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The human endogenous cannabinoid system (ECS) regulates key physiological processes and alterations in its signaling pathways, and endocannabinoid levels are associated with diseases such as neurological and neuropsychiatric conditions, cancer, pain and inflammation, obesity, and metabolic and different immune related disorders. Immune system cells express the G-protein coupled cannabinoid receptor 1 (CB 1 ), but its functional role has not been fully understood, likely due to the lack of appropriate tools. The availability of novel tools to investigate the role of CB 1 in immune regulation might contribute to identify CB 1 as a potential novel therapeutic target or biomarker for many diseases. Herein, we report the development and validation of the first fluorescent small molecule probe to directly visualize and quantify CB 1 in blood and tonsil immune cells by flow cytometry and confocal microscopy. We coupled the cannabinoid agonist HU210 to the fluorescent tag Alexa Fluor 488, generating a fluorescent probe with high affinity for CB 1 and selectivity over CB 2 . We validate HU210-Alexa488 for the rapid, simultaneous, and reproducible identification of CB 1 in human monocytes, T cells, and B cells by multiplexed flow cytometry. This probe is also suitable for the direct visualization of CB 1 in tonsil tissues, allowing the in vivo identification of tonsil CB 1 -expressing T and B cells. This study provides the first fluorescent chemical tool to investigate CB 1 expression and function in human blood and tonsil immune cells, which might well pave the way to unravel essential features of CB 1 in different immune and ECS-related diseases.

Recurrent respiratory tract infections (RRTIs) are the first leading cause of community- and nosocomial-acquired infections. Antibiotics remain the mainstay of treatment, enhancing the potential to develop antibiotic resistances. Therefore, the development of new alternative approaches to prevent and treat RRTIs is highly demanded. Daily sublingual administration of the whole heat-inactivated polybacterial preparation (PBP) MV130 significantly reduced the rate of respiratory infections in RRTIs patients, however, the immunological mechanisms of action remain unknown. Herein, we study the capacity of MV130 to immunomodulate the function of human dendritic cells (DCs) as a potential mechanism that contribute to the clinical benefits. We demonstrate that DCs from RRTIs patients and healthy controls display similar ex vivo immunological responses to MV130. By combining systems biology and functional immunological approaches we show that MV130 promotes the generation of Th1/Th17 responses via receptor-interacting serine/threonine-protein kinase-2 (RIPK2)- and myeloid-differentiation primary-response gene-88 (MyD88)-mediated signalling pathways under the control of IL-10. In vivo BALB/c mice sublingually immunized with MV130 display potent systemic Th1/Th17 and IL-10 responses against related and unrelated antigens. We elucidate immunological mechanisms underlying the potential way of action of MV130, which might help to design alternative treatments in other clinical conditions with high risk of recurrent infections., (© 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Allergen-specific immunotherapy (AIT) is the single disease-modifying treatment for allergy. Clinical trials show AIT to be safe and effective for many patients; however, it still faces problems related to efficacy, safety, long treatment duration and low patient adherence. There has been intensive research to develop alternative strategies, including novel administration routes, adjuvants or hypoallergenic molecules. Promising results are reported for some of them, but clinical progress is still moderate. Allergoids conjugated to nonoxidized mannan from  Saccharomyces cerevisiae  have emerged as a novel concept of vaccine targeting dendritic cells (DCs). Preclinical human and animal models demonstrated that allergoids conjugated to mannan enhance allergen uptake, promote healthy responses to allergens by inducing Th1 and T regulatory (Treg) cells, and show clinical efficacy in veterinary medicine. Dose-finding phase II clinical trials in humans are currently ongoing. We review the current stage of allergoids conjugated to mannan as next generation vaccines for AIT., Competing Interests: C. Benito-Villalvilla declares that she has no competing interests. I. Soria is employee of Inmunotek S.L. J.L. Subiza is the CEO-President of Inmunotek S.L. O. Palomares received lecture fees from Allergy Therapeutics, Amgen, AstraZeneca, Inmunotek, Novartis, Sanofi-Genzyme and Stallergenes and participated in advisory boards from Novartis and Sanofi-Genzyme.

Background: Apart from active allergen avoidance, immunotherapy is regarded as the most effective form of treatment available for type I allergies. Such treatments involve the administration of allergen preparations in various forms and by various routes. Virus-like particles (VLPs) offer a very effective platform for immunization with the allergen and are characterized by high immunogenicity, low allergenicity and high clinical efficacy. Formulations that include Toll-like receptor ligands, T cell stimulatory epitopes and/or depot-forming adjuvants appear to enhance activation of the relevant immune cells. Short nucleotide sequences including CpG motifs have also been intensively explored as potent stimulators of dendritic cells and B cells., Methods: The present paper is based on a systematic literature search in PubMed and MEDLINE, and focuses on the pertinent immunological processes and on clinical data relating to use of VLPs and CpG motifs for the treatment of allergic rhinitis (AR)., Results: Many published studies have reported positive clinical results following administration of VLPs, either alone or in combination with CpG motifs and, in some cases, even in the absence of the allergen-specific allergen., Conclusions: These results indicate that VLPs modulate immune responses in ways which underline their exceptional promise as a platform for the immunotherapy of allergic disorders. However, clinical evaluations remain limited, and further large-scale and longer-term studies will be necessary to substantiate the efficacy and safety of these novel therapies., Competing Interests: L. Klimek has received research support for the execution of studies, and fees for consultancy work, lectures and/or training activities, and/or remuneration as a member of a Scientific Advisory Board from ALK-Abelló, Allergopharma, Bencard, Bionorica, Biomay, Boehringer Ingelheim, Cytos, Circassia, HAL, GSK, Leti, Lofarma, MEDA/Mylan, Novartis, Stallergenes and Roxall. T. Kündig is developing VLPs for clinical use. M.F. Kramer and S. Guethoff are staff members of Bencard Allergie GmbH/Allergy Therapeutics plc; this company is developing VLPs for clinical use. C.B. Schmidt-Weber has received research support, fees for consultancy work, lectures, and travel costs from Allergopharma, Leti Pharma, Bencard, Regeneron, Leo Pharma, Novartis and owns shares of Pls Design and Tolerogenics. T. Jakob has received research support from ALK-Abello, Allergopharma, Cosmetics Europe, Novartis and Thermo Fisher Scientific; and lecturing and consulting fees from ALK-Abello, Allergy Therapeutics/Bencard, Allergopharma, Leti and Novartis. M. Bachmann is developing VLPs for clinical use. E. Jensen-Jarolim, O. Palomares and M.O. Mohsen declare that they have no competing interests.

