Improving diagnostic tests for food allergy with recombinant allergens

Food allergy is one important manifestation of atopic allergy. Primary food allergy mainly affects young children (class I), whereas adults frequently develop food allergy as a consequence of an inhalant sensitization (class II). At present, the diagnostic instrument for proving class II food allergy is not satisfactory. Skin tests as well as serological tests are in general neither very specific nor highly sensitive because they depend on food extracts, which differ in their content of individual allergens, vary between manufacturers, and even between different batches. Since the presence of food allergen-specific IgE antibodies does not always correlate with clinical symptoms against the respective food, oral provocation tests (ideally double-blind placebo-controlled food challenges) have to be performed to validate serological diagnosis or skin tests. However, oral provocation tests are connected to several practical problems and include a specific risk for the allergic patient. Applying DNA technology, up to 40 food allergens have been produced in recombinant form, which implies standardized quality and unlimited quantity of the respective proteins. Hence, such molecules might be used to solve problems of clinical and molecular allergology in diagnosis, research, and therapy of class II food allergies. First experiments with recombinant food allergens in this respect appear very promising.     

Focus: Allergic Diseases and Type II Immunity: Allergy Prevention: An Overview of Current Evidence

Background: There has been a rapid rise in allergic disorders across the globe. This has increased research into the determinants of allergy development, to identify factors that may be manipulated to mitigate risk. An opportune window in immunological development appears to exist in early life whereby certain exposures may promote or prevent the development of an allergic disposition. Furthermore, factors that affect the composition and diversity of the microbiome in early life have been explored. In this review, we discuss current literature and recommendations relating to exposures that may prevent allergy development or promote tolerance. Risk factors and recommendations: Delivery by caesarean section, omission of breastfeeding, vitamin D insufficiency, and environmental exposures, such as cigarette smoke exposure, all increase the risk of an allergic predisposition. Dietary diversity during pregnancy, lactation, and in infancy is protective. Breastfeeding for at least 4 months reduces the risk of eczema. Recommendations for food-allergen exposure has shifted from delayed introduction to early introduction as a tolerance-inducing strategy. Supplements such as probiotics and vitamins during pregnancy and infancy have yet to produce conclusive results for allergy prevention. Emollient use in infancy has not been shown to be protective against eczema or food allergy.     

Exploring the Determinants of the Perceived Risk of Food Allergies in Canada

Food allergies are emerging health risks in much of the Western world, and some evidence suggests prevalence is increasing. Despite lacking scientific consensus around prevalence and management, policies and regulations are being implemented in public spaces (e.g., schools). These policies have been criticized as extreme in the literature, in the media, and by the non-allergic population. Backlash appears to be resulting from different perceptions of risk between different groups. This article uses a recently assembled national dataset (n = 3,666) to explore how Canadians perceive the risks of food allergy. Analyses revealed that almost 20% self-report having an allergic person in the household, while the average respondent estimated the prevalence of food allergies in Canada to be 30%. Both of these measures overestimate the true clinically defined prevalence (7.5%), indicating an inflated public understanding of the risks of food allergies. Seventy percent reported food allergies to be substantial risks to the Canadian population. Multivariate logistic regression models revealed important determinants of risk perception including demographic, experience-based, attitudinal, and regional predictors. Results are discussed in terms of understanding emerging health risks in the post-industrial era, and implications for both policy and risk communication.     

Absence of Foxp3+ Regulatory T Cells during Allergen Provocation Does Not Exacerbate Murine Allergic Airway Inflammation

Regulatory T cells (Tregs) play a non-redundant role in maintenance of immune homeostasis. This is achieved by suppressing both, priming of naïve cells and effector cell functions. Although Tregs have been implicated in modulating allergic immune responses, their influence on distinct phases of development of allergies remains unclear. In this study, by using bacterial artificial chromosome (BAC)-transgenic Foxp3-DTR (DEREG) mice we demonstrate that the absence of Foxp3⁺ Tregs during the allergen challenge surprisingly does not exacerbate allergic airway inflammation in BALB/c mice. As genetic disposition due to strain specificity may contribute significantly to development of allergies, we performed similar experiment in C57BL/6 mice, which are less susceptible to allergy in the model of sensitization used in this study. We report that the genetic background does not influence the consequence of this depletion regimen. These results signify the temporal regulation exerted by Foxp3⁺ Tregs in limiting allergic airway inflammation and may influence their application as potential therapeutics.     

