Дисертації з теми "Proteine allergeniche"

The general aim of the activities conducted in the framework of my PhD were to learn modern techniques for analyzing proteins by mass spectrometry (MS) and then to apply these techniques and approaches to the analysis of allergenic proteins contained in foods. My research activities were conducted at the Laboratory of Protein Chemistry of CRIBI, University of Padua, where previously I have conducted the research for my Thesis for the Doctor degree in Pharmaceutical Biotechnologies. During the fist year of my PhD I have concluded the Thesis project on the amyloid aggregation of -lactalbumin, a model protein utilized for investigating molecular aspects of protein amyloidogenesis. The results of this research were quite interesting and indeed they have been published in an international journal. During the first two years I acquired a solid knowledge on several aspects of the MS methodology and I was able to learn the theory and practice of several modern techniques and approaches in this ambit. The specific aim was to analyze allergenic proteins contained in complex matrices as foods and to this aim several proteins were extracted and purified from several food samples. The research has been focused mostly on the allergenic proteins from milk and eggs, known to cause widespread allergies. The proteins of interest were analyzed by using several chromatographic and electrophoretic techniques and also by means of HPLC connected to a tandem MS electrospray instrument. I was able to show that MS techniques can be used to identify allergenic proteins even when contained in very complex mixtures. Therefore, these MS techniques perhaps can be used as an alternative to the immunochemical methods nowadays in use for detecting allergens. I have also analyzed the chemical modifications that allergenic proteins suffer during several industrial treatments of foods, including heat treatment. During the third year of my PhD I spent a six months period at the Biochemistry Laboratory of the Imperial College in London, being involved in a project aimed to study in a large scale the proteins of the mosquito Anopheles gambiae. The MS analyses were focused on the proteins responsible of the mating behaviour of A. gambiae, hoping to identify a target for controlling the behaviour of this vector of the malaria disease. Summing up, besides the publication dealing with amyloid aggregates of beta- lactalbumin, this PhD Thesis is composed by a major part dealing with MS analysis of allergenic proteins and by a minor one dealing with MS analysis of proteins from A. gambiae.

MS Analysis of Allergenic Proteins. Food allergy is a significant worldwide public health issue. Proteins from cow's milk, chicken eggs, soybean and peanuts are the most frequent allergens contained in the complex foods prepared by industrial processes. Allergenic proteins can induce allergic reaction in their native structural state or upon chemical or conformational changes induced by the industrial treatments. Nowadays, the identification of allergenic proteins in foods is conducted by using immunochemical methods such as ELISA tests, but these techniques suffer from several limitations due to cross-reactivity and false negative results. Indeed, alterations in the allergen’s structure or chemical modifications can prevent the interaction with the antibody, thus causing misleading data. Since allergens are toxic even in trace amounts, there is a need for reliable and sensitive analytical methods for allergenic proteins. The purpose of this PhD project was to develop procedures for the identification of these proteins in food samples by using mass spectrometry (MS), likely overcoming some limitations of the immunochemical assays. Indeed, the MS approach for identifying proteins makes use of data pertaining to the amino acid sequence of the protein, while immunochemical methods are linked to the integrity of the three-dimensional structure of proteins. In order to test immunochemical approaches, polyclonal antibodies raised against the main allergenic proteins of milk (α-lactalbumin and β-lactoglobulin) and eggs (ovomucoid, ovalbumin and lysozyme) were purchased. Preliminary studies were performed in order to check the quality of the antibodies, in terms of specificity of recognition and cross-reactivity. Moreover, the responses of the antibodies using as antigens the purified commercial proteins and the same proteins contained in complex food matrices after thermal treatment were checked. Since allergenic proteins usually are contained in complex mixtures of huge amounts of other proteins, the methodology nowadays named “targeted proteomics” was considered very appropriate. By this approach, a protein contained in a complex mixture can be identified by a MS