Novel murine mAbs define specific and cross-reactive epitopes on the latex profilin panallergen Hev b 8.

The production of specific antibodies able to recognize allergens from different sources or block interactions between allergens and antibodies mediating allergic reactions is crucial for developing successful tools for diagnostics and therapeutics. Panallergens are highly conserved proteins present in widely different species, implicated in relevant cross-reactions. The panallergen latex profilin (Hev b 8) has been associated with the latex-food-pollen syndrome. We generated five monoclonal IgGs and one IgE from murine hybridomas against recombinant Hev b 8 and evaluated their interaction with this allergen using ELISA and biolayer interferometry (BLI). Affinity purified mAbs exhibited high binding affinities towards rHev b 8, with KD1 values ranging from 10-10 M to 10-11 M. Some of these antibodies also recognized the recombinant profilins from maize and tomato (Zea m 12 and Sola l 1), and the ash tree pollen (Fra e 2). Competition ELISA demonstrated that some mAb pairs could bind simultaneously to rHev b 8. Using BLI, we detected competitive, non-competitive, and partial-competition interactions between pairs of mAbs with rHev b 8, suggesting the existence of at least two non-overlapping epitopes on the surface of this allergen. Three-dimensional models of the Fv of 1B4 and 2D10 IgGs and docking simulations of these Fvs with rHev b 8 revealed these epitopes. Furthermore, these two mAbs inhibited the interaction of polyclonal IgE and IgG4 antibodies from profilin-allergic patients with rHev b 8, indicating that the mAbs and the antibodies present in sera from allergic patients bind to overlapping epitopes on the allergen. These mAbs can be useful tools for immune-localization studies, immunoassay development, or standardization of allergenic products.

Allergen component analysis as a tool in the diagnosis and management of occupational allergy.

We are now in the epoch of "molecular allergology" and numerous clinically relevant allergenic molecules are available improving the performance of in vitro allergen tests and allergen detection methods. This review is focusing on characterized occupational allergens and their implementation into the in vitro diagnosis for occupational allergy and in allergen detection methods. More than 400 occupational agents are identified and documented as being 'respiratory sensitizers', but currently only a limited number of them are characterized on the molecular level and available for routine diagnosis as native or recombinant allergens. One exception, however, is natural rubber latex (NRL) from Hevea brasiliensis still remaining an important occupational allergen source. Characterization of 15 NRL allergens led to the development of assays for the determination of allergen content of NRL materials and the implementation of component-resolved diagnosis (CRD) for specific IgE antibody measurement. Microarray or singleplex using recombinant or native allergens are reliable tools for NRL allergy diagnosis. In addition, NRL allergy is an excellent model for improving extract-based specific IgE measurement by amplification of NRL extract preparation with stable recombinant major allergen rHev b 5. Despite the many efforts to characterize the occupationally relevant wheat allergens for baker's asthma, the most frequently occurring forms of occupational asthma, the results are highly diverse. Wheat sensitization profiles of bakers showed great interindividual variability and no wheat allergen could be classified as the major allergen. For diagnosis of baker's asthma, a whole wheat extract is still the best option for specific IgE determination. But single wheat allergens might help to discriminate between wheat-induced food allergy, grass pollen allergy and baker's asthma. For workplace-related allergens like coffee, wood, soybean, seafood and moulds allergens are characterized and few of them are available, but their relevance for occupational sensitization routes should be verified in the further studies.

Misleading Allergens in the Diagnosis of Latex Allergy: Profilin and Cross-Reactive Carbohydrate Determinants.

BACKGROUND: It has been suggested that latex-specific IgE analysis may lead to false-positive results, especially in patients with pollen allergy. In the present study, the reasons underlying clinically irrelevant latex-specific IgE positivity were investigated. METHODS: Thirty patients with latex allergy (group 1), 89 patients sensitised to aeroallergens (group 2a), and 98 healthy individuals without allergy (group 2b) were enrolled. Participants from all 3 groups were subjected to skin prick tests with aeroallergens including latex, latex-specific IgE analysis (ImmunoCAP), and nasal provocation test with latex. All cases demonstrating positive latex-specific IgE also underwent specific IgE tests (ImmunoCAP) with latex profilin, birch pollen profilin, peach lipid transfer protein, and pineapple bromelain as cross-reactive carbohydrate determinants. RESULTS: Comparison of the atopic and healthy control groups showed that the rate of positive latex-specific IgE was significantly higher in group 2a. Latex profilin-, birch pollen profilin-, and bromelain-specific IgE were remarkably higher in group 2a. CONCLUSION: False positivity to latex-specific IgE in ImmunoCAP analysis may be observed in approximately 19% of patients with pollen allergy. Profilins and bromelain are the main contributors to clinically irrelevant positive latex-specific IgE.

