Failure to comply with prescribed regimens is a major reason for the failure of treatment programs. This study investigated factors which are related to compliance with prescribed diet revision therapy for food allergies in school-aged children. Forty-five children, aged 6 to 12 years, who were under a physician's care for food allergies, formed the sample. The Health Belief Model was used as the basis for a questionnaire devised to measure these factors. The development of the Diet Revision Therapy Parent Questionnaire involved a pilot test and revisions; the resulting instrument consists of 38 items organized into 4 subtests, of which one 7-item subtest is to be considered optional. The 38-item DRTPQ has a full scale internal consistency reliability of .87, and a composite reliability of .61 for the four subscales. The canonical correlation between 3 types of subjective ratings of compliance and the 4 subtests is .80, with 64% shared variance between these sets of variables. A discriminant function of 3 subtests of the DRTPQ proved capable of discriminating diet therapy dropouts from continuing subjects with 88.9% accuracy. These three subtests measured: 1. Parent and family life factors, such as the amount of perceived interference in normal routines, 2. Child's attitudes to the treatment and his/her normal behavior with respect to cooperation with parental demands, and 3. Belief in the benefits to be derived from the treatment. A fourth category of items measured perceived severity of the condition and perceived susceptibility to illness but proved not to predict compliance in this sample, although it may be useful in clinical practice. Suggestions for interventions to aid compliance are outlined. / Education, Faculty of / Educational and Counselling Psychology, and Special Education (ECPS), Department of / Graduate

Food allergies within the United States and Europe have become exceedingly prevalent in children, adolescents, and adults. In the United States alone, over 12 million people have been diagnosed with at least one food allergy. If the allergen is accidentally consumed, the food allergy sufferer may suffer a life-threatening condition known as anaphylaxis. More than 200 U.S. food allergic consumers die annually as a result of anaphylaxis due to ingestion of food allergens. To safely manage food allergies, food allergy sufferers and stakeholders (such as caregivers) require clear and complete ingredient information to avoid certain allergens in packaged food products. Despite U.S. legislative mandates designed to assist the food allergic consumers and stakeholders to make safe food selection choices, food allergy labeling on consumer packaged food products remains unclear. To properly manage food allergy, it is imperative that food allergy stakeholders have access to clear and unambiguous food allergen ingredients located on the food product labels. The purpose of this research was to capture the impact of current United States food allergen labeling guidelines on U.S. food allergy stakeholders. With the elaboration likelihood framework, this research aimed to better understand how the congruence of allergen ingredient information on the Product Label Claim on the front of the package and Nutrition Facts Panel information on the back of the package affected food allergic consumers and stakeholders in evaluation of product packaging allergen information. A total of 223 food allergy stakeholders were administered four surveys depicting food allergen labeling variations found on U.S. food product labels. Seventeen hypotheses were formulated and analyzed with ANCOVA and t tests to measure the impact of congruence and elaboration of the food allergen messages on the food product labels, with a specific focus on purchase intention. Findings indicated that food allergy stakeholders had difficulties using and understanding existing food allergen labels under current U.S. food allergen labeling guidelines. The results also showed confusion among U.S. food allergic stakeholders with safe food selection choices when the label claims did not align with the actual allergen ingredient information. Research, public policy, and managerial implications were thoroughly discussed.

