IgE Immunoreactivity Research Articles

The Manifold Bioactivity and Immunoreactivity of Microbial Proteins of Cow and Human Mature Milk in Late Lactation.

Simple SummaryThe debate over the validity and benefits of breastfeeding children after the age of 1 and the superiority of human over cow’s milk is still ongoing. The recommendation of exclusive breastfeeding for about 6 months, followed by continued breastfeeding as a complementary food source for 1 year or longer, seems justified under many circumstances. The microbiological parameters of the milk play a vital role in this respect. So far, the focus has been on the qualitative profile of the microbiota, bacterial interactions with milk compounds, and the metabolites produced by bacteria. However, the role of bacterial proteins in milk, according to the authors’ knowledge, has been analyzed. It is reported that due to the disruption of the regulatory axis of the immune system in the course of hypersensitivity, organisms may give rise to decreased IgA-mediated (physiological) and increased IgE-mediated (hypersensitive) responses even to host gut microbiota proteins. In this publication, the aim was to compare whether the bacterial proteins in the mature human milk of late lactation and cow’s milk of different breeds can determine the different immunoreactive and bioactive properties of milk.(1) Human milk (HM) is a source of many microorganisms, whose structure contains microbial protein (MP). In addition to the known health-promoting properties of HM, many activities, including immunoreactivity, may result from the presence of MP. Cow’s milk (CM)-derived MP may be 10 times more abundant than MP derived from HM. (2) Raw cow’s milk samples of Holstein and Jersey breeds, commercially available pasteurized milk, and milk from three human donors in the late lactation phase were subjected to chemical and microbiological analyzes. Microorganisms from the milk material were recovered, cultured, and their activities were tested. MPs were extracted and their immunoreactivity was tested with human high IgE pooled sera. The milk types were subjected to simulated digestion. Milk and microbial proteins were identified with LCMS and subjected to an in silico analysis of their activities. Their antioxidant potential was analysed with the DPPH method. (3) The MP of HM shows a stronger IgE and IgG immunoreactivity in the tests with human sera compared to the MP of CM (p = 0.001; p = 0.02, respectively). There were no significant differences between the microbes in the MP of different cattle breeds. The MS-identification and in silico tests of milk and microbial proteins confirmed the presence of MP with immunoreactivity and antioxidant potential. (4) MPs possess a broad bioactive effect, which was determined by an in silico tools. The balance between an MP’s individual properties probably determines the raw material’s safety, which undoubtedly requires further research.

Carbonylation induced by antibiotic and pesticide residues on casein increases its IgE binding and allergenicity

This work aimed to evaluate the effect of carbonylation induced by tetracyclines, β-lactams, fluoroquinolones, and pyrethroids in caseins of bovine origin on their immunoreactivity and allergenicity. Using a spectrophotometric method, ELISA, dot-blot, and an IgE-mediated milk allergy mouse model, we confirmed that antibiotics and pesticides at their maximum residue limit, promoted the in vitro carbonylation of caseins (among 5.0 ± 0.01 and 67.5 ± 0.70 nmol of carbonyl/mg of protein); furthermore, carbonylations greater than 19 nmol significantly increase the in vitro IgE immunoreactivity of caseins (average OD among 0.63–1.50) regarding the negative control (average OD: 0.56). On the other hand, sensitized mice exposed to oxidized caseins showed increased clinical scores (2–5), positive skin tests, and footpad swelling (0.28–0.59 mm) compared to the negative control (1–2; negative skin tests; 0.1 mm, respectively), denoting increased allergenicity. These results suggest that casein carbonylation increases their IgE immunoreactivity and allergenicity, a fact that could be explained by the resistance to the digestion promoted by carbonylation and by conformational changes in the random coil casein structure, which can expose cryptic epitopes or neoepitopes.

Biomarkers for diagnosis and prediction of therapy responses in allergic diseases and asthma.

