Ramon Bencharitiwong

Mechanisms underlying differential food allergy response to heated egg

BACKGROUND Egg white proteins are usually subjected to heating, making them edible for the majority of children with egg allergy. OBJECTIVE We sought to investigate the underlying mechanisms responsible for the reduced allergenicity displayed by heat-treated egg white allergens. METHODS C3H/HeJ mice were orally sensitized with ovalbumin (OVA) or ovomucoid and challenged with native or heated proteins to evaluate their allergenicity. Immunoreactivity was assessed by immunoblotting using sera from children with egg allergy. In vitro gastrointestinal digestion of native and heated OVA and ovomucoid was studied by SDS-PAGE and liquid chromatography. Intestinal uptake of intact native and heated OVA and ovomucoid by human intestinal epithelial (Caco-2) cells was investigated. Rat basophil leukemia cells passively sensitized with mouse serum and human basophils passively sensitized with serum from children with egg allergy were used to assess the effector cell activation by heated, digested, and transported OVA and ovomucoid. RESULTS Heated OVA and ovomucoid did not induce symptoms of anaphylaxis in sensitized mice when administered orally. Heating did not completely destroy IgE-binding capacity of OVA or ovomucoid but enhanced in vitro digestibility of OVA. Digestion of both OVA and ovomucoid diminished mediator release in rat basophil leukemia assay and basophil activation. Heating of allergens prevented transport across human intestinal epithelial cells in a form capable of triggering basophil activation or T-cell activation. CONCLUSION Heat treatment reduces allergenicity of OVA and ovomucoid. This is partially a result of the enhanced gastrointestinal digestibility of heated OVA and the inability of heated OVA or ovomucoid to be absorbed in a form capable of triggering basophils.

Effect of heat treatment on milk and egg proteins allergenicity

Heating destroys many conformational epitopes and reduces allergenicity of some foods. IgE‐epitope binding has been shown to be different among patients who outgrew their cows milk or hens egg allergy and those who did not. A significant proportion of milk‐ or egg‐allergic children are tolerant to these foods in their baked forms. We sought to explore the effects of heating on milk and egg proteins and to evaluate for differences in immunolabeling among children with regard to reactivity to heated milk or egg.

Egg-white-specific IgA and IgA2 antibodies in egg-allergic children: is there a role in tolerance induction?

Decreased serum food‐specific IgA antibodies have been associated with allergic disease in cross‐sectional, case–control studies. The purpose of this study was to prospectively compare egg‐white‐(EW)‐specific IgA and IgA2 levels between egg‐allergic children and children tolerating egg.

The role of casein-specific IgA and TGF-β in children with food protein-induced enterocolitis syndrome to milk

Food protein‐induced enterocolitis syndrome (FPIES) is a gastrointestinal hypersensitivity disorder with a poorly understood pathophysiology and no biomarkers to aid in diagnosis.

Humoral and cellular responses to casein in patients with food protein–induced enterocolitis to cow's milk

Background: Food protein–induced enterocolitis syndrome (FPIES) is a non–IgE‐mediated food allergy manifesting within 1 to 4 hours of food ingestion with repetitive emesis and lethargy. Objective: We sought to characterize immune responses to casein in children with FPIES caused by cows milk (CM). Methods: Total IgE and IgM, CM‐specific IgG, and casein‐specific IgE, IgG, IgG4, and IgM levels, as well as immunoglobulin free light chains, were measured in both patients with active and those with resolved CM‐FPIES. Proliferating casein/T‐effector cell counts were measured in children with CM‐FPIES, children with IgE‐mediated CM allergy, and those tolerating CM. Cytokine concentrations in the supernatants were quantified. Serum cytokine and tryptase levels were measured before and after a positive oral food challenge (OFC) result and compared with levels in those with a negative OFC result. Results: We found low levels of CM and casein‐specific IgG and casein‐specific IgG4 in patients with CM‐FPIES versus those tolerating CM (P < .05). Although we found both a high CD4+ T cell–proliferative response and TH2 cytokines production after casein stimulation in children with CM‐FPIES, results were similar to those in control subjects. Significantly lower secretion of IL‐10 and higher secretion of IL‐9 by casein‐stimulated T cells were found in patients with CM‐FPIES versus those with IgE‐mediated CM allergy. Lower baseline serum levels of IL‐10 and higher tryptase levels were found in active CM‐FPIES versus resolved CM‐FPIES. We found a significant increase in serum IL‐10 and IL‐8 levels after a positive OFC result. Conclusions: We confirm the paucity of humoral response in patients with CM‐FPIES. IL‐10 might play a key role in acquisition of tolerance in patients with CM‐FPIES. Increased serum IL‐8 levels in patients with active FPIES suggest neutrophil involvement. Elevated baseline serum tryptase levels in patients with active FPIES suggest low‐grade intestinal mast cell activation or increased mast cell load. GRAPHICAL ABSTRACT Figure. No caption available.

