Co-sensitization to the three non-homologous major cashew allergens Ana o 1, Ana o 2 and Ana o 3 is caused by IgE cross-reactivity.

BACKGROUND: Cashew nuts often cause strong allergic reactions, even exceeding those of peanuts. Ana o 1 (vicilin), Ana o 2 (legumin) and Ana o 3 (2S albumin) are major cashew allergens. Co-sensitization to all three non-homologous cashew nut allergens has been observed. We hypothesize that this might be due to IgE cross-reactivity. METHODS: IgE cross-inhibitions were performed with Ana o 1-3 using sera from cashew nut allergic patients. Related hazelnut allergens Cor a 11, 9 and 14 were used as controls. For comparison, IgE cross-reactivity between the hazelnut allergens was investigated using sera from hazelnut allergic patients. RESULTS: Median percentages of cross-inhibitions between Ana o 1-3 were 84-99%. In comparison, medians of cross-inhibitions between hazelnut allergens were 33-62%. The IC50 values revealed the highest IgE affinity to Ana o 3 and Cor a 14. Hazelnut legumin Cor a 9 inhibited IgE-binding to Ana o 1, 2, and 3 with median percentages of 75%, 56%, and 48%, respectively. No cross-reactivity was observed between allergenic vicilins or between 2S albumins from cashew and hazelnut. In silico identified potentially cross-reactive peptides of Ana o 3 overlapped with previously reported IgE epitopes of all three allergens. CONCLUSIONS: IgE with high affinity to Ana o 3 that cross-reacts with the other two major non-homologous cashew nut allergens might be responsible for the high allergenic potency of cashew nut. These cross-reactive IgE comprises the major fraction of specific IgE in cashew allergic patients, and might be responsible for cross-reactivity between unrelated tree nuts.

The lipid interaction capacity of Sin a 2 and Ara h 1, major mustard and peanut allergens of the cupin superfamily, endorses allergenicity.

BACKGROUND: Sin a 2 (11S globulin) and Ara h 1 (7S globulin) are major allergens from yellow mustard seeds and peanut, respectively. The ability of these two allergens to interact with lipid components remains unknown. OBJECTIVE: To study the capacity of Sin a 2 and Ara h 1 to interact with lipid components and the potential effects of such interaction in their allergenic capacity. METHODS: Spectroscopic and SDS-PAGE binding assays of Sin a 2 and Ara h 1 with different phospholipid vesicles and gastrointestinal and endolysosomal digestions in the presence or absence of lipids were performed. The capacity of human monocyte-derived dendritic cells (hmoDCs) to capture food allergens in the presence or absence of lipids, the induced cytokine signature, and the effect of allergens and lipids to regulate TLR2-L-induced NF-kB/AP-1 activation in THP1 cells were analyzed. RESULTS: Sin a 2 and Ara h 1 bind phosphatidylglycerol (PG) acid but not phosphatidylcholine (PC) vesicles in a pH-dependent manner. The interaction of these two allergens with lipid components confers resistance to gastrointestinal digestion, reduces their uptake by hmoDCs, and enhances their stability to microsomal degradation. Mustard and peanut lipids favor a proinflammatory environment by increasing the IL-4/IL-10 ratio and IL-1ß production by hmoDCs. The presence of mustard lipids and PG vesicles inhibits TLR2-L-induced NF-kB/AP-1 activation in THP1 cells. CONCLUSION: Sin a 2 and Ara h 1 interact with lipid components, which might well contribute to explain the potent allergenic capacity of these two clinically relevant allergens belonging to the cupin superfamily.

EAACI Molecular Allergology User's Guide.

