Gastrointestinal immunopathology of food protein-induced enterocolitis syndrome and other non-immunoglobulin E-mediated food allergic diseases.

OBJECTIVE: To provide a concise summary of the current literature regarding gastrointestinal immunopathology of food protein-induced enterocolitis syndrome (FPIES) and other non-immunoglobulin E (IgE)-mediated food allergic diseases. DATA SOURCES: Data were extracted from PubMed, MEDLINE, and ScienceDirect databases. STUDY SELECTIONS: Original articles, review articles, and guidelines published in the past 5 years in peer-reviewed journals were first summarized. The original articles cited were then reviewed and relevant results were extracted. RESULTS: Patients with FPIES and non-IgE-mediated food allergic diseases developed vomiting, diarrhea, and food aversion expelled food allergen from their bodies. Aside from T helper type 2 (TH2) immunity, TH1, TH17, innate immunity, and epithelial mucosal barrier defect were also found to be important in the pathogenesis. Eosinophils, widely identified in the biopsy samples, were key players or were late-recruited cells for tissue repairs in those diseases. Intestinal dysbiosis and their metabolites stimulated enterochromaffin cells or enteroendocrine cells to produce serotonin, interfering with intestinal motility and subsequently affecting brain function. FPIES and non-IgE-mediated food allergic diseases were likely part of the atopic march. Allergic inflammation in intestinal mucosa might result in subsequent inflammation in the airway mucosa, suggesting the theory of "one mucosa, one disease." CONCLUSION: The immune responses of FPIES and non-IgE-mediated food allergic diseases were not limited to the gastrointestinal tract, but also trigger wider inflammatory responses beyond it. Further research will be required to determine the systemic effect and intestinal microbiome of those diseases.

Hydrophobicity drives receptor-mediated uptake of heat-processed proteins by THP-1 macrophages and dendritic cells, but not cytokine responses.

Although an impact of processing on immunogenicity of food proteins has clearly been demonstrated, the underlying mechanisms are still unclear. We applied 3 different processing methods: wet heating (60 °C) and low- or high-temperature (50 °C or 130 °C, respectively) dry-heating in absence or presence of reducing sugars, to ß-lactoglobulin (BLG), lysozyme and thyroglobulin, which represent dietary proteins with different pI or molecular weight. Uptake of the soluble fraction of the samples was tested in two types of, genetically homogeneous, antigen-presenting cells (macrophages and dendritic cells derived from THP-1 monocytes). This revealed a strong correlation between the uptake of the different protein samples by macrophages and dendritic cells, and confirmed the key role of hydrophobicity, over aggregation, in determining the uptake. Several uptake routes were shown to contribute to the uptake of BLG by macrophages. However, cytokine responses following exposure of macrophages to BLG samples were not related to the levels of uptake. Together, our results demonstrate that heat-treatment-induced increased hydrophobicity is the prime driving factor in uptake, but not in cytokine production, by THP-1 macrophages.

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.

Duodenal bacterial proteolytic activity determines sensitivity to dietary antigen through protease-activated receptor-2.

Microbe-host interactions are generally homeostatic, but when dysfunctional, they can incite food sensitivities and chronic diseases. Celiac disease (CeD) is a food sensitivity characterized by a breakdown of oral tolerance to gluten proteins in genetically predisposed individuals, although the underlying mechanisms are incompletely understood. Here we show that duodenal biopsies from patients with active CeD have increased proteolytic activity against gluten substrates that correlates with increased Proteobacteria abundance, including Pseudomonas. Using Pseudomonas aeruginosa producing elastase as a model, we show gluten-independent, PAR-2 mediated upregulation of inflammatory pathways in C57BL/6 mice without villus blunting. In mice expressing CeD risk genes, P. aeruginosa elastase synergizes with gluten to induce more severe inflammation that is associated with moderate villus blunting. These results demonstrate that proteases expressed by opportunistic pathogens impact host immune responses that are relevant to the development of food sensitivities, independently of the trigger antigen.

Food-Related Contact Dermatitis, Contact Urticaria, and Atopy Patch Test with Food.

