Molecular and immunological characterization of wheat allergens

Bäcker Asthma bezeichnet eine IgE-vermittelte Überempfindlichkeitsreaktion, die durch Inhalation von Weizenmehl ausgelöst wird. Der Begriff weist auf eine berufsbezogene Erkrankung hin, von denen 4-25% betroffen sind. Das Erkennen des auslösenden Allergens und die Vermeidung der Allergenbelastung könnte die Symptome verringern. In der Vergangenheit konnten viele Allergene isoliert werden, aber es sind keine Tests verfügbar um zwischen der Weizen-abhängigen Nahrungsmittelallergie und Bäcker Asthma zu unterscheiden. Constantin et al. präsentierte in ihrer kürzlich publizierten Studie einen Allergen-Microarray basierend auf gereinigten, rekombinanten Proteinen. Isolierung, Identifizierung und Charakterisierung weiterer Allergene, die in Bäcker Asthma involviert sind, trägt zu einer Verbesserung der Methodik bei, welche sich zu einer effektiven Methode für die Krankheitsdiagnose in der Zukunft entwickeln könnte. Bezogen auf diese Vorstellung, war das Hauptziel meiner Diplomarbeit die molekulare, strukturelle und immunologische Charakterisierung von neuen Allergenen, die an Bäcker Asthma beteiligt sind. Die isolierten IgE-reaktiven cDNA Klone konnten durch Sequenzanalysen folgendermaßen identifiziert werden: Thioredoxin h (Klon 37), Glutathiontransferase (Klon 38), 1-Cysperoxiredoxin (Klon 112), Profilin (Klon 123) und Dehydrin (Klon 126). Die rekombinanten Proteine wurden in E.coli als C-terminal hexahistidin-markierte Proteine exprimiert und das Molekulargewicht wurde ermittelt. Ein Dot-Blot Experiment wurde durchgeführt um die IgEReaktivität der rekombinanten Proteine zu untersuchen. 1-Cys-peroxiredoxin wurde stark von Serum-IgE von 35.7% der Patienten, die an Bäcker Asthma leiden, erkannt. Darüber hinaus konnte mittels Untersuchung der Histaminfreisetzung eine stark allergene Wirkung in 21% der Patienten nachgewiesen werden. Im Gegensatz dazu wurde Thioredoxin h von keinen der Allergiker erkannt und keine Histaminfreisetzung induziert. Es gibt einen Bedarf an rekombinanten Weizenallergenen, die speziell von Bäcker Asthma Patienten erkannt werden, um die Diagnose zu verbessern, die Notwendigkeit von Provokationstests zu verringern und sie möglicherweise in der Immuntherapie von IgEvermittelten Allergie einsetzen zu können.Baker’s asthma is an IgE-mediated hypersensitivity disease caused by inhalation of wheat flour. The term still indicates the problem specific to a particular profession, from which 4- 25% are affected. Identification of the workspace agent could minimize the symptoms by avoiding exposure to the allergen. In the past, many allergens could be isolated, but there is no test available to discriminate between wheat-dependent food allergy and Baker’s asthma. Constantin et al. presented in a recently published study an allergen microarray based on purified recombinant proteins. Further isolation, identification and characterization of allergens involved in Baker’s asthma will improve this method which could become an effective tool for the diagnosis of the disease in the future. Based on this idea, the main aim of my diploma thesis was the molecular, structural and immunological characterization of new allergens involved in Baker’s asthma. The isolated IgE-reactive cDNA clones could be identified by sequence analysis as follows: Thioredoxin h (clone 37), Glutathione transferase (clone 38), 1-Cys-peroxiredoxin (clone 112), Profilin (clone 123) and Dehydrin (clone 126). Recombinant proteins were expressed in E.coli as C-terminally hexahistidine-tagged proteins and the molecular weight was determined. A dot-blot experiment which is an effective method to study IgE-reactivity of recombinant proteins, was performed. 1-Cys-peroxiredoxin was strongly recognized by serum IgE from 35.7% of patients suffering from Baker’s asthma. Furthermore, in histamine release assays, an important method for evaluating allergenicity of allergens, we could show strong allergenic activity in 21% of patients. In contrast, thioredoxin h was recognized by none of the allergics and only induced a weak degranulation in the histamine release assay. There is a need of recombinant wheat allergens, specifically recognized by baker’s asthma patients, to use them for diagnosis, reduce provocation testing and possibly immunotherapy of IgE-mediated allergy