Profilin, a Change in the Paradigm.

Profilin is a protein that is present in all eukaryotic cells and is responsible for cross-reactivity between pollen, latex, and plant foods. It has been classically acknowledged as a minor or nearly irrelevant allergen, although recent data are changing this conception. The objective of this manuscript is to provide a comprehensive review of published data on the role of this ubiquitous allergen in pollen, latex, and plant food allergy. The patterns of recognition of this minor allergen follow a north-south gradient. Although present in all pollens and vegetables, profilin is significantly associated with allergy to grass pollen and to Cucurbitaceae fruits. Heb v 8, the latex profilin, is usually a marker of profilin allergy in plant food-allergic patients, although it has no clinical relevance in latex allergy. Sensitization to profilin jeopardizes the diagnosis of pollen allergy and selection of immunotherapy, and although component-resolved diagnosis can identify its impact, there are no tailored treatments available. In recent years, several new publications have shown how profilin should be taken into account and, under certain circumstances, considered a marker of severity, an allergen capable of inducing respiratory symptoms, and, in its natural purified form, a potential candidate for etiological treatment of food allergy. Current data on profilin strongly support the need for a shift in the previously accepted paradigm for this allergen. More research should be done to assess the real clinical impact of sensitization in specific populations and to develop therapeutic strategies.

Decrease in the rate of sensitization and clinical allergy to natural rubber latex.

BACKGROUND: In the 1980s, a striking increase in natural rubber latex (NRL) allergy was seen. Since then, many measures have been taken to prevent NRL allergy. OBJECTIVES: To investigate changes in the prevalence of NRL sensitization/clinical NRL allergy over time from 2002 to 2013. METHODS: All patients prick tested for NRL at the Department of Dermatology and Allergy Centre, Odense University Hospital were included in this study (n = 8580). In NRL-sensitized patients, the clinical relevance was evaluated for NRL. Furthermore, concomitant positive prick test results for birch pollen were recorded, together with food-related symptoms and sensitization in a subgroup of patients. RESULTS: The prevalence of NRL sensitization declined from 6.1% in 2002-2005 to 1.9% in 2006-2009, and then to 1.2% in 2010-2013 (p < 0.0001). The prevalence of clinical NRL allergy declined from 1.3% in 2002-2005 to 0.5-0.6% in 2006-2013 (p < 0.004). Among the NRL-sensitized patients, 64% had a concomitant positive prick test reaction to birch pollen, and 52% had a history of reaction to oral intake of related fruits or vegetables. CONCLUSION: Our study showed a statistically significant decline in the number of patients sensitized/clinically allergic to NRL. Many of the NRL-sensitized patients without clinical allergy to NRL had concomitant birch pollen sensitization, and reported food-related symptoms.

Cross-reactivity between cassava and latex in a colombian patient with an anaphylactic reaction

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Cross-reactivity syndromes: presentation of two cases and review of the literature.

Cross-reactivity has important consequences in some immune disorders, including allergic and autoimmune diseases, which can affect both diagnostic and therapeutic approaches. One of the most common cross-reactivity syndromes is pollen-food syndrome (PFS). The patient is sensitized with pollen by the airways and exhibits an allergic reaction to food antigen with a structural similarity to the pollen. PFS usually presents with pruritus and swelling of the mouth and throat during or just after ingestion of fresh, uncooked fruits and vegetables. Latex fruit syndrome is another cross-reactivity syndrome. It is the association of latex allergy and allergy to plant foods, which affects up to 50% of latex-allergic patients. Here, we present two cases with crossreactivity syndrome.

