Allergen-specific Manner Research Articles

Peanut allergen inhibition prevents anaphylaxis in a humanized mouse model.

Peanut-induced allergy is an immunoglobulin E (IgE)-mediated type I hypersensitivity reaction that manifests symptoms ranging from local edema to life-threatening anaphylaxis. Although there are treatments for symptoms in patients with allergies resulting from allergen exposure, there are few preventive therapies other than strict dietary avoidance or oral immunotherapy, neither of which are successful in all patients. We have previously designed a covalent heterobivalent inhibitor (cHBI) that binds in an allergen-specific manner as a preventive for allergic reactions. Building on previous in vitro testing, here, we developed a humanized mouse model to test cHBI efficacy in vivo. Nonobese diabetic-severe combined immunodeficient γc-deficient mice expressing transgenes for human stem cell factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3 developed mature functional human mast cells in multiple tissues and displayed robust anaphylactic reactions when passively sensitized with patient-derived IgE monoclonal antibodies specific for peanut Arachis hypogaea 2 (Ara h 2). The allergic response in humanized mice was IgE dose dependent and was mediated by human mast cells. Using this humanized mouse model, we showed that cHBI prevented allergic reactions for more than 2 weeks when administered before allergen exposure. cHBI also prevented fatal anaphylaxis and attenuated allergic reactions when administered shortly after the onset of symptoms. cHBI impaired mast cell degranulation in vivo in an allergen-specific manner. cHBI rescued the mice from lethal anaphylactic responses during oral Ara h 2 allergen-induced anaphylaxis. Together, these findings suggest that cHBI has the potential to be an effective preventative for peanut-specific allergic responses in patients.

Covalent heterobivalent inhibitor effectively inhibits anaphylaxis to peanut allergen in a humanized mouse model

Abstract Peanut-induced allergy is an IgE-mediated type I hypersensitivity reaction that manifests symptoms ranging from local edema to life-threatening anaphylaxis. While there are treatments for symptoms in allergic patients resulting from allergen exposure, there are few preventive therapies other than avoidance and no allergen specific therapies. We have previously designed a covalent heterobivalent inhibitor (cHBI) that binds in an allergen-specific manner as a preventive for allergic reactions. Building on previous in vitro testing, in this report we have developed a humanized mouse model to test cHBI efficacy in vivo. Humanized mice (NSG-SCF/GM-CSF/IL-3) developed mature functional human mast cells in various tissues and developed robust anaphylactic reactions when passively sensitized with human IgE monoclonal antibodies specific for peanut allergen. We found that the allergic response is IgE dose-dependent and is mediated by human mast cells indicated by elevated tryptase serum levels and the upregulation of mast cell degranulation markers. Using the validated humanized mouse model, we showed that cHBI inhibited IgE-mediated anaphylaxis and human mast cell degranulation. We demonstrated that cHBI effectively inhibits anaphylaxis for up to 14 days, and cHBI inhibition is specific to peanut allergen. Importantly, cHBI rescued the mice from lethal anaphylactic response during oral peanut-induced anaphylaxis. These findings suggest that cHBI has the potential to be an effective preventative for peanut food allergy in patients.

Toll-like receptor ligand-peptide conjugates in the immunomodulatory response of food allergy

Covalent conjugation of allergens to Toll-Like Receptor (TLR) ligands lead to structures that enhance allergen interaction with antigen presenting cells and modulate the immune response. They can present peptide epitopes in an allergen-specific manner, with potential applications for designing vaccines and developing a tolerance response in food allergy (FA). We aimed to address the potential applicability of TLR4/TLR7 ligands and Pru p 3 allergen conjugates in FA immunotherapy.

Human cystatin SN is an endogenous protease inhibitor that prevents allergic rhinitis

Protease allergens disrupt epithelial barriers to exert their allergenicity. Cystatin SN (encoded by CST1) is an endogenous cysteine protease inhibitor upregulated in nasal epithelia in patients with allergic rhinitis (AR). We sought to investigate the protective effect of human cystatin SN on AR symptoms using pollen-induced AR mouse models. We performed an invitro protease activity assay to evaluate the effect of recombinant human cystatin SN (rhCystatin SN) on Japanese cedar (JC) or ragweed proteases. Ahuman nasal epithelial cell line, RPMI 2650, was used to examine tight junction (TJ) disruption invitro. Mice were sensitized and nasally challenged with JC or ragweed pollens with or without rhCystatin SN to examine the effect of rhCystatin SN on AR symptoms and the epithelial barrier invivo. Because mice lack CST1, we generated transgenic (Tg) mice expressing human CST1 under control of its genomic control region (hCST1-Tg mice) to examine the role of cystatin SN in physiologically expressed conditions. rhCystatin SN inhibited JC but not ragweed protease activities and prevented JC-induced but not ragweed-induced TJ disruption invitro. Exogenous administration of rhCystatin SN ameliorated JC-induced but not ragweed-induced sneezing and nasal TJ disruption invivo. Furthermore, hCST1-Tg mice showed decreased JC-induced but not ragweed-induced sneezing symptoms and nasal TJ disruption compared with wild-type mice. Human cystatin SN suppresses AR symptoms through inhibiting allergen protease activities and protecting the nasal TJ barrier in an allergen-specific manner. We propose that upregulation of nasal endogenous protease inhibitors, including cystatin SN, is a novel therapeutic strategy for protease allergen-induced AR.