The availability of allergen molecules ('components') from several protein families has advanced our understanding of immunoglobulin E (IgE)-mediated responses and enabled 'component-resolved diagnosis' (CRD). The European Academy of Allergy and Clinical Immunology (EAACI) Molecular Allergology User's Guide (MAUG) provides comprehensive information on important allergens and describes the diagnostic options using CRD. Part A of the EAACI MAUG introduces allergen molecules, families, composition of extracts, databases, and diagnostic IgE, skin, and basophil tests. Singleplex and multiplex IgE assays with components improve both sensitivity for low-abundance allergens and analytical specificity; IgE to individual allergens can yield information on clinical risks and distinguish cross-reactivity from true primary sensitization. Part B discusses the clinical and molecular aspects of IgE-mediated allergies to foods (including nuts, seeds, legumes, fruits, vegetables, cereal grains, milk, egg, meat, fish, and shellfish), inhalants (pollen, mold spores, mites, and animal dander), and Hymenoptera venom. Diagnostic algorithms and short case histories provide useful information for the clinical workup of allergic individuals targeted for CRD. Part C covers protein families containing ubiquitous, highly cross-reactive panallergens from plant (lipid transfer proteins, polcalcins, PR-10, profilins) and animal sources (lipocalins, parvalbumins, serum albumins, tropomyosins) and explains their diagnostic and clinical utility. Part D lists 100 important allergen molecules. In conclusion, IgE-mediated reactions and allergic diseases, including allergic rhinoconjunctivitis, asthma, food reactions, and insect sting reactions, are discussed from a novel molecular perspective. The EAACI MAUG documents the rapid progression of molecular allergology from basic research to its integration into clinical practice, a quantum leap in the management of allergic patients.

IgE, IgG4 and IgA specific to Bet v 1-related food allergens do not predict oral allergy syndrome.

BACKGROUND: Birch pollen-associated plant food allergy is caused by Bet v 1-specific IgE, but presence of cross-reactive IgE to related allergens does not predict food allergy. The role of other immunoglobulin isotypes in the birch pollen-plant food syndrome has not been investigated in detail. METHODS: Bet v 1-sensitized birch pollen-allergic patients (n = 35) were diagnosed for food allergy by standardized interviews, skin prick tests, prick-to-prick tests and ImmunoCAP. Concentrations of allergen-specific IgE, IgG1, IgG4 and IgA to seven Bet v 1-related food allergens were determined by ELISA. RESULTS: Bet v 1, Cor a 1, Mal d 1 and Pru p 1 bound IgE from all and IgG4 and IgA from the majority of sera. Immunoglobulins to Gly m 4, Vig r 1 and Api g 1.01 were detected in <65% of the sera. No significant correlation was observed between plant food allergy and increased or reduced levels of IgE, IgG1, IgG4 or IgA specific to most Bet v 1-related allergens. Api g 1-specific IgE was significantly (P = 0.01) elevated in celeriac-allergic compared with celeriac-tolerant patients. Likewise, frequencies of IgE (71% vs 15%; P = 0.01) and IgA (86% vs 38%; P = 0.04) binding to Api g 1.01 were increased. CONCLUSION: Measurements of allergen-specific immunoglobulins are not suitable for diagnosing Bet v 1-mediated plant food allergy to hazelnut and Rosaceae fruits. In contrast, IgE and IgA to the distantly related allergen Api g 1 correlate with allergy to celeriac.

Update of the WHO/IUIS Allergen Nomenclature Database based on analysis of allergen sequences.

The IUIS Allergen Nomenclature Sub-Committee, under the auspices of the World Health Organization and the International Union of Immunological Societies, maintains the systematic nomenclature of allergenic proteins and publishes a database of approved allergen names on its Web site, www.allergen.org. In this paper, we summarize updates of allergen names approved at the meetings of the committee in 2011 through 2013. These changes reflect recent progress in identification, cloning, and sequencing of allergens. The goals of this update were to increase consistency in the classification of allergens, isoallergens, and variants and in the incorporation of the evolutionary classification of proteins into allergen nomenclature, while keeping changes of established names to a minimum in the interest of continuity. Allergens for which names have been updated include respiratory allergens from birch and ragweed pollen, midge larvae, and horse dander; food allergens from peanut, cow's milk, and tomato; and cereal grain allergens. The IUIS Allergen Nomenclature Sub-Committee encourages researchers to use these updated allergen names in future publications.

The performance of a component-based allergen microarray for the diagnosis of kiwifruit allergy.