Plant cell-made protein antigens for induction of Oral tolerance

The gut associated lymphoid tissue has effective mechanisms in place to maintain tolerance to food antigens. These can be exploited to induce antigen-specific tolerance for the prevention and treatment of autoimmune diseases and severe allergies and to prevent serious immune responses in protein replacement therapies for genetic diseases. An oral tolerance approach for the prevention of peanut allergy in infants proved highly efficacious and advances in treatment of peanut allergy have brought forth an oral immunotherapy drug that is currently awaiting FDA approval. Several other protein antigens made in plant cells are in clinical development. Plant cell-made proteins are protected in the stomach from acids and enzymes after their oral delivery because of bioencapsulation within plant cell wall, but are released to the immune system upon digestion by gut microbes. Utilization of fusion protein technologies facilitates their delivery to the immune system, oral tolerance induction at low antigen doses, resulting in efficient induction of FoxP3⁺ and latency-associated peptide (LAP)⁺ regulatory T cells that express immune suppressive cytokines such as IL-10. LAP and IL-10 expression represent potential biomarkers for plant-based oral tolerance. Efficacy studies in hemophilia dogs support clinical development of oral delivery of bioencapsulated antigens to prevent anti-drug antibody formation. Production of clinical grade materials in cGMP facilities, stability of antigens in lyophilized plant cells for several years when stored at ambient temperature, efficacy of oral delivery of human doses in large animal models and lack of toxicity augur well for clinical advancement of this novel drug delivery concept.     

Autoimmunity and the microbiome: T‐cell receptor mimicry of “self” and microbial antigens mediates self tolerance in holobionts

I propose a T‐cell receptor (TcR)‐based mechanism by which immunity mediates both “genetic self” and “microbial self” thereby, connecting microbiome disease with autoimmunity. The hypothesis is based on simple principles. First, TcR are selected to avoid strong cross‐reactivity with “self,” resulting in selection for a TcR repertoire mimicking “genetic self.” Second, evolution has selected for a “microbial self” that mimics “genetic self” so as to share tolerance. In consequence, our TcR repertoire also mimics microbiome antigenicity, providing a novel mechanism for modulating tolerance to it. Also, the microbiome mimics the TcR repertoire, acting as a secondary immune system. I call this TcR‐microbiome mimicry “holoimmunity” to denote immune tolerance to the “holobiont self.” Logically, microbiome‐host mimicry means that autoimmunity directed at host antigens will also attack components of the microbiome, and conversely, an immunological attack on the microbiome may cross‐react with host antigens producing “holoautoimmunity.”

     

Probiotics: Potent Immunomodulatory Tool Against Allergy

Probiotics are defined as live microbial food ingredients that produce several beneficial effects to human health. Probiotic bacteria have been mostly investigated in the prevention of and treatment for different gastrointestinal diseases and allergies. It is not fully clear how probiotics exert their beneficial effects on health, but one of the most probable mechanisms of action is the modulation of immune responses via the mucosal immune system of the gut. Commensal bacteria in the gastrointestinal tract play an integral role in both innate and humoral immunity. It is well established that this protective role can be maintained or modulated by the ingestion of probiotics. More recently, it has been shown that specific probiotic strains can influence the secretion of cytokines to help direct naïve T-helper cells toward either a Th1-dominant, cell-mediated immune response or toward a Th2-dominant, humoral immune response. This paper will review current knowledge of the Th1/Th2 model of humoral immunity as well as introduce how strain-specific probiotics can be used therapeutically to help balance this immune response and therefore help prevent allergy.     

益生菌、益生元和后生元对食物过敏的影响和作用机制

食物过敏(food allergy,FA)的发病率在近二十年来持续上升,已经严重影响患者的生活质量.生命早期接触外源性抗原少而导致的免疫耐受限制是FA的主要原因.微生物-宿主的相互作用与FA密切相关,健康的微生物菌群促进宿主在生命早期建立成熟的免疫系统,减少FA的易感性,因此,改善肠道菌群和调节机体免疫在治疗FA中至关...     