In vivo induction of regulatory T cells promotes allergen tolerance and suppresses allergic contact dermatitis

Allergic contact dermatitis (ACD) is a common T-cell mediated inflammatory skin condition, characterized by an intensely pruritic rash at the site of contact with allergens like poison ivy or nickel. Current clinical treatments use topical corticosteroids, which broadly and transiently suppress inflammation and symptoms of ACD, but fail to address the underlying immune dysfunction. Here, we present an alternative therapeutic approach that teaches the immune system to tolerate contact allergens by expanding populations of naturally suppressive allergen-specific regulatory T cells (Tregs). Specifically, biodegradable poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA) microparticles were engineered to release TGF-β1, Rapamycin, and IL-2, to locally sustain a microenvironment that promotes Treg differentiation. By expanding allergen-specific Tregs and reducing pro-inflammatory effector T cells, these microparticles inhibited destructive hypersensitivity responses to subsequent allergen exposure in an allergen-specific manner, effectively preventing or reversing ACD in previously sensitized mice. Ultimately, this approach to in vivo Treg induction could also enable novel therapies for transplant rejection and autoimmune diseases.

Allergen-encoding bone marrow transfer inactivates allergic T cell responses, alleviating airway inflammation.

Memory Th2 cell responses underlie the development and perpetuation of allergic diseases. Because these states result from immune dysregulation, established Th2 cell responses represent a significant challenge for conventional immunotherapies. New approaches that overcome the detrimental effects of immune dysregulation are required. We tested whether memory Th2 cell responses were silenced using a therapeutic approach where allergen expression in DCs is transferred to sensitized recipients using BM cells as a vector for therapeutic gene transfer. Development of allergen-specific Th2 responses and allergen-induced airway inflammation was blocked by expression of allergen in DCs. Adoptive transfer studies showed that Th2 responses were inactivated by a combination of deletion and induction of T cell unresponsiveness. Transfer of BM encoding allergen expression targeted to DCs terminated, in an allergen-specific manner, Th2 responses in sensitized recipients. Importantly, when preexisting airway inflammation was present, there was effective silencing of Th2 cell responses, airway inflammation was alleviated, and airway hyperreactivity was reversed. The effectiveness of DC-targeted allergen expression to terminate established Th2 responses in sensitized animals indicates that exploiting cell-intrinsic T cell tolerance pathways could lead to development of highly effective immunotherapies.

Comparison of two oral probiotic preparations in a randomized crossover trial highlights a potentially beneficial effect of Lactobacillus paracasei NCC2461 in patients with allergic rhinitis

BackgroundThere is promising but conflicting evidence to recommend the addition of probiotics to foods for prevention and treatment of allergy. Based on previous studies with fermented milk containing Lactobacillus paracasei NCC2461, we aimed to compare the effect of a powder form of the latter probiotic with the effect of a blend of Lactobacillus acidophilus ATCC SD5221 and Bifidobacterium lactis ATCC SD5219 in patients with allergic rhinitis.MethodsA double-blind, randomized, cross-over study, involving 31 adults with allergic rhinitis to grass pollen, was performed outside the grass pollen season (registration number: NCT01233154). Subjects received each product for 4-weeks in two phases separated by a wash-out period of 6 to 8 weeks. A nasal provocation test was performed before and after each 4-week product intake period, and outcome parameters (objective and subjective clinical symptoms; immune parameters) were measured during and/or 24 hours after the test.ResultsOut of the 31 subject enrolled, 28 completed the study. While no effect was observed on nasal congestion (primary outcome), treatment with NCC2461 significantly decreased nasal pruritus (determined by VAS), and leukocytes in nasal fluid samples, enhanced IL-5, IL-13 and IL-10 production by peripheral blood mononuclear cells in an allergen specific manner and tended to decrease IL-5 secretion in nasal fluid, in contrast to treatment with the blend of L. acidophilus and B. lactis.ConclusionsDespite short-term consumption, NCC2461 was able to reduce subjective nasal pruritus while not affecting nasal congestion in adults suffering from grass pollen allergic rhinitis. The associated decrease in nasal fluid leukocytes and IL-5 secretion, and the enhanced IL-10 secretion in an allergen specific manner may partly explain the decrease in nasal pruritus. However, somewhat unexpected systemic immune changes were also noted. These data support the study of NCC2461 consumption in a seasonal clinical trial to further demonstrate its potentially beneficial effect.

