Tesi sul tema "Allergen microarrays"

Les outils d’aide au diagnostic utilisant l’intelligence artificielle (IA) aideront très prochainement les praticiens à proposer une médecine plus personnalisée et de précision pour les patients. Les maladies auto-immunes et allergiques (MAIA) sont le parfait exemple de maladies au diagnostic complexe pouvant bénéficier de ces outils. Les anticorps antinucléaires (ANA) sur les cellules épithéliales humaines (HEp-2) sont la référence pour le dépistage et le diagnostic des maladies auto-immunes (MAI). Pour une harmonisation des pratiques de laboratoires et cliniques, une lecture et une classification automatiques des aspects d’ANA observés sur cellules HEp-2 par immunofluorescence indirecte (IIF) respectant la classification recommandée par l’International Consensus on Antinuclear Antibody Patterns (ICAP) sont des exigences croissantes. A partir d’une collection complète d’images de cellules HEp-2 du CHU de Bordeaux et en utilisant une méthodologie d’apprentissage supervisé, un système de classification automatique des aspects d’IFI pour les images de cellules HEp-2 a été développé à partir des recommandations de l’ICAP et adaptées aux pratiques locales. Il repose sur un classificateur pour les aspects du noyau seulement (16 aspects et jusqu’à 2 aspects par image) et un second classificateur pour les aspects du cytoplasme seulement. Fort de résultats prometteurs, le système proposé devrait contribuer à la reconnaissance automatique des aspects d’ANA permettant des tests quantitatifs réflexes ciblés sur quelques auto-anticorps afin de faciliter in fine un diagnostic efficace et précis des MAI. Les puces à allergènes, permettent de rechercher simultanément jusqu’à 300 IgE spécifiques et s’intègrent dans une démarche diagnostique ascendante des allergies où, à partir d’une analyse la plus large possible, nous cherchons ensuite à déterminer quel(s) allergène(s) est (sont) susceptible(s) d’expliquer les symptômes du patient. Néanmoins, la masse de données produites dépasse la capacité d’analyse de l’utilisateur moyen et le grand nombre de résultats obtenus peut masquer ceux qui sont réellement pertinents cliniquement. Une base de données a été constituée à partir de 4271 résultats de puces (Société Française d’Allergologie), et de vingt-cinq données démographiques et cliniques. Un data challenge international a permis l’obtention de premiers modèles capables de prédire les profils allergiques des patients. Un outil plus complet et adapté à la pratique quotidienne est en cours de développement. L’outil pourra procurer au clinicien une probabilité d’allergie moléculaire par famille de protéines à partir de la puce à allergènes et un nombre très restreint de données cliniques ou démographiques, limitant ainsi les délais diagnostiques et le recours aux tests de provocation orale. Les outils d’aide aux diagnostics utilisant les technologies dites d’IA participent notamment à l’amélioration de l’efficience des techniques actuelles pour libérer du temps vis-à-vis de tâches répétitives et à faible valeur ajoutée. Ils sont généralement mal perçus par les praticiens considérant perdre leur expertise, voire être remplacés par les algorithmes. Particulièrement forte en Biologie Médicale, cette amélioration touche directement à la fonction de Biologiste Médical. Pour tenter de mieux comprendre, nous nous sommes intéressés au lien de confiance, s’il peut en être un, entre le praticien et l’outil d’aide au diagnostic. Les notions de fiabilité et de véracité ont pu être discutées. Une enquête nationale auprès des biologistes médicaux pratiquant l’IFI sur cellules HEp-2 a permis de révéler une réticence avec des raisons liées aux performances et à une méconnaissance des systèmes. Le déploiement et l’adhésion faisant l’unanimité de stratégies similaires dans le domaine de la cytologie une fois les performances constatées, montre un réel intérêt. [...]