Modern health care requires a proactive and individualized response to diseases, combining precision diagnosis and personalized treatment. Accordingly, the approach to patients with allergic diseases encompasses novel developments in the area of personalized medicine, disease phenotyping and endotyping, and the development and application of reliable biomarkers. A detailed clinical history and physical examination followed by the detection of IgE immunoreactivity against specific allergens still represents the state of the art. However, nowadays, further emphasis focuses on the optimization of diagnostic and therapeutic standards and a large number of studies have been investigating the biomarkers of allergic diseases, including asthma, atopic dermatitis, allergic rhinitis, food allergy, urticaria and anaphylaxis. Various biomarkers have been developed by omics technologies, some of which lead to a better classification of distinct phenotypes or endotypes. The introduction of biologicals to clinical practice increases the need for biomarkers for patient selection, prediction of outcomes and monitoring, to allow for an adequate choice of the duration of these costly and long‐lasting therapies. Escalating healthcare costs together with questions about the efficacy of the current management of allergic diseases require further development of a biomarker‐driven approach. Here, we review biomarkers in diagnosis and treatment of asthma, atopic dermatitis, allergic rhinitis, viral infections, chronic rhinosinusitis, food allergy, drug hypersensitivity and allergen immunotherapy with a special emphasis on specific IgE, the microbiome and the epithelial barrier. In addition, EAACI guidelines on biologicals are discussed within the perspective of biomarkers.

Proteomic and Bioinformatic Investigations of Heat-Treated Anisakis simplex Third-Stage Larvae.

Anisakis simplex third-stage larvae are the main source of hidden allergens in marine fish products. Some Anisakis allergens are thermostable and, even highly processed, could cause hypersensitivity reactions. However, Anisakis proteome has not been studied under autoclaving conditions of 121 °C for 60 min, which is an important process in the food industry. The aim of the study was the identification and characterization of allergens, potential allergens, and other proteins of heat-treated A. simplex larvae. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify 470 proteins, including allergens—Ani s 1, Ani s 2, Ani s 3, Ani s 4, Ani s 5—and 13 potential allergens that were mainly homologs of Anisakis spp., Ascaris spp., and Acari allergens. Ani s 2, Ani s 3, Ani s 5, and three possible allergens were found among the top 25 most abundant proteins. The computational analysis allowed us to detect allergen epitopes, assign protein families, and domains as well as to annotate the localization of proteins. The predicted 3D models of proteins revealed similarities between potential allergens and homologous allergens. Despite the partial degradation of heated A. simplex antigens, their immunoreactivity with anti-A. simplex IgG antibodies was confirmed using a Western blot. In conclusion, identified epitopes of allergenic peptides highlighted that the occurrence of Anisakis proteins in thermally processed fish products could be a potential allergic hazard. Further studies are necessary to confirm the IgE immunoreactivity and thermostability of identified proteins.

053 Myosin heavy chain, a novel allergen for fish allergy in patients with atopic dermatitis

The frequency of fish allergy (FA) has been increasing since seafood cuisine such as raw fish has become popular. FA is caused by many allergens, among which parvalbumin was most frequently documented; however, little is known about other allergens in a real-world setting. In this study we explored IgE-reactivity with fish allergens using FA patients’ serum. Western blot analysis revealed that a newly found protein of approximately 250kD was recognized by serum IgE in five out of six FA patients but not in control individuals. The protein of interest separated on the two-dimensional gel electrophoresis was identified as myosin heavy chain (MYHC) by LS-MS/MS. The IgE immunoreactivity was reproduced when used newly synthesized recombinant MYHC. The patients’ serum IgE, which had been pre-absorbed with fish extracts, lost the reactivity to MYHC recombinant protein, strongly suggesting the antigen specificity for MYHC. Finally, the basophil activation test revealed rsMYHC- dependent IgE reactivity as well as pathogenetic relevance. Results from the present study implicated that FA patients were heterogenous in their allergen specificities since some showed positive for parvalbumin but not for MYHC, and the others showed vice versa. All the FA patients with MYHC-specific IgE had atopic dermatitis at present or in the past. Therefore, impaired permeability barrier might have contributed to the sensitization of this high molecular allergen, MYHC, after touching fish by bear hands. Because raw fish cuisine is popular not only in Japan but also outside of Japan, the risk of FA may be increasing.

