise-induced anaphylaxis (WDEIA). Recently, a missing mutant wheat cultivar at 1B chromosome Glu-B3 and closely linked Gli-B1 loci was bred. This cultivar (ω5D) has a deficiency in ω-5 and γ-gliadins as well as some low-molecular-weight glutenins. We evaluated specific immunoglobulin E (sIgE) reactivity of the ω5D in WDEIA patients compared to wild-type cultivar.MethodsSerum samples from 14 WDEIA and 7 classic wheat allergy patients were used to compare the allergenicity of ω5D and wild-type cultivars using immunoglobulin E immunoblotting, enzyme-linked immunosorbent assay (ELISA), and ImmunoCAP inhibition assays.ResultsImmunoblotting revealed that ω5D extracts had less sIgE binding to gliadins and glutenins in WDEIA sera than wild-type extracts. Immunoblot inhibition assay for gliadin sIgE reactivity also showed that ω5D gliadins had less allergenicity than wild-type gliadins. ELISA inhibition assay showed stronger allergenicity of gliadins than glutenins, although they had cross-reactivity. This assay also showed that the 50% inhibitory concentrations (IC50) of ω5D extracts against gliadin- or glutenin-sIgE reactivity were approximately 4-fold higher in WDEIA patients than those of wild-type extracts. The inhibition capacity of ω5D gliadins against recombinant ω-5 gliadin-sIgE reactivity was also lower in WDEIA patients than that of wild-type.ConclusionsThe allergenicity of the ω5D cultivar is markedly lower for WDEIA patients in the sIgE inhibition tests. These results suggest that the ω5D cultivar may be a safe alternative for WDEIA patients.

Characterization and epitope prediction of phosphopyruvate hydratase from Penaeus monodon (black tiger shrimp).

Shellfish allergies constitute an important cause of food-induced anaphylactic reactions, which pose challenges to food safety and human health worldwide. In the present study, the specific IgE (sIgE) binding characteristics of different shrimp proteins of black tiger shrimp (Penaeus monodon) to the sera of eight shrimp-allergic patients from China were studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and nanoliquid chromatography time-of-flight mass spectrometry. According to the PLGS scores (>2000) and the sequence coverage (>40%), eight proteins with sIgE binding activity were identified, including myosin heavy chain type 1 (K4Q4N8), hemocyanin (G1AP69 and Q95V28), phosphopyruvate hydratase (O96656), arginine kinase (C7E3T4), tropomyosin (A1KYZ2), sarcoplasmic calcium binding protein (H7CHW2) and glyceraldehyde-3-phosphate dehydrogenase (A0A097BQP2). Among these eight proteins, phosphopyruvate hydratase was a prevalent IgE-binding protein among these Chinese patients with binding observed in 100% of sera. Moreover, 13 peptides were predicted as epitopes of phosphopyruvate hydratase. These new details help us to understand the crustacean IgE-binding proteins especially Penaeus monodon IgE-binding proteins, that would cause allergic reaction to Chinese patients. And our findings may provide essential information to improve allergy prevention and clinical treatment to shrimp allergy in China. PRACTICAL APPLICATION: This research may have diagnostic and therapeutic value for shrimp allergies in China.

IgE-binding residues analysis of the house dust mite allergen Der p 23

House dust mites (HDMs) are one of the major causes of allergies in the world. The group 23 allergen, Der p 23, from Dermatophagoides pteronyssinus, is a major allergen amongst HDM-sensitized individuals. This study aims to determine the specific immunoglobulin E (sIgE) binding frequency and IgE-binding residues of recombinant Der p 23 (rDer p 23) allergen amongst a cohort of consecutive atopic individuals in a tropical region. We performed site-directed mutagenesis and carried out immuno-dot blot assays using 65 atopic sera. The immuno-dot blot assays results indicated that the two residues K44 and E46 which are located at the N-terminal region are the major IgE-binding residues. The rDerp-23 sIgE titers are strongly correlated to the number of IgE-binding residues for rDer p 23 (P < 0.001). Atopic individuals who were only sensitized to HDM have a significantly higher number of IgE-binding residues than the individuals who were polysensitized to HDM and other crude allergens (P < 0.05). Individuals with allergic multimorbidity and moderate-to-severe allergic rhinitis also have a higher number of IgE-binding residues compared to those with single allergic disease and mild allergic rhinitis. The results prompt us to hypothesize that the individuals who have a higher number of IgE-binding residues may face a bigger challenge to be treated through immunotherapy due to the complexity in designing an effective hypoallergen with a high number of IgE-binding residues. We propose that the development of a refined molecular diagnostic assay, which includes alanine substitution of surface-exposed residues could be a more precise diagnostic strategy to identify all the IgE-binding residues of a major allergen for an atopic individual and the development could be another new dimension in allergy diagnosis and allergen immunotherapy treatment.

