Allergenicity assessment of ß-lactoglobulin ferulic acid-glucose conjugates.

We prepared the ß-lactoglobulin (BLG)-ferulic acid (FA)-glucose (Glu) conjugates by alkaline method and Maillard reaction to assess the allergenicity. FA and Glu can form a ternary covalent conjugate with BLG, as evidenced by the shortening of SEC retention time, upward migration of SDS-PAGE protein bands, considerable decrease in free amino and sulfhydryl content, and changes in multistructure. BLG-Glu-FA conjugates weakly bound to immunoglobulin E in allergic sera was weak, reduced interleukin 4 and tumor necrosis factor α levels in RBL-2H3 cells and histamin and interleukin 6 secretion levels in KU812 cells, and inhibited the nuclear factor-κB signaling pathway. In vivo experiments showed that the conjugates regulated T-cell homeostasis in mouse splenic and mesenteric lymphocytes and attenuated splenic and duodenal immune injury. Therefore, the conjugates of BLG with FA combined with Glu altered the epitope structure and exhibited low allergenicity.

Reducing the Allergenicity of ß-Lactoglobulin by Covalent Modification with Different Contents of Epigallocatechin Gallate (EGCG): In Vitro and In Vivo Studies.

ß-Lactoglobulin (ßLG) is a major allergen in bovine milk protein. This study was designed to investigate changes in ßLG structure, digestibility, and allergenicity induced by covalent binding modification with different contents of (-)-epigallocatechin 3-gallate (EGCG). The reaction of EGCG conjugation with ßLG reached saturation at a molar ratio of 1:60 ßLG:EGCG. Conjugation with EGCG altered the ßLG structure, decreased IgE-binding capacity, and increased digestibility in a dose-dependent manner. In vivo studies showed that covalent conjugation with EGCG can reduce ßLG-induced allergic symptoms with reducing levels of IgE, histamine, and mast cell protease-1 (mMCP-1) and the percentage of sensitized mast cells. Allergenicity was reduced more effectively in saturated ßLG-EGCG conjugates compared to semisaturated conjugates. Observed changes in IFN-γ, IL-4, IL-5, IL-10, and TGF-ß levels suggested that ßLG-EGCG conjugates were able to promote Th1/Th2 immune balance. These findings further our understanding of the relationship between the degree of polyphenol conjugation and the allergenicity of food allergens.

Peptide Characterization and Functional Stability of a Partially Hydrolyzed Whey-Based Formula over Time.

Human clinical trials have shown that a specific partially hydrolyzed 100% whey-based infant formula (pHF-W) reduces AD risk in the first yeast of life. Meta-analyses with a specific pHF-W (pHF-W1) confirm a protective effect while other meta-analyses pooling different pHF-W show conflicting results. Here we investigated the molecular composition and functional properties of the specific pHF-W1 as well as the stability of its manufacturing process over time. This specific pHF-W1 was compared with other pHF-Ws. We used size exclusion chromatography to characterize the peptide molecular weight (MW), a rat basophil degranulation assay to assess the relative level of beta-lactoglobulin (BLG) allergenicity and a preclinical model of oral tolerance induction to test prevention of allergic sensitization. To analyze the exact peptide sequences before and after an HLA binding assay, a mass cytometry approach was used. Peptide size allergenicity and oral tolerance induction were conserved across pHF-W1 batches of production and time. The median MW of the 37 samples of pHF-W1 tested was 800 ± 400 Da. Further oral tolerance induction was observed using 10 different batches of the pHF-W1 with a mean reduction of BLG-specific IgE levels of 0.76 log (95% CI = -0.95; -0.57). When comparing pHF-W1 with three other formulas (pHF-W2 3 and 4), peptide size was not necessarily associated with allergenicity reduction in vitro nor oral tolerance induction in vivo as measured by specific IgE level (p < 0.05 for pHF-W1 and 2 and p = 0.271 and p = 0.189 for pHF-W3 and 4 respectively). Peptide composition showed a limited overlap between the formulas tested ranging from 11.7% to 24.2%. Furthermore nine regions in the BLG sequence were identified as binding HLA-DR. In conclusion, not all pHF-Ws tested have the same peptide size distribution decreased allergenicity and ability to induce oral tolerance. Specific peptides are released during the different processes used by different infant formula producers.

