No description available.

This study was conducted to develop and validate cleaning procedures for different processing equipment of varying complexity and to determine the efficacy of two different allergen tests. Following introduction of selected allergens to processing equipment, two treatments were applied - water wash or scrub/sanitize ? and a no clean was also evaluated. The equipment used consisted of a slicer, grinder, injector, vacuum tumbler, and plastic lugs. To introduce the allergen to the slicer, nine ready-to-eat hams were used. One hundred twenty-two kilograms of pork trim were ground, and a milk allergen was incorporated into the meat. The injector was contaminated with a food allergen by injecting boneless pork loins with a marinade containing soy flour. The slicer, grinder, injector, tumbler, and lugs were then subjected to randomized treatments. The results showed that the water wash and scrub/sanitize treatments did not differ significantly among the pieces of equipment tested. This study supported that both water wash and scrub/sanitize treatments can effectively removed allergens to a level below the industry threshold of 5 ppm.

Seafood including crustaceans, on ingestion, are known to provoke gastrointestinal as well as systemic allergic reactions. Crustaceans are aquatic arthropods with a chitinous exoskeleton and include shrimp, lobster, prawn and crab. Earlier studies in our laboratory have led to the identification and characterization of three allergens from shrimp, designated as Sa-I, Sa-I1 and Sa-III. The former two were shown to be heat stable proteins with a mol. wt. of 8.4 and 34 kDa respectively, while Sa-III was identified as tRNA Arg and TRNATyr ). Sa-II was found to be the major allergen contributing to more than 50% of the allergenic activity. There are several reports on the existence of cross-reactivity among atopic allergens, in particular food allergens. It is well known that individuals with shrimp allergy often complain of adverse reactions following the ingestion of other re1ated crustaceans. Recognition of crustacea as a group causing adverse reactions in sensitive individuals has a basis in the close phylogenetic relationship of shrimp, lobster, crab and prawn. Thus, one could expect appreciable similarity in the IgE binding epitopes of the offending allergens from related crustaceans. The present study was, therefore, aimed towards the identification of the major cross-reacting crustacean allergen and localization of its IgE binding epitopes. Cross-reactivity among a1lergens from shrimp, prawn, crab and lobster was evaluated by immunochemical methods. Antigenic cross-reactivity was established by immunodiffusion using shrimp-specific rabbit IgG. Competitive ELlSA inhibition experiments using sera of shrimp sensitive patients revealed a high degree of allergenic cross-reactivity between different crustaceans. SDSPAGE and immunoblot analysis using the sera of shrimp sensitive patients have identified a 34 kDa protein as the cross-reacting crustacean allergen. Using shrimp as a model system and Sa-II as a representative crustacean allergen, further studies were carried out to get an insight into the structural and molecular basis of allergenic cross-reactivity. The strategies adopted were, (1) to raise allergen specific anti-idiotypic antibodies and explore the possibility of using these anti-idiotypic antibodies as surrogate allergens for diagnosis of crustacea allergy and (2) to identify the IgE binding epitopes on the major shrimp allergen Sa-II, which may be shared by the 34 kDa allergen from the related crustaceans. In order to explore idiotypic, anti-idiotypic and anti-anti-idiotypic responses to Sa-II, Balb/c mice were immunized with affinity purified human idiotypic antibodies directed against the purified allergen. This resulted in the production of anti-idiotypic antibodies which were quantitated using rabbit idiotypic antibodies raised against the same allergen. The mouse anti-idiotypic antibodies recognized shrimp-specific human idiotypic antibodies of the IgE isotype from 18 of 20 individuals, and IgG antibodies from 14 of 20 shrimp sensitive patients. Immunization of Balb/ c mice with affinity purified, allergen-specific anti-idiotypic antibodies induced anti-allergen IgE and IgG responses in the absence of the allergen. The induction of anti-anti-idiotypic antibodies functionally identical to allergen-specific idiotypic antibodies confirmed that the anti-idiotypic antibodies generated, are indeed a mirror image of the allergen. The present study thus