Degradation of anti‐nutritional factors and reduction of immunoreactivity of tempeh by co‐fermentation with Rhizopus oligosporus RT‐3 and Actinomucor elegans DCY‐1

SummaryThe effects of solid‐state fermentation with four filamentous fungi (Rhizopus oligosporus, Actinomucor elegans, Neurospora crassa and Rhizopus oryzae) and their combination on the degradation of protein allergens and anti‐nutritional factors (ANFs) of tempeh were evaluated. Glycinin, β‐conglycinin, trypsin inhibitor and flatulence‐causing oligosaccharides were significantly hydrolysed by all assayed strains but the level of reduction of ANFs depended on the strain. ELISA was conducted to evaluate IgE immunoreactivity using sera from soy sensitive individuals. Results revealed that co‐fermentation of R. oligosporus and A. elegans resulted in the highest reduction in IgE immunoreactivity for all sera used and showed greatest degradations on α’, α, β, acidic subunits (100% hydrolysed) and extensive hydrolysis of flatulence‐causing oligosaccharides. Tempeh fermented with R. oligosporus and A. elegans displayed high level of soluble protein and peptide (<10 kD) content. Co‐fermentation of R. oligosporus and A. elegans was proved to be effective in producing hypoallergenic tempeh.

Proteins derived from Prunus armeniaca kernel are possible to cause Immunoglobulin E reactivity in human sera

Allergic diseases are always caused by protein molecules and in most cases arise in individuals with documented immunologic reactions to foods, pollen, or plant IgE. However, proteins derived from herbal medicine which act as an allergen have not been widely studied. Here, we aim to confirm if the herbal medicine Prunus armeniaca kernel at the proteomic level triggers IgE immunoreactivity. If it does, we can hypothesize what components of protein molecules in the herbal medicine Prunus armeniaca kernel react with and bind to IgE. To test this hypothesis, we utilized proteomic tools and SDS-PAGE stained with Coomassie blue and identified proteins using LC-MS/MS. We also assessed the degree of plasma IgE reactivity in an isolated protein of Prunus armeniaca kernel using western blots of human IgE and an enzyme immunoassay panel, the Allergy-Q test, in 400 human sera. As a result, we found three sera which were positive candidates as Prunus armeniaca kernel IgE binding proteins. The results imply that Prunus armeniaca kernel can act as an allergen given the identification of IgE binding proteins among the isolated proteins of Prunus armeniaca kernel.

Molecular fingerprinting of complex grass allergoids: size assessments reveal new insights in epitope repertoires and functional capacities

Background Subcutaneous allergen immunotherapy (SCIT) is a well-documented treatment for allergic disease which involves injections of native allergen or modified (allergoid) extracts. The use of allergoid vaccines is a growing sector of the allergy immunotherapy market, associated with shorter-course therapy. The aim of this study was the structural and immunological characterisation of group 1 (Lol p 1) IgG-binding epitopes within a complex mix grass allergoid formulation containing rye grass.Methods HP-SEC was used to resolve a mix grass allergoid preparation of high molecular weight into several distinct fractions with defined molecular weight and elution profiles. Allergen verification of the HP-SEC allergoid fractions was confirmed by mass spectrometry analysis. IgE and IgG immunoreactivity of the allergoid preparations was explored and Lol p 1 specific IgG-binding epitopes mapped by SPOT synthesis technology (PepSpot™) with structural analysis based on a Lol p 1 homology model.Results Grass specific IgE reactivity of the mix grass modified extract (allergoid) was diminished in comparison with the mix grass native extract. A difference in IgG profiles was observed between an intact mix grass allergoid preparation and HP-SEC allergoid fractions, which indicated enhancement of accessible reactive IgG epitopes across size distribution profiles of the mix grass allergoid formulation. Detailed analysis of the epitope specificity showed retention of six Lol p 1 IgG-binding epitopes in the mix grass modified extract.Conclusion The structural and immunological changes which take place following the grass allergen modification process was further unravelled revealing distinct IgG immunological profiles. All epitopes were mapped on the solvent exposed area of Lol p 1 homology model accessible for IgG binding. One of the epitopes was identified as an ‘immunodominant’ Lol p 1 IgG-binding epitope (62-IFKDGRGCGSCFEIK-76) and classified as a novel epitope. The results from this study support the concept that modification allows shorter-course therapy options as a result of providing an IgG epitope repertoire important for efficacy. Additionally, the work paves the way to help further develop methods for standardising allergoid platforms.

Fractionation and identification of the allergic proteins in Aspergillus species.