Diagnostic tools based on artificial intelligence (AI) and capable of integrating several types of data, will be crucial in the next coming years in helping practitioners provide more personalized, precision medicine for patients. Autoimmune and allergic diseases are perfect examples of complex, multi-parametric diagnostics that could benefit from such tools. Antinuclear antibodies (ANA) on human epithelial cells (HEp-2) are important biomarkers for the screening and diagnosis of autoimmune diseases. For harmonization of biological practices and clinical management, automatic reading and classification of ANA immunofluorescence patterns for HEp-2 images according to the nomenclature recommended by the International Consensus on Antinuclear Antibody Patterns (ICAP) seems to be a growing requirement. In our study, an automatic classification system for Indirect Immunofluorescence (IIF) patterns of HEp-2 cells images was developed using a supervised learning methodology, based on a complete collection of HEp-2 cell images from Bordeaux University Hospital labelled accordingly to ICAP recommendations and local practices. The system consists of a classifier for nucleus patterns only (16 patterns and allowing recognition of up to two aspects per image) and a second classifier for cytoplasm aspects only. With this contribution to the automation of ANA in medical biology laboratories, it will enable reflex quantitative tests targeted on a few autoantibodies, ultimately facilitating efficient and accurate diagnosis of autoimmune diseases. Allergen microarrays, enable the simultaneous detection of up to 300 specific IgE antibodies and are part of a bottom-up diagnostic approach in which, on the basis of the broadest possible analysis, we then seek to determine which allergen(s) is (are) likely to explain the patient's symptoms. However, the mass of data produced by this single analysis is beyond the analytical capacity of the average user and the large number of results obtained simultaneously can mask those that are truly clinically relevant. A database of 4271 patients (Société Française d'Allergologie) was created, including allergen microarrays data and twenty-five demographic and clinical data. This database allowed the development of the first models capable of predicting patients' allergic profiles thanks to an international data challenge. The best F1-scores were around 80%. A more comprehensive tool adapted to daily practice is currently under development. Based essentially on microarrays data and a very few clinical and demographic data, it will be able to provide clinicians with a probability of molecular allergy by protein family, thus limiting diagnostic delays and the need for oral provocation tests. Diagnostic tools using so-called AI technologies are helping to improve the efficiency of current techniques, leveraging locks for repetitive, low-value-added tasks. These tools are generally poorly perceived by practitioners, who feel that they are losing their expertise, and even that they are being replaced by algorithms. This impression is particularly strong in Medical Biology, where this improvement directly affects the function of the Medical Biologist. In an attempt to better understand this, we took a closer look at the relationship of trust, if there can be one, between the practitioner and the diagnostic tool. The concepts of reliability and veracity were discussed. Thanks to a survey of medical biologists working on the analysis of aspects of HEp-2 cells, a certain reticence can be highlighted, with reasons linked to performance scores and unfamiliarity with the systems. The deployment and commitment to similar strategies in the field of biological hematology shows real interest once performance has been established. The development of two diagnostic tools for autoimmune and allergic diseases is laying the foundations for improved results and lasting integration into a more personalized, precision medicine

Allergic reactions are pathological immune reactions against common, harmless environmental proteins, termed “allergens”. Type I hypersensitivity reactions are mediated by IgE antibodies; these reactions are the most prevalent, affecting all ages and both genders to almost the same extent. This form of allergic reaction is distributed widely affecting more than 25% of the population in the industrial world. The increased incidence of IgE-mediated allergy is thought to be a combination of environmental and genetic factors, each account for about 50%. The effects associated with this disease may reduce quality of life, daily activities, and in some cases threaten life, hence affecting work and leisure time, increasing healthcare and medication costs. IgE-mediated allergy is defined a complex innate and adoptive immune response to natural harmless environmental allergens in which allergen- specific IgE antibodies are predominantly produced. In addition, it is believed that other immunoglobulin isotypes might be involved in atopic diseases (mainly IgG4), although the roles of these antibody isotype are still debatable whether they are pathogenic or protective antibodies. Generally, diagnostic procedures for IgE-mediated allergy begin with the patient’s history, clinical symptoms, physical examination, in vivo provocation tests, and are eventually confirmed with laboratory investigations. In this thesis we develop a microarray technology for use in-house for comprehensive investigations of immunoglobulin reactivity profiles against numerous allergens in atopic disease. Several printing and blocking buffers were developed in-house to preserve functionality of the arrayed proteins, minimise background noise, and increase signal intensity. The new diagnostic platform was validated by set of quality controls, then applied on large-scale applications (allergic patients, general populations, suspected asthmatics and healthy control individuals) to measure total and specific IgE antibodies. Application of the developed antibody microarray platform identified the difficulty of setting cutoffs using a “healthy control group”. We tested both the 75th and the 95th percentiles of the healthy control group as a cut-off point for total IgE levels that resulted in 39.3ng/ml and 70.68ng/ml respectively. When the 75th percentile was selected, the number of participants in the allergy patients classed as positive above the cutoff points was 92 out of 105 (87.6%). However, if the 95th percentile was selected, the number of positive patients was 63 out of 105 (60%) in the same group. On the other hand, the developed allergen microarray platform was used to immunoprofile allergen specific immunoglobulin subtypes in the four different cohorts against 70 SPT allergens. MeV software was used to present the reactivity profiles of the immunoglobulin subtypes semi-quantitatively that resulted in the hierarchical clustering according to allergen species. The reactivity profiles of allergen specific IgE in the allergic group recognized multiple allergens while specific IgE in the healthy control group was negative except for one serum sample. Specific IgE in the random population was lower than in the suspected asthmatics although it recognized allergens from different species (ingested, inhaled, venom). Similarly, this multi-allergen microarray platform was adapted to investigate the roles of other antibody isotypes (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) in IgE- mediated allergy. The study revealed no roles for these isotype except IgG4 that showed multiple recognition patterns in serum samples of healthy individuals and allergic patients. A comparison between microarray results and provocation testing (SPT) was performed for the samples from suspected asthmatics. To enable direct comparison, IgE:IgG4 ratio was utilized as IgE levels alone would not be expected to correlate with wheal and flare reaction. The IgE:IgG4 ratio showed good correlation with the six allergens tested by SPT (r=0.9299, p<0.0001). In addition, the multi-allergen chip can detect additional responses outside the standard six SPT- allergens performed routinely, and moreover reports on IgG4 responses that are never assessed by SPT, but are potentially important to the likelihood of asthma and allergy. Overall, the results indicated that the new developed microarray platform is highly specific, sensitive and reproducible. Subsequently, this high-throughput microarray platform can work as a diagnostic tool for monitoring the development of allergic diseases, predicting allergic reactions particularly for polysensitised patients, evaluating patient response to treatments, and eventually allowing the classification of allergic individuals. In conclusion, a new high-throughput multi-allergen platform has been developed that will certainly improve the future of allergy diagnosis.