Nigella sativa (black cumin) seed extract alleviates symptoms of allergic diarrhea in mice, involving opioid receptors

The incidence of food hypersensitivity and food allergies is on the rise and new treatment approaches are needed. We investigated whether N. sativa, one of its components, thymoquinone, or synthetic opioid receptor (OR)-agonists can alleviate food allergy. Hence, ovalbumin (OVA)-sensitized BALB/c-mice were pre-treated either with a hexanic N. sativa seed extract, thymoquinone, kappa-(U50'4889) or mu-OR-agonists (DAMGO) and subsequently challenged intra-gastrically with OVA. All 4 treatments significantly decreased clinical scores of OVA-induced diarrhea. N. sativa seed extract, thymoquinone, and U50'488 also decreased intestinal mast cell numbers and plasma mouse mast cell protease-1 (MMCP-1). DAMGO, in contrast, had no effect on mast cell parameters but decreased IFNγ, IL-4, IL-5, and IL-10 concentration after ex vivo re-stimulation of mesenteric lymphocytes. The effects on allergy symptoms were reversible by OR-antagonist pre-treatment, whereas most of the effects on immunological parameter were not. We demonstrate that N. sativa seed extract significantly improves symptoms and immune parameters in murine OVA-induced allergic diarrhea; this effect is at least partially mediated by thymoquinone. ORs may also be involved and could be a new target for intestinal allergy symptom alleviation. N. sativa seed extract seems to be a promising candidate for nutritional interventions in humans with food allergy.     

Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity

Peanut allergy is one of the most life-threatening food allergies and one of the serious challenges facing the peanut and food industries. Current proposed solutions focus primarily on ways to alter the immune system of patients allergic to peanut. However, with the advent of genetic engineering novel strategies can be proposed to solve the problem of peanut allergy from the source. The objectives of this study were to eliminate the immunodominant Ara h 2 protein from transgenic peanut using RNA interference (RNAi), and to evaluate the allergenicity of resulting transgenic peanut seeds. A 265-bp-long PCR product was generated from the coding region of Ara h 2 genomic DNA, and cloned as inverted repeats in pHANNIBAL, an RNAi-inducing plant transformation vector. The Ara h 2-specific RNAi transformation cassette was subcloned into a binary pART27 vector to construct plasmid pDK28. Transgenic peanuts were produced by infecting peanut hypocotyl explants with Agrobacterium tumefaciens EHA 105 harbouring the pDK28 construct. A total of 59 kanamycin-resistant peanut plants were regenerated with phenotype and growth rates comparable to wild type. PCR and Southern analyses revealed that 44% of plants stably integrated the transgene. Sandwich ELISA performed using Ara h 2-mAbs revealed a significant ( P <  0.05) reduction in Ara h 2 content in several transgenic seeds. Western immunobloting performed with Ara h 2-mAb corroborated the results obtained with ELISA and showed absence of the Ara h 2 protein from crude extracts of several transgenic seeds of the T₀ plants. The allergenicity of transgenic peanut seeds expressed as IgE binding capacity was evaluated by ELISA using sera of patients allergic to peanut. The data showed a significant decrease in the IgE binding capacity of selected transgenic seeds compared to wild type, hence, demonstrating the feasibility of alleviating peanut allergy using the RNAi technology.     

Food allergy enhances allergic asthma in mice

Background

Atopic march refers to the typical transition from a food allergy in early childhood to allergic asthma in older children and adults. However the precise interplay of events involving gut, skin and pulmonary inflammation in this process is not completely understood.

Objectives

To develop a mouse model of mixed food and respiratory allergy mimicking the atopic march and better understand the impact of food allergies on asthma.

Methods

Food allergy to ovalbumin (OVA) was induced through intra-peritoneal sensitization and intra-gastric challenge, and/or a respiratory allergy to house dust mite (HDM) was obtained through percutaneous sensitization and intra-nasal challenges with dermatophagoides farinae (Der f) extract. Digestive, respiratory and systemic parameters were analyzed.

Results

OVA-mediated gut allergy was associated with an increase in jejunum permeability, and a worsening of Der f-induced asthma with stronger airway hyperresponsiveness and pulmonary cell infiltration, notably eosinophils. There was overproduction of the pro-eosinophil chemokine RANTES in broncho-alveolar lavages associated with an enhanced Th2 cytokine secretion and increased total and Der f-specific IgE when the two allergies were present. Both AHR and lung inflammation increased after a second pulmonary challenge.

Conclusion

Gut sensitization to OVA amplifies Der f-induced asthma in mice.     