BACKGROUND: Allergy to kiwifruit is increasingly reported across Europe. Currently, the reliability of its diagnosis by the measurement of allergen-specific IgE with extracts or by skin testing with fresh fruits is unsatisfying. OBJECTIVE: To evaluate the usefulness of a component-based allergen microarray for the diagnosis of kiwifruit allergy in a large group of patients. METHODS: With an allergen microarray, we measured specific IgE and IgG4 levels to a panel of nine kiwifruit allergens in sera of 237 individuals with kiwifruit allergy. Sera from 198 allergic patients without kiwifruit allergy served as controls. Furthermore, we determined the extent of sensitization to latex. RESULTS: The panel of kiwifruit allergens showed a diagnostic sensitivity of 66%, a specificity of 56% and a positive predictive value of 73%. Sera from kiwifruit-allergic patients contained significantly more frequently Act d 1-specific IgE than sera from control patients. Furthermore, 51% of the positive sera contained IgE directed to a single allergen, namely Act d 1 (45%), Act d 9 (27%) or Act d 7 (13%). Within the control group, 36% sera recognized a single allergen. Out of those, 48% were positive to the cross-reactive glycoallergen Act d 7, 43% to the profilin Act d 9 and only 5% to Act d 1. Allergen-specific IgG4 levels did not differ between kiwifruit-allergic and -tolerant patients. Kiwifruit- and latex-allergic patients contained Hev b 11-specific IgE significantly more frequently than latex-allergic patients without kiwifruit allergy. CONCLUSIONS: Act d 1 can be considered a marker allergen for genuine sensitization to kiwifruit. We demonstrated that a component-based kiwifruit allergen microarray would improve the prognostic value of in vitro diagnostic tests.

Kiwifruit Act d 11 is the first member of the ripening-related protein family identified as an allergen.

BACKGROUND: Kiwifruit is an important cause of food allergy. A high amount of a protein with a molecular mass compatible with that of Bet v 1 was observed in the kiwifruit extract. OBJECTIVE: To identify and characterize kirola, the 17-kDa protein of green kiwifruit (Act d 11). METHODS: Act d 11 was purified from green kiwifruit. Its primary structure was obtained by direct protein sequencing. The IgE binding was investigated by skin testing, immunoblotting, inhibition tests, and detection by the ISAC microarray in an Italian cohort and in selected Bet v 1-sensitized Austrian patients. A clinical evaluation of kiwi allergy was carried out. RESULTS: Act d 11 was identified as a member of the major latex protein/ripening-related protein (MLP/RRP) family. IgE binding to Act d 11 was shown by all the applied testing. Patients tested positive for Act d 11 and reporting symptoms on kiwifruit exposure were found within the Bet v 1-positive subset rather than within the population selected for highly reliable history of allergic reactions to kiwifruit. Epidemiology of Act d 11 IgE reactivity was documented in the two cohorts. IgE co-recognition of Act d 11 within the Bet v 1-like molecules is documented using the microarray IgE inhibition assay. CONCLUSIONS: Act d 11 is the first member of the MLP/RRP protein family to be described as an allergen. It displays IgE co-recognition with allergens belonging to the PR-10 family, including Bet v 1.

Dissection of immunoglobulin E and T lymphocyte reactivity of isoforms of the major birch pollen allergen Bet v 1: potential use of hypoallergenic isoforms for immunotherapy.

We dissected the T cell activation potency and the immunoglobulin (Ig) E-binding properties (allergenicity) of nine isoforms of Bet v 1 (Bet v 1a-Bet v 1l), the major birch pollen allergen. Immunoblot experiments showed that Bet v 1 isoforms differ in their ability to bind IgE from birch pollen-allergic patients. All patients tested displayed similar IgE-binding patterns toward each particular isoform. Based on these experiments, we grouped Bet v 1 isoforms in three classes: molecules with high IgE-binding activity (isoforms a, e, and j), intermediate IgE-binding (isoforms b, c, and f), and low/no IgE-binding activity (isoforms d, g, and 1). Bet v 1a, a recombinant isoform selected from a cDNA expression library using IgE immunoscreening exhibited the highest IgE-binding activity. Isoforms a, b, d, e, and 1 were chosen as representatives from the three classes for experimentation. The potency of each isoallergen to activate T lymphocytes from birch pollen-allergic patients was assayed using peripheral blood mononuclear cells, allergen-specific T cell lines, and peptide-mapped allergen-specific T cell clones. Among the patients, some displayed a broad range of T cell-recognition patterns for Bet v 1 isoforms whereas others seemed to be restricted to particular isoforms. In spite of this variability, the highest scores for T cell proliferative responses were observed with isoform d (low IgE binder), followed by b, 1, e, and a. In vivo (skin prick) tests showed that the potency of isoforms d and 1 to induce typical urticarial type 1 reactions in Bet v 1-allergic individuals was significantly lower than for isoforms a, b, and e. Taken together, our results indicate that hypoallergenic Bet v 1 isoforms are potent activators of allergen-specific T lymphocytes, and Bet v 1 isoforms with high in vitro IgE-binding activity and in vivo allergenicity can display low T cell antigenicity. Based on these findings, we propose a novel approach for immunotherapy of type I allergies: a treatment with high doses of hypoallergenic isoforms or recombinant variants of atopic allergens. We proceed on the assumption that this measure would modulate the quality of the T helper cell response to allergens in vivo. The therapy form would additionally implicate a reduced risk of anaphylactic side effects.