Poverty and long distances are two reasons why some people in the third world countries hasdifficulties seeking medical help. A solution to the long distances could be if the medical carewas more mobile and diagnostically tests could be performed on site in villages. A new pointof-care test based on a small blood shows promising results both in run time and mobility.However, the method still needs more advanced equipment for analysis of the resultingmicroarray. This study has investigated the potential to perform the analysis within asmartphone application, performing all steps from image capturing to a diagnostic result. Theproject was approach in two steps, starting with implementation and selection of imageanalysis methods and finishing with implementing those results into an Android application.A final application was not developed, but the results gained from this project indicates that asmartphone processing power is enough to perform heavy image analysis within a sufficientamount of time. It also imply that the resolution in the evaluated images taken with a Nexus 6together with an external macro lens most likely is enough for the whole analysis, but furtherwork must be done to ensure it.

[ Truncated abstract ] Atopic diseases such as asthma are thought to be driven to a significant extent by T helper memory cells which are programmed to respond in a harmful way to environmental allergens (e.g. house dust mite). Previous studies in humans and in animal models have established that activation of TH2 cytokine genes in T memory responses to allergens is central to the disease process. However, only a subset of atopics harbouring a TH2-memory response phenotype manifests clinical symptoms of disease. Moreover, clinical trials with TH2 antagonists in atopic patients have proven disappointing, suggesting underlying complexities in disease pathogenesis which escape regulation via these approaches. It was thus hypothesised that additional genes involved in the activation program of allergen-specific T memory cells which are central to disease pathogenesis remain unidentified. The aim of the current study was to identify such novel genes by applying microarray technology to survey genome-wide expression patterns in an in vitro model of allergen-driven human T cell activation. In contrast to previous human microarray studies in this area focusing on mitogen activated T cell lines and clones, the current study avoided the use of strong activation stimuli which have the potential to distort patterns of gene expression, and reports for the first time the findings of microarray analysis of house dust mite allergen-driven acute gene activation in recirculating T memory cells harvested from the peripheral blood of human atopics. ... Finally, methodology was established to investigate the function of the novel atopy-associated genes. In loss-of-function experiments, expression of DACT1 and CAMK2D was silenced in primary T cell responses driven by bacterial superantigens, a model system for studying T cell responses under conditions which mimic antigen-specific activation. Whilst silencing DACT1 and CAMK2D expression did not influence classical readouts of T cell function including proliferation and cytokine production, microarray profiling was employed to identify putative downstream transcriptional targets of each gene. The experimental strategy and optimised methodology presented herein can now be employed to investigate the molecular circuitry linking the novel atopy-associated genes to the T cell activation process. In conclusion, several novel genes associated with allergen-driven T memory responses in atopics have been first described in this thesis and represent logical candidates for more detailed immunological and genetic studies related to the pathogenesis of atopic diseases.

Allergic responses are mainly mediated by immunoglobulin E (IgE) and mast cells or basophils expressing the high-affinity IgE receptor FcεRI. Cross-linking of FcεRI and IgE complexes with allergen induces basophil degranulation and release of inflammatory chemical mediators, leading to clinical symptoms. Common allergy diagnostic tests such as ImmunoCAP, focused on the measurement of specific IgE in patients, commonly lead to misdiagnosis. The allergen-specific IgE in patients’ sera might not always lead to FcεRI cross-linking on mast cells or basophils, resulting in no related clinical symptoms, as observed in some food allergies. In order to mimic the allergic response and generate an in vitro diagnostic device to address these issues, a basophil-microarray platform that couples the diversity of a protein array with the biological output of basophilic cells is being developed. This platform allows testing of up to five thousand allergens using a drop of patient’s blood. In this study, the optimisation steps and preliminary results are presented. The platform in development relies upon the use of a humanised rat basophilic leukaemia (RBL) cell line RBL-703/21 and different methods to measure the levels of basophil activation. ß-hexosaminidase assay showed that the human FcεRI expressing RBL-703/21 cell line was able to bind human IgE in the presence of anti-IgE/allergens and led to degranulation. Fibronectin has shown to greatly avoid cell losses during experiments, calcium ionophore A23187 is an unsuitable positive control due to a fast down regulation of VLA-4 and subsequent detachment of cells. The commercial Ca2+ probe (fluo-4, AM) was shown to efficiently measure the intracellular calcium flux upon activation in the micro-well plate, but due to the lack of a detection system, it can not to be used in the microarray. Two reporter plasmids encoding GFP or DsRed with an NFAT promoter region were transfected into RBL-703/21s and optimised. Both reporter systems were able to detect the presence of functional allergen-specific IgE in the sera of patients, and showed the expected bell–shaped dose response curves. Results correlated with those measured by clinical diagnostic methods. The fluorescence system developed using reporter genes does not need further processing, making it an ideal system for the future development of basophil microarray platforms.