provides evidence that anti-idiotypic antibodies raised against allergen-specific idiotypic antibodies may substitute for the original allergen in the induction of allergen-specific idiotypic antibodies. The demonstration of shared idiotopes on IgG and IgE antibodies in the sera of shrimp sensitive patients supports the use of allergen-specific anti-idiotypic antibodies as surrogate allergens. These anti-anti-idiotypic antibodies not only recognized Sa-II, but also the 34 kDa allergen from prawn, lobster and crab. Cross-reactivity studies using polyclonal sera of shrimp sensitive patients and Sa-II anti-anti-idiotypic antibodies have attributed the allergenic cross-reactivity observed among the related crustaceans to the presence of highly conserved IgE binding epitopes on the 34 kDa crossreacting allergen from shrimp, crab, lobster and prawn. In order to identify the igE binding epitopes on Sa-11, it was subjected to limited tryptic digestion and the peptides were separated by reverse phase HPLC. Amino acid sequence analysis of these peptides and several other peptides generated by Asp N and Lys C treatment revealed an 861 homology with the muscle protein tropomyosin from the fruit fly Drosophila melanogaster, suggesting that the major shrimp allergen is tropomyosin. To establish that Sa-II is indeed tropomyosin, the latter was isolated from shrimp and its physicochemical and immunochemical properties were compared with those of Sa-II. Both tropomyosin and Sa-II had the same molecular mass and focused in the isoelectric pH range of 4.8-5.4. In the presence of 6 M urea, the mobility of both Sa-I1 and shrimp tropomyosin shifted to give an apparent molecular mass of 50 kDa, which is a characteristic property of tropomyosins. Shrimp tropomyosin bound to specific IgE antibodies in the sera of shrimp sensitive patients as assessed by competitive ELISA inhibition and immunoblot analysis. Tropomyosin, similar to Sa-I1 was subjected to limited tryptic digestion and the tryptic maps of both Sa-II and tropomyosin as obtained by reverse phase HPLC were found to be super imposable. Dot blot immunoassay and competitive ELISA inhibition assay using the sera of shrimp sensitive patients identified two peptides, 6 and 9 that exhibited allergenic activity. Both the peptides were purified to homogeneity and sequenced. Peptide 6 is a nonapeptide corresponding to the amino adds 153-161 and peptide 9 has 17 amino acids corresponding to the aminoacid residues 50-66. The peptides individually blocked upto 50% the binding of allergen-specific IgE to hropomyosin. Sa-II specific mouse anti-anti-idiotypic antibodies recognized not only tropomyosin, but also the two allergenic peptides, thus confirming that these peptides represent the major IgE binding epitopes. The IgG binding activity was found to be associated with peptides 6 and 9 as assessed by dot blot immunoassay using the sera of shrimp sensitive patients. Thus, it was found that both IgG and IgE binding epitopes on shrimp tropomyosin are identical. Tropomyosins from both phylogenetically related and unrelated species were assessed for allergenic activity using the sera of shrimp sensitive patients. It was found that allergenic activity was associated with tropomyosins from related crustaceans and from Drosophila melanogaster which shares 86% homology with shrimp tropornyosin. However, tropomyosins from totally unrelated species like yeast, chicken, bovine, rat, rabbit and human did not exhibit allergenic activity. A comparison of the amino acid sequence of shrimp tropomyosin in the region of IgE binding epitopes with the corresponding regions of bopomyosins from different species confirmed lack of allergenic cross-reactivity. The allergenic peptides 6 and 9 were able to inhibit the binding of tropomyosins from related crustaceans to shrimp tropomyosin-specific IgE antibodies to the same extent, confirming the presence of highly conserved IgE binding epitopes. It has been established for the first time that the major crustacean allergen is the heat stable muscle protein, tropomyosin, and extensive cross-reactivity between different members of crustacea is due to the presence of highly conserved IgE binding epitopes on tropomyosins from these sources. Thus, from the present study, information with respect to the amino acid sequence of tropomyosin and localization of its 1gE binding epitopes, could be used to design synthetic peptides corresponding to the B cell and T cell epitopes which would find application in the diagnosis and desensitization of individuals allergic to crustacea.