Dietary intervention for oral allergy syndrome as a treatment in orofacial granulomatosis: a new approach?

BACKGROUND: Orofacial granulomatosis (OFG) is a chronic granulomatous condition of the mouth, face and lips. Recent work demonstrates a high rate of atopy and silver birch sensitisation from skin prick testing (SPT). Oral allergy syndrome (OAS) is an acute oro-pharyngeal IgE mediated reaction, triggered by foods that cross react with pollens, most commonly silver birch. The aim of this study was to determine if patients with OFG and positive SPT to common OAS associated pollens responded to avoidance of cross reactive foods. METHODS: Patients with OFG and positive SPT to silver birch, grass, mugwort, ragweed and latex were required to avoid cross reacting foods, for 6 weeks and, in those who responded, for a total of 12 weeks. All had standardized oral examinations and were given severity scores (SS) at each appointment. RESULTS: Twenty two of 47 (47%) patients had one or more positive SPT and 13/22 completed 6 weeks on the diet. No difference was seen in SS between weeks 0 (14.62 ± 11.16) and 6 (13.31 ± 10.33; P = 0.656). Six of 14 (43%) had significantly improved SS (week 0; 19.17 ± 12.95, week 6; 10.83 ± 4.99, P = 0.027). Five completed 12 weeks and no further improvement was seen (week 6; 11 ± 5.57, week 12; 10.4 ± 9.94; P = 0.068). Two patients required no further treatments. CONCLUSIONS: On an intention to treat basis, only 2/14 patients improved and required no further intervention. Whilst this diet cannot be recommended routinely, the improvement seen in some patients raises questions about the role of OAS in patients with OFG.

Profilin cross-reactive panallergen causes latex sensitization in the pediatric population allergic to pollen.

BACKGROUND: Component-resolved diagnostics (CRD) has been demonstrated to be an excellent new tool for improving the current diagnosis of allergies, and it allows differentiation between polysensitization and cross-reactivity. OBJECTIVE: To demonstrate the role of cross-reactive pollen allergens in pediatric patients living in areas with large amounts of airborne grass pollen grains who are sensitive to grass pollen and latex. METHODS: Serum samples were obtained from 106 children between 3 and 14 years of age diagnosed with allergies to pollen based on clinical history, skin prick tests, and specific immunoglobulin E (IgE). None of them had allergy symptoms to latex or fruits. From these 106 children, 56 patients revealed positive results to Phleum-specific major allergens but not to cross-reactive allergens. The other 50 patients who showed positive specific IgE to Phleum-specific major allergens and to cross-reactive pollen allergens also showed positive results to latex allergens. CRD was carried out by specific IgE quantification using a fluoro-enzyme immunoassay (ImmunoCAPT System). RESULTS: Results demonstrated a positive significant relationship between the specific IgE to Hev b 8 and Phl p 12 and also between the specific IgE to Hev b 8 and latex extract in the group of patients sensitized to species-specific and cross-reactive Phleum allergens. Positive significant relationships were also found between profilin and avocado or peach sensitizations. No other latex allergens gave positive results. CONCLUSION: The apparent sensitization to latex in pediatric patients allergic to grass pollen is caused by the cross-reactive profilin panallergen; however, it is appears not to be clinically relevant.

Molecular allergens in the diagnosis of latex allergy.

BACKGROUND: Molecular allergens enable the definition of sensitization profiles in allergic patients. AIM: To validate the most helpful allergens for the diagnosis of latex allergy in different clinical situations. METHODS: 130 patients suspected to be allergic to latex with positive IgE against natural rubber latex (NRL) have been studied: 97 were confirmed as latex allergic (among which 55 professionally exposed to latex and 35 with a peranaesthetic anaphylactic shock) and 33 were only sensitized to latex without clinical allergy. Each serum was tested for IgE against 9 recombinant latex allergens and bromelain using Phadia ImmunoCAP 250. RESULTS: rHev b 6.01, 6.02, 2 and 5 were the major allergens in the allergic population. An excellent correlation (94%) was observed between IgE against rHev b 6.01 and latex prick test positivities. IgE against rHev b 1, 3 and 5 were more frequent and their levels significantly higher in patients with peranaesthetic anaphylactic shock. Among the asymptomatic patients (29/33 allergic to pollen), NRL IgE positivity is explained by the presence of anti-rHev b 8 and/or anti-carbohydrate IgE. CONCLUSIONS: rHev b 6.01 and rHev b 5 specific IgE are of major interest to confirm latex allergy diagnosis. rHev b 5 is particularly useful in case of monosensitization where clinical symptoms and latex skin prick tests may be discordant, rHev b1 and rHev b 3 are interesting to document multi-operated and peranaesthetic latex allergy. Finally, rHev b 8 is a helpful marker to highlight latex/pollen cross-reactivity which improves the specificity of the serological tests.

