Release of soluble protein from peanut (Arachis hypogaea, Leguminosae) and its adsorption by activated charcoal.

Peanut (Arachis hypogaea, Leguminosae) allergy is a major cause of food-induced anaphylaxis. The potential use of activated charcoal (AC) to adsorb and reduce the bioavailability of peanut protein allergens for use in the moderation of hypersensitivity reactions was investigated. The rate and extent of protein release from peanut and the adsorption of the solubilized protein by AC was determined under physiological pH values and confirmed in vivo using a porcine animal model system. Peanut proteins were adsorbed with equal efficiency at pH 2 and 7 and are completely removed from solution by an AC/protein ratio of approximately 80:1. This suggests that AC can bind protein under gastric (pH 2) or intestinal (pH 7) conditions. The rapid adsorption of soluble peanut allergens and the continuous binding of allergens released from peanut particulate material suggest the potential efficacy of using AC for gastric decontamination and possible elimination of a biphasic allergic reaction.

Adsorption of peanut (Arachis hypogaea, Leguminosae) proteins by activated charcoal.

The binding of peanut protein allergens to activated charcoal (AC), used medically for gastric decontamination following the ingestion of toxic substances, was investigated for potential clinical application. Crude peanut extract (CPE) or purified peanut protein allergens Ara h 1 and 2 were co-incubated with AC under a variety of conditions followed by centrifugation to remove the AC and adsorbed protein. The resulting supernatant solution was analyzed for unadsorbed protein by gel electrophoresis and quantitative protein assay. The extent of protein adsorption by a known amount of AC was determined. Protein binding to AC was rapid and irreversible. The extent of adsorption was unaffected by pH, but was optimal near physiological salt concentrations. Denatured proteins, or those of larger molecular weight, required more AC than smaller or native proteins. The extent of protein binding increased with temperature, supporting the concept that protein molecules diffuse into vacant pores of appropriate size on the charcoal surface.

Diet regulates the development of gut-associated lymphoid tissue in neonatal piglets.

BACKGROUND: Controversy exists concerning diet-induced changes to gut epithelia and immunocytes that occur during weaning. Furthermore, studies on dietary effects on the development of the neonatal immune system, especially gut-associated lymphoid tissue (GALT), are lacking. OBJECTIVE: The purpose of this study was to investigate growth and development, intestinal morphology, and GALT immune maturation in sow-reared littermates in comparison with early-weaned piglets fed a casein-based liquid diet. METHOD: Piglets were breast fed by the sow or were weaned at 48 h to a casein-based diet (formula) that provided the amount of nutrient requirements recommended by the National Research Council. RESULTS: Gross physical appearance and visual inspection of the gastrointestinal tract and other organs at necropsy revealed normal organogenesis in both cohorts. On postnatal day 21, body weight, liver and kidney weight relative to body weight, small intestine length, and weight-to-length ratio were greater in formula-fed piglets as compared with sow-reared piglets (p<0.05). The CD21+ B lymphocyte component of GALT and spleen was reduced in the formula-fed piglets. This was associated with lower circulating IgG and IgM levels in the formula-fed as compared with the breast-fed neonatal piglets (p<0.001). CONCLUSIONS: Feeding a casein-based formula to newborn piglets may compromise the development of GALT and systemic immune system. Further, the neonatal piglet model may be used to identify the effects of dietary factors on the development of the neonatal immune system.

Comparison of physiological and in vitro porcine gastric fluid digestion.