Methacholine (MCh) and mannitol challenges are tests used to assess airway responsiveness. It has been shown that airway responsiveness to direct bronchoconstrictors like MCh tends to increase following exposure to allergen but the response to mannitol an indirect stimuli, is not known. Furthermore, the provocative concentration causing a 20% decrease in Forced Expiratory Volume in one second (FEV1) for adenosine 5’ monophosphate (AMP) correlates better to sputum eosinophilia than MCh PC 20. Hence, we hypothesized that airway responsiveness will be greater when measured with mannitol than MCh. We studied airway responsiveness to MCh and mannitol first at 3 hours and then later at 24 hours after allergen challenge. The 3-hour study yielded results contrary to our hypothesis therefore a twenty-four hour study was undertaken. Ten mild atopic asthmatics who had a positive MCh challenge and an allergic response to allergen extracts such as cat, horse, and house dust mite completed the 3-hour study. Eleven mild atopic asthmatics with the criteria above completed the 24-hour study. Both studies were non-blinded, randomized clinical trials. Airway responsiveness to MCh was quantitated by changes in PC20. Airway responsiveness to mannitol was quantitated as PD15 in the 3-hour study and dose response ratio (DRR) in the 24-hour study. In both studies, the allergen challenges were separated by 14 days. Fractional exhaled nitric oxide measurements (FENO) were collected in both studies at varying time points to track airway inflammation. In the 3-hour study, the geometric mean MCh PC20 decreased significantly after allergen exposure from 0.88 mg/ml to 0.50 mg/ml (p = 0.02) indicating airway responsiveness to MCh increased. Conversely, the geometric mean mannitol PD15 increased significantly from 174 mg to 284 mg (p =0.02) indicating a decrease in airway responsiveness to mannitol. In the 24-hour study, the geometric mean MCh PC20 again decreased significantly from 5.9 mg/ml to 2.2 mg/ml (p= 0.01) after allergen exposure. The mannitol DRR increased significantly from 63 mg/∆%FEV1 to 158 mg/∆%FEV1 (p = 0.03). FENO levels increased significantly in MCh arm but not mannitol arm. That is pre allergen challenge versus 24 hours after allergen challenge (for MCh arm: 26 ppb pre to 55 ppb post; for mannitol arm: 31 ppb pre to 39 ppb post). In conclusion, at three and twenty-four hours after allergen challenge, a time when the airways are more responsive to MCh, there is a significant decrease in airway responsiveness to mannitol.

Universiẗat, Diss., 2007--Frankfurt (Main).

No description available.