We have recently reported that grass pollen allergoids conjugated with nonoxidized mannan of Saccharomyces cerevisae using glutaraldehyde results in a novel hypoallergenic mannan-allergen complex with improved properties for allergen vaccination. Using this approach, human dendritic cells show a better allergen uptake and cytokine profile production (higher IL-10/IL-4 ratio) for therapeutic purposes. Here we aim to address whether a similar approach can be extended to dogs using canine dendritic cells. Six healthy Spanish Greyhound dogs were used as blood donors to obtain canine dendritic cells (DC) derived from peripheral blood monocytes. Allergens from Dermatophagoides farinae mite were polymerized and conjugated with nonoxidized mannan. Nuclear magnetic resonance (NMR), gel electrophoresis (SDS-PAGE), immunoblotting and IgE-ELISA inhibition studies were conducted to evaluate the main characteristics of the allergoid obtained. Mannan-allergen conjugate and controls were assayed in vitro for canine DC uptake and production of IL-4 and IL-10. The results indicate that the conjugation of D. farinae allergens with nonoxidized mannan was feasible using glutaraldehyde. The resulting product was a polymerized structure showing a high molecular weight as detected by NMR and SDS-PAGE analysis. The mannan-allergen conjugate was hypoallergenic with a reduced reactivity with specific dog IgE. An increase in both allergen uptake and IL-10/IL-4 ratio was obtained when canine DCs were incubated with the mannan-allergen conjugate, as compared with the control allergen preparations (unmodified D. farinae allergens and oxidized mannan-allergen conjugate). We conclude that hypoallergenic D. farinae allergens coupled to nonoxidized mannan is a novel allergen preparation suitable for canine allergy immunotherapy targeting dendritic cells., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Allergy is a major public health problem with a high socio-economic impact. The number of allergic patients is expected to reach to four billion within two decades when the World's population reaches to 10 billion. Our knowledge on the molecular mechanisms underlying allergic diseases and allergen tolerance induction had significant advances during the last years. Nowadays, it is well accepted that the generation and maintenance of allergen-specific regulatory T cells (Tregs) and regulatory B cells (Bregs) and the involvement of their suppressive cytokines and surface molecules are essential for the induction of allergen tolerance. These mechanisms play essential roles for the restoration of healthy immune responses to allergens in allergen-specific immunotherapy (AIT) and healthy immune response during high-dose antigen exposure in beekeepers and cat owners. AIT remains as the only disease-modifying and curative treatment for allergic diseases and represents a perfect model to investigate the antigen-specific immune responses in humans. A large number of clinical trials demonstrated AIT as an effective treatment in many patients, but it still faces several drawbacks in relation to efficacy, safety, long duration, and patient adherence. Novel strategies to overcome these inconveniences, such as the development of novel adjuvants and alternative routes of administration are being developed. The better understanding of the molecular mechanism governing the generation of Treg and Breg cells during allergen tolerance might well open new avenues for alternative therapeutic interventions in allergic diseases and help better understanding of other immune-tolerance-related diseases., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Tonsils have an active role in immune defence and inducing and maintaining tolerance to allergens. Vitamins A, D, and E, and antimicrobial peptide LL-37 may have immunomodulatory effects. We studied how their serum levels were associated with allergy status, intratonsillar/nasopharyngeal virus detection and intratonsillar expression of T cell- and innate immune response-specific cytokines, transcription factors and type I/II/III interferons in patients undergoing tonsillectomy., Methods: 110 elective tonsillectomy patients participated. Serum levels of vitamins A, 25(OH)D, and E, LL-37 and allergen-specific IgE as well as nasopharyngeal/intratonsillar respiratory viruses were analyzed. The mRNA expression of IFN-α, IFN-β, IFN-γ, IL-10, IL-13, IL-17, IL-28, IL-29, IL-37, TGF-β, FOXP3, GATA3, RORC2 and Tbet in tonsils were analyzed by quantitative RT-PCR., Results: The median age of the patients was 16 years (range 3-60), 28% of subjects had atopy, and 57% carried ≥1 respiratory virus in nasopharynx. Detection of viruses decreased by age. Higher vitamin A levels showed borderline significance with less viral detection (P = 0.056). Higher 25(OH)D was associated with less allergic rhinitis and atopy (P < 0.05) and higher vitamin E with less self-reported allergy (P < 0.05). In gene expression analyses, 25(OH)D was associated with higher IL-37, vitamin A with higher IFN-γ and vitamin E with less IL-28 (P < 0.05). LL-37 was associated with less FOXP3, RORC2 and IL-17 in tonsils (P < 0.05)., Conclusions: Vitamin D and E levels were associated with less allergic disorders. Vitamin A was linked to antiviral and vitamin D with anti-inflammatory activity. LL-37 and was linked to T regulatory cell effects.

Background: Allergen immunotherapy (AIT) is the only curative treatment for allergy. AIT faces pitfalls related to efficacy, security, duration, and patient compliance. Novel vaccines overcoming such inconveniences are in demand., Objectives: We sought to study the immunologic mechanisms of action for novel vaccines targeting dendritic cells (DCs) generated by coupling glutaraldehyde-polymerized grass pollen allergoids to nonoxidized mannan (PM) compared with glutaraldehyde-polymerized allergoids (P) or native grass pollen extracts (N)., Methods: Skin prick tests and basophil activation tests with N, P, or PM were performed in patients with grass pollen allergy. IgE-blocking experiments, flow cytometry, confocal microscopy, cocultures, suppression assays, real-time quantitative PCR, ELISAs, and ELISpot assays were performed to assess allergen capture by human DCs and T-cell responses. BALB/c mice were immunized with PM, N, or P. Antibody levels, cytokine production by splenocytes, and splenic forkhead box P3 (FOXP3)(+) regulatory T (Treg) cells were quantified. Experiments with oxidized PM were also performed., Results: PM displays in vivo hypoallergenicity, induces potent blocking antibodies, and is captured by human DCs much more efficiently than N or P by mechanisms depending on mannose receptor- and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-mediated internalization. PM endorses human DCs to generate functional FOXP3(+) Treg cells through programmed death ligand 1. Immunization of mice with PM induces a shift to nonallergic responses and increases the frequency of splenic FOXP3(+) Treg cells. Mild oxidation impairs these effects in human subjects and mice, demonstrating the essential role of preserving the carbohydrate structure of mannan., Conclusions: Allergoids conjugated to nonoxidized mannan represent suitable vaccines for AIT. Our findings might also be of the utmost relevance to development of therapeutic interventions in other immune tolerance-related diseases., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Immunotherapy for treating IgE-mediated allergies requires high doses of the corresponding allergen. This may result in undesired side effects and, to avoid them, hypoallergenic allergens (allergoids) polymerized with glutaraldehyde are commonly used. Targeting allergoids to dendritic cells to enhance cell uptake may result in a more effective immunotherapy. Allergoids coupled to yeast mannan, as source of polymannoses, would be suitable for this purpose, since mannose-binding receptors are expressed on these cells. Conventional conjugation procedures of mannan to proteins use oxidized mannan to release reactive aldehydes able to bind to free amino groups in the protein; yet, allergoids lack these latter because their previous treatment with glutaraldehyde. The aim of this study was to obtain allergoids conjugated to mannan by an alternative approach based on just glutaraldehyde treatment, taking advantage of the mannoprotein bound to the polymannose backbone. Allergoid-mannan glycoconjugates were produced in a single step by treating with glutaraldehyde a defined mixture of allergens derived from Phleum pratense grass pollen and native mannan (non-oxidized) from Saccharomyces cerevisae. Analytical and structural studies, including 2D-DOSY and (1)H-(13)C HSQC nuclear magnetic resonance spectra, demonstrated the feasibility of such an approach. The glycoconjugates obtained were polymers of high molecular weight showing a higher stability than the native allergen or the conventional allergoid without mannan. The allergoid-mannan glycoconjugates were hypoallergenic as detected by the IgE reactivity with sera from grass allergic patients, even with lower reactivity than conventional allergoid without mannan. Thus, stable hypoallergenic allergoids conjugated to mannan suitable for using in immunotherapy can be achieved using glutaraldehyde. In contrast to mannan oxidation, the glutaraldehyde approach allows to preserve mannoses with their native geometry, which may be functionally important for its receptor-mediated recognition.