Thesis (MSc Consumer Sc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Food allergies in children are a growing problem and have various effects on these children, their households, the community and the economy. No medication or cure is currently available to consumers suffering from food-allergies. Medical teams are aware of the patients' need for support, but information is lacking and understanding of their basic needs and problems neglected. Two qualitative research methods, namely the focus group technique and case studies were used to investigate the emotional and physical environment of children allergic to wheat, soy, cow's milk and egg respectively. Five focus group meetings were conducted with the caregivers of food-allergic children. Two during the pilot study and three in the main study. A total of twenty three participants attended the three meetings of the main study. A meeting schedule was developed and the meetings carefully moderated. After the data had been obtained from the focus group meetings, there was a need for more in-depth information and understanding. Seven of the participants of the focus groups were identified, through developed criteria, to be further investigated as case studies. Five units of investigation were identified for the case studies. It was the (i) caregiver, (ii) allergic child, (iii) other children, (iv) "non-caregiver parent" and (v) household. Some of the matters that were investigated in both the focus groups and case studies were health consciousness, menu planning, recipe needs and use, reading of food labels, hidden allergens and the social and emotional effects of having and being a food-allergic child. A great need for better support and educational programs to assist these households were identified. Very few of the households were health conscious, while they all experienced problems with menu planning, identifying hidden allergens and suffered from social and or emotional problems. Very few participants made use of recipes or identified a need for allergen-free recipes. After having gained insight in the daily lives of these households, four - virtually identical, two-week menu cycles were developed to suit the life style requirements and restrictions of these children. The same recipes were used, but allergens omitted, replacements made and the recipes adapted as necessary. Product scouting was done for hidden allergens in selected popular convenience foods, especially those from the lowest level of the USDA Food Guide Pyramid. Hidden allergens appeared to be a problem when food is prepared and bought for the food allergic child. It should be taken into account that despite the information stated on the food label undeclared allergens might still be present in food. The two-week menu cycle for the child allergic to cow's milk was then quantitatively evaluated for nutritional adequacy by FoodFinder ™ 2 - a software program, for children aged between seven and ten years. All the macronutrients and micronutrients identified by the National Survey in 1999 as insufficient «67% of the RDA) among South African children aged between one and nine years, were evaluated as well as those mentioned in the WHO Dietary and Health Goals. All the nutrients were sufficient, except for calcium and Vitamin D. Supplementation is advised. The sensory acceptability of the menu-items in the two-week menu cycles were not evaluated. A vast number of issues pertaining to the food-allergic child require further research. A great need exists for the development of educational programs to support the households with food-allergic children. The identification of hidden allergens and the development of reliable sensory testing models for food-allergic children are all areas that should urgently be researched further. / AFRIKAANSE OPSOMMING: Voedsel-allergieë by kinders is 'n groeiende probleem met 'n verskeidenheid effekte op dié kinders, hulle huishoudings, die gemeenskap en die ekonomie. Tans is geen medikasie of kuur teen voedsel-allergieë beskikbaar nie. Mediese spanne is bewus van die behoefte aan sorg en leiding wat deur dié spesifieke groep verbruikers benodig word, maar inligting oor hulle behoeftes en probleme is ontoereikend. Twee kwalitatiewe navorsingsmetodes, naamlik die fokusgroeptegniek en gevallestudies, is gebruik om die emosionele en fisiese omgewing van kinders allergies vir onderskeidelik grane, soja, melk en eiers te ondersoek. Tydens 'n voorloperstudie is twee fokusgroepvergaderings gehou. Vir die hoofstudie is drie fokusgroepvergaderings met versorgers van kinders met 'n voedsel-allergie gehou met 'n totaal van drie en twintig deelnemers. 'n Vergaderingskedule is ontwikkel en die vergaderings is sorgvuldig gemodereer. Nadat die data van die fokusgroepe ingesamel is, was daar behoefte aan meer in diepte inligting en begrip. Aan die hand van ontwikkelde kriteria is sewe van die deelnemers aan die fokus groepe as gevallestudies geselekteer. Vyf eenhede van ondersoek is geïdentifiseer vir die gevallestudies. Dit is: (i) die allergiese kind, (ii) versorger, (iii) ander kinders, (iv) die "nie-versorgende" ouer en (v) die huishouding. Sommige van die aspekte wat in beide die fokusgroepe en gevallestudies ondersoek is, was gesondheidsbewustheid, spyskaartbeplanning, die gebruik en behoefte aan resepte, lees van voedseletikette, verskuilde allergene en die sosiale en emosionele effek van 'n voedsel-allergie op die kind sowel as die ouers. 