Background and Purpose:Allergy is an undesired immune response to non-pathogenic agents. However, some opportunistic microorganisms such as fungi can also cause allergy. Among those fungi, hyphae form of Aspergillus strains including A. fumigatus, A. flavus, and A. niger could be mentioned. In this study, we aimed to separate allergic proteins from Aspergillus strains and determine their identity.Materials and Methods:Standard species of Aspergillus strains were cultivated in optimized conditions and the mycelium was separated by centrifugation. The fungal cells were lysed through physical methods such as freeze-thawing and grinding to prepare a suitable protein extract. The protein concentration was measured by Bradford method and the electrophoretic pattern of the extract was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were fractionated by ammonium sulfate precipitation and anion exchange chromatography using fast protein liquid chromatography (FPLC) system. The IgE immunoreactivity of the sensitized patients and controls was studied using the fractionated proteins by enzyme-linked immunosorbent assay (ELISA). Following SDS-PAGE, proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes and the strips were blotted with allergic patients' and controls' sera. The immunoreactive bands were excised from colloidal coomassie-stained SDS-PAGE gels and studied by mass spectroscopy methods.Results:Among the studied species, A. fumigatus showed stronger IgE reactivity and more IgE reactive protein bands than others did. The proteins with higher molecular weights showed stronger immunoreactivity in Western blotting. Receiver operating characteristic curve analysis demonstrated a correlation between the results of the applied ELISA methods. One of the most prominent IgE-reactive proteins was confirmed to be 45 kDa mycelia catalase. Conclusion:Our findings confirmed that high molecular weight proteins might play a major role in allergy and IgE reactivity to Aspergillus species. Moreover, the results showed that precipitation and chromatographic methods are applicable for fractionation of fungal proteins such as mycelial catalase.

The prevalence of wheat and spelt sensitivity in a randomly selected Australian population

To determine the prevalence of wheat sensitivity in a randomly selected Australian population, a study was carried out on sera samples of the Geelong Osteoporosis Study (GOS) age-stratified cohorts of men and women who were randomly selected from electoral rolls for the Barwon Statistical Division (n = 1145). The human sera were analysed by ELISA-based method using the ImmunoCAP 100 instrument (Phadia, Sweden). IgE from human sera were bound to the wheat (f4) and milk (f2) allergens that have been previously coated on the ImmunoCAPs. The number of IgE wheat and milk RAST positive individuals was determined. In order to relate the increased IgE immuno-reactivity to allergen symptoms, a questionnaire was established and sent to the blood donors and 974 individuals responded. Of these (n = 974) 147 individuals (15.1%) reported symptoms caused by wheat consumption, 179 (15.6%) and 112 (9.8%) sera showed RAST positive results (IgE > 60 response units) in wheat and milk RAST tests, respectively. However, only 2...

Reduction of IgE Immunoreactivity of Whole Peanut (Arachis hypogaea L.) After Pulsed Light Illumination

Pulsed light (PL), a novel food processing and preservation technology, has been shown in literature to reduce allergen levels on peanut, soybean, almond and shrimp protein extracts. This study investigated how PL affected the immunoreactivity of whole peanut kernels at two sample-to-lamp distances (7 and 10 cm) and a PL frequency of 3 pulses/s. A combination of different illumination durations and distances were tested to explore the effective conditions for PL roasting of whole peanuts. After the PL treatments, crude protein of the whole peanuts was extracted and compared to that of the raw peanuts (control) in vitro for allergen reactivity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, dot blot, and enzyme-linked immunosorbent assay (ELISA) with pooled plasma of allergenic patients. The SDS-PAGE, Western blot, dot blot results all showed that PL was capable of mitigating all major allergens of whole peanut kernels to an undetectable level. Indirect ELISA results showed 80 % signal reduction for the PL-treated peanut as compared to raw peanut. A closer distance (7 cm) between the PL lamp and the sample resulted in stronger reduction of IgE immunoreactivity than 10 cm.