Identification of clinically relevant cross-sensitization between Soliadgo virgaurea (goldenrod) and Hevea brasiliensis (natural rubber latex).

BACKGROUND: Solidago virgaurea (goldenrod) is a perennial weed from which no allergens have been identified. A high latex content in its leaves has been reported. Although not an airborne allergen, it may be an important occupational sensitizer. OBJECTIVE: To identify allergenic proteins in goldenrod and to determine whether they cross-react with Hevea brasiliensis latex. METHODS: Potential cross-reactive allergens in latex and goldenrod were investigated by immunoblot inhibition and ImmunoCAP inhibition analyses using serum from patients with clinically evident goldenrod and/or latex allergy. Cross reactivity between latex allergens and goldenrod proteins was studied using recombinant Hev b 1, 3, 4, 5, 6.01, 6.02, 8, 9, or 11 in ImmunoCAP inhibition analyses. RESULTS: Immunoglobulin (Ig) E antibodies from individuals with goldenrod allergy bound extracted goldenrod proteins ranging from 20 kDa to 130 kDa in Western blots. Evidence for latex and goldenrod cross reactivity was identified by ImmunoCAP and immunoblot inhibition experiments using serum from patients with strongly positive concomitant latex and goldenrod-specific IgE antibody responses. Observed latex-goldenrod cross reactivity could not be ascribed to any of the recombinant major latex allergens evaluated. CONCLUSIONS: H brasiliensis latex and goldenrod contain cross-reactive and unique allergenic proteins. Exposure to goldenrod may sensitize patients to latex and vice versa.

Component-resolved diagnosis from latex allergy by microarray.

BACKGROUND: A positive specific IgE (sIgE) result for latex does not always mirror the clinical situation and is frequently found in individuals without overt latex allergy. OBJECTIVE: We sought to investigate the potential of component-resolved diagnosis (CRD) of latex allergy by microarray and to assess whether the technique allows discriminating genuine allergy from asymptomatic sensitization. METHODS: Twenty-six healthy controls without a history of latex allergy with a negative latex sIgE and skin test, 22 latex-allergic patients with a compelling history of latex allergy with a positive latex sIgE and prick test and 20 latex-sensitized individuals with a frequent asymptomatic exposure to natural rubber latex-containing devices with a negative latex skin test but a positive sIgE were also included. CRD was performed with the ImmunoCAP ISAC microarray and traditional singleplexed ImmunoCAP. RESULTS: In all patients, the diagnosis of latex allergy could be established by the combination of recombinant latex components present on the microarray (Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02). Over three-quarters of our patients were sensitized for Hev b 5 and/or Hev b 6.02. Some patients also displayed reactivity for Hev b 1 and/or Hev b 3. In contrast, none of the individuals sensitized to natural rubber latex or control individuals demonstrated IgE reactivity for rHev b 1, rHev b 3, rHev b 5 or rHev b 6.02. Three-quarters of the patients sensitized to latex displayed a positive microarray result for recombinant latex profilin (rHev b 8). In contrast to the results obtained by traditional ImmunoCAP for bromelain, almost no sensitization for cross-reactive carbohydrates was demonstrated by bromelain spotted on the microarray. CRD by traditional singleplexed ImmunoCAP showed highly comparable results. CONCLUSION: CRD by microarray is a reliable tool for diagnosing latex allergy. In addition, the technique allows discrimination between genuine allergy and sensitization. CRD by microarray can improve the diagnosis of IgE-mediated latex allergy by discriminating between genuine allergy and sensitization. CRD by microarray is a reliable tool to diagnose latex allergy. In addition, the technique allows discrimination between a genuine allergy and simple sensitization.