A main focus in healthcare is the active search for alternative strategies to antibiotics, both for prophylactic and therapeutical interventions, due to the accelerated and widespread increase in antibiotic resistance. This problem is more marked for patients with recurrent infections, in which the risk for antibiotic resistance and adverse effects is higher and can be life-threatening. Although antibiotics remain the mainstay of treatment for infectious diseases, prophylactic vaccines via the mucosal route in defined populations of patients with recurrent infections has gained use in recent years. Concomitantly, relevant advances in the formulation and administration of these vaccines driven by an increased knowledge of mucosal immunity have expanded their use, although still in its infancy. These drugs target both the innate and adaptive immune systems, at the actual point of entry of most pathogens. A fascinating new application of the concepts of trained immunity may open novel studies in their potential uses, given the paradoxically simultaneous pro-tolerogenic and boosting effector effects on diverse immune cells for different antigens. Here we delineate an updated review on the immunomodulatory mechanisms of mucosal polybacterial vaccines., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Endo-β-1,3-glucanases are widespread enzymes with glycosyl hydrolitic activity involved in carbohydrate remodelling during the germination and pollen tube growth. Although members of this protein family with allergenic activity have been reported, their effective contribution to allergy is little known. In this work, we identified Fra e 9 as a novel allergenic β-1,3-glucanase from ash pollen. We produced the catalytic and carbohydrate-binding domains as two independent recombinant proteins and characterized them from structural, biochemical and immunological point of view in comparison to their counterparts from olive pollen. We showed that despite having significant differences in biochemical activity Fra e 9 and Ole e 9 display similar IgE-binding capacity, suggesting that β-1,3-glucanases represent an heterogeneous family that could display intrinsic allergenic capacity. Specific cDNA encoding Fra e 9 was cloned and sequenced. The full-length cDNA encoded a polypeptide chain of 461 amino acids containing a signal peptide of 29 residues, leading to a mature protein of 47760.2 Da and a pI of 8.66. An N-terminal catalytic domain and a C-terminal carbohydrate-binding module are the components of this enzyme. Despite the phylogenetic proximity to the olive pollen β-1,3-glucanase, Ole e 9, there is only a 39% identity between both sequences. The N- and C-terminal domains have been produced as independent recombinant proteins in Escherichia coli and Pichia pastoris, respectively. Although a low or null enzymatic activity has been associated to long β-1,3-glucanases, the recombinant N-terminal domain has 200-fold higher hydrolytic activity on laminarin than reported for Ole e 9. The C-terminal domain of Fra e 9, a cysteine-rich compact structure, is able to bind laminarin. Both molecules retain comparable IgE-binding capacity when assayed with allergic sera. In summary, the structural and functional comparison between these two closely phylogenetic related enzymes provides novel insights into the complexity of β-1,3-glucanases, representing a heterogeneous protein family with intrinsic allergenic capacity.

Background: The thaumatin-like protein (TLP) Ole e 13 in raw olive fruit is responsible for occupational allergy in olive oil mill workers. However, these workers do not experience allergic symptoms after ingestion of edible olive., Objectives: To analyze the presence of IgE-reactive TLP in raw and edible olive fruit and to assess the allergenic potency of both sources., Methods: The content of TLP in raw and edible olive fruit protein extracts was analyzed using immunoblotting with sera from allergic patients and with olive TLP-specific IgG. The structural and immunological stability of TLP were assayed using immunoblotting after treatment of both raw olive and purified TLP with 0.25 M NaOH solution for 24 hours. Olive pollen extract was investigated by immunoblotting for TLP content., Results: The TLP contained in raw olive fruit was not present in edible olives as a result of maceration before human consumption. No TLP was detected in olive pollen using specific IgG or sera from patients allergic to olive fruit. Sera from patients allergic to olive pollen did not react with purified TLP., Conclusions: IgE-reactive TLP is not present in edible olive, thus explaining the low number of patients allergic to this highly consumed fruit. Patients allergic to olive pollen are not sensitized toTLP and, therefore, not expected to be at risk of food allergy to olive fruit or TLP plant sources.

Background: The generation and maintenance of allergen-specific T-cell tolerance is a key step in healthy immune responses to allergens and successful allergen-specific immunotherapy. Breaking of peripheral T-cell tolerance to allergens can lead to the development of allergies, but the mechanisms are not completely understood., Objective: We sought to identify molecular mechanisms that break allergen-specific T-cell tolerance in human subjects., Methods: Proliferative responses of allergen-specific T cells from tonsils and peripheral blood were measured by using tritiated thymidine incorporation and carboxyfluorescein succinimidyl ester (CFSE) dilution experiments. Cytokine levels in cell-free supernatants were quantified by using the cytometric bead array, and mRNA expression of transcription factors and cytokines was determined by using quantitative PCR. Myeloid dendritic cells (DCs) were characterized by using flow cytometry., Results: In allergic patients the immune profile of the tonsils represents the atopic status of patients, with low expression of the TH1 cell-specific transcription factor T-bet and the cytokine IFN-γ, as well as IL-10. Human tonsils show very low levels of allergen-induced T-cell proliferation, thus representing a very suitable in vivo model to assess mechanisms of breaking allergen-specific T-cell tolerance. Triggering of Toll-like receptor (TLR) 4 or TLR8 and the proinflammatory cytokines IL-1β or IL-6 break allergen-specific T-cell tolerance in human tonsils and peripheral blood through a mechanism dependent on the adaptor molecule myeloid differentiation primary response gene (88) (MyD88). In particular, myeloid DCs and stimulations that activate them broke the tolerance of allergen-specific CD4(+) T cells, whereas plasmacytoid DCs and stimulations that activate them, such as TLR7 and TLR9, did not have any effect. Tolerance-breaking conditions induced by different molecular mechanisms were associated with a mixed cytokine profile with a tendency toward increased levels of IL-13 and IL-17, which are TH2 and TH17 cytokines, respectively., Conclusion: Certain innate immune response signals and proinflammatory cytokines break allergen-specific CD4(+) T-cell tolerance in normally unresponsive subjects, which might lead to the development or exacerbation of allergic diseases after encountering microbes or inflammatory conditions., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Immunoglobulin (Ig) E-mediated food allergy is a type 2 helper T cell (TH2)-dependent disease whose prevalence is increasing in industrialized countries as a direct consequence of reduced tolerance to food antigens. The generation of regulatory T cells (Treg) is a key component of oral tolerance, and compelling experimental evidence has demonstrated that functional allergen-specific Treg cells play a major role in healthy immune responses to allergens and clinically successful allergen-specific immunotherapy. In the particular case of IgE-mediated food allergy, further investigations are required to firmly demonstrate the role of Treg cells during desensitization, induction of tolerance, or both, and several studies have also suggested a key role for these cells in healthy responses to food allergens. Treg cells are able to suppress the sensitization and effector phases of allergic reactions via several mechanisms of action based on multiple soluble and surface-binding molecules. Our knowledge of the mechanisms governing the generation of food allergen-specific Treg cells in the gastrointestinal mucosa, including the specific dendritic cell subsets involved in such processes, has increased significantly over the last decade. The identification of alternative tissues where oral tolerance to food allergens might occur in vive is crucial, not only for a better understanding of the pathophysiology of food allergy, but also for the development of alternative therapeutic interventions. Recent findings demonstrate that oral tolerance can be induced in the tonsils through generation and maintenance of functional allergen-specificTreg cells. Further investigation in this area could pave the way for novel treatments of food allergy and other immune tolerance-related diseases.