'n Groot behoefte aan beter ondersteuning en opvoedkundige programme, om hierdie huishoudings te help, is geïdentifiseer. Baie min van die huishoudings was werkilk gesondheidsbewus, terwyl hulle almal probleme met spyskaartbeplanning, identifisering van verborge allergene en sosioemosionele probleme ondervind het. Weinig van die deelnemers het gebruik gemaak van resepte of het 'n behoefte aan allergeen-vrye resepte ondervind. Nadat insig in die daaglikse lewenstyl van hierdie huishouding verkry is, is vier amper-identiese twee-weekse siklusspyskaarte ontwikkel wat die lewenstyl en behoeftes van hierdie huishoudings aanspreek. Dieselfde resepte was gebruik vir al vier twee-weeksiklusspyskaarte, met die verskil dat verskillende allergene vervang is, na gelang van die allergie verskillende behoefte. Marknavorsing is gedoen op alledaagse produkte en is hoofsaaklik gefokus op die produkte wat die basis van die voedselpiramide uitmaak. Daar is bevind dat verskeulde allergene voorkom in produkte waar dit nie verwag word nie, en soms selfs nie op die voedseletikette verklaar word nie. Die twee-weeksiklusspyskaart van die melk-allergiese kind is kwantitatief ge-evalueer vir toereikendheid van die dieet vir kinders tussen die ouderdomme sewe tot tien jaar op die Foodfinder TM 2 - sagteware program. Die makro- en mikronutriente wat volgens 'n nasionale opname in 1999 as problematiese «67% van die RDA) vir kinders in die ouderdomsgroep een tot nege, uitgewys is, is geneem en die voedingstowwe is ook teen die Werêld Gesondheidsorganisasie (WHO) se dieetdoelwitte getoets. AI die nutriënte was voldoende volgens die analise, behalwe kalsium en Vitamen D. Supplementasie word aanbeveel vir dié twee nutriënte. Die sensoriese aanvaarbaarheid van die spyskaartitems in die siklusspyskaart is nie getoets nie. Verskeie aspekte wat vorendag gekom het, benodig verdere navorsing. 'n Groot behoefte aan die ontwikkeling van opvoedkundige programme, wat die huishoudings met voedsel-allergiese kinders ondersteun, bestaan. Die identifikasie van verskuilde allergene en die ontwikkeling van 'n beproefte sensoriese toetsmodel vir voedsel-allergiese kinders is potensiële gebiede wat dringend verder nagevors behoort te word.

Background: Food allergy, an abnormal immunologic response to food protein, has an estimated prevalence of 6% in young children and 3.7% in adults in the U.S. The only proven therapy for food allergy is strict elimination of the offending allergens. As a result, caregivers and patients could experience constant anxiety and stress that affects their quality of life. Additionally, food allergy can lead to significant economic impact on the health care system, since severe reactions often lead to ED visits and hospitalizations. Objectives: The first major objective was to determine the economic burden of Food Allergy and Anaphylaxis (FAA) patients in the U.S. by estimating the direct medical and indirect costs. The second principal objective involved assessing the Health Related Quality of Life (HRQL) of food allergic patients by measuring their health utilities and disease specific quality of life. Methods: Economic burden was estimated by measuring certain direct medical and indirect costs from a societal perspective. Costs were estimated using a bottom-up approach -- calculating the average cost of illness per patient and multiplying it by reported prevalence estimates. FAA patients with an emergency department (ED) visit, office based physician visit, outpatient department visit, and hospital admission were identified from a list of federally administered databases using ICD-9 codes. Sensitivity analyses were conducted to measure the robustness of the estimates. The cross-sectional HRQL study measured health utilities in food allergic adults and children, and quality of life in allergic adults using EQ-5D and FAQL-AF questionnaires respectively. These questionnaires were administered in an online survey format. Regression models were specified to explore the deviations in HRQL scores between patients with different disease related characteristics. Results: The findings reveal that for a given year (2007), direct medical costs worth $225 million and indirect costs worth $115 million were incurred. Owing to the irregularities in the reporting and diagnosis of food allergy, these values might be an underestimation. Simulations from probabilistic sensitivity analysis generated mean direct medical costs of $307 million and indirect costs of $203 million. Survey responses were collected online for eight months, during which 45 adults and 94 parents (acting as proxy for their food allergic child) responded. Adults reported a mean utility of 0.874 compared to 0.918 for children. Gender, number of food allergies and frequency of carrying epinephrine device had significant impacts on HRQL scores. An effect size of 0.003 was estimated comparing health utilities of food allergic adults with the general U.S. population. Conclusions: This was the first research to examine economic burden of FAA, and elucidate health utilities for food allergic patients. A large proportion of costs were incurred due to ambulatory visits. Effect size calculation revealed that health utilities of food allergic patients were very similar to the general U.S. population.