New allergies after cord blood transplantation

Background aimsUmbilical cord blood transplantation (CBT) is an effective treatment for benign and malignant diseases. Late effects of CBT are not well described in the literature. In the present study, we present our experience of new-onset allergies in long-term survivors after CBT.MethodsAfter an initial patient had a severe peanut allergic reaction after CBT, all CBT patients were prospectively followed for new allergy development. Fifty patients received CBT between March 2006 and June 2011.ResultsThe median follow-up after CBT was 447 days (range, 12–2022). At the time of analysis, 30 patients were alive, with 3-year survival of 55.5%; median follow-up of surviving patients was 910 days (range, 68–2022). The allergic syndrome developed in five patients, with the cumulative incidence of new allergies at 2 years of 18.4% (95% confidence interval, 10.8–26). The median time to onset of new allergy after transplantation was 298 days (range, 250–809).ConclusionsAllergy development has been linked to a delayed maturation of the immune system in several studies. We present the first case series of patients who had new allergies after CBT. Further study of this novel complication as well as counseling of patients after CBT would be important.     

Gut microbiome and the risk factors in central nervous system autoimmunity

Humans are colonized after birth by microbial organisms that form a heterogeneous community, collectively termed microbiota. The genomic pool of this macro-community is named microbiome. The gut microbiota is essential for the complete development of the immune system, representing a binary network in which the microbiota interact with the host providing important immune and physiologic function and conversely the bacteria protect themselves from host immune defense. Alterations in the balance of the gut microbiome due to a combination of environmental and genetic factors can now be associated with detrimental or protective effects in experimental autoimmune diseases. These gut microbiome alterations can unbalance the gastrointestinal immune responses and influence distal effector sites leading to CNS disease including both demyelination and affective disorders. The current range of risk factors for MS includes genetic makeup and environmental elements. Of interest to this review is the consistency between this range of MS risk factors and the gut microbiome. We postulate that the gut microbiome serves as the niche where different MS risk factors merge, thereby influencing the disease process.     

Assessment of allergenicity of diploid and hexaploid wheat genotypes: identification of allergens in the albumin/globulin fraction

Wheat is an important part of the daily diet of millions of people. However, this staple food is also responsible for food allergies. Ancient cultivars of wheat are gaining interest today but nothing is known about their allergenicity. Many wheat proteins have been reported as causative food allergens, including some prolamin-type gluten proteins, and salt soluble proteins of the albumin/globulin (A/G) type. The objective of this work is to obtain information about the allergenicity of the salt soluble A/G fraction of an ancient diploid cultivar compared with a standard hexaploid bread wheat cultivar using 20 sera from patients with wheat allergy. Differences in the IgE reactivity of sera towards the two genotypes were quantified by ELISA. Qualitative differences in IgE-binding proteins were searched after 1D or 2D electrophoresis. For most of the sera, the concentration in A/G specific IgE was higher for the hexaploid T. aestivum (cv Récital) than for the diploid T. monococcum (cv Engrain). The analysis of 2D spots revealed by immunoblotting leads to the identification by mass spectrometry of 39 IgE-binding proteins, some of them unknown until now as wheat allergens. Numerous allergens were identified, differences observed between Engrain and Récital will be discussed.     

Elevated Antigen-Driven IL-9 Responses Are Prominent in Peanut Allergic Humans

Food allergies, and peanut allergy in particular, are leading causes of anaphylactic fatalities worldwide. The immune mechanisms that underlie food allergy remain ill-defined and controversial, in part because studies in humans typically focus on analysis of a limited number of prototypical Th1/Th2 cytokines. Here we determine the kinetics and prevalence of a broad panel of peanut-driven cytokine and chemokine responses in humans with current peanut allergy vs those with stable, naturally occurring clinical tolerance to peanut. Our primary focus is identification of novel indicators of immune dysregulation. Antigen-specific cytokine mRNA and protein responses were elicited in primary culture via peanut or irrelevant antigen (Leishmania extract, milk antigens) mediated stimulation of fresh peripheral blood cells from 40 individuals. Peanut extract exposure in vitro induced a broad panel of responses associated with Th2/Th9-like, Th1-like and Th17-like immunity. Peanut-dependent Type 2 cytokine responses were frequently found in both peanut allergic individuals and those who exhibit clinical tolerance to peanut ingestion. Among Th2/Th9-associated cytokines, IL-9 responses discriminated between allergic and clinically tolerant populations better than did commonly used IL-4, IL-5 or IL-13 responses. Comparison with responses evoked by unrelated control antigen-mediated stimulation showed that these differences are antigen-dependent and allergen-specific. Conversely, the intensity of IL-12, IL-17, IL-23 and IFN-γ production was indistinguishable in peanut allergic and peanut tolerant populations. In summary, the ability to generate and maintain cytokine responses to peanut is not inherently distinct between allergic and peanut tolerant humans. Quantitative differences in the intensity of cytokine production better reflects clinical phenotype, with optimally useful indicators being IL-9, IL-5, IL-13 and IL-4. Equivalent, and minimal, Ag-dependent pro-inflammatory cytokine levels in both healthy and peanut allergic volunteers argues against a key role for such cytokines in maintenance of clinical tolerance to food antigens in humans.     