Background: The 11S globulin Sin a 2 is a marker to predict severity of symptoms in mustard allergic patients. The potential implication of Sin a 2 in cross-reactivity with tree nuts and peanut has not been investigated so far. In this work, we studied at the IgG and IgE level the involvement of the 11S globulin Sin a 2 in cross-reactivity among mustard, tree nuts and peanut., Methods: Eleven well-characterized mustard-allergic patients sensitized to Sin a 2 were included in the study. A specific anti-Sin a 2 serum was obtained in rabbit. Skin prick tests (SPT), enzyme-linked immunosorbent assay (ELISA), immunoblotting and IgG or IgE-inhibition immunoblotting experiments using purified Sin a 2, Sin a 1, Sin a 3, mustard, almond, hazelnut, pistachio, walnut or peanut extracts were performed., Results: The rabbit anti-Sin a 2 serum showed high affinity and specificity to Sin a 2, which allowed us to demonstrate that Sin a 2 shares IgG epitopes with allergenic 11S globulins from tree nuts (almond, hazelnut, pistachio and walnut) but not from peanut. All the patients included in the study had positive skin prick test to tree nuts and/or peanut and we subdivided them into two different groups according to their clinical symptoms after ingestion of such allergenic sources. We showed that 11S globulins contain conserved IgE epitopes involved in cross-reactivity among mustard, tree nuts and peanut as well as species-specific IgE epitopes., Conclusions: The allergenic 11S globulin Sin a 2 from mustard is involved in cross-reactivity at the IgE level with tree nuts and peanut. Although the clinical relevance of the cross-reactive IgE epitopes present in 11S globulins needs to be investigated in further detail, our results contribute to improve the diagnosis and management of mustard allergic patients sensitized to Sin a 2.

Receptors made visible: The described biotin-tagged small-molecule probes with excellent affinities for the CB(1) and CB(2) cannabinoid receptors (CB(1)R and CB(2)R) enable direct visualization of these receptors in native cellular systems, including neurons, microglia, and immune cells. This method could overcome some of the limitations of current methodologies and may help to dissect the complexity of the endogenous cannabinoid system., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This work investigates the resistance to proteolysis and heating of the yellow mustard (Sinapis alba L.) allergens Sin a 1 (2S albumin), Sin a 3 (nonspecific lipid transfer protein, LTP), and Sin a 4 (profilin) to explain their potential capability to induce primary sensitization at the gastrointestinal level. Sin a 1 and Sin a 3 resisted gastric digestion showing no reduction of the IgE reactivity. Intestinal digestion of Sin a 1 and Sin a 3 produced a limited proteolysis but retained significant IgE-binding reactivity. Sin a 1 was stable after heating, and although Sin a 3 was modified, most of its structure was recovered after cooling back. These two allergens would be therefore able to sensitize by ingestion. Sin a 4 was completely digested by gastric treatment and its conformational structure markedly modified at 85 °C. Thus, this allergen can be described as a nonsensitizing mustard allergen.

Substantial progress in understanding mechanisms of immune regulation in allergy, asthma, autoimmune diseases, tumors, organ transplantation, chronic infections, and pregnancy is in an exciting developmental phase that might lead to a variety of targeted therapeutic approaches. Recent progress in the interaction between immune/inflammatory cell subsets through cytokines, particularly the extension of the knowledge on reciprocal regulation and counterbalance between subsets of T(H)1, T(H)2, T(H)9, T(H)17, T(H)22, T follicular helper cells and different subsets of regulatory T cells, as well as corresponding and co-orchestrating B-cell, natural killer cell, dendritic cell, and innate lymphoid cell subsets, offers new possibilities for immune intervention. Studies on new subsets confirm the important role of T cells in the instruction of tissue cells and also demonstrate the important role of feedback regulation for the polarization toward distinct T-cell subsets. T(H)17 and T(H)22 cells are 2 emerging T(H) cell subsets that link the immune response to tissue inflammation; IL-17A and IL-17F and IL-22 are their respective prototype cytokines. Although both cytokines play roles in immune defense to extracellular bacteria, IL-17 augments inflammation, whereas IL-22 plays a tissue-protective role. This review focuses on current knowledge on T(H)17 and T(H)22 cells and their role in inflammation, with special focus on the mechanisms of their generation and driving and effector cytokines, as well as their role in host defense, autoimmunity, and allergic diseases., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: Tonsils are strategically located in the gateway of both alimentary and respiratory tracts representing the first contact point of food and aeroallergens with the immune system. Tonsillectomy removes only the palatine tonsils and sometimes adenoids. Lingual tonsil is anatomically big and remains lifelong intact., Objective: The aim of this study was to demonstrate cellular and molecular mechanisms of oral tolerance induction to food and aeroallergens in human tonsils., Methods: Tonsil allergen-specific FOXP3(+) regulatory T (Treg) cells, plasmacytoid dendritic cells (pDCs), and myeloid dendritic cells were characterized by flow cytometry and suppressive assays. Intracellular staining, [(3)H]-thymidine incorporation, and carboxy-fluorescein succinimidyl ester dilution experiments were performed. Tonsil biopsies were analyzed by confocal microscopy., Results: CD4(+)FOXP3(+) Treg cells and pDCs constitute important T- and dendritic cell-compartments in palatine and lingual tonsils. Tonsil pDCs have the ability to generate functional CD4(+)CD25(+)CD127(-)FOXP3(+) Treg cells with suppressive property from naive T cells. CD4(+)FOXP3(+) Treg cells proliferate and colocalize with pDCs in vivo in T-cell areas of lingual and palatine tonsils. Tonsil T cells did not proliferate to common food and aeroallergens. Depletion of FOXP3(+) Treg cells enables the allergen-induced proliferation of tonsil T cells, indicating an active role of Treg cells in allergen-specific T-cell unresponsiveness. High numbers of major birch pollen allergen, Bet v 1-specific CD4(+)FOXP3(+) Treg cells, are identified in human tonsils compared with peripheral blood. A positive correlation between the percentages of FOXP3(+) Treg cells and pDCs is observed in tonsils from nonatopic individuals., Conclusion: Functional allergen-specific Treg cells are identified both in lingual and in palatine tonsils., (Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Advancing our understanding of mechanisms of immune regulation in allergy, asthma, autoimmune diseases, tumor development, organ transplantation, and chronic infections could lead to effective and targeted therapies. Subsets of immune and inflammatory cells interact via ILs and IFNs; reciprocal regulation and counter balance among T(h) and regulatory T cells, as well as subsets of B cells, offer opportunities for immune interventions. Here, we review current knowledge about ILs 1 to 37 and IFN-γ. Our understanding of the effects of ILs has greatly increased since the discoveries of monocyte IL (called IL-1) and lymphocyte IL (called IL-2); more than 40 cytokines are now designated as ILs. Studies of transgenic or knockout mice with altered expression of these cytokines or their receptors and analyses of mutations and polymorphisms in human genes that encode these products have provided important information about IL and IFN functions. We discuss their signaling pathways, cellular sources, targets, roles in immune regulation and cellular networks, roles in allergy and asthma, and roles in defense against infections., (Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Allergy is a Th2-mediated disease that involves the formation of specific IgE antibodies against innocuous environmental substances. The prevalence of allergic diseases has dramatically increased over the past decades, affecting up to 30% of the population in industrialized countries. The understanding of mechanisms underlying allergic diseases as well as those operating in non-allergic healthy responses and allergen-specific immunotherapy has experienced exciting advances over the past 15 years. Studies in healthy non-atopic individuals and several clinical trials of allergen-specific immunotherapy have demonstrated that the induction of a tolerant state in peripheral T cells represent a key step in healthy immune responses to allergens. Both naturally occurring thymus-derived CD4+CD25+FOXP3+ Treg and inducible type 1 Treg inhibit the development of allergy via several mechanisms, including suppression of other effector Th1, Th2, Th17 cells; suppression of eosinophils, mast cells and basophils; Ab isotype change from IgE to IgG4; suppression of inflammatory DC; and suppression of inflammatory cell migration to tissues. The identification of the molecules involved in these processes will contribute to the development of more efficient and safer treatment modalities.