BACKGROUND: In previous studies, the major peanut allergen Ara h 1 was digested in vitro using pepsin and porcine gastric fluid. The results suggested that in vivo gastric digestion of allergen protein can be modeled accurately by peptic hydrolysis in vitro. In the current investigation, studies were designed to follow the gastrointestinal (GI) digestion of peanut allergens under true physiological conditions. In vitro digestion with porcine gastric fluid was compared with actual physiological digestion of peanut allergens in the porcine digestive tract in vivo. METHODS: Analysis of physiologic digestion was performed in piglets administered a 20-gram bolus of peanut meal followed by periodic sampling and analysis of GI contents. The pH was monitored, and digesta were analyzed by SDS-PAGE and immunoblot analysis. RESULTS: Peanut meal initially neutralized stomach contents to a pH of approximately 7, which was subsequently acidified by HCl secretion within 30 min. Acidification to pH 2-4 resulted in active pepsin digestion of soluble protein in the stomach. Soluble intact protein/allergens were rapidly degraded to pepsin-resistant peptides in the stomach followed by hydrolysis of these fragments in the small intestine. Particulate material was evident in both the stomach and small intestine that could contribute to continued release of peanut allergens Ara h 1, 2 and 3. CONCLUSIONS: Porcine gastric digestion of peanut proteins resembles true physiological digestion only under optimal physiologic conditions. Soluble proteins are rapidly digested and insoluble material continues to release IgE-reactive proteins throughout the GI tract. GI digestion of food allergens can play a prominent role when assessing allergens within the context of a food matrix or meal and during the sensitization phase of IgE-mediated allergy.

Allergenic characteristics of a modified peanut allergen.

Attempts to treat peanut allergy using traditional methods of allergen desensitization are accompanied by a high risk of anaphylaxis. The aim of this study was to determine if modifications to the IgE-binding epitopes of a major peanut allergen would result in a safer immunotherapeutic agent for the treatment of peanut-allergic patients. IgE-binding epitopes on the Ara h 2 allergen were modified, and modified Ara h 2 (mAra h 2) protein was produced. Wild-type (wAra h 2) and mAra h 2 proteins were analyzed for their ability to interact with T-cells, their ability to bind IgE, and their ability to release mediators from a passively sensitized RBL-2H3 cell line. Multiple T-cell epitopes were identified on the major peanut allergen, Ara h 2. Ara h 2 amino acid regions 11-35, 86-125, and 121-155 contained the majority of peptides that interact with T-cells from most patients. The wAra h 2 and mAra h 2 proteins stimulated proliferation of T-cells from peanut-allergic patients to similar levels. In contrast, the mAra h 2 protein exhibited greatly reduced IgE-binding capacity compared to the wild-type allergen. In addition, the modified allergen released significantly lower amounts of beta-hexosaminidase, a marker for IgE-mediated RBL-2H3 degranulation, compared to the wild-type allergen.

Intralesional immunotherapy of warts with mumps, Candida, and Trichophyton skin test antigens: a single-blinded, randomized, and controlled trial.

BACKGROUND: Warts occur commonly in humans. Destructive modalities are generally the first physician-administered therapy. Other treatment options include immunotherapy. Intralesional immunotherapy using mumps, Candida, or Trichophyton skin test antigens has proved efficacy in the treatment of warts. OBJECTIVES: To determine rates of wart resolution in response to injection of antigen alone, antigen plus interferon alfa-2b, interferon alfa-2b alone, and normal saline; and to compare response according to viral type, major histocompatibility complex antigens, and peripheral blood mononuclear cell proliferation to autologous human papillomavirus antigen before and after injection. DESIGN: Randomized, single-blinded, placebo-controlled, clinical trial. SETTING: Medical school-based dermatology department. PATIENTS: Two hundred thirty-three patients clinically diagnosed as having 1 or more warts. Main Outcome Measure Clinical resolution of warts in response to intralesional immunotherapy. RESULTS: Responders were observed in all treatment arms, but were significantly more likely to have received antigen (P<.001). Resolution of distant untreated warts was observed, and was significantly more likely in subjects receiving antigen (P<.001). Interferon did not significantly enhance the response rate (P = .20) and did not differ from normal saline (P = .65). No viral type or major histocompatibility complex antigen correlated with response or lack of response (P>.99 and P = .86, respectively). A positive peripheral blood mononuclear cell proliferation assay result (2 times pretreatment levels) was significantly more likely among responders (P = .002). While there was no significant difference in response based on sex (P = .56), older subjects (>40 years) were less likely to respond (P = .01). CONCLUSIONS: Intralesional immunotherapy using injection of Candida, mumps, or Trichophyton skin test antigens is an effective treatment for warts, as indicated by significantly higher response rates and distant response rates in subjects receiving antigen. Viral type and major histocompatibility complex antigens did not seem to influence treatment response. Response is accompanied by proliferation of peripheral blood mononuclear cells to human papillomavirus antigens, suggesting that a human papillomavirus-directed cell-mediated immune response plays a role in wart resolution.