Embora alguns estudos tenham considerado a possibilidade da existência de uma relação direta entre alergia alimentar e alterações de comportamento, são escassas as evidências que sustentem esta hipótese. Relatamos neste trabalho que, após desafio oral com o antígeno, camundongos sensibilizados com ovalbumina (OVA) apresentaram maiores níveis de ansiedade, maiores níveis séricos de corticosterona e aumento da imunorreatividade para Fos no núcleo paraventricular do hipotálamo (PVN) e no núcleo central da amígdala (CeA), áreas cerebrais relacionadas com emotividade. Os neurônios ativados pela alergia alimentar no PVN e no CeA são capazes de produzir fator liberador de corticotrofina (CRF). Os dados também demonstraram que a alergia alimentar leva à ativação do núcleo do trato solitário (NTS). Além disso, observamos que animais imunizados com OVA desenvolveram aversão à ingestão de uma solução contendo clara de ovo. Um tratamento com anticorpo anti-IgE ou a indução de tolerância oral bloquearam tanto o desenvolvimento da aversão à dieta contendo o antígeno quanto a expressão de c-fos no sistema nervoso central (SNC). Para investigar o modo pelo qual se dá a comunicação entre o cérebro e o intestino de animais com alergia alimentar, tratamos camundongos neonatalmente com capsaicina visando a destruição de fibras sensoriais do tipo C. O tratamento com capsaicina, embora não tenha impedido o desenvolvimento da aversão, diminuiu a sua magnitude. Ademais, este tratamento bloqueou completamente a ativação do PVN e diminuiu a expressão de c-fos induzida pela alergia alimentar no núcleo do trato solitário (NTS). Contudo, o tratamento com capsaicina não modificou a imunorreatividade para Fos no CeA de animais imunizados desafiados por via oral com o antígeno. Estes resultados demonstram claramente que a alergia alimentar influencia a atividade do SNC e o comportamento animal. Além disso, os dados obtidos evidenciam a participação de mecanismos dependentes de IgE e das fibras do tipo C na comunicação entre cérebro e o intestino de animais com alergia alimentar. De modo geral, além de enfatizar a relevância da integração entre os sistemas imune e nervoso na elaboração de respostas adaptativas, este estudo fornece subsídios para uma melhor compreensão de possíveis desordens de natureza psicológica em pacientes alérgicos. / Although many authors have considered the possibility of a direct interaction between food allergy and behavioral changes, the evidence supporting this hypothesis is elusive. Here we show that after oral OVA challenge allergic mice present higher levels of anxiety, increased serum corticosterone, and increased Fos expression in the paraventricular nucleus of the hypothalamus (PVN) and in the central nucleus of the amygdala (CeA), which are both emotionality-related brain areas. Food allergy-activated neurons in the PVN and in the CeA are able to produce CRF. We found that food allergy also induced enhanced Fos immunoreactivity in the nucleus of tractus solitarii (NTS) of OVA-immunized animals. Besides that, OVA-immunized animals developed aversion to an antigen-containing solution. Treatment with anti-IgE antibody, or induction of oral tolerance abrogated both food aversion and the expression of c-fos in the central nervous system. In order to investigate the brain-gut communication in allergic animals, we have employed destruction of sensory C fibers by neonatal capsaicin treatment. Although this treatment did not block development of food aversion, it decreased the magnitude of such aversion. Moreover, we observed that while the degree of Fos staining in the NTS of allergic mice was only diminished by neonatal capsaicin, it was completely blocked in the PVN. However, capsaicin did not modify food alergy-induced c-fos expression in the CeA. Besides establishing a direct relationship between brain function and food allergy, our findings provide evidence showing that IgE-dependent mechanisms and the sensory C fibers play an important role in food allergy signaling to the mouse brain. Finally, this study creates a solid ground for understanding the etiology of psychological disorders in allergic patients.