Currently food allergy diagnostic tests are not able to predict clinical reactivity in sensitized patients (those with specific IgE against a particular allergen). Traditionally, allergy diagnostic tests used complete extracts of allergenic sources containing multiple molecules, some allergic and others not. This greatly limits the accuracy in the diagnosis and identification of possible allergic reactions to different foods by the existence of cross reactivity. Through the last decades, thanks to advances in the characterization of allergens at molecular level, the concept of Component-Based Diagnosis has been developed. This is based on the reasoning that the presence of specific IgE for the protein actually responsible for the allergic response should be detected and not the one against a mixture of molecules. Furthermore, the study of IgE and IgG4 recognition at epitope level using microarrays of synthetic peptides has been described as a useful tool for diagnosis, prognosis and development of a therapy for food allergy. The hypothesis of this thesis is that these new methodologies can improve the diagnosis of shellfish and lipid transfer protein (LTP) allergy. The aim of this thesis is to characterize clinically and at a molecular level these two types of food allergies, using these new methodologies. Regarding shellfish allergy, we found that tropomyosin, sarcoplasmic protein binding of calcium and myosin light chain are the allergens associated with clinical reactivity, i.e, are more common in shrimp allergic patients than in tolerant individuals sensitized shrimp. On the other hand, arginine kinase and hemocyanin allergens would be more involved in the phenomena of cross-reactivity with other arthropods (mites and/or cockroach). Additionally the synthetic peptide microarray has identified differential recognition of IgE and IgG4 epitopes among allergic and tolerant individuals. Regarding allergy to LTP, allergenic proteins ubiquitously distributed in the plant kingdom, we observed that patients suffer from reactions with a wide range of plant foods to which are sensitized, being peach the most common one. Furthermore, these patients have a variety of clinical symptoms from very mild to very severe and life-threatening as in the case of anaphylaxis, which can be attributed to allergens from different families. The component-based diagnosis in a microarray format that includes a diverse panel of allergenic proteins from different families is useful for diagnosing these patients, since the only proteins identified as responsible for the clinical symptoms are the LTPs, although the symptoms are diverse and sometimes more closely resemble to those caused by other allergens such as profilins or homologues of Bet v 1. Moreover, it offers an overview of positive and negative sensitivities in these patients in a single trial, with its multiplex properties. Cases of anaphylaxis with a cofactor involvement, such as NSAIDs, were frequently observed in these patients. The simultaneous presence of these drugs with the food allergen triggers allergic reactions in the individual that would not occur without the presence of the drug or would have been of less severity. We have developed a preliminary in vitro model based on the basophil activation test that allowed us to observe the in vitro effect observed in vivo. We observed an increase of degranulation/activation of basophils when stimulation is done with food in the presence the drug, compared to when stimulated only with food. In this thesis we can conclude that both the component-based diagnosis and epitope mapping are useful tools for the characterization of food allergy to shellfish proteins and LTP, and that they should be considered to improve the efficiency of diagnosis of these two types of food allergies.