Enhanced Uptake of Processed Bovine ß-Lactoglobulin by Antigen Presenting Cells: Identification of Receptors and Implications for Allergenicity.

SCOPE: ß-lactoglobulin (BLG) is a major cow milk allergen encountered by the immune system of infants fed with milk-based formulas. To determine the effect of processing on immunogenicity of BLG, this article characterized how heated and glycated BLG are recognized and internalized by APCs. Also, the effect of heat-induced structural changes as well as gastrointestinal digestion on immunogenicity of BLG is evaluated. METHODS AND RESULTS: The binding and uptake of BLG from raw cow milk and heated either alone (BLG-H) or with lactose/glucose (BLG-Lac and BLG-Glu) to the receptors present on APCs are analyzed by ELISA and cell-binding assays. Heated and glycated BLG is internalized via galectin-3 (Gal-3)and scavenger receptors (CD36 and SR-AI) while binding to the receptor for advanced glycation end products (R AGE) does not cause internalization. Receptor affinity of BLG is dependent on increased hydrophobicity, ß-sheet exposure and aggregation. Digested glycated BLG maintained binding to sRAGE and Gal-3 but not to CD36 and SR-AI, and is detected on the surface of APCs. This suggests a mechanism via which digested glycated BLG may trigger innate (via RAGE) and adaptive immunity (via Gal-3). CONCLUSIONS: This study defines structural characteristics of heated and glycated BLG determining its interaction with APCs via specific receptors thus revealing enhanced immunogenicity of glycated versus heated BLG.

Bovine ß-lactoglobulin-induced passive systemic anaphylaxis model using humanized NOG hIL-3/hGM-CSF transgenic mice.

Food allergy is a common disease caused by intake of allergen-containing foods, such as milk, eggs, peanuts and wheat. Systemic anaphylaxis is a severe hypersensitive allergic reaction resulting from degranulation of mast cells or basophils after cross-linking of surface high-affinity IgE receptors (Fcε-RI) with allergen-specific IgE and allergens. In this study, we developed a novel human mast cell/basophil-engrafted mouse model that recapitulates systemic anaphylaxis triggered by ß-lactoglobulin (BLG), a major allergen found in cow's milk. Human CD34+ hematopoietic stem cells were transferred into NOG (non-Tg) or NOG hIL-3/hGM-CSF transgenic (Tg) mice. After 14-16 weeks, bovine BLG-specific human IgE was intravenously injected into humanized mice, followed by intravenous or oral bovine BLG exposure 1 day later. Body temperature in Tg, but not in non-Tg, mice gradually decreased within 10 min, and 80% of Tg mice died within 1 h by intravenous BLG exposure. Serum histamine levels and anaphylaxis scores in Tg mice were markedly increased compared to non-Tg mice. Furthermore, these allergic symptoms were significantly inhibited by epinephrine treatment of the Tg mice. Therefore, the current NOG hIL-3/hGM-CSF Tg mouse model may be useful for development of novel anaphylaxis drugs for treatment of food allergies and for safety assessment of low-allergenicity extensively hydrolyzed cow's milk whey protein-based infant formulas.

Analysis of altered miRNA profiling in the colon of a mouse model with ß-lactoglobulin allergy.