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.

Wong Ho Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 140-153). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Acknowledgements --- p.III / Abstract --- p.IV / 摘要 --- p.VI / List of Tables --- p.VIII / List of Figures --- p.IX / List of Abbreviations --- p.XI / Table of Contents --- p.XIII / Chapter 1 --- General introduction --- p.1 / Chapter 2 --- Literature reviews --- p.4 / Chapter 2.1 --- LRP and winged bean --- p.4 / Chapter 2.1.1 --- Nutritional values of crop plants --- p.4 / Chapter 2.1.2 --- Lysine-rich protein (LRP) --- p.7 / Chapter 2.1.2.1 --- Identification of lysine-rich protein (LRP) --- p.7 / Chapter 2.1.2.2 --- Cloning cDNA for WBLRP --- p.7 / Chapter 2.1.2.3 --- Transgenic Expression of LRP in other plants --- p.8 / Chapter 2.1.3 --- Unknowns remained --- p.8 / Chapter 2.2 --- Food allergy and gastro-immunity --- p.10 / Chapter 2.2.1 --- What is allergy? 一 A brief introduction --- p.10 / Chapter 2.2.2 --- Food allergy and its symptoms --- p.12 / Chapter 2.2.3 --- Gastrointestinal immunity --- p.13 / Chapter 2.2.4 --- Possible mechanism of food allergy --- p.16 / Chapter 2.2.5 --- Available tests and limitations --- p.18 / Chapter 2.2.6 --- Radioallergosorbent test (RAST) --- p.19 / Chapter 2.2.7 --- Digestibility test --- p.20 / Chapter 2.2.8 --- Betv-1 Allergen Family --- p.21 / Proteins --- p.23 / Chapter 2.3 --- Pathogenesis-related proteins --- p.23 / Chapter 2.3.1 --- Defense-related proteins and pathogenesis-related proteins (PRs) --- p.23 / Chapter 2.3.2 --- Class 10 PR proteins (PR-10s) --- p.25 / Chapter 2.3.3 --- The expression patterns of PR-10s --- p.27 / Chapter 2.3.3.1 --- Pathogens-induced and signal-induced expression --- p.27 / Chapter 2.3.3.2 --- Spatially- and developmentally-regulated expression --- p.28 / Chapter 2.3.3.3 --- Other induction patterns --- p.29 / Chapter 2.3.4 --- Functions ofPR-10s --- p.30 / Chapter 2.4 --- Development of hypotheses and experiments --- p.32 / Chapter 3 --- Materials and methods --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Materials --- p.38 / Chapter 3.2.1 --- Chemicals --- p.38 / Chapter 3.2.2 --- Apparatus and commercial kits --- p.39 / Chapter 3.2.3 --- Vectors and bacterial strains --- p.39 / Chapter 3.2.4 --- Plant and animal materials --- p.40 / Chapter 3.2.5 --- Computer software --- p.40 / Chapter 3.3 --- Purification of LRP --- p.41 / Chapter 3.3.1 --- Purification of LRP from winged bean --- p.41 / Chapter 3.3.1.1 --- Extraction of total protein --- p.41 / Chapter 3.3.1.2 --- Differential pI precipitation --- p.41 / Chapter 3.3.1.3 --- Determination of the pI point of LRP --- p.42 / Chapter 3.3.1.4 --- Native tricine-PAGE and gel elution --- p.42 / Chapter 3.3.2 --- Purification from E. coli --- p.45 / Chapter 3.3.2.1 --- Construction of pET vector expressing recombinant LRP (rLRP) --- p.45 / Chapter 3.3.2.2 --- Expression of rLRP --- p.50 / Chapter 3.3.2.3 --- Purification by gel electrophoresis and gel band elution --- p.50 / Chapter 3.4 --- Anti-serum production --- p.52 / Chapter 3.5 --- Allergy tests --- p.53 / Chapter 3.5.1 --- Pepsin digestion --- p.53 / Chapter 3.5.1.1 --- Determination of optimal concentration of pepsin --- p.53 / Chapter 3.5.1.2 --- Pepsin digestion of allergenic and non-allergenic model proteins --- p.55 / Chapter 3.5.1.3 --- Pepsin digestion of LRP and immunodetection --- p.55 / Chapter 3.5.2 --- Trypsin digestion --- p.56 / Chapter 3.5.2.1 --- Determination of optimal trypsin concentration --- p.56 / Chapter 3.5.2.2 --- Trypsin digestion of