analysis of a peptide fragment that is specific for the protein of interest and contained in the very complex mixture of a tryptic digest of a protein sample. The procedure involves specific labelling and isolation of the specific peptide, named “proteotypic”. To this aim, tryptophan (Trp) residues in proteins were modified by reaction with 2,4-dinitrophenyl-sulfenyl chloride (DNPS-Cl), that leads to a Trp-derivative with the DNPS label attached at 2-position of the indole nucleus. The selection of Trp(DNPS)- peptides from the complex mixture of a tryptic digest of a protein sample was achieved by exploiting the significant change in hydrophobicity and retention time of DNPS-modified peptides in a reverse-phase HPLC column. Moreover, DNPS-labelled Trp-peptides were isolated by hydrophobic interaction chromatography, as well as by immunoaffinity chromatography using a column prepared with anti-DNP antibodies. The “targeted proteomics” procedure was optimised using a mixture of model proteins and then applied to identify a protein allergen contained in a raw bakery product. Overall, it was demonstrated that the novel procedure of selective labelling and isolation of Trp-peptides allows a considerable simplification of the fingerprinting/MS approaches nowadays used for the identification of proteins in proteomics research. Other Research Activities. During the PhD course I had the opportunity to collaborate with other members of the lab in a couple of additional projects, partly as a continuation of previous research conducted for the doctoral thesis. Documentation of this activity is herewith included as an Appendix at the end of this PhD Thesis. The molecular properties of the complex formed by α-lactalbumin with oleic acid were investigated in detail. This complex appears to be very interesting, since it has been shown to display cellular toxicity specifically for cancer cells. It was shown that the protein in the complex is in an oligomeric state, at variance from previous statements that the protein was monomeric. Moreover, it was shown the oleic acid can interact also with other proteins, including apomyoglobin. The main conclusion of this work was that the protein moiety serves as a carrier of the otherwise poorly soluble fatty acid, thus leading to an enhancement of its water solubility and consequently of its intrinsic cytotoxic properties. A manuscript rescrubbing these results is in an advanced state of preparation. Enterocin AS-48 is a 70-residue circular polypeptide produced by Enterococcus faecalis displaying a wide antibacterial activity. Limited proteolysis of AS-48 was used to prepare a linear form of this enterocin, as well as 38- and 55-residue fragments. Nicked AS-48 showed a lower helicity by far-ultraviolet circular dichroism and a reduced stability to thermal denaturation, but it was active against the sensitive bacteria assayed. The fragments also partly retained the biological activity of the intact protein. These results indicate that the circularization phenomenon is not required for the antibacterial activity, but it is crucial for the stabilization of the native structural state. This research was published in FEBS Lett. (2008).
Analisi di proteine allergeniche mediante spettrometria di massa. Le allergie alimentari rappresentano ormai una problematica clinica di livello mondiale. Tra i prodotti alimentari considerati pericolosi per il loro elevato contenuto in proteine allergeniche troviamo il latte bovino, le uova, la soia e le arachidi. Le proteine allergeniche possono scatenare reazioni allergiche sia mantenendo la loro struttura nativa, sia in seguito a modifiche chimiche e conformazionali indotte dai processi industriali. I metodi d’elezione applicati per l’identificazione di proteine allergeniche negli alimenti sono rappresentati dai saggi immunochimici come i test ELISA. Tali metodi presentano però numerose limitazioni causate da fenomeni di cross reattività e da falsi positivi. Inoltre, alterazioni nella struttura delle proteine allergeniche o eventuali modifiche chimiche possono modificare l’interazione con gli anticorpi specifici, invalidando i risultati. Dal momento che gli allergeni sono tossici anche in tracce, è necessario sviluppare dei metodi analitici efficaci e affidabili per la loro identificazione. Lo scopo di questo progetto di tesi è stato quello di sviluppare delle procedure per l’identificazione di proteine allergeniche mediante spettrometria di massa (MS) che possano superare i limiti metodici dei saggi immunologici. Oltretutto, l’identificazione delle proteine mediante MS si basa sull’analisi della sequenza amminoacidica di quest’ultime, mentre i saggi immunochimici sono strettamente dipendenti dall’integrità della struttura tridimensionale della proteina antigenica. Al fine di testare la validità