Peptide Release after Simulated Infant In Vitro Digestion of Dry Heated Cow's Milk Protein and Transport of Potentially Immunoreactive Peptides across the Caco-2 Cell Monolayer.

Dry heating of cow’s milk protein, as applied in the production of “baked milk”, facilitates the resolution of cow’s milk allergy symptoms upon digestion. The heating and glycation-induced changes of the protein structure can affect both digestibility and immunoreactivity. The immunological consequences may be due to changes in the peptide profile of the digested dry heated milk protein. Therefore, cow’s milk protein powder was heated at low temperature (60 °C) and high temperature (130 °C) and applied to simulated infant in vitro digestion. Digestion-derived peptides after 10 min and 60 min in the intestinal phase were measured using LC-MS/MS. Moreover, digests after 10 min intestinal digestion were applied to a Caco-2 cell monolayer. T-cell epitopes were analysed using prediction software, while specific immunoglobin E (sIgE) binding epitopes were identified based on the existing literature. The largest number of sIgE binding epitopes was found in unheated samples, while T-cell epitopes were equally represented in all samples. Transport of glycated peptide indicated a preference for glucosyl lysine and lactosyl-lysine-modified peptides, while transport of peptides containing epitope structures was limited. This showed that the release of immunoreactive peptides can be affected by the applied heating conditions; however, availability of peptides containing epitopes might be limited.

Serum IgE against galactose-alpha-1,3-galactose is common in Laotian patients with snakebite envenoming but not the major trigger for early anaphylactic reactions to antivenom.

Snake antivenom is the only specific treatment for snakebite envenoming, but life-threatening anaphylaxis is a severe side effect and drawback for the use of these typically mammalian serum products. The present study investigates the hypotheses whether serum IgE antibodies against the epitope galactose-alpha-1,3-galactose (α-gal) located on the heavy chain of non-primate mammalian antibodies are a possible cause for hypersensitivity reactions to snake antivenom. Serum samples from 55 patients with snakebite envenoming were obtained before administration of snake antivenom and tested for serum IgE (sIgE) against α-gal and total IgE. Early anaphylactic reactions (EARs) during the first 3 h after antivenom administration were classified into mild, moderate or severe and correlated with the presence of sIgE against α-gal. Fifteen (27%) out of 55 patients (37 male, 18 female, median 34 years, range 9–90 years) developed EARs after antivenom administration. Eleven, three and one patients had mild, moderate and severe EARs, respectively. Serum IgE against α-gal was detected in 17 patients (31%); in five (33%) out of 15 patients with EARs and in 12 (30%) out of 40 patients without EAR (Odds Ratio = 1.2; 95%-confidence interval: 0.3–4.2) with no correlation to severity. Although the prevalence of serum IgE against α-gal was high in the study population, very high levels of total IgE in the majority of patients question their clinical relevance and rather indicate unspecific sIgE binding instead of allergy. Lack of correlation between α-gal sIgE and EARs together with significantly increased total IgE levels suggest that sIgE against α-gal is not the major trigger for hypersensitivity reactions against snake antivenom.

Allergen Immunotherapy-Induced Immunoglobulin G4 Reduces Basophil Activation in House Dust Mite-Allergic Asthma Patients.

It is unclear if allergen immunotherapy (AIT) can reduce allergy effector cell activation. We evaluated the basophil response during Dermatophagoides pteronyssinus (Der p) subcutaneous immunotherapy (SCIT) and its relationship to allergen-specific immunoglobulin G4 (sIgG4) in allergic rhinitis and/or asthma patients. The study included 55 subjects, of which 35 cases received Der p SCIT and 20 controls received standard medications. Symptom and medication scores (SMSs), sIgG4 levels, specific immunoglobulin E (sIgE) levels, allergen-induced basophil activation tests (BATs) in whole blood, and BAT inhibition assays in serum were determined at weeks 0, 4, 12, 16, 52, and 104 of SCIT. Levels of Der p sIgG4 in SCIT patients significantly increased after 12 weeks of treatment compared to week 0. Serum obtained from SCIT patients significantly inhibited basophil activation after 12 weeks of treatment. Removal of immunoglobulin G4 (IgG4) antibodies at week 104 reduced the ability of serum to block basophil activation. An increase of Der p sIgG4 rather than reduction of Der p sIgE correlated with the reduction of basophil activation during SCIT. The sIgG4 antibodies may compete with sIgE binding to allergens to form an immunoglobulin E (IgE)–allergen complex. SCIT reduced the sensitivity of allergen-triggered basophil activation in Der p allergic rhinitis and/or asthma patients through induction of sIgG4.