Metacarpophalangeal joint loads during bonobo locomotion: model predictions vs. proxies

The analysis of internal trabecular and cortical bone has been an informative tool for drawing inferences about behaviour in extant and fossil primate taxa. Within the hand, metacarpal bone architecture has been shown to correlate well with primate locomotion; however, the extent of morphological differences across taxa is unexpectedly small given the variability in hand use. One explanation for this observation is that the activity-related differences in the joint loads acting on the bone are simply smaller than estimated based on commonly used proxies (i.e. external loading and joint posture), which neglect the influence of muscle forces. In this study, experimental data and a musculoskeletal finger model are used to test this hypothesis by comparing differences between climbing and knuckle-walking locomotion of captive bonobos (Pan paniscus) based on (i) joint load magnitude and direction predicted by the models and (ii) proxy estimations. The results showed that the activity-related differences in predicted joint loads are indeed much smaller than the proxies would suggest, with joint load magnitudes being almost identical between the two locomotor modes. Differences in joint load directions were smaller but still evident, indicating that joint load directions might be a more robust indicator of variation in hand use than joint load magnitudes. Overall, this study emphasizes the importance of including muscular forces in the interpretation of skeletal remains and promotes the use of musculoskeletal models for correct functional interpretations

Musculoskeletal models of a human and bonobo finger: parameter identification and comparison to in vitro experiments

Introduction: Knowledge of internal finger loading during human and non-human primate activities such as tool use or knuckle-walking has become increasingly important to reconstruct the behaviour of fossil hominins based on bone morphology. Musculoskeletal models have proven useful for predicting these internal loads during human activities, but load predictions for non-human primate activities are missing due to a lack of suitable finger models. The main goal of this study was to implement both a human and a representative non-human primate finger model to facilitate comparative studies on metacarpal bone loading. To ensure that the model predictions are sufficiently accurate, the specific goals were: (1) to identify species-specific model parameters based on in vitro measured fingertip forces resulting from single tendon loading and (2) to evaluate the model accuracy of predicted fingertip forces and net metacarpal bone loading in a different loading scenario. Materials & Methods: Three human and one bonobo (Pan paniscus) fingers were tested in vitro using a previously developed experimental setup. The cadaveric fingers were positioned in four static postures and load was applied by attaching weights to the tendons of the finger muscles. For parameter identification, fingertip forces were measured by loading each tendon individually in each posture. For the evaluation of model accuracy, the extrinsic flexor muscles were loaded simultaneously and both the fingertip force and net metacarpal bone force were measured. The finger models were implemented using custom Python scripts. Initial parameters were taken from literature for the human model and own dissection data for the bonobo model. Optimized model parameters were identified by minimizing the error between predicted and experimentally measured fingertip forces. Fingertip forces and net metacarpal bone loading in the combined loading scenario were predicted using the optimized models and the remaining error with respect to the experimental data was evaluated. Results. The parameter identification procedure led to minor model adjustments but considerably reduced the error in the predicted fingertip forces (root mean square error reduced from 0.53/0.69 N to 0.11/0.20 N for the human/bonobo model). Both models remained physiologically plausible after the parameter identification. In the combined loading scenario, fingertip and net metacarpal forces were predicted with average directional errors below 6◦ and magnitude errors below 12%. Conclusions. This study presents the first attempt to implement both a human and nonhuman primate finger model for comparative palaeoanthropological studies. The good agreement between predicted and experimental forces involving the action of extrinsic flexors—which are most relevant for forceful grasping—shows that the models are likely sufficiently accurate for comparisons of internal loads occurring during human and non-human primate manual activities

Biochemical, Biophysical and IgE-Epitope Characterization of the Wheat Food Allergen, Tri a 37

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Aplicación del enfoque fisiográfico para el relevamiento de suelos semidetallado en el departamento Guaraní, Misiones