Background: Patients allergic to mustard are frequently sensitized to peach., Objective: To identify and analyse new yellow mustard allergens that could be involved in IgE cross-reactivity., Methods: Sera from mustard-allergic patients with symptoms to peach were studied. Mass spectrometry analyses provided sequences of IgE-reactive proteins. cDNAs encoding Sin a 3 and Sin a 4 were amplified by polymerase chain reaction, cloned and sequenced. The recombinant allergens were obtained in Pichia pastoris and Escherichia coli, respectively, and used for ELISA, immunoblotting and inhibition experiments. Sequence alignment was used to identify common IgE epitopes., Results: Sin a 3- and Sin a 4-specific cDNAs encode for mature proteins of 92 and 131 amino acids that belong to nsLTP and profilin protein families, respectively. Sin a 3 and Sin a 4 showed 54% and 80% identity with allergenic nsLTP from peach and profilin from melon, respectively. Both recombinant allergens were IgE-reactive in ELISA and immunoblotting. Peach pulp and peel, and melon extracts nearly abolished the IgE binding to recombinant Sin a 3 or recombinant Sin a 4 in immunoblotting., Conclusion: Sin a 3 (nsLTP) and Sin a 4 (profilin) were identified as new mustard allergens and showed IgE cross-reactivity with fruits such as peach or melon, respectively. The knowledge of these two allergens will contribute towards better understand with cross-reactivity between mustard and other plant food allergens, and their availability will provide physicians with useful tools for molecular diagnosis.

Forkhead box P3 (FOXP3)(+)CD4(+)CD25(+) inducible regulatory T (iT reg) cells play an important role in immune tolerance and homeostasis. In this study, we show that the transforming growth factor-beta (TGF-beta) induces the expression of the Runt-related transcription factors RUNX1 and RUNX3 in CD4(+) T cells. This induction seems to be a prerequisite for the binding of RUNX1 and RUNX3 to three putative RUNX binding sites in the FOXP3 promoter. Inactivation of the gene encoding RUNX cofactor core-binding factor-beta (CBFbeta) in mice and small interfering RNA (siRNA)-mediated suppression of RUNX1 and RUNX3 in human T cells resulted in reduced expression of Foxp3. The in vivo conversion of naive CD4(+) T cells into Foxp3(+) iT reg cells was significantly decreased in adoptively transferred Cbfb(F/F) CD4-cre naive T cells into Rag2(-/-) mice. Both RUNX1 and RUNX3 siRNA silenced human T reg cells and Cbfb(F/F) CD4-cre mouse T reg cells showed diminished suppressive function in vitro. Circulating human CD4(+) CD25(high) CD127(-) T reg cells significantly expressed higher levels of RUNX3, FOXP3, and TGF-beta mRNA compared with CD4(+)CD25(-) cells. Furthermore, FOXP3 and RUNX3 were colocalized in human tonsil T reg cells. These data demonstrate Runx transcription factors as a molecular link in TGF-beta-induced Foxp3 expression in iT reg cell differentiation and function.

The Gel/Gas/Phr family of fungal beta(1,3)-glucanosyltransferases plays an important role in cell wall biogenesis by processing the main component beta(1,3)-glucan. Two subfamilies are distinguished depending on the presence or absence of a C-terminal cysteine-rich domain, denoted "Cys-box." The N-terminal domain (NtD) contains the catalytic residues for transglycosidase activity and is separated from the Cys-box by a linker region. To obtain a better understanding of the structure and function of the Cys-box-containing subfamily, we identified the disulfide bonds in Gas2p from Saccharomyces cerevisiae by an improved mass spectrometric methodology. We mapped two separate intra-domain clusters of three and four disulfide bridges. One of the bonds in the first cluster connects a central Cys residue of the NtD with a single conserved Cys residue in the linker. Site-directed mutagenesis of the Cys residue in the linker resulted in an endoplasmic reticulum precursor that was not matured and underwent a gradual degradation. The relevant disulfide bond has a crucial role in folding as it may stabilize the NtD and facilitate its interaction with the C-terminal portion of a Gas protein. The four disulfide bonds in the Cys-box are arranged in a manner consistent with a partial structural resemblance with the plant X8 domain, an independent carbohydrate-binding module that possesses only three disulfide bonds. Deletion of the Cys-box in Gas2 or Gas1 proteins led to the formation of an NtD devoid of any enzymatic activity. The results suggest that the Cys-box is required for proper folding of the NtD and/or substrate binding.

Ole e 9 is an olive pollen allergen belonging to group 2 of pathogenesis-related proteins. The protein is composed of two immunological independent domains: an N-terminal domain (NtD) with 1,3-beta-glucanase activity, and a C-terminal domain (CtD) that binds 1,3-beta-glucans. We have determined the three-dimensional structure of CtD-Ole e 9 (101 amino acids), which consists of two parallel alpha-helices forming an angle of approximately 55 degrees , a small antiparallel beta-sheet with two short strands, and a 3-10 helix turn, all connected by long coil segments, resembling a novel type of folding among allergens. Two regions surrounded by aromatic residues (F49, Y60, F96, Y91 and Y31, H68, Y65, F78) have been localized on the protein surface, and a role for sugar binding is suggested. The epitope mapping of CtD-Ole e 9 shows that B-cell epitopes are mainly located on loops, although some of them are contained in secondary structural elements. Interestingly, the IgG and IgE epitopes are contiguous or overlapped, rather than coincident. The three-dimensional structure of CtD-Ole e 9 might help to understand the underlying mechanism of its biochemical function and to determine possible structure-allergenicity relationships.

Background: Among allergenic spices, mustard represents 1 of the most important in terms of allergenic potency and widespread use. An 11S globulin (Sin a 2) has been isolated from yellow mustard seeds and identified as a new major allergen., Objective: Cloning and sequencing a cDNA encoding a Sin a 2 subunit and producing the allergen as a recombinant protein., Methods: Sin a 2 subunit-encoding cDNA was amplified by polymerase chain reaction, cloned, and sequenced. The allergen was produced as a recombinant protein in Escherichia coli and used for enzyme-linked immunosorbent assay, immunoblotting, and inhibition experiments. Sera from patients with mustard allergy and an anti-polyhistidine monoclonal antibody were used., Results: Sin a 2-specific cDNA comprises an open reading frame that encodes a protein of 510 amino acids, in which the first 23 residues correspond to the signal peptide. Sequence alignment with other allergenic 11S globulins showed levels of sequence identity ranging between 27% and 38%. Three peptides described as epitopes in Ara h 3 were moderately conserved in Sin a 2. Approximately 87% of the IgE binding to natural Sin a 2 was inhibited by the recombinant allergen using sera from patients with mustard allergy., Conclusion: The recombinant 11S globulin from yellow mustard seeds produced in E coli retained the IgE-binding capability of the natural allergen., Clinical Implications: The availability of Sin a 2 sequence and its recombinant production could help to develop future therapeutic approaches and might well open new investigation lines to resolve whether 11S globulins are proteins implicated in cross-reactivity processes involving mustard seeds.