Ubiquitin E3 Ligase A20 Contributes to Maintaining Epithelial Barrier Function

Background and Aims: Epithelial barrier dysfunction is involved in the pathogenesis of allergic diseases; the mechanism is to be further understood. Ubiquitin E3 ligase A20 (A20) plays a role in maintaining the homeostasis in the body. This study aims to investigate the role of A20 in maintaining the epithelial barrier function. Methods: Human intestinal epithelial cell line, Caco-2 cells, was cultured to monolayers to test the endocytosis and degradation of a model allergen, ovalbumin (OVA). The role of A20 in the endosome/lysosome fusion in epithelial cells was tested with A20-sufficient and A20-deficient Caco-2 cells and visualized by immunocytochemistry. Results: Caco-2 cells could endocytose exogenous allergens (OVA) in culture. The endocytic OVA was degraded in A20-sufficient Caco-2 cells via the mechanism of endosome/lysosome fusion, while the A20-deficient Caco-2 monolayers converted the OVA to the basal compartment of transwells, which conserved the antigenicity reflected by that it induced T cell proliferation in an allergen-specific manner. A20 was required in the fusion of endosomes and lysosomes. Conclusion: A20 contributes to maintaining the epithelial barrier function.

T cell killing by tolerogenic dendritic cells protects mice from allergy

It is well established that allergy development can be prevented by repeated low-dose exposure to contact allergens. Exactly which immune mechanisms are responsible for this so-called low zone tolerance (LZT) is not clear, although CD8⁺ suppressor T cells are known to have a role. Here, we show that TNF released by tolerogenic CD11⁺CD8⁺ DCs located in skin-draining lymph nodes is required and sufficient for development of tolerance to contact allergens in mice. DC-derived TNF protected mice from contact allergy by inducing apoptosis in allergen-specific effector CD8⁺ T cells via TNF receptor 2 but did not contribute to the generation and function of the regulatory T cells associated with LZT. The TNF-mediated killing mechanism was induced in an allergen-specific manner. Activation of tolerogenic DCs by LZT CD8⁺ suppressor T cells and enhanced TNF receptor 2 expression on contact allergen-specific CD8⁺ effector T cells were required for LZT. Our findings may explain how tolerance protects from allergic diseases, which could allow for the development of new strategies for allergy prevention.

Allergen‐specific IgE testing in the diagnosis of food allergy and the event of a positive match in the bioinformatics search

Current documents on risk assessment of genetically modified foods recommend including IgE-binding tests on sera from allergic patients. However, there is no generally accepted recommendation on technical aspects of the testing procedures or on the interpretation of the results, despite that fact that both false positive and false-negative results may be caused by variability of the test procedures. The present article discusses the state-of-the-art of serological test procedures for qualitative and quantitative determination of specific IgE and interpretation of test results. It is emphasized that the use of sera from clinically well-characterized subjects is of high importance. In the case of a positive test result, the biological activity of the detected IgE antibodies, i. e., the potential to trigger mediator release from basophils or mast cells in an allergen-specific manner, should be taken into account. However, present data also indicate that validation of such mediator release tests is required, both in terms of experimental protocols and with respect to correlation of the test results with the clinical situation. Further studies are also required to prove the usefulness of targeted serum screening, i. e., the testing of gene products from organisms not known to be allergenic with sera from subjects allergic to related species.

Theophylline Suppresses IL-5 and IL-13 Production, and Lymphocyte Proliferation upon Stimulation with House Dust Mite in Asthmatic Children

Background: Although theophylline has long been used as a medication for bronchial asthma (BA), much remains to be elucidated about its action on Th2 cells, which play a critical role in the development of BA. Objective: This study aimed to clarify the effect of theophylline on Th2 cells in an allergen-specific manner in children with BA. Methods: Peripheral blood mononuclear cells (PBMCs) from 32 children with BA were stimulated with house dust mite (HDM) or a purified HDM allergen Der f 1 in the presence or absence of theophylline at a therapeutic concentration (5–20 µg/ml). The proliferation of lymphocytes and the secretion of IL-5 and IL-13 were measured to estimate the influence of theophylline. Results: Theophylline at a concentration of 20 µg/ml significantly suppressed lymphocyte proliferation induced by 6-day culture with HDM or Der f 1. It also significantly reduced the production of IL-5 and IL-13 upon stimulation with HDM or Der f 1. Suppression of cytokine production and lymphocyte proliferation increased with concentrations of theopylline between 5 and 20 µg/ml. Conclusion: Theophylline suppresses the proliferation of lymphocytes and the production of proinflammatory Th2 cytokines, IL-5 and IL-13 induced by stimulation of PBMCs with HDM in children with BA.