Se estudia la relación paisaje-suelo en el departamento Guaraní, Misiones, aplicando el enfoque fisiográfico. Para la clasificación de las unidades de paisaje se utilizó el Modelo Digital de Elevaciones y parámetros del terreno derivados. En cada unidad se realizan descripciones del sitio (relieve, vegetación, uso del suelo) y el reconocimiento de perfiles de suelo. Los resultados permitieron diferenciar cinco unidades a nivel de Gran Paisaje: La Meseta central constituye el relieve más antiguo, con lomas muy amplias y pendientes suaves, donde dominan suelos rojos profundos (Ultisoles y Oxisoles), y se desarrolla el cultivo de té y yerba mate; las Estribaciones de la Meseta central es un sector más erosionado, con pendientes mayores y dominancia de Alfisoles rojos y Ultisoles, siendo un área de reserva ecológica; El Lomerío comprende lomadas de menor extensión y mayores pendientes que las anteriores, donde dominan Alfisoles rojos y afloramientos rocosos. Entre las lomadas se desarrollan valles en “U”, donde se reconocen Alfisoles pardos y Molisoles. El uso de los suelos es para la producción de té, yerba mate, ganadería y forestación en las lomas y cultivos anuales en los valles; La Serranía se caracteriza por cerros fuertemente disectados con laderas de pendientes pronun ciadas, donde los valles son estrechos y en forma de “V”. Dominan los Entisoles y afloramientos rocosos en sectores cuspidales o de mayor pendiente, mientras que en áreas planas, se desarrollan Molisoles. El uso de los suelos es para producción de yerba mate, tabaco, citronela y forestación, siendo la unidad que presenta mayor cobertura de vegetación natural; El valle del Río Uruguay presenta Entisoles en sectores de pendiente y Molisoles en terrazas, donde se producen cultivos anuales. El análisis fisiográfico realizado será de utilidad para el relevamiento de suelos y la confección de la Carta a escala semidetallada del departamento.Fil: Moretti, Lucas M. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Cerro Azul; ArgentinaFil: Tenti Vuegen, Leonardo M. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Suelos. ArgentinaFil: Barbaro, S.E. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Cerro Azul; ArgentinaFil: Hopechek, L.A. Provincia de Misiones. Ministerio del Agro y la Producción; ArgentinaFil: Lanfranco, M. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi; ArgentinaFil: Alvarenga, F.A. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Cerro Azul; ArgentinaFil: Florentín, J. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Cerro Azul; ArgentinaFil: Pahr, Norberto Manuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; ArgentinaFil: Von Wallis, Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; ArgentinaFil: Rodriguez, Darío M. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Suelos; ArgentinaFil: Schulz, Guillermo A. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Suelos; ArgentinaFil: Escobar, Dardo. Ministerio de Agricultura, Ganadería y Pesca; ArgentinaFil: Ybarra, Diego Daniel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Corrientes; ArgentinaFil: Perucca, S. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Agencia de Extensión Rural Río Cuarto; ArgentinaFil: Iwasita, Bárbara Eloisa. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Cerro Azul; Argentin