OBJECTIVES: The differences in the expression profiles of colonic miRNAs between ß-lactoglobulin (ß-Lg) allergic mice and normal mice were analyzed to investigate the important role of the miRNA regulation mechanism in the pathogenesis of cow's milk allergy. METHODS: The present study performed Illumina sequencing to characterize the miRNA profile changes in mouse colon responding to ß-Lg challenge. Target genes were predicted by TargetScan 50 and miRanda 3.3a algorithms and assessed by GO and KEGG analysis. The expression levels of selected miRNAs and cytokine production were verified by cell transfection and quantitative RT-PCR. RESULTS: A total of 15 miRNAs were diversely expressed between the colon of the normal and ß-Lg-sensitized mice (P < 0.05, fold change of >1.50 or <0.67), including six up-regulated miRNAs and nine down-regulated miRNAs, among which seven miRNAs were validated using qRT-PCR. GO enrichment and KEGG pathway analyses further revealed that biological process, protein binding, cytoplasm and the pathways of cancer were significantly enriched, which were closely connected to the allergic inflammation development. Additionally, six key functional interaction pairs in ß-Lg allergy were identified in miRNA prediction algorithms and verified using qRT-PCR. CONCLUSIONS: We can conclude that our results suggested that the miRNAs regulation network participated in the pathogenesis of cow's milk allergy.

Glycation of ß-lactoglobulin combined by sonication pretreatment reduce its allergenic potential.

ß-lactoglobulin (ß-Lg) was treated through different ultrasonic power and subsequently glycated with galactose to investigate its structural changes and immunological properties, and then evaluated by high-resolution mass spectrometry, enzyme-linked immunosorbent assay and basophil histamine release test. Ultrasonication combined with glycation (UCG) modification significantly reduced the IgE/IgG-binding capacity, and the release of ß-hexosaminidase, histamine and interleukin-6, accompanied with changes in the secondary and tertiary structures. The decrease in the allergenicity of ß-Lg depended not only on the glycation of K47, 60, 83, 91 and 135 within the linear epitopes, but also on the denaturation of conformational epitopes, which was supported by the glycation-induced alterations of the secondary and tertiary structures. This study confirmed that UCG modification is a promising method for decreasing the allergenic potential of allergic proteins, which is likely to develop a practical technology to produce hypo-allergenic milk.

Influence of ultrasonic pretreatment on the structure, antioxidant and IgG/IgE binding activity of ß-lactoglobulin during digestion in vitro.

The influence of ultrasonic pretreatment on the structure, antioxidant activity, and IgG/IgE binding activity of ß-lactoglobulin (ß-Lg) during digestion in vitro were studied by spectroscopy, chromatography and ELISA. After ß-Lg was treated by ultrasonic prior to digestion in vitro, the treated ß-Lg showed high intrinsic fluorescence emission, more of the hydrolytic products and high antioxidant activity, which depend on the proteolysis promoted by ultrasonic, and produced many small-molecule antioxidant peptides. Native ß-Lg was resistant to gastric digestion and retained its allergenicity. However, the allergenicity of ultrasonicated ß-Lg after gastric digestion was increased due to ultrasonic promotes the production of peptides with intact structure and immunogenicity. Subsequent gastrointestinal digestion, ultrasonicated ß-Lg has a diametrically opposite results because the increase of small peptides with the decreasing of immunogenicity. Therefore, the structural changes of ß-Lg by ultrasonic and gastrointestinal digestion were responsible for improving the antioxidant activity and reducing the IgG/IgE binding activity.

Immune Effects of the Nitrated Food Allergen Beta-Lactoglobulin in an Experimental Food Allergy Model.

Food proteins may get nitrated by various exogenous or endogenous mechanisms. As individuals might get recurrently exposed to nitrated proteins via daily diet, we aimed to investigate the effect of repeatedly ingested nitrated food proteins on the subsequent immune response in non-allergic and allergic mice using the milk allergen beta-lactoglobulin (BLG) as model food protein in a mouse model. Evaluating the presence of nitrated proteins in food, we could detect 3-nitrotyrosine (3-NT) in extracts of different foods and in stomach content extracts of non-allergic mice under physiological conditions. Chemically nitrated BLG (BLGn) exhibited enhanced susceptibility to degradation in simulated gastric fluid experiments compared to untreated BLG (BLGu). Gavage of BLGn to non-allergic animals increased interferon-γ and interleukin-10 release of stimulated spleen cells and led to the formation of BLG-specific serum IgA. Allergic mice receiving three oral gavages of BLGn had higher levels of mouse mast cell protease-1 (mMCP-1) compared to allergic mice receiving BLGu. Regardless of the preceding immune status, non-allergic or allergic, repeatedly ingested nitrated food proteins seem to considerably influence the subsequent immune response.