Production of a chimeric allergen derived from the major allergen group 1 of house dust mite species in Nicotiana benthamiana

Plants are widely accepted as a general platform for the large-scale production of recombinant proteins, which has been demonstrated by the successful expression of various exogenous proteins. Using plants as a bioreactor for mass production of target proteins for vaccines is thought to show the most potential. This study explores whether a chimeric allergen R8, derived from the major allergen group 1 of house dust mites species (Dermatophagoides farinae and Dermatophagoides pteronyssinus), is expressed in tobacco. The highly efficient and useful Tobacco mosaic virus RNA-based overexpression (TRBO) vector was used to investigate expression of the R8 molecule in tobacco by agroinfection. Presence of R8 was detected using SDS-PAGE and Western blotting. Purified allergens were characterized using IgE-binding activity assay and allergen-specific immunotherapy (ASIT) in murine asthmatic models. The recombinant R8 was successfully expressed in tobacco leaves. The pro-peptide was observed in the herbaceous leaf extracts. This protein exhibits properties similar to the parental allergen ProDer f 1 expressed in Escherichia coli or tobacco with respect to IgE immunoreactivity. R8 also rectifies imbalance of TH1/TH2 cells. An herbaceous plant expression system model allows mass production of R8, which might be used in the future for diagnosis of asthma or production of a candidate vaccine for allergen-specific immunotherapy of asthma.

The sensitivity to different types of milk in patients with Ulcerative colitis

Event Abstract Back to Event The sensitivity to different types of milk in patients with Ulcerative colitis Irina Besu1*, Aleksandra Konic-Ristic2, Njegica Jojic3, Dragan Popovic4 and Zorica Juranic1 1 Institute of Oncology and Radiology of Serbia, Experimental Oncology, Serbia 2 Institute for Medical Research, Department of Nutrition and Metabolism, Serbia 3 KBC Zvezdara, Department of Gastroenterology and Hepatology, Serbia 4 Clinical Center of Serbia, Institute of Gastroenterology and Hepatology, Serbia Objective: Ulcerative colitis (UC) is a form of inflammatory bowel disease, which is treated as an autoimmune disease. The aim of this study was to determine the prevalence of the increased levels of serum antibodies to different types of milk: fresh cow’s milk (FCM), fresh goat’s milk (FGM), boiled cow’s milk (BCM) and boiled goat’s milk (BGM) in patients with UC. Methods: Seventeen patients with UC (15 with proven increased, mainly IgG and IgE immunity to cow’s milk proteins (CMP) and 2 without it) were included in this research. Levels of serum IgA, IgG and IgE antibodies to FCM, FGM, BCM and BGM were determined by ELISA test. The statistical analysis of data was performed by Wilcoxon rank sum test. Results: The levels of serum anti-FCM IgA, IgG and IgE antibodies were significantly higher than levels of serum anti-BCM (p=0.0313; p=0.0256; p=0.0029) respectively. Statistical analysis of IgG and IgE immunoreactivity to BGM and BCM showed that the levels of anti-BCM IgG and IgE antibodies were significantly higher in comparison to levels of anti-BGM IgG and IgE antibodies (p=0.0066; p=0.0005). The level of serum anti-FCM IgG antibody was significantly higher than level of serum anti-FGM (p=0.0125). The level of IgE immunity to FGM is significantly higher than level of IgE immunity to BGM (p=0.0231). Conclusion: These results indicate that patients with UC with increased immunity to CMP, could consider the use of BGM as the alternative protein source. Keywords: Immunoreactivity, Milk, antibody, IgA, IgG, IgE Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Besu I, Konic-Ristic A, Jojic N, Popovic D and Juranic Z (2013). The sensitivity to different types of milk in patients with Ulcerative colitis. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00805 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 19 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Irina Besu, Institute of Oncology and Radiology of Serbia, Experimental Oncology, Belgrade, Serbia, irina.besu@ncrc.ac.rs Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Irina Besu Aleksandra Konic-Ristic Njegica Jojic Dragan Popovic Zorica Juranic Google Irina Besu Aleksandra Konic-Ristic Njegica Jojic Dragan Popovic Zorica Juranic Google Scholar Irina Besu Aleksandra Konic-Ristic Njegica Jojic Dragan Popovic Zorica Juranic PubMed Irina Besu Aleksandra Konic-Ristic Njegica Jojic Dragan Popovic Zorica Juranic Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Modulation of IgE immunoreactivity to broad bean proteins after food processing in a Moroccan population