Identification of Der f 23 as a new major allergen of Dermatophagoides farinae

House dust mites (HDM) are common allergen sources worldwide. At present, 32 of the 37 internationally recognized HDM allergen groups have been identified in Dermatophagoides farinae. The present study study describes the identification of the first known D. farinae Group 23 allergen (Der f 23). Recombinant Der f 23 protein (rDer f 23) was cloned, expressed and purified. The open reading frame of rDer f 23 was 525 base pairs and encoded a 174-amino acid protein (GenBank accession no., KU166910.1). ELISAs indicated that 72/129 HDM allergic serum samples (55.8%) had specific immunoglobulin E (sIgE) binding activity to rDer f 23. Additionally, 3/10 patients with HDM allergies (30%) exhibited positive skin prick test reactions to rDer f 23. IgE western blot analysis data suggested that only 4/11 HDM allergic sera had a positive sIgE binding result. Sequence homology analysis revealed an extra P2 region (Ser56-Thr117) in Der f 23 that was not present in the D. pteronyssinus homolog, which may affect sIgE binding. Der f 23ΔP2 demonstrated binding with HDM allergic sera, whereas the P2 peptide alone did not. The sIgE binding ability of Der f 23 ΔP2 (Der f 23 with a truncated P2 region) was more marked compared with that of Der f 23 in an IgE ELISA. These data indicate that P2 region in Der f 23 attenuates IgE binding ability. In conclusion, the results of the present study indicate that Der f 23 is a major HDM allergen with predominantly conformational sIgE binding epitopes. The allergenic identification of Der f 23 and its inclusion in World Health Organization/International Union of Immunological Societies database contributes to the theoretical basis underlying the diagnosis and treatment of HDM allergic diseases.

Functional and Immunoreactive Levels of IgG4 Correlate with Clinical Responses during the Maintenance Phase of House Dust Mite Immunotherapy.

Allergen-specific immunotherapy for house dust mite allergy is effective, but there are no validated biomarkers reflecting or predicting the clinical efficacy. We aimed to investigate the relationship between clinical outcomes and functional responses of allergen-specific IgG4 (sIgG4) and specific IgE (sIgE) during Dermatophagoides pteronyssinus s.c. allergen immunotherapy (SCIT) in allergic rhinitis and/or asthma patients. Combined symptom medication scores (SMS), D. pteronyssinus-sIgG4 levels, D. pteronyssinus-sIgE levels, and the serum inhibitory capacity against D. pteronyssinus-sIgE facilitated allergen binding to B cells (IgE-FAB) were determined during the updosing (week 0, 4, 12, and 16) and maintenance (week 52, 104, and 156) phase of SCIT. We found that SCIT patients had a significant improvement in SMS from week 52 to 156 compared with medication-treated control subjects (p < 0.05). Levels of D. pteronyssinus-sIgG4 in SCIT patients showed a significant increase from week 12 to 156 (p < 0.05). Serum obtained from SCIT patients significantly inhibited D. pteronyssinus-sIgE binding to B cells after 16 wk (p < 0.01). Significantly lower levels of D. pteronyssinus-sIgE were observed in SCIT patients after 52 wk (p < 0.05). A significant relationship was demonstrated between SMS and IgE-FAB or D. pteronyssinus-sIgG4 during the maintenance phase according to linear regression analysis. In conclusion, D. pteronyssinus-sIgG4 level and D. pteronyssinus IgE-FAB are associated with clinical efficacy in the maintenance phase rather than the updosing phase of SCIT. Immunologic tolerance can be induced with SCIT when maintenance phase is achieved.