A wide variety of foods may cause or aggravate skin diseases such as contact dermatitis, contact urticaria, or atopic dermatitis (AD), both in occupational and private settings. The mechanism of action underlying allergic disease to food, food additives, and spices may be immunologic and non-immunologic. The classification and understanding of these reactions is a complex field, and knowledge of the possible reaction patterns and appropriate diagnostic test methods is essential. In addition, certain foods may cause worsening of atopic dermatitis lesions in children. The atopy patch test (APT) is a well-established, clinically useful tool for assessing delayed type reactions to protein allergens in patients and may be useful to detect protein allergens relevant for certain skin diseases. The APT may even detect sensitization against allergens in intrinsic atopic dermatitis patients, who show negative skin prick test and negative in vitro IgE test results against these allergens. Native foods, SPT solutions on filter paper, and purified allergens in petrolatum have been used for APT. The European Task Force on Atopic Dermatitis (ETFAD) has worked on standardizing this test in the context of AD patients, who are allergic to aeroallergens and food. This recommended, standardized technique involves test application at the upper back of children and adults; use of large, 12-mm Finn chambers; avoidance of any pre-treatment such as tape stripping or delipidation; standardized amounts of purified allergens in petrolatum; and use of the standardized ETFAD reading key. The APT may not be the best working or best standardized of all possible skin tests, but it is the best test that we currently have available in this niche.

The Effect of Digestion and Digestibility on Allergenicity of Food.

Food allergy prevalence numbers are still on the rise. Apart from environmental influences, dietary habits, food availability and life-style factors, medication could also play a role. For immune tolerance of food, several contributing factors ensure that dietary compounds are immunologically ignored and serve only as source for energy and nutrient supply. Functional digestion along the gastrointestinal tract is essential for the molecular breakdown and a prerequisite for appropriate uptake in the intestine. Digestion and digestibility of carbohydrates and proteins thus critically affect the risk of food allergy development. In this review, we highlight the influence of amylases, gastric acid- and trypsin-inhibitors, as well as of food processing in the context of food allergenicity.

Animal models of allergen-specific immunotherapy in food allergy: Overview and opportunities.

Food allergy is an adverse reaction to otherwise harmless proteins in food. The disease is a major health problem of growing concern, affecting approximately 5%-8% of young children and 2%-4% of adults. No accepted strategy exists for prevention and treatment of food allergy, and strict avoidance of the offending food is presently the only viable management option. Living with food avoidance may have a huge impact on the quality of life of food allergic patients, with daily fear of serious or even fatal reactions. The urgent need for safe and efficient food allergy treatment options has led to massive research efforts to develop and improve strategies for food allergy immunotherapeutic approaches. A first step in developing new and improved strategies of immunotherapy often involves the use of animal models. In present review, we provide an overview of animal studies of allergen-specific immunotherapy highlighting opportunities and challenges for each approach. The presented models, almost exclusively performed in mice, assess therapeutic efficacy and immunological outcomes following oral, intraperitoneal, subcutaneous, epicutaneous, and sublingual administration of native allergens, or preparations of hydrolyzed allergen, T-cell-directed peptides, or allergen with immunomodulatory adjuvants. Recently, approaches using immune cell therapy have demonstrated efficacy. Current models mainly assess anaphylaxis as the primary clinical outcome. With the increased appreciation that food allergy is a heterogeneous disease presenting different phenotypes, there is a continued need to develop new disease-relevant therapeutic models of food allergy.

Regulatory Immune Mechanisms in Tolerance to Food Allergy.

Oral tolerance can develop after frequent exposure to food allergens. Upon ingestion, food is digested into small protein fragments in the gastrointestinal tract. Small food particles are later absorbed into the human body. Interestingly, some of these ingested food proteins can cause allergic immune responses, which can lead to food allergy. So far it has not been completely elucidated how these proteins become immunogenic and cause food allergies. In contrast, oral tolerance helps to prevent the pathologic reactions against different types of food antigens from animal or plant origin. Tolerance to food is mainly acquired by dendritic cells, epithelial cells in the gut, and the gut microbiome. A subset of CD103+ DCs is capable of inducing T regulatory cells (Treg cells) that express anti-inflammatory cytokines. Anergic T cells also contribute to oral tolerance, by reducing the number of effector cells. Similar to Treg cells, B regulatory cells (Breg cells) suppress effector T cells and contribute to the immune tolerance to food allergens. Furthermore, the human microbiome is an essential mediator in the induction of oral tolerance or food allergy. In this review, we outline the current understanding of regulatory immune mechanisms in oral tolerance. The biological changes reflecting early consequences of immune stimulation with food allergens should provide useful information for the development of novel therapeutic treatments.