Hydrophobicity and aggregation, but not glycation, are key determinants for uptake of thermally processed ß-lactoglobulin by THP-1 macrophages.

The aim of this study is to investigate the immunological relevance of modifications of food protein structure due to thermal processing. We investigated the uptake of ß-lactoglobulin, treated with 3 different processing methods, by THP-1 macrophages: wet heating (60 °C in solution) and high- or low-temperature (130 °C or 50 °C, respectively) dry heating, combined with either of 8 types of saccharides or without saccharide. The processing method that was applied significantly affected the uptake of BLG by THP-1 macrophages, while the type of saccharide only had an influence in high-temperature dry heated samples. A set of physicochemical parameters of processed samples was determined, to determine the samples' molecular weight, hydrophobicity, amyloid-like structure, surface charge and secondary structure. Analysis of protein structure alterations indicated the uptake to be linked to the wet heating processing method and percentage of α-helix structure, amyloid-like structures, polymers, and hydrophobicity. We hypothesize that both amyloid-like structures and molecular weight were related to the increased hydrophobicity and therefore postulate that the exposure of hydrophobic regions is the leading physicochemical characteristic for the observed uptake of wet heated BLG samples by THP-1 macrophages. This work demonstrates how differential thermal processing of foods, through protein modification, can have an impact on its interaction with the immune system.

Taurine administration prevents the intestine from the damage induced by beta-lactoglobulin sensitization in a murine model of food allergy.

BACKGROUND: Allergy to cow's milk proteins has often been associated with dysfunction of the intestinal mucosa caused by chronic inflammation in infants. This study evaluated the protective effect of taurine on intestinal damage induced by beta-lactoglobulin (ß-Lg) in Balb/c mice used as an animal model of allergy to cow's milk proteins. METHODS: Balb/c mice were treated with taurine administered orally by gavage (3mmol/kg/day) or intraperitoneally (100mg/kg/day) for two weeks, then sensitized intraperitoneally with ß-Lg. The electrophysiological parameters: active ion transport of chloride (Short-circuit current: Isc) and the passive ion permeability (Conductance: G) were measured ex vivo in Ussing chamber by intestine challenge with ß-Lg. Histological study was used to assess gut inflammation. Serum levels of TNF-α and IL-6 were measured. Serum IgG and IgE anti-ß-Lg were determined by ELISA. RESULTS: Compared with sensitized mice, ß-Lg challenge of intestinal epithelium of taurine-pre-treated mice in Ussing chamber did not influence the intensity of Isc, nor produce any changes in the G, reflecting a reduction in the secretory response and epithelial permeability. Histological and morphometric analysis showed that taurine reduced the intestinal damage and limited intestine retraction caused by ß-Lg sensitization. No statistically significant difference in the serum levels of TNF-α or IL-6 was found after oral or intraperitoneal administration of taurine. Treatment with taurine significantly decreased the IgG (p<0.001) and IgE anti ß-Lg levels (p<0.05). CONCLUSIONS: These results have for the first time provided evidence that pre-treatment with taurine appears to prevent intestinal damage induced by ß-Lg.

Reducing the allergenic capacity of ß-lactoglobulin by covalent conjugation with dietary polyphenols.

To help produce hypoallergenic food, this study investigated reducing the allergenicity and improving the functional properties of bovine ß-lactoglobulin (ßLG) by covalent conjugation with (-)-epigallo-catechin 3-gallate (EGCG) and chlorogenic acid (CA). The covalent bond between the polyphenols and the amino acid side-chains in ßLG was confirmed by MALDI-TOF-MS and SDS-PAGE. Structural analysis by fluorescence spectroscopy, circular dichroism (CD) and Fourier transform infrared (FTIR) indicated that the covalent conjugate of EGCG and CA led to the changed protein structure of ßLG. Western blot analysis and enzyme-linked immunosorbent assay indicated that conjugation of ßLG with these polyphenols was effective in reducing the IgE-binding capacity of ßLG. The conjugates maintained the retinol-binding activity without denaturation the protein and enhanced the thermal stability with high antioxidant activity. The study provides an innovative approach to producing hypoallergenic food.