Characterization of allergens involved in IgE-mediated wheat allergy

Ziel: Ziel meiner Dissertation war die Charakterisierung von IgE-reaktiven cDNA Klonen, welche von einer cDNA Bank aus Weizen isoliert wurden. Nachdem die Weizenallergene rekombinant hergestellt wurden war es unser Ziel, die Allergene sowohl biochemisch, biophysikalisch als auch immunologisch charakterisieren und für die Allergiediagnostik verwenden. Material und Methoden: Eine cDNA Bank aus Weizen (Triticum aestivum) wurde hergestellt und mit Serum IgE Antikörpern von Patienten mit Bäcker Asthma oder Weizen-abhängigen Nahrungsmittelallergie gescreent um neue Weizenallergene zu entdecken. Nachdem die Proteine in einem prokaryotischem (E.coli Bakterien) oder eukaryotischem (Bakulovirus-infizierte Insektenzellen) Expressionssystem rekombinant hergestellt wurden, wurden sie mittels Massenspektrometrie, Zirkulardichroismus, Größenausschluss-Chromatographie und chemischem Crosslinking biochemisch analysiert. RAST-basierende, nicht-denaturierende Dot-Blot Experimente wurden verwendet, um deren IgE-Reaktivität zu bestimmen und basophile Degranulierungsexperimente wurden durchgeführt um die allergene Wirkung der Proteine zu untersuchen. Zusätzlich wurden Allergen-spezifische Hasenantikörper verwendet um den Verlauf der Proteinexpression im Weizensamen, die Kreuzreaktivität, die Stabilität unter gastrischen oder duodenalen Bedingungen, als auch deren Hitzestabilität in verschiedenen Brotsorten nach dem Backen, zu untersuchen. Resultate: Im Zuge meiner Dissertation habe ich fünf verschiedene Bäcker Asthma- Allergene (Thioredoxin h, Glutathione transferase, 1-Cys-peroxiredoxin, Profilin und Dehydrin) charakterisiert, wobei 1-Cys-peroxiredoxin aufgrund der Prävalenz in der IgE Erkennung, den Ergebnissen der basophilen Degranulationstests und auch der immunologischen Kreuzreaktivität als das wichtigste Allergen unter ihnen beschrieben wurde. Des Weiteren haben wir ein neues Weizennahrungsmittelallergen, Alpha Purothionin (Tri a 37) identifiziert und charakterisiert. Dieses neue Allergen ist ein serologisches Markerallergen für die schwere Weizennahrungsmittelallergie. Fazit: Durch die Verwendung von rekombinanten Weizenallergenen in Microarray-basierenden Komponenten-auflösenden Diagnosverfahren kann eine detaillierte Diagnose und eine zusätzliche Unterscheidung der verschiedenen klinischen Manifestationen der Weizenallergien gemacht werden.Objectives: Aim of my thesis was the characterization of IgE-reactive cDNA clones which were isolated from a wheat seed cDNA library. After recombinant production of wheat allergens, my aim was to characterize them regarding biochemical, biophysical and immunological properties and for the use in diagnosis of allergy. Material and Methods: A cDNA library (Triticum aestivum) from wheat seeds was constructed and screened with serum IgE antibodies from patients with baker's asthma or wheat-dependent food allergy to identify novel wheat allergens. After recombinant protein expression using a prokaryotic system (E.coli cells) or a eukaryotic system (baculovirus-infected insect cells), allergens were biochemically analyzed using mass spectrometry, circular dichroism spectroscopy, size exclusion chromatography and chemical crosslinking. RAST-based, non-denaturing dot blot experiments were used to determine their IgE-reactivity and basophil degranulation experiments were performed to investigate the allergenic activity. Additionally, allergen-specific rabbit antibodies were raised to study the course of protein expression during wheat seed maturation, cross-reactivity, their stability when subjected to gastric or duodenal digestion, as well as their heat stability in different bread sorts. Results: In the context of my thesis, five baker's asthma allergens (thioredoxin h, glutathione transferase, 1-Cys-peroxiredoxin, profilin and dehydrin) were characterized. The cross-reactive allergen 1-Cys-peroxiredoxin was identified as the most relevant allergen according to prevalence of IgE-recognition and basophil degranulation assays. Additionally, we identified and characterized a new wheat food allergen alpha purothionin (Tri a 37) which was found to serve as a serologic marker for the diagnosis of severe wheat food allergy. Conclusion: The use of recombinant wheat allergens in microarray-based component-resolved diagnostic tests allows a detailed diagnosis and may distinguish between various clinical manifestations of wheat allergies.submitted by Sandra PahrAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2014OeBB(VLID)171634

Molecular and immunological characterization of Tri a 36, a low molecular weight glutenin, as a novel major wheat food allergen

Abstract Wheat is an essential element in our nutrition but one of the most important food allergen sources. Wheat allergic patients often suffer from severe gastrointestinal and systemic allergic reactions after wheat ingestion. In this study, we report the molecular and immunological characterization of a new major wheat food allergen, Tri a 36. The cDNA coding for a C-terminal fragment of Tri a 36 was isolated by screening a wheat seed cDNA expression library with serum IgE from wheat food-allergic patients. Tri a 36 is a 369-aa protein with a hydrophobic 25-aa N-terminal leader peptide. According to sequence comparison it belongs to the low m.w. glutenin subunits, which can be found in a variety of cereals. The mature allergen contains an N-terminal domain, a repetitive domain that is rich in glutamine and proline residues, and three C-terminal domains with eight cysteine residues contributing to intra- and intermolecular disulfide bonds. Recombinant Tri a 36 was expressed in Escherichia coli and purified as soluble protein. It reacted with IgE Abs of ∼80% of wheat food-allergic patients, showed IgE cross-reactivity with related allergens in rye, barley, oat, spelt, and rice, and induced specific and dose-dependent basophil activation. Even after extensive in vitro gastric and duodenal digestion, Tri a 36 released distinct IgE-reactive fragments and was highly resistant against boiling. Thus, recombinant Tri a 36 is a major wheat food allergen that can be used for the molecular diagnosis of, and for the development of specific immunotherapy strategies against, wheat food allergy.</jats:p