A descriptive pilot study of cytokine production following stimulation of ex-vivo whole blood with commercial therapeutic feline hydrolyzed diets in individual healthy immunotolerant cats.

BACKGROUND: Hydrolyzed diets are used in companion animals for the diagnosis and treatment of adverse food reaction. Similarly, hydrolyzed formulas are used in human infants with severe inflammatory bowel disease or milk allergy, and these must meet the standard of hypoallergenicity through rigorous testing. Unfortunately, no standards are currently applied to hydrolyzed veterinary therapeutic diets, and data for the immunogenicity of feline diets is also not available. Therefore, the main aim of this pilot study was to determine if ex-vivo whole blood stimulation assays could be used to characterize the cytokine response to hydrolyzed commercial diets in a small number of individual healthy immunotolerant cats. This approach has also been used to investigate cytokine production in response to cow milk protein in humans and currently similar studies do not exist in companion animals. Nine healthy cats previously eating the same basal diet were divided into groups and fed one of three hydrolyzed diets exclusively for 6 weeks. Heparinized whole blood was collected from each cat before and after the feeding trial. Ex-vivo whole blood stimulation assays were performed using crude extracts of the basal diet as a positive control, as this diet contained the same proteins present in the hydrolyzed diet but were intact, saline as a negative control, and each cat's respective hydrolyzed diet. Supernatants were collected and analyzed for tumor necrosis factor-alpha, interleukin-10 (IL-10), and interleukin-4 using enzyme-linked immunosorbant assay. RESULTS: Seven cats produced detectable amounts of the anti-inflammatory cytokine IL-10 upon stimulation with the basal diet. Two cats produced detectable amounts of IL-10 upon stimulation with a hydrolyzed soy-based diet and one cat produced a detectable amount of IL-10 upon stimulation with a hydrolyzed chicken-based diet (>125 pg/mL). CONCLUSIONS: Results from this pilot study suggest that in some healthy immunotolerant cats, some hydrolyzed diets may elicit a similar cytokine response compared to their basal diet, which contained the same proteins intact. Therefore, animals may be able to recognize and react to some hydrolyzed forms of tolerated proteins, and may also suggest IL-10 as a target for investigation as a potential marker for dietary tolerance in cats, however further studies would be necessary to corroborate this. Further studies are also needed to determine if this would also be the same in immunologically naïve, sensitized and clinically hypersensitized cats.

In vitro digestibility and IgE reactivity of enzymatically cross-linked heterologous protein polymers.

Homologous and heterologous cross-linked polymers of whey protein isolate (WPI), soy protein isolate (SPI) and casein (CN) and their binary mixtures, viz., WPI+SPI, WPI+CN and SPI+CN, were produced using transglutaminase, and their in vitro IgE reactivity and digestibility under simulated gastro-intestinal conditions were studied. The results showed that the IgE reactivity of protein components in heterologous polymers was significantly lower than that in homologous polymers, suggesting that each protein component masked the IgE-reactive epitopes in the other protein component more effectively in heterologous polymers than in homologous polymers. In vitro digestion under simulated gastro-intestinal conditions revealed that both homologous and heterologous polymers were less digestible than untreated proteins, but the peptides released during the time course of digestion were less IgE-reactive. The results of this study indicate that hypoallergenic protein products could be produced by transglutaminase-mediated heterologous polymerization of protein mixtures.

Presence of intraepithelial food antigen in patients with active eosinophilic oesophagitis.

BACKGROUND: Although eosinophilic oesophagitis (EoE) is putatively mediated by an abnormal response to food antigen, the oesophagus is considered relatively impermeable to large molecules. AIM: To assess whether food antigens penetrate the oesophageal mucosa in patients with EoE. METHODS: Anti-gliadin staining was performed in three groups: active EoE, inactive EoE and EoE patients on a low or gluten free diet. To appraise the specificity of our results, we also performed gliadin staining on six patients without oesophageal disease who were consuming gluten. The groups with EoE on gluten also underwent endoscopic infusion with gluten containing soy sauce and repeat biopsies during the endoscopy. We measured eosinophil density, dilated intercellular spaces (on a 0-4+ scale) and gliadin in oesophageal mucosa by immunofluorescence. RESULTS: Patients with active EoE had significantly greater epithelial density of anti-gliadin staining when compared to inactive EoE (P < 0.0065) and gluten-free patients (P < 0.0008) at baseline and after soy infusion. Gliadin was not detected in non-EoE control patients. The distribution of gliadin was both cytoplasmic and nuclear. There was good correlation of dilated intercellular spaces grade and total gliadin staining intensity (r = 0.577, P = 0.0077). Acute oesophageal perfusion of a commercial gliadin-rich soy sauce did not lead to an increase in gliadin staining in active or inactive EoE. CONCLUSION: These findings suggest, although do not prove, that antigen penetration in active eosinophilic oesophagitis might be facilitated by impairment of epithelial integrity.