Human Allergen-Specific IgG Subclass Antibodies Measured Using ImmunoCAP Technology.

BACKGROUND: Knowledge of human IgG subclass antibody responses to various allergens has been hampered by a lack of reliable standardized assays. The aim here was to develop quantitative immunoassays for human IgG1, IgG2, and IgG3 antibodies using ImmunoCAP® technology and to evaluate their application. METHODS: Enzyme conjugates with isotype-specific monoclonal antibodies and calibrators composed of purified myeloma paraproteins were developed for each assay and used together with other standardized assay reagents for the Phadia® 100 instrument. The calibrators were adjusted to the international reference preparation IRP 67/86. The assays were characterized and used together with other standard ImmunoCAP assays to measure antibodies to various allergens in preliminary studies. RESULTS: The new assays had limits of quantitation of 1.0 (IgG1), 4.6 (IgG2), and 0.04 mgA/L (IgG3), and coefficients of variation of <20%. Only some minor cross-reactivity with IgG2 was observed for the specific IgG1 assay. The specific IgG2 assay showed a bias for the allotype G2m(23) and compensation factors were used to adjust the measured concentrations accordingly. Preliminary studies indicated a strong and stable IgG4 antibody response to ß-lactoglobulin in healthy individuals, a high IgG1 and even higher IgG2 antibody response to house dust mite in sensitized and nonsensitized subjects, and a mixed IgG subclass response to venom allergens in allergic patients with increasing IgG4 antibody levels during venom immunotherapy. CONCLUSIONS: The new research assays are valuable tools for immunological studies, enabling the characterization of antibody profiles using a standardized approach, and facilitating data interpretation and the comparison of results across studies.

T-cell epitope-containing hypoallergenic ß-lactoglobulin for oral immunotherapy in milk allergy.

BACKGROUND: Optimally hydrolyzed ß-Lactoglobulin (ßLg) is a promising milk oral immunotherapy (OIT) candidate with respect to showing reduced B-cell reactivity but retaining the T-cell epitope. To demonstrate that an edible hypoallergenic ßLg hydrolysate containing the T-cell epitope is suitable for OIT. We tested how chymotrypsin affected the retention of the T-cell epitope of ßLg when preparing ßLg hydrolysates using food-grade trypsin. METHODS: We investigated the effect of chymotrypsin activity on the formation of the T-cell epitope-containing peptide of ßLg (ßLg102-124 ) and prepared an edible ßLg hydrolysate containing ßLg102-124 using screened food-grade trypsins. B-cell reactivity was determined using immunoassays in which ELISA was performed with anti-ßLg rabbit IgG and Western blotting was performed with a milk-specific IgE antiserum. RESULTS: In ßLg hydrolysis performed by varying the activity of trypsin and chymotrypsin, chymotrypsin activity inhibited the formation of ßLg102-124 with an increase in hydrolysis time in a dose-dependent manner. ßLg102-124 was generated by two of five food-grade trypsins used at a ratio of 1:50 (w/w, enzyme/substrate) for 20 h at 40°C. The edible ßLg hydrolysate retained ßLg102-124 and showed a reduction in molecular weight distribution and antigenicity against IgG and IgE. CONCLUSIONS: Chymotrypsin activity inhibited the formation of ßLg102-124 in the trypsin hydrolysate of ßLg. This ßLg trypsin hydrolysate is a novel candidate for peptide-based OIT in cow's milk allergy for safely inducing desensitization.

Thymic Stromal Lymphopoietin Induction Is Mediated by the Major Whey Proteins α-Lactalbumin and ß-Lactoglobulin through the NF-κB Pathway in Immune Cells.