Polyphosphate kinase 1, a central node in the stress response network of Mycobacterium tuberculosis, connects the two-component systems MprAB and SenX3–RegX3 and the extracytoplasmic function sigma factor, sigma E

Polyphosphate (poly P) metabolism regulates the stress response in mycobacteria. Here we describe the regulatory architecture of a signal transduction system involving the two-component system (TCS) SenX3-RegX3, the extracytoplasmic function sigma factor sigma E (SigE) and the poly P-synthesizing enzyme polyphosphate kinase 1 (PPK1). The ppk1 promoter of Mycobacterium tuberculosis is activated under phosphate starvation. This is attenuated upon deletion of an imperfect palindrome likely representing a binding site for the response regulator RegX3, a component of the two-component system SenX3-RegX3 that responds to phosphate starvation. Binding of phosphorylated RegX3 to this site was confirmed by electrophoretic mobility shift assay. The activity of the ppk1 promoter was abrogated upon deletion of a putative SigE binding site. Pull-down of SigE from M. tuberculosis lysates of phosphate-starved cells with a biotinylated DNA harbouring the SigE binding site confirmed the likely binding of SigE to the ppk1 promoter. In vitro transcription corroborated the involvement of SigE in ppk1 transcription. Finally, the overexpression of RseA (anti-SigE) attenuated ppk1 expression under phosphate starvation, supporting the role of SigE in ppk1 transcription. The regulatory elements identified in ppk1 transcription in this study, combined with our earlier observation that PPK1 is itself capable of regulating sigE expression via the MprAB TCS, suggest the presence of multiple positive-feedback loops in this signalling circuit. In combination with the sequestering effect of RseA, we hypothesize that this architecture could be linked to bistability in the system that, in turn, could be a key element of persistence in M. tuberculosis.

Application of the 16-kDa buckwheat 2 S storage albumin protein for diagnosis of clinical reactivity

The 16-kDa protein of buckwheat (BW) has been implicated as a major allergen in BW allergy. To characterize the 16-kDa allergen and evaluate its clinical significance as an indicator of BW allergy. Complementary DNA from the 16-kDa allergen was cloned and expressed in Escherichia coli. Allergenicity was confirmed with IgE immunoblotting or with an enzyme-linked immunosorbent assay. The clinical utility of the recombinant protein (r16 kDa) for diagnosis of BW reactivity was evaluated in 18 BW-allergic and in 20 asymptomatic BW-sensitized subjects. The 16-kDa allergen, composed of 127 amino acids, has 50% homology to the reported 8-kDa BW allergen, which belongs to the 2 S storage albumin. The r16-kDa protein can inhibit specific IgE (sIgE) antibody binding to the native BW 16-kDa allergen but minimally inhibited sIgE binding to crude BW extract. Approximately 77.8% of patients with the BW allergy produced sIgE antibodies to the r16-kDa protein, compared with a complete lack of reactivity in the 20 asymptomatic BW-sensitized subjects. The areas of the receiver operating characteristic curves for the skin prick test (mean, 0.93; 95% confidence interval, 0.85 to approximately 1.01; P < .001) and the rl6-kDa enzyme-linked immunosorbent assay (mean, 0.93; 95% confidence interval, 0.84 to approximately 1.01; P < .001) were higher than the area of the BW IgE measurement curve determined by ImmunoCAP (a system for assaying serum IgE) (mean, 0.80; 95% confidence interval, 0.66 to approximately 0.94; P = .002). The 16-kDa allergen belongs to the 2 S storage albumin. Measurement of rl6-kDa sIgE was more discriminating than measurement of ImmunoCAP sIgE in whole BW extracts for the diagnosis of clinical reactivity to BW.

Characterization of Buckwheat 19-kD Allergen and Its Application for Diagnosing Clinical Reactivity

Background: The 19-kD protein of buckwheat (BW) has been suggested to be a major allergen, but its characteristics and clinical significance are poorly defined. Methods: cDNA of the 19-kD BW allergen was cloned and expressed in Escherichia coli. Allergenicity and cross-allergenicity were confirmed by inhibition immunoblotting or by ELISA inhibition. The recombinant (r19-kD) protein was assessed for clinical utility in the diagnosis of BW reactivity in 18 BW-allergic and 19 BW-asymptomatic sensitized subjects using receiver operating characteristic analysis. Results: The 19-kD BW allergen, which is composed of 135 amino acids, has a weak homology to the vicilin-like allergens of cashew (Ana o 1), English walnut (Jug r 2) and 7 S globulin from Sesamum indicum. The r19-kD protein can inhibit sIgE binding to native 19-kD BW allergen. The maximum percentage inhibition of sIgE binding to crude BW extract was 56%. About 83.3% of the BW allergy patients had sIgE bound to r19-kD protein, compared to only 1 of the 19 BW-asymptomatic sensitized subjects. The areas under the receiver operating characteristic curves for the skin prick tests [0.925 (95% confidence interval: 0.839–1.012), p < 0.001] as well as r19-kD protein sIgE ELISAs [0.860 (95% confidence interval: 0.725–0.995), p <0.001] were higher than that of BW sIgE coated allergen particle test results [0.803 (95% confidence interval: 0.661–0.945), p = 0.002]. Conclusions: The 19-kD BW allergen may be the major allergen from BW. For the diagnosis of clinical reactivity to BW, the r19-kD protein sIgE ELISA test was more discriminative than the coated allergen particle sIgE measurement using whole BW extract.