Actualment els mètodes diagnòstics de l'al•lèrgia alimentària no són capaços de predir la reactivitat clínica dels pacients sensibilitzats (els que tenen IgE específica davant un determinat al•lergen). Tradicionalment les proves diagnòstiques de l'al•lèrgia han utilitzat extractes complets de fonts al•lergèniques que contenen múltiples molècules, algunes al•lergèniques i altres no. Això limita enormement la precisió en el diagnòstic i la possibilitat d'identificar reaccions al•lèrgiques a diferents aliments per l'existència de reactivitats creuades. Gràcies a la caracterització dels al•lèrgens a nivell molecular, s'ha desenvolupat el concepte del Diagnòstic Basat en Components que es basa en el raonament de detectar la presència d'IgE específica per a la proteïna realment responsable de la resposta al•lèrgica i no per una mescla de molècules. Addicionalment, l'estudi del reconeixement IgE i IgG4 a nivell d'epítops amb microarrays de pèptids sintètics pot ser una eina útil per al diagnòstic, pronòstic i desenvolupament d'una teràpia per l'al•lèrgia alimentària. La hipòtesi d'aquesta tesi és que aquestes noves metodologies poden millorar el diagnòstic de l'al•lèrgia al marisc i a les proteïnes de transferència de lípids (LTP), presents en múltiples aliments vegetals. L'objectiu és doncs caracteritzar clínicament i a nivell molecular aquests dos tipus d'al lèrgies alimentàries, utilitzant aquestes noves metodologies. Respecte a l'al•lèrgia al marisc, els al lèrgens tropomiosina, proteïna sarcoplàsmica d'unió de calci i la cadena lleugera de miosina s'associen amb la reactivitat clínica a la gamba. D'altra banda, els al•lèrgens arginina quinasa i hemocianina estarien més implicats en fenòmens de reactivitat creuada amb altres artròpodes. Addicionalment, amb el microarray de pèptids sintètics s'ha pogut identificar un reconeixement diferencial d'epítops IgE i IgG4 entre pacients al•lèrgics i tolerants. Respecte a l'al•lèrgia a les LTP, els pacients pateixen reaccions amb un ampli ventall d'aliments vegetals, sent el préssec el més freqüent, amb una gran diversitat de símptomes clínics, que poden atribuir-se a al•lèrgens de diferents famílies. El diagnòstic basat en components en el format d'un microarray que inclou proteïnes al•lergèniques de diferents famílies és útil per al diagnòstic d'aquests pacients, ja que permet identificar que les úniques proteïnes responsables els símptomes clínics són les LTP, encara que els símptomes siguin molt variats i en algunes ocasions s'assemblin més als provocats per altres al•lèrgens com les profilines o els homòlegs de Bet v 1. En aquests pacients són freqüents els casos d'anafilàxia en què està involucrat un cofactor, com els antiinflamatoris no esteroïdals. La presència del fàrmac amb l'al•lergen alimentari desencadena reaccions al•lèrgiques que sense el fàrmac no es donarien o serien de menor severitat. Hem desenvolupat un model preliminar in vitro basat en el test d'activació de basòfils que ens ha permès observar in vitro l'efecte observat in vivo. En conclusió, el diagnòstic basat en components i el mapatge d'epítops són eines útils per a la caracterització de l'al•lèrgia alimentària al marisc i a les proteïnes LTP, i s'han de considerar per millorar l'eficiència del diagnòstic d'aquests dos tipus d'al•lèrgies alimentàries.

Methods to measure allergen-specific immunoglobulin E (sIgE) at nasal level in patients with allergic rhinitis are currently of great interest for many reasons, such as the evidence for local production of IgE in the nasal mucosa. Nasal secretions are easily collected with various non-invasive procedures and suitable for IgE measurements. Nevertheless, IgE “classic” assays, based on immunoenzymatic methods and allergen extracts, require large volumes and are inefficient in detecting the low amounts of IgE in NasSec. In this study, IgE antibodies to 15 allergen molecules of Dermatophagoides pteronyssinus and Dermatophagoides farinae were tested with an allergen microarray in nasal secretions of 30 mite sensitized, allergic rhinitis patients and 29 healthy, not-sensitized controls. Nasal IgE to major allergen molecules (nDer p 1, nDer f 1, rDer p 2, rDer f 2, rDer p 23) identified the mite-allergic patients with 90% sensitivity and 100% specificity. Therefore, testing nasal IgE to allergen molecules by a microarray approach may play a role in the diagnostic work-up of patients with allergic rhinitis.

Tese de mestrado, Controlo da Qualidade e Toxicologia dos Alimentos, Universidade de Lisboa, Faculdade de Farmácia, 2014
Food allergies are a common issue in western countries. In the last decade, these diseases has increased significantly, and nowadays it is estimated that affects 2-8% of the population. Within the food allergies, plant food is the most frequent in adult population and the most part of the plant food allergens belong to protein families with defense or storage functions. Among plant food allergies there is a special interest in tree nut allergy. In the course of history, nuts have been part of the diet around the world. Tree nuts have a high nutritional value and they are very important in the human diet. However, in the developed world, the allergic reactions caused by tree nuts represent one of the first causes of food allergies in children and the first in adults. Understanding the mechanism by which a harmless protein to the organism is capable of inducing an allergic response is the basis to prevent and treat this type of disease. Until now, in food allergy, the only possible treatment is avoiding the consumption of the culprit food. Although, the existence of cross-reactivity between allergens and the specific sensitization profiles of each patient, makes it difficult to know which foods are related and which ones the patient should avoid. In order to develop safe and effective immunotherapy, it is necessary to characterize the allergens involved both at molecular and immunological level. The major allergens described in tree nuts are 7S vicilins, 11S legumins, 2S albumins, lipid transfer proteins (LTPs) and thaumatin-like proteins (TLPs). In this thesis, the allergenic molecular basis of these proteins was studied in order to try to understand the possible mechanisms that are mediating sensitization and cross-reactivity and the prevalence of these proteins in a Spanish population, with the use of protein microarrays.