Fungi are one of the major organisms that cause allergic disease in human. A number of proteins from fungi have been found to be allergenic or possess immunostimulatory properties. Identifying and characterizing allergens from fungal genomes will help facilitate our understanding of the mechanism underlying host-pathogen interactions in allergic diseases. Currently, there is a lack of tools that allow us to rapidly and accurately predict allergens from whole genomes. In the context of whole genome annotation, allergens are rare compared to non-allergens and thus the data is considered highly skewed. In order to achieve a confident set of predicted allergens from a genome, false positive rates must be lowered. Current allergen prediction tools often produce many false positives when applied to large-scale data set such as whole genomes, and thus lower the precision. Moreover, the most accurate tools are relatively slow because they use sequence alignment to construct feature vectors for allergen classifiers. This dissertation presents computational approaches in characterizing the allergen repertoire in fungal genomes as part of the whole genome studies of Alternaria, an important allergenic/opportunistic human pathogenic fungus and necrotrophic plant parasite. In these studies, the genomes of multiple Alternaria species were characterized for the first time. Functional elements (e.g. genes, proteins) were first identified and annotated from these genomes using computational tools. Protein annotation and comparative genomics approaches revealed the link between Alternaria genotypes and its prolific saprophytic lifestyle that provides at least a partial explanation for the development of pathological relationships between Alternaria and humans. A machine learning based tool (Allerdictor) was developed to address the neglected problem of allergen prediction in highly skewed large-scale data sets. Allerdictor exhibited high precision over high recall at fast speed and thus it is a more practical tool for large-scale allergen annotation compared with existing tools. Allerdictor was then used together with a comparative genomics approach to survey the allergen repertoire of known allergenic fungi. We predicted a number of mold allergens that have not been experimentally characterized. These predicted allergens are potential candidates for further experimental and clinical validation. Our approaches will not only facilitate the study of allergens in the increasing number of sequenced fungal genomes but also will be useful for allergen annotation in other species and rapid prescreening of synthesized sequences for potential allergens. / Ph. D.

No description available.

Cette thèse a pour objectif d’identifier et caractériser des biomarqueurs liés aux traitements d’immunothérapie allergénique (ITA). En particulier, le travail s’est porté sur les biomarqueurs prédictifs de l’efficacité du traitement : leur estimation avant la prise médicamenteuse permettrait d’évaluer les bénéfices cliniques à l’issue de l’ITA. À partir d’échantillons de sérum collectés avant ITA et provenant de patients inclus dans une étude clinique contrôlée, randomisée et dirigée contre les pollens de graminées, une méthode d’analyse protéomique différentielle (la 2D-DIGE) a permis d’identifier une protéine comme candidat biomarqueur prédictif de l’efficacité de l’ITA. Dans un premier temps, les variants moléculaires de la protéine identifiée ont été caractérisés. L’analyse en spectrométrie de masse de la protéine a permis d’identifier les modifications post-Traductionnelles associées aux isoformes plus abondants dans le sérum des patients pour lesquels une réponse positive à l’ITA est observée. Une approche protéomique de quantification relative a été mise en œuvre par LC-MS/MS sans marquage, et a permis d’identifier des peptides de la protéine d’intérêt, portant des modifications post-Traductionnelles spécifiques, associés à un bénéfice clinique accru en fin d’ITA. Dans un deuxième temps, l’implication de la fétuine-A dans la physiopathologie de l’allergie a été étudiée. Des modèles cellulaires humains ont mis en évidence le caractère essentiel des acides sialiques portés par la protéine pour l’activation de la voie TLR4, dans les mécanismes de l’immunité innée initiant la réaction allergique. In vivo, les modèles murins d’asthme allergique développés ont en revanche montré la fonction anti-Inflammatoire de la protéine. La fétuine-A a donc un rôle ambivalent, mais est indubitablement liée à la régulation de l’inflammation allergique. La validation des candidats biomarqueurs peptidiques de la protéine dans d’autres cohortes cliniques représente une future étape clé, qui permettrait d’envisager l’usage de ces marqueurs en clinique, pour améliorer la sélection des patients les plus susceptibles de répondre à l’ITA. / This thesis aimed at identifying and characterizing predictive biomarkers associated with allergen-Specific immunotherapy (AIT) efficacy. In particular, we were focused on biomarkers predictive of AIT efficacy: quantifying such markers before treatment would allow estimating the final clinical benefit. For this purpose, serum samples were collected before AIT from patients included in a double-Blind, placebo controlled clinical study against grass pollen allergy. Their analysis by means of differential proteomics (2D-DIGE) pointed out a protein, named fetuin-A, as a candidate biomarker predictive of AIT efficacy. First, the protein fetuin-A isoforms were extensively characterized by mass spectrometry, and specific post-Translational modifications (PTMs) were associated to the isoforms more abundant in sera from patients positively responding to AIT. A second proteomic approach allowed identifying fetuin-A peptides, differentially expressed among groups of patients. Some peptides from the candidate protein, bearing specific post-Translational modifications (PTMs), are associated with an increased clinical benefit at the end of the treatment. In a second part, we studied the involvement of the fetuin-A during the course of allergic inflammation. Human cellular assays highlighted that sialic acids on the protein PTMs are essential to activate the TLR4 pathway, and inducing innate immune mechanisms linked to allergy. In vivo, murine models of allergic asthma showed an opposite anti-Inflammatory function of the protein. Thus, the protein fetuin-A is still ambivalent, but is undoubtedly linked to the regulation of allergic inflammation. Validation of peptides candidate biomarkers in larger clinical cohorts is of utmost interest, since using such biomarkers in clinics would improve patients’ selection and therefore clinical benefit from the treatment.