Peanut protein allergens: the effect of roasting on solubility and allergenicity.

BACKGROUND: A contributing factor to food allergen stability is heat resistance. Peanut allergens in particular are resistant to heat, which results in their decreased solubility upon routine extraction and may have a profound influence on their continued presence in the digestive tract. Although there have been a number of studies characterizing soluble extracts of raw and roasted proteins, the relative solubility of the insoluble material following routine extraction for residual allergen characterization has not been investigated. The effects of various treatments on the re-solubilization and subsequent allergenicity of this insoluble peanut protein material are presented here. METHODS: Various methods to resolubilize the insoluble protein material were used, including pH, proteases and glycosidases. Protease digestion of nonextractable peanut proteins with pepsin, chymotrypsin and trypsin was performed in appropriate buffers as previously optimized for peanut proteins. Glycosidase activity in the presence of protease inhibitors was performed at pH 2. Digested samples were then subjected to SDS-PAGE/Western blot analysis using serum IgE from peanut-sensitive individuals. RESULTS: Progressive roasting of peanuts resulted in a significant decrease in protein solubility. The acidic proteins were resolubilized moderately at high pH, with solubility decreasing as pH approached the pI of the protein. However, at pH 2 the solubility increased dramatically. More extensive resolubilzation was observed with amylase treatment, presumably due to cleavage of glycoside of glycoproteins. The protein released into solution had a high IgE-binding capacity. While amylase was effective at resolubilizing this material, digestive tract proteases were not. CONCLUSION: The presence of these insolubilized peanut proteins provides a continuous source of major allergens to the gastrointestinal mucosal immune system.

Soy immunotherapy for peanut-allergic mice: modulation of the peanut-allergic response.

BACKGROUND: Allergen-specific immunotherapy (IT) is an effective therapeutic modality to prevent further anaphylactic episodes in patients with insect sting hypersensitivity and is being investigated for peanut allergy. So far, peanut-specific IT has been unsuccessful because of the side effects of therapy. Soybean seed storage proteins share significant homology with the respective peanut allergens. OBJECTIVE: This study was undertaken in mice to investigate whether specific doses of soybean would desensitize peanut-allergic mice. METHODS: C3H/HeJ mice were sensitized to peanut with 3 intraperitoneal (IP) injections of crude peanut extract. The mice were desensitized by IP injections with either crude peanut or soybean extract for 4 weeks, 3 times a week. Controls included placebo desensitization with PBS and naive mice. After 2 weeks of rest, mice were challenged IP with crude peanut extract. Thirty minutes later, symptom scores and body temperatures were recorded. Serum immunoglobulins, peanut-induced splenocyte proliferation, and secreted cytokines were measured before and after desensitization. RESULTS: The clinical symptoms in the soybean- and peanut-desensitized animals were markedly reduced compared with the placebo-treated mice. Specific IgG1 levels to crude peanut were significantly lower in the soy IT group than in the peanut IT group. The cellular response to crude peanut was also downregulated in the soy IT group, as shown by decreased peanut-specific stimulation indices and a cytokine profile skewed toward a T H 1 response. CONCLUSIONS: Soy IT can be used to desensitize/downregulate peanut-specific response in peanut-allergic mice and could provide a new therapeutic intervention for peanut allergy.