Interference of cross-reactive carbohydrates in the determination of specific IgE in alcohol drinkers and strategies to minimize it: the example of latex.

BACKGROUND: Cross-reactive carbohydrate determinants (CCDs) are N-glycans in plant and invertebrate proteins that interfere with specific IgE determinations. The prevalence of IgE to Man2XylFucGlcNAc2 (MUXF), the CCD from bromelain, may be increased in heavy drinkers. OBJECTIVE: To further investigate the relationship of alcohol consumption to CCD specific IgE. Latex was used as an example for investigating CCD interference with in vitro allergy testing and how to minimize the interference by using nonglycosylated recombinant allergens and inhibition assays. METHODS: We determined the levels of IgE to CCD markers (MUXF and ascorbate oxidase) and natural rubber latex in 270 adults without a history of latex allergy (73 abstainers or occasional drinkers, 76 light drinkers, 47 moderate drinkers, and 74 heavy drinkers). In cases with latex reactivity, we performed inhibition assays with MUXF and screened for IgE to a panel of recombinant latex allergens. Fourteen-day serologic follow-up was available for a subset of individuals. RESULTS: Moderate to heavy drinkers displayed an increased prevalence of IgE to CCD markers. The presence of CCD specific IgE was closely associated with latex IgE reactivity. Inhibition studies and the absence of reactivity to nonglycosylated recombinant latex allergens indicated CCD interference in latex IgE determinations. Serum levels of specific IgE decreased with alcohol abstention. CONCLUSIONS: In this population, alcohol consumption is associated with an increased prevalence of IgE reactivity to natural rubber latex due to CCD interference. The use of nonglycosylated recombinant allergens and inhibition assays may help to minimize CCD interference in populations in which IgE to CCDs is common.

Latex allergy: a model for therapy.

Allergy to natural rubber latex products emerged as an important clinical condition following an increase in the use of latex gloves for barrier protection in the early 1980s. In addition to latex glove users, other high-risk groups with different latex exposure include spina bifida patients and others with multiple surgical procedures. Subjects with fruit and vegetable allergy are also at risk due to cross-reactive allergens. Following the significant advances in the identification and characterization of common aeroallergens, latex allergy was well placed to become an excellent model of therapy. Awareness of latex allergy and modes of sensitization enabled epidemiological studies to inform allergen avoidance initiatives, substantially reducing inadvertent exposure in major hospitals in Western countries. Spina bifida is often identified in utero or soon after birth, allowing vigorous latex allergen avoidance with enhanced efficacy of primary prevention. However, changing demographics of latex allergy and technological revolution in countries such as China and India are predicted to unleash a second wave of latex allergy reemphasizing the incentive for improved manufacturing procedures for latex products. The desirable high tensile strength and elasticity of natural rubber latex have made the commercial identification of good alternatives very difficult but this would also be attractive for primary prevention. In addition, an effective specific immunotherapy regimen would be valuable for selected high-risk atopic individuals. Current subcutaneous and sublingual immunotherapy schedules have been tested for treatment of latex allergy with evidence of efficacy but the risks of adverse events are high. For such potent allergens as latex, hypoallergenic but T cell-reactive preparations are required for clinical use. Identification of allergenic components of latex products, with generation of monoclonal antibodies and recombinant allergens, allowed sequence determination and mapping of T cell and B cell epitopes. Together, these reagents and data facilitated improved diagnostics and investigation of novel-specific therapeutics. Potential hypoallergenic latex preparations identified include modified non-IgE-reactive allergen molecules and short T cell epitope peptides. The co-administration of adjunct therapies such as anti-IgE or corticosteroids and of appropriate adjuvants for induction of regulatory T cell response offers promise for clinically effective, safe latex-specific vaccines.

Identification of immunoglobulin E (IgE)-binding epitopes and recombinant IgE reactivities of a latex cross-reacting Indian jujube Ziz m 1 allergen.