Reversal of the adult IgE high responder phenotype in mice by maternally transferred allergen-specific monoclonal IgG antibodies during a sensitive period in early ontogeny.

IgE is an important trigger in allergy and asthma, diseases whose development is suggested to depend on an initial sensitization in early life. While induction of murine IgE responses requires both a genetically based IgE high responder phenotype and defined experimental conditions, maternally transferred IgG can override these prerequisites and suppress IgE formation in an allergen-specific manner. Here, we show that maternally transferred monoclonal IgG, irrespective of their subclass and recognized epitopes, induce IgE unresponsiveness, which is effective for parenteral immunization with bee venom phospholipase A2 as well as for airway-immunization with nebulized ovomucoid-containing ovalbumin. This IgE suppression is detectable in the offspring during the first 4 months of life, but not thereafter and not in the dams. However, when the initial immunization at an age of 3 or 4 months was followed by further application of both allergens via their respective routes, IgE suppression persisted up to an age of more than one year. If applicable to man, these findings may allow the development of a new strategy for the prevention of allergy and asthma by maternally transferred or neonatally injected allergen-specific mAb in combination with natural or prophylactic exposure to the respective allergens during early childhood.

Detection of Multiple Allergen-specific IgEs on Microarrays by Immunoassay with Rolling Circle Amplification

First described in 1967, the radio allergo sorbent test (RAST) has been the standard technique for measuring allergen-specific IgE antibodies in serum (1). An updated version of the RAST test, termed CAP (Pharmacia), has been introduced (2). In clinical practice, CAP results must be interpreted with care. The diagnostic performance of CAP varies in an allergen-specific manner, and CAP scores do not always correlate with clinical severity (3)(4). CAP sensitivity, specificity, and positive predictive values agree well with skin prick tests (SPTs) for house dust mites and grasses, but poorly with tests for cat dander and peanuts (5). Microarray technology potentially offers advantages in diagnostic applications such as allergy testing because the amount of reagent required, and thus the cost per assay, is greatly reduced (6). This approach has been difficult to reduce to practice, however, because the extremely small volumes (∼0.5–5 nL) of sample used to create spots on these microarrays require extremely sensitive methods of analyte detection (7). We have used rolling circle amplification (RCA) (8) for the detection of antibody bound to antigen (9). In this “immunoRCA”, the 5′ end of a RCA primer is attached to an antibody; thus, in the presence of circular DNA, DNA polymerase, and nucleotides, the rolling circle reaction produces a concatamer of circular DNA sequence copies that remain attached to the antibody. The amplified DNA can be detected by hybridization of complementary oligonucleotide probes. ImmunoRCA, therefore, represents a novel approach for signal amplification of antibody-antigen recognition events on microarrays. ImmunoRCA can detect IgE in a format using high-density microarrays of anti-human IgE printed on glass slides by a pin-tool type microarraying robot (9). Here, we describe the production of microarrays of multiple allergens and demonstrate the utility of these microarrays in combination with immunoRCA to simultaneously detect …

Regulation of allergen-specific immune responses by CD4+ CD45R+ cells in patients with allergic contact dermatitis

Circulating T lymphocytes from 20 patients with an immediate patch test reaction were tested for proliferative responses in vitro to contact allergens during both immediate and delayed skin reactions. T lymphocytes collected during the immediate patch test reaction responded specifically to the challenge allergens in the presence of autologous monocytes. Subset analysis revealed that the proliferation pattern was dominated by the CD4+CD29+ T-cell subpopulation, whereas CD8+ or CD4+CD45R+ cells did not respond. A similar pattern in T-cell subset proliferation was observed when cells were collected during a positive delayed skin reaction. In contrast, in the case of a negative delayed skin reaction, proliferative responses of lymphocytes to the specific allergens were dominated by CD45R+ cells. The latter T-lymphocyte subset could greatly suppress in an allergen-specific manner the proliferation of CD29+ autologous cells. The in vitro allergen-specific proliferation of CD29+ or CD45R+ cells was restricted by the major histocompatibility complex class II (HLA-DR) gene products. It is suggested that allergen-specific immune responses take place in the induction and evolution of an immediate patch test reaction into a delayed one.