Peanut protein allergens: gastric digestion is carried out exclusively by pepsin.

BACKGROUND: A major characteristic of many food allergens, including Ara h 1, a major peanut allergen, is their resistance to gastric digestion. One estimate of the allergenic potential of a possible protein allergen is its stability under simulated gastric conditions. OBJECTIVE: Because the rate and extent of digestion of allergenic proteins will affect the severity of any subsequent allergic response, it is important to correlate protein allergen digestion in simulated gastric fluid with that in actual gastric fluid. METHODS: A major peanut allergen, Ara h 1, was digested in vitro by using both pepsin and porcine gastric fluid. Several comparisons between the 2 sets of proteolytic conditions were assessed including pH optima and the effect of temperature, denaturants, and specific enzyme inhibitors. RESULTS: In vitro digestion of Ara h 1 with pepsin and porcine gastric fluid resulted in virtually identical hydrolysis patterns as observed on SDS-PAGE. The protease activity of both pepsin and gastric fluid were inhibited at high pH and in the presence of pepstatin. However, both remained active in 4 mol/L urea and at 60 degrees C. CONCLUSIONS: Protein digestion in the porcine stomach is carried out by pepsin. In vivo gastric digestion is modeled accurately by peptic hydrolysis. Digestion conditions in vivo are comparable to experimental conditions in vitro provided that the acidic nature of the stomach contents is optimal for characterization of the allergen under standard pepsin digestion conditions. Additional experimentation using crude food extracts, both in the presence and absence of a complete meal, is needed to elucidate the complete physiologic nature of food allergen digestion.

Diet and the development of atopic disease.

PURPOSE OF REVIEW: The present review addresses the current literature regarding the impact of diet and the development of atopic disease. A search of the literature was carried out covering the following topics: diet and nutrition combined with immediate hypersensitivity, atopy, atopic disease, atopic dermatitis, and food allergy. RECENT FINDINGS: The search results identified a significant contribution in the form of reviews considering this important topic, which ultimately led to the author's recommendation of these reviews to impress upon readers the impact of the atopy triad: atopic dermatitis to allergic rhinitis and asthma. SUMMARY: A great deal of information exists in the pathomechanisms of atopic disease that will affect the classification of allergic and non-allergic atopic diseases. Increasing data on the genetic, humoral and cellular forms associated with these diseases will provide more clear-cut diagnostic criteria, treatment regimens and a more strict definition of the disease variants.

Sensitization and allergic response and intervention therapy in animal models.

A review is presented of 3 murine models and a swine neonatal model used to investigate immunotherapeutic options. In Model 1, mutation of linear IgE-binding epitopes of Ara h 1 for the preparation of a hypoallergenic Ara h 1 is discussed with respect to expression in transgenic tobacco plants and correct folding following expression in the pET16b construct. In Model 2, the mutations of Ara h 1 were assessed for use as an immunotherapeutic agent. Although some protective benefit was observed with the modified Ara h 1 protein, animals desensitized with heat-killed E. coil preparations showed increased protection to challenge. In Model 3, soybean homologs to peanut proteins were investigated to determine if soybean immunotherapy can potentially provide benefit to peanut-allergic subjects. Although some protection was provided, additional experimentation with respect to optimal doses for sensitization and challenge will need to be investigated. In Model 4, the neonatal swine model was used to profile different foods (low to moderate to high sensitizing) similar to food allergies in humans. Evidence suggests such feasiblity; however, threshold levels for sensitization and allergic responses will need additional study. In summary, murine and swine animal models are being used to address immunotherapeutic avenues and investigation into the mechanisms of food-allergic sensitization.

Food biotechnology: is this good or bad? Implications to allergic diseases.