allergenic and non-allergenic model proteins --- p.57 / Chapter 3.5.2.3 --- Trypsin digestion of LRP and immuno-detection --- p.57 / Chapter 3.5.3 --- Pepsin and trypsin digestion --- p.58 / Chapter 3.5.3.1 --- Digestions of allergenic model proteins --- p.58 / Chapter 3.5.3.2 --- Digestion of LRP --- p.58 / Chapter 3.5.4 --- IgE binding tests --- p.58 / Chapter 3.6 --- Physiology studies --- p.59 / Chapter 3.6.1 --- Preparation for the studies --- p.59 / Chapter 3.6.1.1 --- Growing winged bean in the field --- p.59 / Chapter 3.6.1.2 --- Growing winged bean in sterile conditions --- p.60 / Chapter 3.6.1.3 --- Production ofLRP-cDNA probe --- p.60 / Chapter 3.6.2 --- Detecting the expression of LRP in winged bean --- p.61 / Chapter 3.6.2.1 --- RNA extraction --- p.61 / Chapter 3.6.2.2 --- RT-PCR and DNA sequencing --- p.62 / Chapter 3.6.2.3 --- RNA electrophoresis and northern blot analysis --- p.63 / Chapter 3.6.2.4 --- Protein extraction --- p.63 / Chapter 3.6.2.5 --- Western blot and immuno-detection --- p.63 / Chapter 3.6.3 --- Expression of LRP in germinating winged bean seeds --- p.64 / Chapter 3.6.3.1 --- Seed germination --- p.64 / Chapter 3.6.3.2 --- Detection of LRP in germinating seeds --- p.64 / Chapter 3.6.4 --- RNase activity test --- p.65 / Chapter 4 --- Results --- p.67 / Chapter 4.1 --- Purification of LRP --- p.67 / Chapter 4.1.1 --- Purification from winged bean --- p.67 / Chapter 4.1.1.1 --- Identification of pI point of LRP --- p.67 / Chapter 4.1.1.2 --- Native tricine PAGE and gel elution --- p.70 / Chapter 4.1.2 --- Purification from E. coli --- p.71 / Chapter 4.1.2.1 --- Construction of pET-LRP vector --- p.71 / Chapter 4.1.2.2 --- Expression of rLRP and gel purification --- p.74 / Chapter 4.2 --- Antiserum production --- p.76 / Chapter 4.3 --- Allergy tests --- p.81 / Chapter 4.3.1 --- Pepsin digestion --- p.81 / Chapter 4.3.2 --- Trypsin digestion --- p.89 / Chapter 4.3.3 --- Pepsin and trypsin digestion --- p.96 / Chapter 4.3.4 --- Human serum IgE binding test --- p.104 / Chapter 4.4 --- Physiological studies --- p.105 / Chapter 4.4.1 --- Samples preparation --- p.105 / Chapter 4.4.2 --- RT-PCR and DNA sequencing --- p.105 / Chapter 4.4.3 --- Expression profile of WBLRP in winged bean somatic organs --- p.108 / Chapter 4.4.4 --- Expression profile ofWBLRP in winged bean flower --- p.111 / Chapter 4.4.5 --- Expression profile ofWBLRP in winged bean maturing seeds --- p.114 / Chapter 4.4.6 --- Expression profile of WBLRP gene in winged bean germinating seeds --- p.117 / Chapter 4.4.7 --- Functional assay of LRP --- p.121 / Chapter 5 --- Discussion --- p.124 / Chapter 5.1 --- LRP purification and antibody production --- p.124 / Chapter 5.2 --- Allergy tests --- p.125 / Chapter 5.3 --- Expression of LRP in WB --- p.131 / Chapter 5.4 --- Functional assay of LRP --- p.134 / Chapter 5.5 --- Hypothesis Testing --- p.135 / Chapter 5.6 --- Future prospective， --- p.136 / Chapter 6 --- Conclusion --- p.138 / Chapter 7 --- References --- p.140

Embora alguns estudos tenham considerado a possibilidade da existência de uma relação direta entre alergia alimentar e alterações de comportamento, são escassas as evidências que sustentem esta hipótese. Relatamos neste trabalho que, após desafio oral com o antígeno, camundongos sensibilizados com ovalbumina (OVA) apresentaram maiores níveis de ansiedade, maiores níveis séricos de corticosterona e aumento da imunorreatividade para Fos no núcleo paraventricular do hipotálamo (PVN) e no núcleo central da amígdala (CeA), áreas cerebrais relacionadas com emotividade. Os neurônios