Non-IgE-mediated gastrointestinal food allergies in children.

Non-IgE-mediated gastrointestinal food allergic disorders (non-IgE-GI-FA) including food protein-induced enterocolitis syndrome (FPIES), food protein-induced enteropathy (FPE), and food protein-induced allergic proctocolitis (FPIAP) are relatively uncommon in infants and young children, but are likely under-diagnosed. Non-IgE-GI-FA have a favorable prognosis, with majority resolving by age 3-5 years. Diagnosis relies on the recognition of symptoms pattern in FPIAP and FPIES and biopsy in FPE. Further studies are needed for a better understanding of the pathomechanism, which will lead eventually to the development of diagnostic tests and treatments. Limited evidence supports the role of food allergens in subsets of constipation, gastroesophageal reflux disease, irritable bowel syndrome, and colic. The immunologic pathomechanism is not fully understood and empiric prolonged avoidance of food allergens should be limited to minimize nutrient deficiency and feeding disorders/food aversions in infants.

Influence of dietary protein on serum metabolites and antioxidant status: A study in Chrysolophus amherstiae.

This experiment was conducted to study the effect of feeding graded levels of dietary crude protein (CP) on serum biochemical profile of Lady Amherst's pheasants (LAP). Eighteen male LAP were randomly distributed into three groups of six each in an experiment based on completely randomized design. The CP content of the diets was 13.4%, 16.5%, and 19.1%, in groups I, II, and III, respectively. Serum concentrations of uric acid was lowest (P < 0.05) in group I. Relationship between serum concentration of uric acid and nitrogen intake was linear (R(2) = 0.39, P < 0.01). Concentrations of other serum metabolites and enzymes were similar among the groups. Serum concentration of triiodothyronine (T3 ) was highest (P < 0.05) in group I, followed by groups II and III. Total antioxidant capacity (TAC) of serum was lower (P < 0.007), whereas serum concentration of malondialdehyde (MDA) was higher (P < 0.001) in group I as compared to groups II and III. Regression of serum concentration of TAC (R(2) = 0.74, P < 0.01) and MDA (R(2) = 0.39, P < 0.05) was polynomial. Heterophil to lymphocyte ratio was higher (P < 0.007) in group I as compared to groups II and III. Relationship between H/L ratio and nitrogen intake was polynomial (R(2) = 0.69, P < 0.05). Cell mediated immune response measured as foot web index was similar among the groups. Based upon the results, it was concluded that a diet containing 16.5% crude protein would be optimum for improving antioxidant defense and the ability of Lady Amherst's pheasant to combat stress. Zoo Biol. 35:346-354, 2016. © 2016 Wiley Periodicals, Inc.

Hypersensitivity linked to exposure of broad bean protein(s) in allergic patients and BALB/c mice.

OBJECTIVE: Broad bean (Vicia faba L.), a common vegetable, belongs to the family Fabaceae and is consumed worldwide. Limited studies have been done on allergenicity of broad beans. The aim of this study was to determine if broad bean proteins have the ability to elicit allergic responses due to the presence of clinically relevant allergenic proteins. METHODS: Simulated gastric fluid (SGF) assay and immunoglobulin E (IgE) immunoblotting were carried out to identify pepsin-resistant and IgE-binding proteins. The allergenicity of broad beans was assessed in allergic patients, BALB/c mice, splenocytes, and RBL-2H3 cells. RESULTS: Eight broad bean proteins of approximate molecular weight 70, 60, 48, 32, 23, 19, 15, and 10 kDa that remained undigested in SGF, showed IgE-binding capacity as well. Of 127 allergic patients studied, broad bean allergy was evident in 16 (12%). Mice sensitized with broad bean showed increased levels of histamine, total and specific IgE, and severe signs of systemic anaphylaxis compared with controls. Enhanced levels of histamine, prostaglandin D2, cysteinyl leukotriene, and ß-hexosaminidase release were observed in the primed RBL-2H3 cells following broad bean exposure. The levels of interleukin IL-4, IL-5, IL-13 and regulated on activation, normal T-cell expressed and secreted were found enhanced in broad bean-treated splenocytes culture supernatant compared with controls. CONCLUSION: This study inferred that broad bean proteins have the ability to elicit allergic responses due to the presence of clinically relevant allergenic proteins.