Mediator release assay for assessment of biological potency of German cockroach allergen extracts

BACKGROUND Cockroach is an important allergen in inner-city asthma. The diagnosis and treatment of cockroach allergy has been impeded by the lack of standardized cockroach extracts. OBJECTIVE We investigated the utility of a mediator release assay based on rat basophil leukemia (RBL) cells for comparing the potency of German cockroach extracts. METHODS RBL cells (line 2H3) transfected with human FcepsilonRI were passively sensitized with sera from subjects with cockroach allergy and stimulated with serial dilutions of 3 commercial cockroach extracts (1:10 weight/volume). In addition, the in-house prepared extract was tested in separate experiments with pooled sera that produced optimal performance in the RBL assay. N-hexosaminidase release (NHR) was used as a marker of RBL cell degranulation and was examined in relation to the intradermal skin test (ID(50)EAL) and serum cockroach-specific and total IgE levels. RESULTS The median cockroach-specific IgE concentration in 60 subjects was 0.72 kU(A)/L (interquartile range, 0.35-2.97 kU(A)/L); 19 sera (responders) produced a minimum 10% NHR to more than 1 extract. Responders had higher median cockroach-specific IgE (7.4 vs 1.0 kU(A)/L) and total IgE (429 vs 300 kU/L) levels than nonresponders. Ranking of extract potency was consistent between the mediator release assay and the ID(50)EAL. For the in-house prepared cockroach extract, the dose-response curves were shifted according to the concentration of the extract. NHR was reproducible between different experiments by using pooled sera. CONCLUSION The mediator release assay measures biologic potency and correlates with the ID(50)EAL. It should be further evaluated to determine whether it could be used to replace intradermal skin test titration for assessing the potency of cockroach extract.

Heating does not decrease immunogenicity of goat's and ewe's milk

URE 1. A, SDS-PAGE (A-1) and Western blot analysis (A-2) with u , and EM allergies and milk-tolerant negative control showed high an d (A-3). ALA, a-Lactalbumin; BLG, b-lactoglobulin; CC, cow’s che ecular weight; SA, serum albumin. extensive amino acid sequence identity of milk proteins (85%100%; see Tables E1 and E2 in this article’s Online Repository at www.jaci-inpractice.org) and similar proteome profiles that imply similarity of protein functions. However, selective GM and ewe’s milk (EM) allergy without CMA (GEMA) have been reported, including anaphylaxis to cheeses from GM and EM in CM-tolerant children. GEMA implies existence of unique GM and EM-allergenic epitopes that are different from CMallergenic epitopes. Among children with CMA, >70% can tolerate extensively heated CM in baked products. We sought to determine the effects of heating on the immunogenicity of GM and EM proteins in children with GEMA. We recruited 17 subjects with milk allergy from Spain, Greece, and the United States; 13 were males; median age was 7.2 years, and 25% to 75% interquartile range (IQR) was 4.0 to 9.9 years. The study was approved by the Institutional Review Boards at The Mount Sinai School of Medicine, Pediatric Hospital “P & A Kiriakou,” and Hospital Infantil Universitario Nino Jesus; informed consent was obtained from the subjects. Eight subjects with CM allergy tolerated extensively heated oral milk challenge but reacted to unheated milk challenge. Nine subjects had history of convincing, immediate allergic symptoms on ingestion of GM or EM or both (Table I). The 8

Presence of functional, autoreactive human milk-specific IgE in infants with cow's milk allergy

Occasionally, exclusively breastfed infants with cows milk allergy (CMA) remain symptomatic despite strict maternal milk avoidance.