As alergias alimentares são um problema comum nos países ocidentais. Na última década, estas doenças têm aumentado significativamente e actualmente é estimado que afectem 2-8% da população. Nas alergias alimentares, a alergia a alimentos vegetais é a mais frequente na população adulta e a maioria dos alergenos de alimentos vegetais pertencem a famílias de proteínas com funções de defesa e armazenamento. Entre as alergias a alimentos vegetais, há um interesse especial na alergia a frutos secos. No decurso da história, os frutos secos têm feito parte da dieta em todo o mundo. Os frutos secos têm um elevado valor nutricional e são muito importantes na dieta humana. Contudo, no mundo desenvolvido, as reacções alérgicas causadas pelos frutos secos, representam uma das primeiras causas de alergia alimentar em crianças e a primeira em adultos. Conhecer o mecanismo pelo qual uma proteína inofensiva ao organismo é capaz de induzir uma resposta alérgica, é a base para prevenir e tratar este tipo de doença. Até agora, na alergia alimentar, o único tratamento possível é evitar o consumo do alimento culpado pela alergia. Todavia, a existência de reactividade-cruzada entre alergenos e os perfis especifícos de sensibilização dos patientes, torna difícil saber que alimentos estão relacionados e quais os alimentos que o paciente deve evitar. De modo a desenvolver imunoterapia segura e eficaz é necessário caracterizar os alergenos envolvidos, tanto a nível molecular como a nível imunológico. Os alergenos maioritários descritos nos frutos secos são vicilinas 7S, leguminas 11S, albuminas 2S, proteínas de transferência de lípidos (LTPs) e proteínas similares a taumatinas (TLPs). Nesta tese, a base molecular alergénica destas proteínas foi estudada de modo a perceber os possíveis mecanismos que medeiam a sensibilização e a reactividade-cruzada e a prevalência destas proteínas numa população Espanhola, com a utilização de microarrays de proteínas

碩士
國立清華大學
生物醫學工程研究所
105
This study reports an integrated automatic microfluidic system for detection of allergy in a microarray platform. Nowadays, allergy is very common in the world. It is a response of the human immune system to antigens in the surroundings. However, detection for allergy requires tedious manual operation, well-trained technicians and a relatively labor-intensive process. In the study, an integrated microfluidic system has been developed by combining several microfluidic techniques and microarray chips. Our previous experiments showed that a microfluidic system for allergy-diagnosis on microarray chips could be realized. It was demonstrated that after mass fabrication, the use of optimized conditions could obtain the similar fluorescence signals to the ones from manual processes and it only took only 20 minutes for the single reaction zone. In order to cut down costs and simplify the process, a new microfluidic system capable of simultaneous multiplex allergy-diagnosis was developed in this work. Experimental results demonstrated that multiplex reaction zones showed similar signals. Not only could it obtain the signals comparable to the on-bench experiments, but it also could increase the applicability of the integrated microfluidic system for automating multiplex detection on allergy microarray chips.

Although exposed to similar environmental stimuli, not all humans develop asthma. Similarly, mouse strains vary in the degree of pathophysiology seen following induction of experimental asthma. A model involving immunization and aerosol challenge with ovalbumin (OVA) was used to investigate factors that may confer strain-dependent resistance or susceptibility to pathology. BALB/c and C57BL/6 mice developed many features of human asthma including inflammation, mucus production and airway obstruction. In contrast, CBA/Ca mice were relatively resistant to development of disease. This was despite the presence of a robust systemic allergic response, as indicated by high levels of OVA-specific and total immunoglobulin and increases in circulating eosinophils comparable to those in BALB/c and C57BL/6 mice. In interleukin (IL)-5 transgenic (Tg) mice the strain specific susceptibility to lung mucus production and airway obstruction was maintained and pathology was greatly accentuated in C57BL/6 and BALB/c but not in CBA/Ca mice. Eosinophils recovered by bronchoalveolar lavage (BAL) from wt and IL-5 Tg CBA/Ca mice lost viability faster than BAL eosinophils from the other two strains and this phenomenon was lung-specific. This may result in less eosinophil accumulation in the lungs of CBA/Ca mice and resistance to asthma-like pathology. Fl hybrids of CBA/Ca mice crossed with either BALB/c or C57BL/6 mice had BAL leukocyte, eosinophil lifespan and cell-free protein profiles similar to those of the respective disease-susceptible parental strains. It is likely that eosinophil apoptosis was not mediated through the extrinsic or receptor mediated pathway. Bcl-2 and Bcl-xL, which both inhibit the intrinsic pathway of apoptosis were highest in BAL eosinophils from the BALB/c strain and this correlated with relatively high IL-5 levels in the lungs. Survivin inhibits apoptosis and expression was significantly higher in BALB/c and C57BL/6 BAL eosinophils than in cells from CBA/Ca mice. This suggests a possible mechanism whereby eosinophils from the asthma-susceptible C57BL/6 and BALB/c mice are more resistant to apoptosis and may account, in part, for the more extensive pathology in these strains. Using global gene expression analysis we identified groups of genes that were differentially regulated in the lungs of mice that are susceptible or resistant to development of asthma-like pathology. 