Numerous pollen allergens have been reported over the last few years. Most of them belong to well-known families of proteins but some others constitute the first member of new allergenic families. Some of the factors that can contribute to the detection and identification of new pollen allergens are: a) advances in the technology tools for molecular analysis; and b) the deep knowledge of many allergenic sources. The combination of these factors has provided vast information on the olive pollen allergogram and the identification of minor allergens that become major ones for a significant population. The close taxonomical relationship between olive tree and ash -both Oleaceae- has permitted to identify Fra e 1 (the Ole e 1-like allergen) in ash pollen and to detect the presence of protein homologues of Ole e 3 and Ole e 6. In the other hand, extensive areas of south Europe are suffering an increasing desertification. As a consequence of this, new botanical species are spontaneously growing in these areas or being used in greening ground programs: Chenopodium album and Salsola kali are some examples recently recognized as allergenic woods. The identification of the complete panel of allergens from the hypersensitizing sources might help to develop more accurate diagnosis, and efficient and safer therapy tools for Type-I allergic diseases.

Olive pollen has a complex allergenic profile, from which more than 10 allergens have been identified and characterized. Some of these belong to well-known protein families and others cannot be included in reported biochemical types. Most of these allergens have been produced by recombinant technology, mainly in Escherichia coli or in Pichia pastoris, and they are good candidates for diagnostic and therapeutic purposes. Diagnosis and immunotherapy of allergy currently use extracts prepared from homogenates of natural sources, which only allow us to detect sensitivity to the complete source. These extracts can be successfully replaced by mixtures with controlled amounts of specific allergenic proteins obtained by recombinant technology in order to define the sensitization profile of individual patients. Recombinant Ole e 1 can be used as a marker for sensitization to Oleaceae. Recombinants Ole e 2 (profilin) and Ole e 3 (polcalcin) can serve as markers of polysensitivity. Finally, recombinant forms of Ole e 6, Ole e 10, and the carboxy-terminal and amino-terminal domains of Ole e 9 would help to detect sensitization to these minority allergens that could be overlooked in the complete olive pollen extract. These recombinant molecules can help provide an accurate diagnosis of sensitivity to individual allergens and, therefore, improve the design of more efficacious allergen-based immunotherapy strategies.

Background: Designing of methods for an accurate diagnosis is a main goal of allergy research. Olive pollen allergy is currently diagnosed using commercially available pollen extracts that do not allow identification of the molecules that elicit the disease., Objective: To analyze the suitability of using the N- and C-terminal domains (NtD and CtD, respectively) of the 1,3-beta-glucanase Ole e 9, a major allergen from olive pollen, for in vitro diagnosis., Methods: Serum samples from 55 olive-allergic patients were assayed using enzyme-linked immunosorbent assay to study hypersensitive patients with IgE reactivity to Ole e 9. The specific IgEs to NtD and CtD, obtained by recombinant technology, were determined by means of immunoblotting, enzyme-linked immunosorbent assay, and inhibition assays., Results: Thirty-one of 33 serum samples from Ole e 9-allergic patients were IgE reactive to recombinant NtD (rNtD) (n = 26 [79%]), recombinant CtD (rCtD) (n = 22 [67%]), or both (n = 17 [52%]). Nine patients (27%) were exclusively reactive to rNtD and 5 (15%) to rCtD. Inhibition assays of IgE binding to Ole e 9 with a mixture of both domains abolished 90% of the binding, whereas 44% and 45% were abolished when rNtD and rCtD were used, respectively., Conclusions: Because sensitization to NtD or CtD of Ole e 9 could be correlated to vegetable food-latex-pollen cross-reactivity processes or to the exacerbation and persistence of asthma, respectively, these molecules could be used in vitro as markers of disease to classify patients and to design a patient-tailored immunotherapy approach.

Background: The allergenicity of the ornamental tree Robinia pseudoacacia, or black locust, is unknown., Objective: To evaluate the prevalence of sensitization to R. pseudoacacia pollen, its possible allergenic cross-reactivity with other common pollens, and the potential implication of pollen panallergens (profilin, polcalcin, and 1,3-beta-glucanase) as a cause of sensitization to R. pseudoacacia pollen., Methods: Skin prick testing with R. pseudoacacia pollen was performed in 149 patients with pollinosis. Nasal challenge with R. pseudoacacia pollen was performed in 10 patients. The prevalence of sensitization to the recombinant forms of profilin (rChe a 2), polcalcin (rChe a 3), and the N-terminal of the 1,3-beta-glucanase (rNtD of Ole e 9) was investigated. Immunoblotting, enzyme-linked immunosorbent assay, and competitive inhibition assays were performed with R. pseudoacacia pollen and recombinant pollen allergens., Results: Sixty-four patients (43%) had positive skin prick test reactions to R. pseudoacacia pollen. Nasal challenge results were positive in 5 sensitized patients and negative in 4 controls and 1 sensitized patient. The allergenic profile of R. pseudoacacia pollen comprises at least the panallergen profilin, a calcium-binding protein, and a 1,3-beta-glucanase. The prevalence of sensitization to rChe a 2, rChe a 3, and rNtD of Ole e 9 was 60%, 33%, and 87%, respectively, among patients sensitized to R. pseudoacacia pollen. Binding of IgE to R. pseudoacacia extract was completely inhibited by Robinia, Chenopodium, Olea, Cupressus, and Lolium extracts., Conclusions: The high prevalence of R. pseudoacacia pollen sensitization in patients with pollinosis is likely to be due to cross-sensitization to panallergens (profilin, polcalcin, and 1,3-beta-glucanase) from other common pollens. This phenomenon may lead to a diagnosis of "allergy mirages."

Background: Clustered severe adverse reactions to immunotherapy with olive pollen extracts have been occasionally reported in areas where olive trees are extensively grown. Allergic patients from these areas, in addition to the major olive pollen allergen Ole e 1, frequently recognize a recently described allergen, Ole e 9., Objective: We aimed to develop an immunoassay to measure Ole e 9 concentration and to study the variability of this allergen in olive pollen extracts., Methods: Monoclonal antibodies (mAb) to Ole e 9 were produced from mice immunized with the pure allergen. One of these mAbs was used to develop a sandwich ELISA with an anti-olive pollen extract rabbit serum as the tracer. Olive pollen batches from several suppliers were analyzed using this method. These batches were also analyzed for Ole e 1 content and biological activity., Results: A 10-fold variation between the extreme values was found for the biological activity of the batches analyzed. Ole e 1 concentration showed a 25-fold variation. Variability of Ole e 9 concentration was extremely high, up to 161 times. The ratio Ole e 1/Ole e 9 varied in a range from 0.6 to 390.4., Conclusion: The availability of a mAb-based ELISA for Ole e 9 made it possible for us to detect an important source of variability in olive pollen batches. This variability may be the cause of outbreaks of adverse reactions in the course of immunotherapy treatments, which have sometimes been observed among olive-allergic patients living in areas with very high levels of airborne olive pollen.