Effect of chemical modifications on allergenic potency of peanut proteins.

BACKGROUND Modification of native peanut extracts could reduce adverse effects of peanut immunotherapy. OBJECTIVE We sought to compare native and chemically modified crude peanut extract (CPE) and major peanut allergens Ara h 2 and Ara h 6 in a mediator-release assay based on the rat basophilic leukemia (RBL) cell line transfected with human Fcε receptor. METHODS Native Ara h 2/6 was reduced and alkylated (RA), with or without additional glutaraldehyde treatment (RAGA). CPE was reduced and alkylated. Sera of subjects with peanut allergy (16 males; median age 7 years) were used for overnight RBL-passive sensitization. Cells were stimulated with 0.1 pg/mL to 10 μg/mL of peanut. β-N-acetylhexosaminidase release (NHR) was used as a marker of RBL degranulation, expressed as a percentage of total degranulation caused by Triton X. RESULTS Median peanut-specific immunoglobulin E was 233 kUA/L. Nineteen subjects were responders, NHR ≥ 10% in the mediator release assay. Responders had reduced NHR by RA and RAGA compared with the native Ara h 2/6. Modification resulted in a later onset of activation by 10- to 100-fold in concentration and a lowering of the maximum release. Modified RA-Ara h 2/6 and RAGA-Ara h 2/6 caused significantly lower maximum mediator release than native Ara h 2/6, at protein concentrations 0.1, 1, and 10 ng/mL (p < 0.001, < 0.001, and < 0.001, respectively, for RA; and < 0.001, 0.026, and 0.041, respectively, for RAGA). RA-CPE caused significantly lower maximum NHR than native CPE, at protein concentration 1 ng/mL (p < 0.001) and 10 ng/mL (p < 0.002). Responders had high rAra h 2 immunoglobulin E (mean, 61.1 kUA/L; p < 0.001) and higher NHR in mediator release assay to native Ara h 2/6 than CPE, which indicates that Ara h 2/6 were the most relevant peanut allergens in these responders. CONCLUSIONS Chemical modification of purified native Ara h 2 and Ara h 6 reduced mediator release in an in vitro assay ∼100-fold, which indicates decreased allergenicity for further development of the alternative candidate for safe peanut immunotherapy.

In vitro assessment of the allergenicity of a novel influenza vaccine produced in dog kidney cells in individuals with dog allergy

BACKGROUND An inactivated influenza vaccine produced in canine kidney cells (MDCK 33016-PF) contains no egg proteins and may be used to immunize egg-allergic patients. Although no major dog allergens were identified in MDCK 33016-PF cells, minor dog allergens might be present and cause reactions in dog-allergic individuals. OBJECTIVE To evaluate the allergenicity of the inactivated influenza vaccine produced in cell culture in a mediator release assay. METHODS Rat basophil leukemia (RBL) cells transfected with human IgE receptor-1 were sensitized with sera from dog-allergic adults with positive skin prick test reactions to dog extract and detectable dog dander IgE and were stimulated with serial dilutions of vaccine and dog dander extract. N-hexosaminidase release (NHR) was used as a marker of RBL cell degranulation. Western blots were performed, and UniCAP was used to measure dog-specific IgE antibody levels. RESULTS The median (interquartile range) level of dog dander IgE was 8.31 kU(A)/L (1.895-14.5 kU(A)/L) and of dog epithelium IgE was 3.19 kU(A)/L (0.835-6.27 kU(A)L). Median (range) maximum NHR (at the first 10-fold dilution) was 0% (0%-1.4%) to vaccine and 10.2% (0%-35.9%) to dog dander (P < .001). In an egg-allergic control subject, the maximum NHR to a vaccine cultured in chick embryo and containing egg protein was 10.2%. IgE antibodies in pooled sera did not bind to vaccine on immunoblots but produced strong binding to dog dander and epithelium extracts. Serum from an egg-allergic control subject strongly bound embryonated egg-derived vaccine. CONCLUSION An influenza vaccine produced in continuous canine kidney cells did not trigger degranulation in RBL cells passively sensitized with human anti-dog IgE.