α-Lactalbumin and ß-lactoglobulin are two major whey proteins that specifically bind immunoglobulin E and are suspected as major allergens causing cow's milk allergy (CMA). Recent studies have shown that thymic stromal lymphopoietin is a critical factor linking at the interface of the body and environment to the T-helper 2 response. However, it is not known whether thymic stromal lymphopoietin expression is changed by α-lactalbumin and ß-lactoglobulin in immune cells. Using RT-PCR and ELISA, the present study was conducted to examine if intravenous injection of α-lactalbumin and ß-lactoglobulin increased pro-inflammatory cytokines, T-helper 2 cytokines, and thymic stromal lymphopoietin expression in several immune cells, including macrophages, mast cells, and keratinocytes. Results showed that α-lactalbumin and ß-lactoglobulin induced thymic stromal lymphopoietin, interleukin-6, and tumor necrosis factor-α expression. It was concluded that the allergenicity of α-lactalbumin and ß-lactoglobulin may be attributed to thymic stromal lymphopoietin induction, T-helper 2 cytokines, and pro-inflammatory cytokines.

Development of ß-lactoglobulin-specific chimeric human IgEκ monoclonal antibodies for in vitro safety assessment of whey hydrolysates.

BACKGROUND: Cow's milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow's milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays. OBJECTIVE: An oligoclonal pool of chimeric human (chu)IgE antibodies against bovine ß-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays. METHODS: Mice were immunized with bovine ß-lactoglobulin, and subsequently the variable domains of dissimilar anti-ß-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains. RESULTS: After sensitization with this pool of anti-ß-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa ß-lactoglobulin, and 5-10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow's milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-ß-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on ß-lactoglobulin, which were also recognized by serum IgEs from cow's milk allergic children. CONCLUSION: Usage of our 'unlimited' source and well-defined pool of ß-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow's milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula.

[The effect of natural whey proteins on mechanisms of blood pressure regulation]. / Wplyw naturalnych bialek serwatki na mechanizmy regulacji cisnienia tetniczego krwi.

Whey is a rich natural source of peptides and amino acids. It has been reported in numerous studies that biological active peptides isolated from cow's milk whey may affect blood pressure regulation. Studies on animals and humans have shown that α-lactalbumin and ß-lactoglobulin obtained from enzymatically hydrolysed whey inhibit angiotensin converting enzyme (ACE), while lactorphins lower blood pressure by normalizing endothelial function or by opioid receptors dependent mechanism. Whey proteins or their bioactive fragments decrease total cholesterol, LDL fraction and triglycerides, thus reducing the risk factors of cardiovascular diseases. The aim of this review is to discuss the effects of whey proteins on the mechanisms of blood pressure regulation.

In search of a tolerance-induction strategy for cow's milk allergies: significant reduction of beta-lactoglobulin allergenicity via transglutaminase/cysteine polymerization.

OBJECTIVE: To explore the use of ß-lactoglobulin polymerized using microbial transglutaminase and heating to identify whether protein polymerization could reduce in vivo allergenicity and maintain in vitro and ex vivo immunoreactivity for use in tolerance-induction protocols. METHODS: Based on previous protocols applied in mice and children, we performed in vivo challenges (using a skin prick test) with native and polymerized ß-lactoglobulin in adult patients with an IgE-mediated allergy to plactoglobulin. In vitro humoral immunoreactivity was analyzed using immunoblotting. Cell-mediated immunoreactivity was analyzed using ex vivo challenges with native and polymerized ß-lactoglobulin and monitored by leukocyte adherence inhibition tests. RESULTS: The skin tests demonstrated that there was a significant reduction in immediate cutaneous reactivity after polymerization. Polymerization did not decrease the immunoblotting detection of s-IgE specific to ß-lactoglobulin. Cell-mediated immunoreactivity, as assessed by ex vivo challenges and leukocyte adherence inhibition tests, did not exhibit significant differences between leukocytes challenged with native versus polymerized ß-lactoglobulin. CONCLUSIONS: The polymerization of ß-lactoglobulin decreased in vivo allergenicity and did not decrease in vitro humoral or ex vivo cell-mediated immunoreactivity. Therefore, we conclude that inducing polymerization using transglutaminase represents a promising technique to produce suitable molecules for the purpose of designing oral/ sublingual tolerance induction protocols for the treatment of allergies.