BackgroundThe aim of this study was to assess the sensitivity profile of the population of Fez and Casablanca in Morocco to dry broad bean (Vicia faba), and to investigate the effect of food processing (heat and/or enzymatic hydrolysis by pepsin) on the human IgE binding capacity to broad bean proteins (BBP). MethodsSera samples from 146 patients with atopic hypersensitivity were recruited in order to evaluate specific IgE levels to native and processed broad bean proteins by ELISA. Under the same conditions, we assessed the immunoreactivity of rabbit IgG obtained by immunisation with native BBP. ResultsHigh IgE levels to BBP were found; in fact, 79.3% of children and 80.4% of adults had positive values. The heat treatment (70°C during 60min) of dry beans proteins showed slight reduction in recognition of these antigens by rabbit IgG (22%) and by human IgE (12%). Pepsin hydrolysis decreased rabbit-IgG recognition by 55% in the first 30min of treatment. In contrast, and under the same conditions, pepsin increased human-IgE recognition with an average of 143% for all patients. However, the combination of the two treatments (heating and pepsin digestion) showed a decrease of 16% in BBP recognition for all patients. ConclusionsThis study demonstrates a high sensitivity of a Moroccan population to broad bean proteins which was resistant to heat and digestion by pepsin.

Clinical and laboratory investigation of oral allergy syndrome to grape.

Oral allergy syndrome (OAS) is occasionally observed following consumption of raw fruits in allergic adults. Since this phenomenon was commonly reported in Khorasan province of Iran; we intended to check if common diagnostic tests could be applied for differential diagnosis of OAS to grapes.IgE reactivity of 84 patients with OAS to grape and 34 patients with OAS to other fruits were analyzed by in vivo and in vitro methods, and the results were compared with those of controls. The patients underwent skin prick test (SPT) with common allergic pollen extracts as well as grape extract. The specific IgE level to grape proteins was determined by an indirect ELISA. The correlation of SPT results with ELISA and western blotting patterns was checked by statistical methods. The results showed a significant correlation of grape SPT diameters with grape specific IgE levels. Furthermore, a significant association of grape SPT results with IgE immunoreactivity of a 10 kDa grape protein, probably lipid transfer protein (LTP) was prominent. Immunoreactivity of other proteins was linked with mild clinical symptoms. The study showed a significant correlation of grape SPT results with grape total extract, as well as its 10 kDa component's IgE reactivity. The results suggested that OAS to grape should not be considered as a main criterion in diagnosis of grape allergy and a combination of grape SPT results with evaluation of IgE reactivity to grape 10 kDa allergen should be considered to achieve a more reliable grape allergy diagnosis.

Heat and pressure treatments effects on peanut allergenicity

Peanut allergy is recognized as one of the most severe food allergies. The aim of this study was to investigate the changes in IgE binding capacity of peanut proteins produced by thermal-processing methods, including autoclaving. Immunoreactivity to raw and thermally processed peanut extracts was evaluated by IgE immunoblot and skin prick test in patients with clinical allergy to peanut. Roasted peanut and autoclaved roasted peanut were selected for IgE ELISA experiments with individual sera, immunoblot experiments with antibodies against peanut allergens (Ara h 1, Ara h 2 and Ara h 3), digestion experiments, and circular dichroism spectroscopy. In vitro and in vivo experiments showed IgE immunoreactivity of roasted peanut proteins decreased significantly at extreme conditions of autoclaving. Circular dichroism experiments showed unfolding of proteins in autoclave treated samples, which makes them more susceptible to digestion. Autoclaving at 2.56atm, for 30min, produces a significant decrease of IgE-binding capacity of peanut allergens.

Increased Allergic Immune Response toSarcoptes scabieiAntigens in Crusted versus Ordinary Scabies

Scabies, a parasitic skin infestation by the burrowing "itch" mite Sarcoptes scabiei, causes significant health problems for children and adults worldwide. Crusted scabies is a particularly severe form of scabies in which mites multiply into the millions, causing extensive skin crusting. The symptoms and signs of scabies suggest host immunity to the scabies mite, but the specific resistant response in humans remains largely uncharacterized. We used 4 scabies mite recombinant proteins with sequence homology to extensively studied house dust mite allergens to investigate a differential immune response between ordinary scabies and the debilitating crusted form of the disease. Subjects with either disease form showed serum IgE against recombinant S. scabiei cysteine and serine proteases and apolipoprotein, whereas naive subjects showed minimal IgE reactivity. Significantly (P < 0.05) greater serum IgE and IgG4 binding to mite apolipoprotein occurred in subjects with crusted scabies than in those with ordinary scabies. Both subject groups showed strong proliferative responses (peripheral blood mononuclear cells) to the scabies antigens, but the crusted scabies group showed increased secretion of the Th2 cytokines interleukin 5 (IL-5) and IL-13 and decreased Th1 cytokine gamma interferon (IFN-gamma) in response to the active cysteine protease. These data confirm that a nonprotective allergic response occurs in the crusted disease form and demonstrate that clinical severity is associated with differences in the type and magnitude of the antibody and cellular responses to scabies proteins. A quantitative IgE inhibition assay identified IgE immunoreactivity of scabies mite antigens distinct from that of house dust mite antigens, which is potentially important for specific scabies diagnosis and therapy.