Expression levels of parvalbumins determine allergenicity of fish species.

BACKGROUND: Parvalbumins are the most important fish allergens. Polysensitization to various fish species is frequently reported and linked to the cross-reactivity of their parvalbumins. Studies on cross-reactivity and its association to the allergenicity of purified natural parvalbumins from different fish species are still lacking. In addition, some studies indicate that dark muscled fish such as tuna are less allergenic. METHODS: Total protein extracts and purified parvalbumins from cod, whiff, and swordfish, all eaten frequently in Spain, were tested for their IgE-binding properties with 16 fish allergic patients' sera from Madrid. The extent of cross-reactivity of these parvalbumins was investigated by IgE ELISA inhibition assays. Additionally, the cDNA sequences of whiff and swordfish parvalbumins were determined. RESULTS: Extractable amounts of parvalbumins from cod were 20 times and from whiff 30 times higher than from swordfish. Parvalbumins were recognized by 94% of the patients in extracts of cod and whiff, but only by 60% in swordfish extracts. Nevertheless, a high cross-reactivity was determined for all purified parvalbumins by IgE inhibition. The amino acid sequence identities of the three parvalbumins were in a range of 62-74%. CONCLUSIONS: The parvalbumins of cod, whiff and swordfish are highly cross-reactive. The high amino acid sequence identity among cod, whiff and swordfish parvalbumins results in the observed IgE cross-reactivity. The low allergenicity of swordfish is due to the low expression levels of its parvalbumin.

Bet v 1 and its homologous food allergen Api g 1 stimulate dendritic cells from birch pollen-allergic individuals to induce different Th-cell polarization.

BACKGROUND: Bet v 1 is the most relevant sensitizing protein for birch pollen (BP)-allergic individuals. Its homologues from plant foods are mainly involved in allergic reactions caused by IgE cross reactivity. We aimed to evaluate the polarizing effect of dendritic cells (DCs) pulsed with Bet v 1, Mal d 1, Api g 1 or Dau c 1 on Th-cell responses. METHODS: Immature DCs were generated from peripheral blood monocytes of BP-allergic and healthy donors by culture with GM-CSF and IL-4 and subsequently pulsed with allergens in combination with maturation factors. Cell surface markers were analysed by FACS. Mature DCs were co-cultured with autologous Th cells and T-cell proliferation and cytokine profiles were determined. RESULTS: In co-culture, mature allergen-pulsed DCs induced autologous Th cells of BP-allergic donors to proliferate significantly more than those of healthy individuals. Exposure of DCs from BP-allergic donors to Bet v 1 resulted in a robust Th2 skewing with significantly higher quantities of IL-5 and elevated IL-13 compared to maturation factors. In contrast, Api g 1-primed DCs from BP allergics significantly enhanced the production of the Th1 cytokine IFN-γ and significantly down-regulated IL-13 compared to maturation factors. In healthy donors, no significant cytokine production could be detected. CONCLUSION: Bet v 1 in contrast to homologous food allergens seems to possess distinct molecular features that enable it to condition DCs from BP-allergic donors to induce allergen-specific T-cell proliferation and Th2 polarization.

Factors influencing the incidence and prevalence of food allergy.

Food allergy is an increasing problem in Europe and elsewhere and severe reactions to food are also becoming more common. As food allergy is usually associated with other forms of allergic sensitisation it is likely that many risk factors are common to all forms of allergy. However the potential severity of the disease and the specific public heath measures required for food allergy make it important to identify the specific risk factors for this condition. Food allergy is unusual in that it often manifests itself very early in life and commonly remits with the development of tolerance. Hypotheses that explain the distribution of food allergy include specific genetic polymorphisms, the nature of the allergens involved and the unique exposure to large quantities of allergen through the gut. Progress has been made in developing more specific and testable hypotheses but the evidence for any of these is still only preliminary. Further collaborative research is required to develop an appropriate public health response to this growing problem.