Focus: Allergic Diseases and Type II Immunity: Strategies for Mast Cell Inhibition in Food Allergy

Mast cells are tissue resident allergic effector cells that drive IgE-mediated food allergies. There are several steps leading to mast cell activation in the context of allergic disease that can be targeted to prevent mast cell activation and degranulation. These include blocking IgE-FcεRI crosslinking and type 2 cytokine receptor activation; modulating cell-surface neural chemical receptors; stabilizing mast cell membranes to prevent co-localization of activating receptors; impeding intracellular signaling; and engaging cell surface inhibitory receptors. This review highlights several ITIM-containing inhibitory mast cell surface receptors that could serve as pharmaceutical targets to prevent mast cell activation and degranulation in the context of food allergy. When activated, these ITIM-containing inhibitory receptors recruit the phosphatases SHP-1, SHP-2, and/or SHIP to dephosphorylate the tyrosine kinases responsible for activation signals downstream of the IgE-FcεRI complex. We describe several members of the Ig and Ig-like inhibitory receptor and C-type lectin inhibitory receptor superfamilies. Fundamental studies exploring the behavior of these receptors within the context of experimental food allergy models are needed. A deeper understanding of how these receptors modulate mast cell-driven food allergic responses will shape future strategies to harness these inhibitory receptors to treat food allergy.     

Allergy and risk of acute lymphoblastic leukemia among children: A nationwide case control study in Greece

Background: Several reports point to inverse associations between allergies and ALL; yet, no study has explored this link using both self-reported-data on allergic history and biomarkers of atopic sensitization. Methods: Clinical information for the variables of interest was available for 252 out of 292 cases of childhood (0–14 years) ALL, newly diagnosed across Greece over a 4.5 year period as well as for 294 hospital controls. Allergen-specific-IgEs, as markers of allergic predisposition, against 24 most prevalent respiratory and food allergens, were determined, using an enzyme immunoassay procedure for 199 children with ALL and 113 controls. Cases were compared with controls through frequency distributions and unconditional multiple logistic regression models to estimate odds ratios (ORs) and 95% confidence-intervals (CIs) regarding associations of allergy with childhood ALL. Results: Self-reported-allergic history overall (OR: 0.49, 95%CI: 0.34–0.72) and practically each one of its main components (respiratory, food, any other clinical allergy) were strongly and inversely associated with ALL. Likewise, the serum IgE inverse association was of the same magnitude (OR: 0.43, 95%CI: 0.22–0.84) mainly contributed by food IgE (OR: 0.39, 95%CI: 0.18–0.83). Conclusion: Beyond the already established inverse association of allergic history with childhood ALL, a same magnitude association is evident when serologic markers of allergic predisposition are used as an alternative measure of allergy. Further research with more appropriate study designs is needed to better understand possible associations between prior allergy and childhood ALL risk.     

A preliminary study of gut dysbiosis in children with food allergy

Gut microbiota of food allergic children was analyzed by high throughput 16S rRNA gene sequencing. Signs of gut dysbiosis, which is likely associated with gut inflammation, was observed in children with food allergies. For example, decreased abundance of genus Akkermansia but increased abundance of Veillonella was found in children with food allergy in comparison with healthy control children.     

Biotechnologies in new high-throughput food allergy tests: why we need them

The increase in prevalence of food allergies generates a need for more accurate and reliable quantitative allergy testing in order to help diagnosis. In this short review, we briefly outline the history of food allergy testing and extend our comments to current multiplex techniques. Particular emphasis is given to new developments in the protein microarray area, where the use of recent advances in biotechnology has the potential to produce high-throughput devices with improved clinical significance.