Background: Trees of the family Oleaceae are important allergen sources, with a strongly varying geographic distribution. For example, olive pollen is an important allergen source in Mediterranean countries, whereas ash pollen dominates in Northern and Central Europe and North America. The aim of this study was to compare the profiles of olive and ash pollen allergens and to study the degree of cross-reactivity using populations of allergic patients selectively exposed to olive or ash pollen., Methods: Olive and ash pollen extracts were analyzed by IgE immunoblotting using sera from Spanish patients highly exposed to olive pollen and Austrian patients without olive but ash pollen exposure. IgE cross-reactivity was studied by qualitative immunoblot inhibition assays and semiquantitative ELISA inhibitions using olive, ash, birch, mugwort, timothy grass pollen extracts and the major olive pollen allergen, Ole e 1., Results: Spanish and Austrian patients exhibited an almost identical IgE-binding profile to olive and ash pollen allergens, with major reactivity directed against Ole e 1, and its homologous ash counterpart, Fra e 1. IgE inhibition experiments demonstrated extensive cross-reactivity between olive and ash pollen allergens. However, whereas cross-reactions between profilins and calcium-binding allergens also occurred between unrelated plant species, cross-reactivity to Ole e 1 was confined to plants belonging to the Oleaceae., Conclusions: Ole e 1 is a marker allergen for the diagnosis of olive and ash pollen allergy., (Copyright (c) 2006 S. Karger AG, Basel.)

Background: Olive pollen is an important cause of allergy in Mediterranean countries. More than 50% of olive-pollen-allergic patients are sensitized against the 1,3-beta-glucanase Ole e 9. To date, prophylactic and therapeutic treatments using purified recombinant allergens have not been studied in animal models of olive pollen allergy., Methods: BALB/c mice were immunized against Ole e 9 combining intraperitoneal injections of the allergen in Al(OH)3 with airway allergen challenges. A prophylactic treatment was performed by intranasal administration of a mixture of the recombinant fragments of the allergen prior to Ole e 9 sensitization. Serum levels of specific IgE, IgG1, IgG2a and IgG2b were measured by ELISA, and total IgE levels by sandwich ELISA. Bronchoalveolar lavage and lungs from mice were collected to study airway inflammation by light microscopy., Results: BALB/c mice immunized against Ole e 9 developed a predominantly Th2-like immune response with allergen-specific immunoglobulin induction and airway inflammation accompanied by the infiltration of eosinophils, lymphocytes, and neutrophils in the lung. Prophylactic treatment by intranasal application of the recombinant fragments of Ole e 9 avoids airway inflammation induced by sensitization with this allergen although the levels of Ole e 9-specific antibodies remain unchanged., Conclusions: Prophylactic intranasal treatment with recombinant fragments of Ole e 9 prevents airway inflammation triggered by immunization to this allergen in a murine model of type I allergy., (Copyright (c) 2006 S. Karger AG, Basel.)

Background: Although mustard seed allergy has been largely reported during the preceding 20 years, currently only 2 allergens, Sin a 1 and Bra j 1, have been identified., Objective: To improve the characterization of the allergenic profile of yellow mustard seeds by reporting the identification and biochemical characterization of an 11S globulin as a new major allergen., Methods: Mustard seed proteins were separated using size exclusion and ion-exchange chromatographic columns, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 2-dimensional polyacrylamide gel electrophoresis. Separation of different polypeptide chains was achieved by reverse-phase high-performance liquid chromatography. Mass spectrometry after tryptic digestion and Edman degradation were used to determine amino acid sequences of peptides. IgE binding assays were performed with 13 serum samples from mustard allergic patients in immunoblotting and enzyme-linked immunosorbent inhibition assays., Results: A protein of 51 kDa was recognized as a major allergen by patients allergic to mustard and called Sin a 2. The allergen was dissociated in 2 chains of 36 and 23 kDa, which also bound IgE. N-terminal end and internal amino acid sequences allowed identification of the new allergen as a seed storage 11S globulin belonging to the Cupin super family. Purified allergen was able to inhibit the IgE binding of sera from allergic patients to mustard seeds extract in up to 55% of the responses., Conclusions: An 11S globulin storage protein has been isolated and identified as a novel major allergen of mustard seeds.

Background: Mustard has been an important cause of food allergy of increasing incidence in the last years. Sin a 1, a storage 2S albumin, is the most relevant allergen from this spice., Methods: Pichia pastoris has been used as host for the recombinant production of the precursor form of Sin a 1 (rproSin a 1). rproSin a 1 was purified to homogeneity by three chromatographic steps: gel filtration, anion exchange and reverse-phase HPLC. Molecular characterization was performed using Edman degradation, mass spectrometry, amino acid composition, and circular dichroism. Immunological properties were analyzed by immunoblotting, ELISA, and ELISA inhibition experiments., Results: We overexpressed rproSin a 1 as a single polypeptide with both large and small chains linked by an internal processed fragment at high yield. The purified rproSin a 1 (>95%) was obtained as a monomeric and soluble protein. rproSin a 1 showed equivalent structural and immunological properties to natural heterodimeric Sin a 1 allergen. rproSin a 1 was recognized by 75% of the patients allergic to mustard. The inhibitory capacity of rproSin a 1 to the total allergenicity of mustard extracts varied from 13 to 83% in different patients, with a mean value of 54%., Conclusions: rproSin a 1 is a good candidate to replace natural allergen in diagnosis protocols of mustard allergy. P. pastoris has been demonstrated to be a suitable expression system for the production of allergenic derivates of Sin a 1 that could be used for immunotherapy purposes in future., (Copyright 2005 S. Karger AG, Basel)

Background: 1,3-beta-glucanases (group 2 of pathogenesis-related proteins) are enzymes widely distributed among higher plants and have been recently proven to be significant allergens., Objective: The aim of this work was to study the potential implication of 1,3-beta-glucanases in cross-reactivities among latex, pollen and vegetable foods., Methods: The cDNA encoding the N-terminal domain (NtD) of Ole e 9, a major allergenic 1,3-beta-glucanase from olive pollen, was amplified by polymerase chain reaction and produced as a recombinant protein in Pichia pastoris (recombinant N-terminal domain, rNtD). Circular dichroism, ELISA, immunoblotting and immunoblotting inhibition experiments were carried out. Sera from olive pollen allergic patients and a rNtD-specific polyclonal antiserum were used., Results: The NtD of Ole e 9 has been produced at high yield in the yeast P. pastoris and possesses 1,3-beta-glucanase activity. The expressed polypeptide conserves IgE and IgG immunodominant epitopes of the whole Ole e 9. A rNtD-specific polyclonal antiserum and sera from olive pollen allergic patients allowed detection of IgG and IgE reactive peptidic epitopes common to 1,3-beta-glucanase Ole e 9 in extracts from ash and birch pollen, tomato, potato, bell-pepper, banana and latex., Conclusion: rNtD and homologous glucanases are new molecules to be used in diagnostic protocols as they could help to identify allergic pollen patients who are at risk for developing allergic symptoms to fruits, vegetables and latex.