Generation of allergen-enriched protein fractions of Hevea brasiliensis latex for in vitro and in vivo diagnosis.

BACKGROUND: The latex of Hevea brasiliensis trees contains a complex proteome that includes a range of allergenic proteins. Current latex extracts that are used for the diagnosis of latex allergy still lack important allergens. We aimed to devise a production process for an improved reagent that would ideally contain the complete latex allergome. METHODS: Latex C-serum was fractionated by ammonium sulfate precipitation, and B- and C-serum proteins were then separated by anion exchange chromatography. Proteins eluting within defined salt concentration ranges were pooled into six final fractions. Fractions were evaluated by two-dimensional electrophoresis and subsequent IgE immunoblot for their spectrum of allergens. The presence of the most important latex allergens in the fractions was checked by Western blot analyses. Each fraction was further evaluated by skin prick test (SPT). RESULTS: Reproducibility of the preparation method was demonstrated with two batches of latex. Comparison of latex B- and C-serum to the six fractions showed a remarkable increase in the number of detectable allergens in the fractions. The presence of the latex allergens Hev b 1-8 and Hev b 13 in the fractions was demonstrated. In SPTs, the fractions produced wheal-and-flare reactions comparable to commercial latex extracts. CONCLUSIONS: This method provides reproducible latex protein fractions of high allergen content for the diagnosis of latex allergy.

Nitric oxide and ocular blood flow in patients with IDDM.

Endothelial dysfunction has been implicated in the pathogenesis of diabetic vascular disorders such as diabetic retinopathy. We hypothesized that either local endogenous nitric oxide (NO) synthesis or local reactivity to endogenous NO might be impaired in patients with IDDM and that this may contribute to the development of diabetic retinopathy. Ten otherwise healthy patients with long-standing IDDM and ten healthy control subjects were studied according to an open randomized two-way cross-over design. Subjects received intravenous infusions of either N(G)-monomethyl-L-arginine, an inhibitor of NO-synthase, or L-arginine, the precursor of NO synthesis, on two separate study days. Ocular hemodynamics were assessed by laser interferometric measurement of fundus pulsations and Doppler sonographic measurement of blood flow velocity in the ophthalmic artery. N(G)-monomethyl-L-arginine decreased fundus pulsations and blood flow velocity in the ophthalmic artery and increased blood pressure in healthy subjects. The responses to NO-synthase inhibition were significantly less in diabetic subjects. In contrast, L-arginine caused a comparable increase in fundus pulsations and decrease in blood pressure in both cohorts. These results indicate that systemic and ocular hemodynamic reactivity to NO-synthase inhibition is reduced in patients with long-standing IDDM, compared with healthy control subjects. Thus, this study indicates that either NO-synthase activity is increased or NO sensitivity is decreased in patients with IDDM and supports the concept of an involvement of the L-arginine-NO system in the pathophysiology of diabetic retinopathy.

Food allergy and its relevance to industrial food proteins.

Food allergies can be defined as IgE-mediated hypersensitivity reactions and are quite distinct from food intolerances, such as Coeliac disease, with symptoms usually appearing within a matter of minutes of exposure. Around 1-2% of adults and up to 5-7% of children suffer from some type of food allergy with foods such as peanuts, tree nuts, wheat and soy, cow's milk, egg, fish and shellfish being responsible for the majority of reactions. The macromolecules recognised by IgE (generally proteinaceous in nature) are termed allergens. A brief description of the allergenic components present in the major allergenic foods is given followed by a short discussion regarding their importance in industrial proteins in the light of recent food labelling legislation for allergens.

Monovalent fusion proteins of IgE mimotopes are safe for therapy of type I allergy.