Ziz m 1 is a major Indian jujube (Zizyphus mauritiana) allergen involved in latex-fruit syndrome, and cDNA of the allergen has been cloned, sequenced and expressed in yeast by our laboratory previously. In this study, we performed an immunoglobulin E (IgE)-binding epitope analysis of Ziz m 1 using overlapping recombinant fragments. Eight overlapping recombinant fragments were generated from the recombinant Ziz m 1 allergen. The fragments were expressed in Escherichia coli and IgE-binding activities were evaluated by sera of latex-Indian jujube-allergic subjects and normal subjects using immunoblotting. Human allergic sera are not able to recognize fragments consisting of amino acid sequences 26-71, 119-280 and 119-291. However, residues at positions 26-199, 26-105, 26-86, 119-320 and 238-330 were found relevant in the IgE-binding. Our results indicate that (72)NISGHCSDCTFLGEE(86) and (292)VWNRYYDLKTNYSSSIILEYVNSGTKYLP(320) of Ziz m 1 are the sequences required for human IgE binding. Four corresponding peptides, (72)NISGHCSDCTE(86), (292)VWNRYYDLKT(301), (300)KTNYSSSIILEY(311) and (309)LEYVNSGTKYLP(320), were synthesized, and these peptides reacted with 70%, 100%, 70% and 70% of 10 allergic sera tested, as revealed by enzyme-linked immunosorbent assay. Sensitization to (292)VWNRYYDLKT(301) correlated significantly with the presence of allergic symptoms (P < 0.001). These findings will be useful in designing diagnostic and therapeutic approaches, thereby contributing to the development of specific immunotherapy for subjects with latex-fruit syndrome.

Modelling diseases: the allergens of Olea europaea pollen.

This study analyzes the influence of the IgE response to certain olive pollen allergens in the modulation of the different clinical phenotypes of allergic disease and their relationship with the level of exposure to pollen and genetic factors. Patients from high-exposure areas had a complex IgE antibody response to allergens of Olea euroapea, which included 3 or more allergens in 75% of cases. The majority allergens were Ole e 1, Ole e 2 (profilin), Ole e 7 (lipid transporting protein), Ole e 9 (glucanase), and Ole e 10. The existence of the antigen HLA-DR2 (15) led to a higher risk of sensitization to Ole e 10 and a greater trend towards the development of severe asthma, which increased in the presence of an anti-profilin IgE. Thirty percent of patients suffering from pollinosis simultaneously presented allergy to vegetable foods. Anti-Ole e 7 IgE was significantly associated with fruit anaphylaxis and anti-profilin IgE was detected in 90% of patients with oral syndrome. Finally, we analyzed the role of glucanase and Ole e 10 as causes of the pollen-latex-fruit syndrome.

Immunological and allergenic responses induced by latex fractions of Calotropis procera (Ait.) R.Br.

Immunological and allergenic responses against the latex of Calotropis procera were investigated in mice by oral and subcutaneous routes. The latex was fractionated according to water solubility and molecular size of its components. The fractions were named as non-dialyzable latex (NDL) corresponding to the major latex proteins, dialyzable latex (DL) corresponding to low molecular size substances and rubber latex (RL) which was highly insoluble in water. Anti-sera against these fractions were assayed for total IgG and IgA titration by ELISA and IgE and IgG(1) were quantified by passive cutaneous anaphylaxis (PCA) in rats and mice, respectively. None of the fractions induced antibodies level increases when mice received latex fractions by oral route and thus, did not develop allergy. Nonetheless, anti-sera of mice sensitized with NDL and RL by subcutaneous route displayed considerable immunological response while DL did not. IgG level augmented consistently against NDL and RL while IgA response was detected only to NDL. NDL and RL induced very strong PCA reactions suggesting that both fractions would contain latex substances involved in allergy. Furthermore, protein analysis of NDL and RL suggests that RL still retain residual proteins abundantly found in NDL that could explain its similar allergenic effect. No IgG(1) reaction was detected in any of the anti-sera tested. According to the results, the proteins of latex of Calotropis procera can provoke allergy by subcutaneous route. The NDL has previously shown to display anti-inflammatory and analgesic activities by intraperitoneal injection. It should be relevant to determine whether NDL could induce such activities when assayed by oral route since it was ineffective to induce allergy by this way.