NMR spectroscopy has been used to determine the solution structure of the precursor form of the recombinant napin BnIb, rproBnIb, a 2S albumin, 109-residue protein from the seeds of Brassica napus. More than 90% of the side-chain proton resonances were unambiguously assigned from the analysis of two-dimensional correlation (COSY), total correlation (TOCSY), and nuclear Overhauser effect (NOESY) spectra. The final structures were computed by using restrained molecular dynamics on the basis of 1316 upper-limit distance constraints derived from NOE cross-correlation intensities. The computed structures exhibited a root-mean-square deviation (RMSD) radius of 0.66 A for the backbone and 1.16 A for the side-chain heavy atoms of the structural core. The resulting structure consists of five amphipathic helices arranged in a right-handed super helix, a folding motif found in other proteins of the prolamin superfamily. As in the case of the mature protein, the recombinant precursor behaves as a plant food allergen. To trace out the origin and characteristics of its allergenic properties, rproBnIb was assayed against simulated gastric fluid and found to be very resistant to proteolysis. Also, heat treatment of the protein followed up to 85 degrees C by circular dichroism showed a very limited unfolding, which was recovered after cooling to 20 degrees C, indicating a high thermal stability. These results suggest that rproBnIb, as other 2S albumins, may be able to reach the gut immune system intact. A comparison of the putative epitopes against IgE antibodies of the three members of the prolamine family [2S albumins, nonspecific lipid transfer proteins (nsLTPs), and alpha-amylase/trypsin inhibitors] indicates that there are not common surfaces of interaction with IgE. Though the epitopes appear to be located in different regions of the proteins, they do comply with the requirements of being solvent-exposed and flexible.

Gas1p is a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae and is a representative of Family GH72 of glycosidases/transglycosidases, which also includes proteins from human fungal pathogens. Gas1p, Phr1-2p from Candida albicans and Gel1p from Aspergillus fumigatus have been shown to be beta-(1,3)-glucanosyltransferases required for proper cell wall assembly and morphogenesis. Gas1p is organized into three modules: a catalytic domain; a cys-rich domain; and a highly O-glycosylated serine-rich region. In order to provide an experimental system for the biochemical and structural analysis of Gas1p, we expressed soluble forms in the methylotrophic yeast Pichia pastoris. Here we report that 48 h after induction with methanol, soluble Gas1p was produced at a yield of approximately 10 mg x L(-1) of medium, and this value was unaffected by the further removal of the serine-rich region or by fusion to a 6 x His tag. Purified soluble Gas1 protein showed beta-(1,3)-glucanosyltransferase activity that was abolished by replacement of the putative catalytic residues, E161 and E262, with glutamine. Spectral studies confirmed that the recombinant soluble Gas1 protein assumed a stable conformation in P. pastoris. Interestingly, thermal denaturation studies demonstrated that Gas1p is highly resistant to heat denaturation, and a complete refolding of the protein following heat treatment was observed. We also showed that Gas1p contains five intrachain disulphide bonds. The effects of the C74S, C103S and C265S substitutions in the membrane-bound Gas1p were analyzed in S. cerevisiae. The Gas1-C74S protein was totally unable to complement the phenotype of the gas1 null mutant. We found that C74 is an essential residue for the proper folding and maturation of Gas1p., (Copyright 2004 FEBS)

Olive tree (Olea europaea) pollen is a main cause of allergy associated with extensive areas of Europe and North America. Ole e 10, a small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from the olive pollen, isolated, and characterized. Circular dichroism analysis gave 17% alpha helix, 33% beta sheet, and 21% beta turn for its secondary structure. Based on amino acid sequences of tryptic peptides, the protein was cloned and sequenced. The allergen consists of a single polypeptide chain of 102 aa, with a signal peptide of 21 residues. Ole e 10 showed homology with the C-terminal domain of another olive allergen, Ole e 9 (1,3-beta-glucanase, 53% identity), with deduced sequences from Arabidopsis thaliana genes (42-46% identity) and with polypeptide segments (Cys boxes) of proteins involved in yeast development (Epd1/Gas-1p/Phr2 families; 42-43% similarity). Ole e 10 showed 55% prevalence for olive-allergic patients and exhibited an IgE response dependent on its conformation. Remarkable IgE cross-reactivity was detected with Ole e 9, but no correlation was observed between the individual IgE responses to both allergens. Ole e 10 shares IgE B cell epitopes with proteins from Oleaceae, Gramineae, Betulaceae, Chenopodiaceae, Cupressaceae, Ambrosia, and Parietaria pollens, latex, and vegetable foods, such as tomato, kiwi, potato, and peach. These data indicate that Ole e 10 is a new pan-allergenic plant protein that shows notable intra- and interspecie IgE cross-reactivity and is a powerful candidate to be involved in pollen-latex-fruit syndrome.

Several allergenic proteins, such as the 1,3-beta-glucanases, have been associated with plant defence responses. Ole e 9 (46 kDa) is a 1,3-beta-glucanase and major allergen from olive pollen, which is a principal cause of allergy in Mediterranean countries. Its C-terminal segment (101 amino acid residues) has been produced as a recombinant polypeptide in the yeast Pichia pastoris. The cDNA encoding the polypeptide was inserted into the plasmid vector pPICZalpha-A and overexpressed in KM71 yeast cells. The recombinant product was purified by size-exclusion chromatography followed by reversed-phase HPLC. Edman degradation, MS and CD were used to determine molecular properties of the recombinant polypeptide, which exhibited 16% alpha-helix and 30% beta-sheet as regular elements of secondary structure. Disulphide bridges of the molecule were determined at positions Cys-14-Cys-76, Cys-33-Cys-94 and Cys-39-Cys-48. The high IgE-binding capability of the recombinant C-terminal segment of Ole e 9 against sera from Ole e 9-sensitive individuals, which was determined by immunoblotting and ELISA inhibition, supported the proper folding of the polypeptide and the maintenance of antigenic properties that it exhibits as a part of the whole allergen. These data indicated that this portion of Ole e 9 constitutes an independent domain, which could be used to study its three-dimensional structure and function, as well as for clinical purposes such as diagnosis and specific immunotherapy. Since it shows sequence similarity with portions of 1,3-beta-glucanases from plant tissues and the Gas/Phr/Epd protein families involved in yeast morphogenesis, we suggest that this domain could play an equivalent functional role within these enzymes.

2S albumin storage proteins from rapeseed (Brassica napus), called napins, consist of two different polypeptide chains linked by disulphide bridges, which are derived by proteolytic cleavage from a single precursor. The precursor form of the napin BnIb (proBnIb) has been cloned using a PCR strategy and sequenced. The amino-acid sequence deduced from the clone includes 31 residues of the small chain and 75 of the large chain, which are connected by the peptide Ser-Glu-Asn. Expression of the cDNA encoding proBnIb has been carried out in the methylotrophic yeast Pichia pastoris. The induced protein was secreted to the extracellular medium at a yield of 80 mg.L(-1) of culture and was purified by means of size-exclusion chromatography and reverse phase-HPLC. Recombinant proBnIb appeared properly folded as its molecular and spectroscopic properties were equivalent to those of the mature heterodimeric protein. As 2S albumin storage proteins from Brassicaceae have been shown to be type I allergy inducers, the immunological activity of the recombinant proBnIb was analysed as a measure of its structural integrity. The immunological properties of the recombinant precursor and the natural napin were indistinguishable by immunoblotting and ELISA inhibition using polyclonal antisera and sera of patients allergic to mustard and rapeseed. In conclusion, the recombinant expression of napin precursors in P. pastoris has been shown to be a successful method for high yield production of homogeneous and properly folded proteins whose polymorphism and complex maturation process limited hitherto their availability.