242, 145 and 42 genes were differentially regulated in the lungs of the C57BL/6, BALB/C and CBA/Ca strains respectively. In C57BL/6 mice, transcripts were significantly enriched for adhesion molecules and we postulate that heightened expression of L-selectin, CD 18, PGSL-1 and LPAM-l on lung eosinophils is responsible for robust recruitment and therefore accumulation of these cells in C57BL/6 mice. 64 genes were differentially regulated only in the asthma-susceptible strains, several of which have not previously been associated witb asthma. The late expression of Chi313, Retnla and Mmp12 correlated with increased expression of IL-10 in the lungs and we hypothesise that this cytokine may be produced by alternatively activated macrophages as part of the resolution of disease. This study identifies several novel genes and mechanisms associated with the modulation of airway inflammation and pathology. The identification of factors that control allergic inflammation may provide novel therapeutic targets for disease intervention.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Although exposed to similar environmental stimuli, not all humans develop asthma. Similarly, mouse strains vary in the degree of pathophysiology seen following induction of experimental asthma. A model involving immunization and aerosol challenge with ovalbumin (OVA) was used to investigate factors that may confer strain-dependent resistance or susceptibility to pathology. BALB/c and C57BL/6 mice developed many features of human asthma including inflammation, mucus production and airway obstruction. In contrast, CBA/Ca mice were relatively resistant to development of disease. This was despite the presence of a robust systemic allergic response, as indicated by high levels of OVA-specific and total immunoglobulin and increases in circulating eosinophils comparable to those in BALB/c and C57BL/6 mice. In interleukin (IL)-5 transgenic (Tg) mice the strain specific susceptibility to lung mucus production and airway obstruction was maintained and pathology was greatly accentuated in C57BL/6 and BALB/c but not in CBA/Ca mice. Eosinophils recovered by bronchoalveolar lavage (BAL) from wt and IL-5 Tg CBA/Ca mice lost viability faster than BAL eosinophils from the other two strains and this phenomenon was lung-specific. This may result in less eosinophil accumulation in the lungs of CBA/Ca mice and resistance to asthma-like pathology. Fl hybrids of CBA/Ca mice crossed with either BALB/c or C57BL/6 mice had BAL leukocyte, eosinophil lifespan and cell-free protein profiles similar to those of the respective disease-susceptible parental strains. It is likely that eosinophil apoptosis was not mediated through the extrinsic or receptor mediated pathway. Bcl-2 and Bcl-xL, which both inhibit the intrinsic pathway of apoptosis were highest in BAL eosinophils from the BALB/c strain and this correlated with relatively high IL-5 levels in the lungs. Survivin inhibits apoptosis and expression was significantly higher in BALB/c and C57BL/6 BAL eosinophils than in cells from CBA/Ca mice. This suggests a possible mechanism whereby eosinophils from the asthma-susceptible C57BL/6 and BALB/c mice are more resistant to apoptosis and may account, in part, for the more extensive pathology in these strains. Using global gene expression analysis we identified groups of genes that were differentially regulated in the lungs of mice that are susceptible or resistant to development of asthma-like pathology. 242, 145 and 42 genes were differentially regulated in the lungs of the C57BL/6, BALB/C and CBA/Ca strains respectively. In C57BL/6 mice, transcripts were significantly enriched for adhesion molecules and we postulate that heightened expression of L-selectin, CD 18, PGSL-1 and LPAM-l on lung eosinophils is responsible for robust recruitment and therefore accumulation of these cells in C57BL/6 mice. 64 genes were differentially regulated only in the asthma-susceptible strains, several of which have not previously been associated witb asthma. The late expression of Chi313, Retnla and Mmp12 correlated with increased expression of IL-10 in the lungs and we hypothesise that this cytokine may be produced by alternatively activated macrophages as part of the resolution of disease. This study identifies several novel genes and mechanisms associated with the modulation of airway inflammation and pathology. The identification of factors that control allergic inflammation may provide novel therapeutic targets for disease intervention.
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Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

The analysis of microarray data is a challenging task because of the large dimensionality and small sample size involved. Although a few methods are available to address the problem of small sample size, they are not sufficiently successful in dealing with microarray data from extremely small (~<20) sample sizes. We propose a method to incorporate information from diverse sources to analyze the microarray data so as to improve the predictability of significant genes. A transformed data set, including statistical parameters, literature mining and gene ontology data, is evaluated. We performed classification experiments to identify potential allergy-related genes. Feature selection is used to identify the effect of features on classifier behaviour. An exploratory and domain knowledge analysis was performed on noisy real-life allergy data, and a subset of genes was selected as positive and negative class. A new set of transformed variables, depending on the mean and standard deviation statistics of the data distribution and other data sources, was identified. Significant allergy- and immune-related genes from the microarray data were selected. Experiments showed that classification predictability of significant genes can be improved. Important features from the transformed variable set were also identified.