ativados pela alergia alimentar no PVN e no CeA são capazes de produzir fator liberador de corticotrofina (CRF). Os dados também demonstraram que a alergia alimentar leva à ativação do núcleo do trato solitário (NTS). Além disso, observamos que animais imunizados com OVA desenvolveram aversão à ingestão de uma solução contendo clara de ovo. Um tratamento com anticorpo anti-IgE ou a indução de tolerância oral bloquearam tanto o desenvolvimento da aversão à dieta contendo o antígeno quanto a expressão de c-fos no sistema nervoso central (SNC). Para investigar o modo pelo qual se dá a comunicação entre o cérebro e o intestino de animais com alergia alimentar, tratamos camundongos neonatalmente com capsaicina visando a destruição de fibras sensoriais do tipo C. O tratamento com capsaicina, embora não tenha impedido o desenvolvimento da aversão, diminuiu a sua magnitude. Ademais, este tratamento bloqueou completamente a ativação do PVN e diminuiu a expressão de c-fos induzida pela alergia alimentar no núcleo do trato solitário (NTS). Contudo, o tratamento com capsaicina não modificou a imunorreatividade para Fos no CeA de animais imunizados desafiados por via oral com o antígeno. Estes resultados demonstram claramente que a alergia alimentar influencia a atividade do SNC e o comportamento animal. Além disso, os dados obtidos evidenciam a participação de mecanismos dependentes de IgE e das fibras do tipo C na comunicação entre cérebro e o intestino de animais com alergia alimentar. De modo geral, além de enfatizar a relevância da integração entre os sistemas imune e nervoso na elaboração de respostas adaptativas, este estudo fornece subsídios para uma melhor compreensão de possíveis desordens de natureza psicológica em pacientes alérgicos. / Although many authors have considered the possibility of a direct interaction between food allergy and behavioral changes, the evidence supporting this hypothesis is elusive. Here we show that after oral OVA challenge allergic mice present higher levels of anxiety, increased serum corticosterone, and increased Fos expression in the paraventricular nucleus of the hypothalamus (PVN) and in the central nucleus of the amygdala (CeA), which are both emotionality-related brain areas. Food allergy-activated neurons in the PVN and in the CeA are able to produce CRF. We found that food allergy also induced enhanced Fos immunoreactivity in the nucleus of tractus solitarii (NTS) of OVA-immunized animals. Besides that, OVA-immunized animals developed aversion to an antigen-containing solution. Treatment with anti-IgE antibody, or induction of oral tolerance abrogated both food aversion and the expression of c-fos in the central nervous system. In order to investigate the brain-gut communication in allergic animals, we have employed destruction of sensory C fibers by neonatal capsaicin treatment. Although this treatment did not block development of food aversion, it decreased the magnitude of such aversion. Moreover, we observed that while the degree of Fos staining in the NTS of allergic mice was only diminished by neonatal capsaicin, it was completely blocked in the PVN. However, capsaicin did not modify food alergy-induced c-fos expression in the CeA. Besides establishing a direct relationship between brain function and food allergy, our findings provide evidence showing that IgE-dependent mechanisms and the sensory C fibers play an important role in food allergy signaling to the mouse brain. Finally, this study creates a solid ground for understanding the etiology of psychological disorders in allergic patients.