Peanut allergy typically results from sensitisation to one or more integral seed storage proteins; Ara h 1, 3 or 2/6. Reactions can be triggered by as little as 3 mg protein in 10% of allergic individuals and are often severe, inducing anaphylaxis which can be fatal. Accidental exposure through unintended presence can therefore be hazardous and foods must be labelled appropriately. Thermal processing is one of the main factors affecting protein properties in food systems, including the formation of Maillard reaction products. Research has suggested a link between the allergenicity of foods and cooking methods employed. A systematic study was undertaken to assess the thermal dependence of these hazard proteins, focusing on changes to solubility and chemical modifications which may alter IgE binding. Proteomic profiling was used to assess the allergenic content of peanut products and develop alternative methods for allergen analysis to support evidence-based application of precautionary allergen labelling. Runner variety peanuts were processed (boiled, fried, roasted) and their protein content determined. Proteins extracts were characterised by 1D- and 2D-polyacrylamide gel electrophoresis, including differential in-gel electrophoresis. Proteomic profiling was undertaken using label-free analysis to assess allergen composition and investigate the formation of Maillard reaction products on the most clinically relevant proteins. Peanut allergen peptide targets were then identified and used to develop label-based quantitation methods and applied to (i) investigate effects of processing on peptide targets and (ii) determine peanut in chocolate products in comparison with a commercial ELISA kit. Orthogonal studies were performed using serum samples from peanut allergic patients obtained from the Manchester Respiratory, Allergy and Thoracic Surgery (ManARTS) Biobank. Patient IgE reactivity to peanut and processed peanut products was assessed by immunoblotting, inhibition ELISA and mediator release assays. Protein solubility was reduced by thermal processing and processed protein required harsh denaturing conditions for extraction. Qualitative analysis highlighted decreased solubility of key allergens, modifications and aggregation after heating. Proteomic profiling identified and quantified different isoforms of the major peanut allergens. The protein content of processed peanuts was reduced by boiling, specifically the 2S albumins, which transferred into the cooking water. The performance of peptides selected for targeted MRM experiments was influenced by thermal processing and the presence of cocoa phenolics. Ara h 2 peptides flanked by arginine were thermostable and may prove more reliable for quantification. Application of microfluidic separation enhanced the efficiency of target ionisation in complex matrices acquiring important sensitivity gains. Maillard modifications to clinically relevant proteins Ara h 2/6 were found within IgE binding domains in raw and processed peanuts. IgE reactivity studies confirmed reduced IgE binding capacity of extensively boiled peanut and hydrolysed protein, but this did not correlate to a reduction in mediator release in poly-sensitised patients. While effective extraction limits the efficacy of analyses, buffers used in MS analyses are more robust in analysing processed protein. Proteomic profiling provides a means of characterising and profiling of allergenic proteins including food ingredients used in clinical applications. Peptides selected for targeted analyses should be validated to assess their suitability in model foods. Cooking waters collected from extensively boiled peanuts may provide an alternative and safer immunotherapy agent for patients predominantly sensitised to Ara h 2/6.