BACKGROUND: Food biotechnology represents advancement in the traditional interspecies and intergeneric breeding methods for improving food supplies worldwide. With respect to safety, foods developed through biotechnology techniques represent one of the most extensively reviewed agricultural advancements in history. OBJECTIVE: To review the relevant issues with respect to foods from genetically modified crops and allergenicity. DATA SOURCES: To impart this information, the author will rely upon his experiences with investigations into food allergy and food allergens, participation in various workshops designed to determine allergenicity of novel proteins introduced into the diet, web sites, issue papers, and articles relevant to the topic. RESULTS: Given that there are no validated methods or models to determine potential allergenicity of novel proteins, criteria have been established based upon characteristics of known food allergens. The combination of genetic and bioinformatics information available from known food allergens applied to foods developed from genetically modified crops to avoid the inadvertent introduction of allergens into foods should pose no significant allergenic concern to individuals with a genetic predisposition to food allergy. Education and sound scientific evaluation provided to the consumer should alleviate any fear of emotionalism as implied by "Frankenfoods." CONCLUSIONS: The estimation that more than two trillion transgenic plants have been grown in 1999 and 2000 alone, with no overt documented adverse food reactions being reported, indicates that genetic modification through biotechnology will not impose immediate significant risks as food allergen sources beyond that of our daily dietary intake of foods from crop plants.

Assessment of protein allergenicity on the basis of immune reactivity: animal models.

Because of the public concern surrounding the issue of the safety of genetically modified organisms, it is critical to have appropriate methodologies to aid investigators in identifying potential hazards associated with consumption of foods produced with these materials. A recent panel of experts convened by the Food and Agriculture Organization and World Health Organization suggested there is scientific evidence that using data from animal studies will contribute important information regarding the allergenicity of foods derived from biotechnology. This view has given further impetus to the development of suitable animal models for allergenicity assessment. This article is a review of what has been achieved and what still has to be accomplished regarding several different animal models. Progress made in the design and evaluation of models in the rat, the mouse, the dog and in swine is reviewed and discussed.

Genetic modification removes an immunodominant allergen from soybean.

The increasing use of soybean (Glycine max) products in processed foods poses a potential threat to soybean-sensitive food-allergic individuals. In vitro assays on soybean seed proteins with sera from soybean-sensitive individuals have immunoglobulin E reactivity to abundant storage proteins and a few less-abundant seed proteins. One of these low abundance proteins, Gly m Bd 30 K, also referred to as P34, is in fact a major (i.e. immunodominant) soybean allergen. Although a member of the papain protease superfamily, Gly m Bd 30 K has a glycine in the conserved catalytic cysteine position found in all other cysteine proteases. Transgene-induced gene silencing was used to prevent the accumulation of Gly m Bd 30 K protein in soybean seeds. The Gly m Bd 30 K-silenced plants and their seeds lacked any compositional, developmental, structural, or ultrastructural phenotypic differences when compared with control plants. Proteomic analysis of extracts from transgenic seed detected the suppression of Gly m Bd 30 K-related peptides but no other significant changes in polypeptide pattern. The lack of a collateral alteration of any other seed protein in the Gly m Bd 30 K-silenced seeds supports the presumption that the protein does not have a role in seed protein processing and maturation. These data provide evidence for substantial equivalence of composition of transgenic and non-transgenic seed eliminating one of the dominant allergens of soybean seeds.

Workshop overview: approaches to the assessment of the allergenic potential of food from genetically modified crops.