By screening phage display random peptide libraries with purified immunoglobulin E (IgE) from birch pollen-allergic patients, we previously defined peptides mimicking natural IgE epitopes (mimotopes) of the major birch pollen allergen Bet v 1. The present study aimed to define a monovalent carrier for the IgE mimotopes to induce protective antibodies directed to the IgE epitopes, suitable for mimotope-specific therapy. We expressed the selected mimotopes as fusion proteins together with streptococcal albumin binding protein (ABP). The fusion proteins were recognized specifically by anti-Bet v 1 human IgE, which demonstrated that the mimotopes fused to ABP resemble the natural IgE epitope. Bet v 1-specific IgG was induced by immunization of BALB/c mice with fusion proteins. These IgG antibodies could inhibit IgE binding to Bet v 1. Skin testing of Bet v 1 allergic mice showed that the ABP mimotope constructs did not elicit type I skin reactions, although they possess IgE binding structures. Our data suggest that IgE mimotopes are safe for epitope-specific immunotherapy of sensitized individuals, when presented in a monovalent form. Therefore, ABP-fused mimotopes are promising candidates for a new type of immunotherapy based on the precise induction of blocking antibodies.

Edible genetically modified microorganisms and plants for improved health.

The development of new strategies for the delivery of vaccine antigens or immune modulators to the mucosal tissue includes innovative approaches such as the use of genetically modified food microorganisms and plants. Even though the 'proof-of-concept' has recently been established for these two systems, key questions mainly related to efficacy and risk of breaking oral tolerance remain to be critically addressed in the immediate future.

Sequence comparisons of the CDR3 hyper-variable loops of human T cell receptors specific for three major T cell epitopes of the birch pollen allergen Bet v 1.

We have analysed the T cell receptor (TCR) alpha and beta chain sequences of 16 human CD4+ T cell clones (TCCs) specific for three important epitopes of the major birch pollen allergen Bet v 1. The TCCs were raised from the peripheral blood of eight patients with birch pollen allergy, showing allergic rhino-conjunctivitis and allergic asthma. The TCCs from these individuals were specific for Bet v 1-derived peptides: amino acids (aa)77-92 (epitope 1), aa93-108 (epitope 2) and aa113-126 (epitope 3). The DNA sequence analysis of the TCRAV and BV regions revealed heterogeneous repertoires for recognition of the peptides. Multiple combinations of AV/AJ and BV/BJ were used. However, some inter-individual restriction was evident. A limited selection of AVS and the normally infrequently used BV1S4 was obvious in TCCs specific for epitope 1. The TCRBV13 was more frequent in TCCs recognizing epitope 3. A very narrow distribution in length could be seen in the CDR3 sequences of the beta chain of TCRs with specificity for epitopes 1 and 2. Inter-individual positional micro-restriction was observed for the aa motif LR in the tCDR3 (epitope 1), for the aa residue M in the alphaCDR3 and for the aa residue G in the betaCDR3 (epitope 3). Our results illustrate clearly that each antigenic peptide derived from a single allergen, is capable of selecting different characteristics in the responding repertoire of TCRs, thus increasing the complexity of allergen-recognition by T lymphocytes. Therefore, our findings limit the potential use of TCR targeted therapeutical strategies in Type I allergy.

T cell clones specific for Bet v I, the major birch pollen allergen, crossreact with the major allergens of hazel, Cor a I, and alder, Aln g I.

Tree pollens are responsible for type I allergies during the flowering season in spring. Pollens from birch, hazel and alder constitute the most important allergen sources in this respect in the northern hemisphere. Human IgE antibodies, specific for the major allergens of these pollens, are known to crossreact, and in general every tree pollen allergic patient is sensitized to these three pollen allergens. In this study we investigated eight T-helper cell clones (CD3+, CD4+, TCR alpha/beta) with specificity for Bet v I, the major birch pollen allergen, as proved by reactivity with purified natural as well as with recombinant allergen. The T cell clones were used to investigate common T cell epitopes of the Bet v I molecule with Cor a I, the major allergen of hazel pollen and Aln g I, the major allergen of alder pollen. All eight T cell clones reacted with all three proteins with different intensity. Moreover, three T cell clones, which were known to react with immunodominant T cell epitopes on the Bet v I molecule, were tested for reactivity with dodecapeptides synthesized according to the corresponding homologous regions of the Cor a I and Aln g I sequence. All the peptides induced strong T cell proliferation, indicating the existence of multiple cross-reacting epitopes. These findings will have an impact on the production of vaccines for immunotherapy of tree pollen allergies.