Proteins are important building blocks of all living organisms. They are composed of a defined sequence of different amino acids, and fold into a specific three-dimensional, ordered structure. The three-dimensional structure largely determines the function of the protein, but protein function always requires motion. Small movements within the protein structure govern the functional properties, and this thesis aims to better understand these discrete protein movements. The motions within the protein structure are governed by thermodynamics, which therefore is useful to predict protein interactions. Nuclear magnetic resonance (NMR) is a powerful tool to study proteins at atomic resolution. Therefore, NMR is the primary method used within this thesis, along with other biophysical techniques such as Fluorescence spectroscopy, Circular Dichroism spectroscopy and in silico modeling. In paper I, NMR in combination with molecular engineering is used to show that the folding of the catalytical subdomains of the enzyme Adenylate kinase does not affect the core of the protein, and thus takes a first step to linking folding, thermodynamic stability and catalysis. In paper II, the structure of the primary allergen from Brazil nut, Ber e 1, is presented along with biophysical measurements that help explain the allergenic potential of the protein. Paper III describes the need for a specific Brazil nut lipid fraction needed to induce an allergenic response. NMR and fluorescence spectroscopy is used to show that there is a direct interaction between Ber e 1 and one or several components in the lipid fraction.

Influence of food allergy and importance of diet therapy for children with atopic dermatitis Atopinis dermatitas yra dažna vaikų lėtinė odos liga, kuri sparčiai plinta visame pasaulyje [304, 369, 370]. Lietuvos mokslininkų duomenimis, atopinis dermatitas nustatytas apie 4-17% vaikų, gyvenančių Lietuvoje [38, 92]. Vienas svarbiausių atopinio dermatito patogenezės veiksnių yra alergija maistui. Įvairūs mokslininkai nurodo, kad 35-80% atopiniu dermatitu sergančių vaikų yra alergiški maistui [13, 40, 82]. Dažniausiai, atsižvelgiant į vaiko amžių, šeimą ir šalies mitybos tradicijas, kiekvienas alergiškas vaikas yra jautrus skirtingiems maisto produktams. Daugelio mokslininkų duomenimis, maisto alergenai – pienas, soja, kiaušiniai, kviečiai, riešutai ir žuvis – apibūdinami kaip vieni pagrindinių, sąlygojančių atopinio dermatito atsiradimą [48, 292]. Dauguma alergijų maistui kyla per pirmuosius 3 – 4 gyvenimo metus, kuomet virškinimo sistemos barjeras yra dar nesubrendęs, o imuninė sistema dar tobulinama gebėjimui toleruoti maistą. Visa tai lemia vaikų virškinimo sistemos reakciją į atskirus maisto produktus ir sukelia įvairius virškinimo sistemos sutrikimus [175, 288, 294]. Kiekvienam atopiniu dermatitu sergančiam vaikui labai svarbu išaiškinti jį alergizuojančius maisto produktus ir skirti dietą, kurioje ne tik nebūtų alergizuojančių maisto produktų, bet kuri užtikrintų pagrindinių maisto medžiagų, vitaminų, mineralinių medžiagų ir energijos reikiamą paros poreikį ir normalų... [to full text]