There is a need to assess the safety of foods deriving from genetically modified (GM) crops, including the allergenic potential of novel gene products. Presently, there is no single in vitro or in vivo model that has been validated for the identification or characterization of potential food allergens. Instead, the evaluation focuses on risk factors such as source of the gene (i.e., allergenic vs. nonallergenic sources), physicochemical and genetic comparisons to known allergens, and exposure assessments. The purpose of this workshop was to gather together researchers working on various strategies for assessing protein allergenicity: (1) to describe the current state of knowledge and progress that has been made in the development and evaluation of appropriate testing strategies and (2) to identify critical issues that must now be addressed. This overview begins with a consideration of the current issues involved in assessing the allergenicity of GM foods. The second section presents information on in vitro models of digestibility, bioinformatics, and risk assessment in the context of clinical prevention and management of food allergy. Data on rodent models are presented in the next two sections. Finally, nonrodent models for assessing protein allergenicity are discussed. Collectively, these studies indicate that significant progress has been made in developing testing strategies. However, further efforts are needed to evaluate and validate the sensitivity, specificity, and reproducibility of many of these assays for determining the allergenicity potential of GM foods.

Monitoring peanut allergen in food products by measuring Ara h 1.

BACKGROUND: Peanut allergy is an important health problem in the United States, affecting approximately 0.6% of children. Inadvertent exposure to peanut is a risk factor for life-threatening food-induced anaphylaxis. OBJECTIVE: The purpose of this investigation was to develop an immunoassay for a major peanut allergen, Ara h 1, to detect peanut allergen in foods so that the risk of inadvertent exposure can be reduced. METHODS: A specific 2-site monoclonal antibody-based ELISA was developed to measure Ara h 1 in foods. The sensitivity of the assay was 30 ng/mL. Ara h 1 was measured in foods (n = 83) with or without peanut and in experiments to optimize allergen yield and to determine peanut contamination in spiked foods. RESULTS: Ara h 1 levels in food products ranged from less than 0.1 microg/g to 500 microg/g. Ara h 1 measured in ng/mL was transformed to microg/g for food products. Peanut butter contained the highest amounts of Ara h 1. Peanut extracts contained from 0.5 to 15 mg Ara h 1/g of peanut depending on the extraction conditions. Optimal extraction of Ara h 1 was obtained by using phosphate buffer with 1 mol/L NaCl and Tween at 60 degrees C. Ara h 1 was not always detected in presence of chocolate under the extraction conditions tested. Spiking experiments showed that the assay could detect approximately 0.1% Ara h 1 contamination of food with ground peanut. There was an excellent correlation between Ara h 1 levels and peanut content measured by using a commercial polyclonal antibody-based ELISA (r = 93, n = 31, P <.001). CONCLUSION: A new sensitive and specific monoclonal antibody-based ELISA was used to monitor Ara h 1 content in food products. This assay should be useful for monitoring peanut contamination in the food manufacturing and processing industry and in developing thresholds for sensitization or allergic reaction in persons with peanut allergy.

Nonmurine animal models of food allergy.

Food allergy can present as immediate hypersensitivity [manifestations mediated by immunoglobulin (Ig)E], delayed-type hypersensitivity (reactions associated with specific T lymphocytes), and inflammatory reactions caused by immune complexes. For reasons of ethics and efficacy, investigations in humans to determine sensitization and allergic responses of IgE production to innocuous food proteins are not feasible. Therefore, animal models are used a) to bypass the innate tendency to develop tolerance to food proteins and induce specific IgE antibody of sufficient avidity/affinity to cause sensitization and upon reexposure to induce an allergic response, b) to predict allergenicity of novel proteins using characteristics of known food allergens, and c) to treat food allergy by using immunotherapeutic strategies to alleviate life-threatening reactions. The predominant hypothesis for IgE-mediated food allergy is that there is an adverse reaction to exogenous food proteins or food protein fragments, which escape lumen hydrolysis, and in a polarized helper T cell subset 2 (Th2) environment, immunoglobulin class switching to allergen-specific IgE is generated in the immune system of the gastrointestinal-associated lymphoid tissues. Traditionally, the immunologic characterization and toxicologic studies of small laboratory animals have provided the basis for development of animal models of food allergy; however, the natural allergic response in large animals, which closely mimic allergic diseases in humans, can also be useful as models for investigations involving food allergy.