Electron-hole liquid in strained SiGe layers of silicon heterostructures

The electron-hole liquid has been found in strained SiGe thin films of Si/Si1−x Gex/Si heterostructures. The density and binding energy of the electron-hole liquid have been determined. Owing to the presence of internal strains in the SiGe layer, the density and binding energy are significantly smaller than the respective quantities for the electron-hole liquid in a bulk single crystal of the solid solution of the same composition. The critical temperature of the transition from the exciton gas to the electron-hole liquid is estimated using the experimental data. The Mott transition (from the exciton gas to electron-hole plasma) occurs above the critical temperatures for high excitation intensities.

Polycrystalline Si1-xGex thin film deposition by rapid thermal chemical vapor deposition

Polycrystalline silicon germanium (poly-Si1−xGe x ) thin films on a-Si film have been deposited by rapid thermal chemical vapor deposition (RTCVD) with SiH4–GeH4–H2. Effect of GeH4/SiH4 and deposition temperature on stoichiometry (x), Si-Ge binding character, composition, hydrogen configuration, crystallinity, preferred orientation, grain size, and surface roughness of poly-Si1−xGe x films has been investigated. Poly-Si1−xGe x deposited on the substrate with amorphous silicon buffer layer on oxide shows better crystallinity and contains the less amount of oxygen than the one deposited directly on the oxide surface. At low temperature region, the Ge–H bond with the small amount of Si–H2 bond is dominant but all hydrogen bonds are desorbed at high temperature. All films have polycrystalline phase and the grain size and (111) orientation increased with increasing deposition temperature in which Ge content also increases at the fixed gas flow rate of GeH4 to total source gas. Poly-Si1−xGe x /Si thin film transistors (TFT) are fabricated and hydrogen during post-hydrogenation process preferentially is attached to Ge dangling bond and the TFT characteristics could be improved.

Two ResD-controlled promoters regulate ctaA expression in Bacillus subtilis.

The Bacillus subtilis ResDE two-component system plays a positive role in global regulation of genes involved in aerobic and anaerobic respiration. ctaA is one of the several genes involved in aerobic respiration that requires ResD for in vivo expression. The ctaAB-divergent promoter regulatory region has three ResD binding sites; A1, A2, and A3. The A2 site is essential for in vivo promoter activity, while binding sites A2 and A3 are required for full ctaA promoter activity. In this study, we demonstrate the role of ResD~P in the activation of the ctaA promoter using an in vitro transcription system. The results indicate that the ctaA promoter (binding sites A2 and A3) has two transcriptional start sites. Binding site A2 was sufficient for weak transcription of the upstream promoter (Pv) by Esigma(A), transcription which was enhanced approximately 1.5-fold by ResD and 5-fold by ResD~P. The downstream promoter (Ps) required both binding sites A2 and A3 and was not transcribed by Esigma(A) with or without ResD~P. RNA polymerase (RNAP) isolated from B. subtilis when cells were at the end of exponential growth (T(0)) or 3, 4, or 5 h into the stationary phase (T(3), T(4), or T( 5), respectively) was used in in vitro transcription assays. Maximal transcription from Ps required T(4) RNAP plus ResD~P. RNAP isolated from a spo0A or a sigE mutant strain was not capable of Ps transcription. Comparison of the Ps promoter sequence with the SigE binding consensus suggests that the ctaA Ps promoter may be a SigE promoter. The collective data from ResD footprinting, in vivo promoter deletion analysis, and in vitro transcription assays suggest that ctaA is transcribed during late exponential to early stationary phases of growth from the Pv promoter, which requires ResD binding site A2, Esigma(A), and ResD~P, and during later stationary phase from Ps, which requires binding sites A2 and A3, ResD~P, and Esigma(E) or a sigma factor whose transcription is dependent on SigE.