[Immunotoxicity and environmental substances].

A well functioning immune system is essential in maintaining integrity of the organism, and malfunction may have severe health consequences. Environmental substances may pose direct toxicity to components of the immune system, often leading to immunosuppression and resulting reduced resistance to infections and tumors. Alternatively, such substances may be recognized by the immune system in a specific fashion, which may result in allergy and autoimmunity. A proper risk assessment of environmental substances in terms of immunotoxicity is necessary. In this manuscript, I reviewed recent three topics about immunotoxicity: (1) IPCS/WHO Guidance for immunotoxicity risk assessment for chemicals, (2) Intestinal immunotoxicity, and (3) Epicutaneous sensitization of food proteins.

Characterization of the allergenic potential of proteins: an assessment of the kiwifruit allergen actinidin.

Assessment of the potential allergenicity (IgE-inducing properties) of novel proteins is an important challenge in the overall safety assessment of foods. Resistance to digestion with pepsin is commonly measured to characterize allergenicity, although the association is not absolute. We have previously shown that specific IgE antibody production induced by systemic [intraperitoneal (i.p.)] exposure of BALB/c strain mice to a range of proteins correlates with allergenic potential for known allergens. The purpose of the present study was to explore further the utility of these approaches using the food allergen, actinidin. Recently, kiwifruit has become an important allergenic foodstuff, coincident with its increased consumption, particularly as a weaning food. The ability of the kiwifruit allergen actinidin to stimulate antibody responses has been compared with the reference allergen ovalbumin, and with the non-allergen bovine haemoglobin. Haemoglobin was rapidly digested by pepsin whereas actinidin was resistant unless subjected to prior chemical reduction (reflecting intracellular digestion conditions). Haemoglobin stimulated detectable IgG antibody production at relatively high doses (10%), but failed to provoke detectable IgE. In contrast, actinidin was both immunogenic and allergenic at relatively low doses (0.25% to 1%). Vigorous IgG and IgG1 antibody and high titre IgE antibody responses were recorded, similar to those provoked by ovalbumin. Thus, actinidin displays a marked ability to provoke IgE, consistent with allergenic potential. These data provide further encouragement that in tandem with analysis of pepsin stability, the induction of IgE after systemic exposure of BALB/c strain mice provides a useful approach for the prospective identification of protein allergens.

The impact of preoperative immunonutrition and other nutrition models on tumor infiltrative lymphocytes in colorectal cancer patients.

AIM: The importance of the alteration of tumor infiltrative lymphocytes (CD4(+), CD8(+), CD16(+), and CD56(+)) in colorectal cancer prognosis is well known. In this study, we analyzed the effect of preoperative immunonutrition and different nutritional models on the clinical condition of colorectal cancer patients. METHODS: Twenty-eight colorectal cancer patients were grouped into 4 groups according to their nutrition regimens randomly and were given immunonutrition (IMN), standard enteral (SE), total parental nutrition (TPN), and normal nutrition (NN) regimens, all of which contained the same calorie-nitrogen content within a 7-day preoperative period. All patients had an endoscopic biopsy before and after the regimen, and the lymphocyte population infiltrating mucosal parts of the resected tumor tissue were evaluated. Immunohistochemical analysis of the tissue specimens was performed by staining with antihuman CD4(+), CD8(+), CD16(+), and CD56(+) antibodies. RESULTS: After nutrition, there were significant increases in each of the 4 groups of CD4(+) and CD8(+) cells within the tumor. Comparing the rates of augmentation, the increased rates of the CD8(+) cells infiltrating the tumor after nutrition in the patients who were fed with IMN were significantly more than the ones in other groups (P = .01). CD16(+) cell infiltration was significantly higher in all groups except the SE and IMN groups. The SE group had increased CD56(+) cell infiltration compared with the other groups. CONCLUSIONS: In the colorectal cancer patients who had nutrition in the 7-day preoperative period, except for the SE nutrition group, there were significant increases of infiltration of CD56(+) cells at the mucosal part of the tumor tissue within the CD4(+) and CD8(+) cell population. When postnutrition values were compared, there was a marked increase of CD8(+) cells in the IMN group.