Humoral immunity to cow’s milk proteins and gliadin within the etiology of recurrent aphthous ulcers?

The goal of this study was to determine the incidence of serum antibodies to gliadin and to cow's milk proteins (CMP) using ELISA test, within patients who have recurrent aphthous ulcers (RAU). Fifty patients with recurrent aphthous ulcers and fifty healthy people were included in this research. Levels of serum IgA and IgG antibodies to gliadin and IgA, IgG and IgE to CMP were determined using ELISA. The levels of serum antigliadin IgA and IgG antibodies were not significantly higher in patients with RAU in comparison with the controls (P = 0.937 and P = 0.1854 respectively). The levels of serum anti-CMP IgA, IgG and IgE antibodies were significantly higher in patients with RAU in comparison with the controls (P < 0.005, P < 0.002 and P < 0.001 respectively). In general, the increased humoral (IgA or IgG) immunoreactivity to CMP was found in 32 of 50 patients, while 17 of them showed the increased levels of both IgA and IgG immunoreactivity to CMP. At the same time, 16 out of 50 patients had IgA, IgG and IgE immunoreactivity to CMP. These results indicate the strong association between high levels of serum anti-CMP IgA, IgG and IgE antibodies and clinical manifestations of recurrent aphthous ulcers.

Effects of food processing on food allergens

Food allergies are on the rise in Western countries. With the food allergen labeling requirements in the US and EU, there is an interest in learning how food processing affects food allergens. Numerous foods are processed in different ways at home, in institutional settings, and in industry. Depending on the processing method and the food, partial or complete removal of the offending allergen may be possible as illustrated by reduction of peanut allergen in vitro IgE immunoreactivity upon soaking and blanching treatments. When the allergen is discretely located in a food, one may physically separate and remove it from the food. For example, lye peeling has been reported to produce hypoallergenic peach nectar. Protein denaturation and/or hydrolysis during food processing can be used to produce hypoallergenic products. This paper provides a short overview of basic principles of food processing followed by examples of their effects on food allergen stability. Reviewed literature suggests assessment of processing effects on clinically relevant reactivity of food allergens is warranted.

Production of native and modified recombinant Der p 1 molecules in tobacco plants

As a complex molecule requiring post-translational processing, it has been difficult to produce the Der p 1 major allergen from the Dermatophagoides pteronyssinus house dust mite in a recombinant form. Here, we tested whether transgenic tobacco plants are suitable to express Der p 1, either as a wild-type molecule or as variants lacking N-glycosylation sites (Gly(-)) and/or cysteine protease activity (Enz(-)). Methods Using Agrobacterium tumefaciens-based transformation, pro Der p 1 molecules bearing mutations within either the N-glycosylation sites (N34Q, N150Q) and/or the cysteine protease-active site (C132V) were expressed in tobacco plants. After purification by ion exchange chromatography, allergens were characterized using immunoblotting, circular dichroism (CD), as well as basophil and T lymphocyte stimulation assays. Four forms of recombinant Der p 1 (i.e. wild-type Gly(+)/Enz(+), as well as Gly(-)/Enz(+), Gly(+)/Enz(-) or Gly(-)/Enz(-) variants) were successfully expressed in tobacco leaves as pro Der p 1 molecules. Spontaneous cleavage of the pro-peptide was observed in tobacco leaf extracts for all forms of recombinant Der p 1 (r Der p 1). CD confirmed that all r Der p 1 molecules, with the exception of the Gly(-)/Enz(-) variant, exhibited secondary structures comparable to the natural protein. A cysteine protease activity was associated only with the Gly(+)/Enz(+) form. All these molecules exhibit a profile similar to natural Der p 1 with respect to IgE immunoreactivity, basophil activation and T cell recognition. A tobacco plant expression system allows the production of various forms of mature Der p 1, which could be used for diagnostic or immunotherapeutic purposes.