博士
臺北醫學大學
醫學科學研究所
97
Allergic rhinitis affects approximately 30% of adults and up to 40% of children in industrialized societies. Medicines available for relief of allergic rhinitis symptoms include antihistamines, decongestants, leukotriene inhibitors, topical hormones and corticosteroids. However, the negative side effects of anti-allergic medicines cause many allergic rhinitis patients to choose traditional Chinese medical treatments - such as taking Chinese herbs or treatment with acupuncture. Acupuncture therapy corrects the equilibrium deviation using the bi-directional regulative actions in treating syndromes by inserting needles into acupoints. We assessed the therapeutic effect of acupuncture in perennial allergic rhinitis patients. We studied the clinical outcomes and gene expression profiles of allergic rhinitis patients who were treated with acupuncture. The patients were treated with acupuncture eight times over a four week period and peripheral blood of these patients was collected at each visit for analysis of gene expression. Eighteen phadiatopTM positive allergic rhinitis patients were analysised by cDNA microarray, and 13 phadiatopTM positive allergic rhinitis patients with 8 phadiatopTM negative rhinitis patients were analysised by Affymetrix human U133A chips. To estimate the therapeutic effect of acupuncture objectively, patients completed the rhinoconjunctivitis quality of life questionnaire (RQLQ) before and after acupuncture therapy. Based upon patient response to the RQLQ in cDNA microarray study, acupuncture therapy significantly improved allergic rhinitis symptoms, including nasal symptoms, non-hay fever symptoms, sleep and practical problems（daily activities）. Additionally, expression of interleukin 1 receptorα(IL1R1) in peripheral blood was significantly decreased at 2 hours, 24 hours and 4 weeks after acupuncture treatment in these patients. In Affymetrix human U133A chips study, The results of the RQLQ and the gene expression profiles were different between the Ph(+) and Ph(-) groups after receiving treatment with acupuncture. Activity, practical problems, and nasal symptoms showed significant improvement in the Ph(+) group versus the Ph(-) group. In addition, genes involved in active immune response, differential of Treg and cell apoptosis, were different in the Ph(+) and Ph(-) groups after acupuncture treatment. To our knowledge, this is the first report of cDNA microarray analysis of differential gene expression in the peripheral blood of allergic rhinitis patients before and after acupuncture treatment. Our data suggest that the balance between T helper 1 (Th1) and T helper 2 (Th2) cell-derived pro-inflammatory versus anti-inflammatory cytokines might be improved by acupuncture treatment. Differential gene expression profiles of Ph(+) and Ph(-) allergic rhinitis patients indicate the distinct physiological responses after receiving acupuncture treatment in these two groups. Our results suggest that personalized medical treatment should be essential for acupuncture treatment in allergic rhinitis patients.

碩士
臺灣大學
獸醫學研究所
98
Allergy has always played an important role in canine skin related diseases. The prevalence of canine allergic dermatitis (CAD) in the canine population was also estimated to be around 3-15%. The purpose of this study is to analyze and understand the type of allergens causing CAD in Taiwan. We analyzed 505 results from Excelsior bio-system’s protein chip microarray 62 item canine allergy test collected from May of 2006 to February of 2008. The result showed 80% of the cases had at least one positive result to the 62 items tested, and 54.06% of the dogs had at least one positive result in the mites group. Our finding shows that mites are the most important group of allergens responsible for CAD in Taiwan. Dermatophagoides pteronyssinus is the top allergen associated with CAD in Taiwan, having a positive rate of 29.50% (149/505), followed by Tyrophagus putrescentiae (24.55%, 124/505)、Aspergillus fumigates (18.02%, 91/505)、Blomia tropicalis (17.82%, 90/505)、and brewer’s yeast (Saccharomyces cerevisiae 17.43%, 88/505). In most countries, Dermatophagoides farinae is more commonly associated with CAD than Dermatophagoides pteronyssinus; however, our protein chip microarray showed that although Dermatophagoides farinae had a high positive rate (17.3%, 86/505), it is still significantly lower than that of Dermatophagoides pteronyssinus (p<0.0001). Other than identifying important allergens causing CAD in Taiwan, we also took brewer’s yeast, the only food allergen that made it to the top five lists further for western blot analysis. Brewer’s yeast is a common food supplement in which it serves rich vitamin B source; its presence is even common in hypoallergenic diets. With western blot analysis, we were able to identify the major proteins responsible for the positive results obtained from protein chip microarray such as triosephosphate isomerase, phosphoglycerate mutase 1, enolase 2, enolase 1 and alpha-glucosidase. Such information may be provided as valuable reference when pet owners choose the right diet for their pets, or manufacturers selecting ingredients for hypoallergenic diets.