dell’approccio immnuchimico, sono stati testati alcuni anticorpi policlonali diretti contro le principali proteine allergeniche di latte α-lattalbumina e β-lattoglobulina) e uova (ovomucoide, ovalbumina e lisozima). Sono stati condotti alcuni studi preliminari per validare la qualità di questi anticorpi, in termini di specificità di riconoscimento della proteina antigenica e della presenza di eventuali fenomeni di cross reattività. Inoltre, è stata valutata la risposta anticorpale usando come antigeni sia le proteine commerciali purificate, sia le stesse proteine contenute in prodotti alimentari prima e dopo trattamento termico. Dato che le proteine allergeniche sono contenute in miscele complesse costituite da altre proteine, è stata considerata estremamente appropriata l’applicazione di una tecnica detta “targeted chromatography”. Secondo questo strategia, è possibile identificare mediante MS una proteina contenuta in una miscela complessa attraverso l’analisi di alcuni frammenti peptidici derivati dalla digestione triptica, che sono specifici della proteina stessa. Questa procedura prevede la modifica chimica e il successivo isolamento di specifici peptidi detti “prototipici”. A tale scopo, i residui di triptofano contenuti nelle proteine sono stati chimicamente modificati mediante una reazione con il composto 2,4- dinitrofenilsulfenil cloruro (DNPS-Cl), che porta alla formazione di un derivato triptofanilico, con il DNPS legato in posizione 2 dell’anello indolico. La selezione dei peptidi modificati con il DNPS-Cl contenuti in una miscela triptica è stata effettuata sfruttando l’aumento di idrofobicità e del tempo di ritenzione di questi peptidi modificati in una colonna HPLC a fase inversa. Inoltre, gli stessi peptidi modificati con DNPS-Cl sono stati isolati mediante cromatografia per immunoaffinità utilizzando una resina derivatizzata con anticorpi monoclonali diretti contro il gruppo DNP. La strategia di ”targeted proteomics” è stata ottimizzata utilizzando una miscela modello di sette proteine e successivamente è stata applicata per l’identificazione di una proteina allergenica contenuta in un prodotto dolciario. È stato inoltre dimostrato che queste nuove procedure di modifica selettiva e di selezione dei peptidi triptofanilici permette di semplificare considerevolmente l’analisi di fingerprinting/MS che è solitamente utilizzata per l’identificazione di proteine nei protocolli di proteomica. Altre attività di ricerca. Durante il periodo di dottorato, ho avuto l’opportunità di collaborare con altri membri del laboratorio in due progetti addizionali come continuazione di un progetto di ricerca precedente. La documentazione relativa a queste attività è riportata in appendice alla tesi di dottorato. Sono state investigate le proprietà molecolari del complesso formato da α-lattalbumina con l’acido oleico. Il complesso appare interessante poiché ha mostrato avere tossicità cellulare diretta selettivamente contro cellule tumorali. È stato dimostrato che la proteina nel complesso ha una struttura oligomerica, diversamente da quanto riportato nelle prime osservazioni, che ipotizzavano fosse in uno stato monometrico. Inoltre, è stato osservato che l’acido oleico interagisce anche con altre proteine, come l’apomioglobina. La principale conclusione di questo lavoro è stata che il motivo oligomerico della proteina veicola l’acido oleico, normalmente poco solubile, favorendo quindi la solubilizzazione dell’acido grasso e conseguentemente della sua proprietà citotossica. È in preparazione un articolo riguardante questi risultati. L’enterocina AS-48, è un polipeptide circolare di 70 residui prodotto da Enterococcus faecalis che mostra a vere attività antibatterica. La proteolisi limitata è stata usata per preparare una forma lineare e due frammenti di questa enterocina. Misure di dicroismo circolare nel lontano ultravioletto hanno dimostrato che la proteina ha una bassa ellitticità e una ridotta stabilità alla denaturazione termica, ma mantiene la sua attività antibatterica., mentre i frammenti presentano un’attività ridotta. Questi risultati indicano che la circolarizzazione è un fenomeno che non è richiesto per l’attività antibatterica, ma è cruciale per la stabilizzazione della struttura nativa. Questa ricerca è stata pubblicata in FEBS Lett. (2008).

2013 - 2014
The great interest of the research activity in food allergies could be attributed to the increase of allergic reactions all over the world not only in infants but even in adult age. As an alternative to the development of an allergen-free diet, many works have been focused on a novel approach for the treatment of allergens: instead of eliminating the allergens from the diet, the immunoresponse can be reduced or even eliminated by inducing some modifications of their molecular structure. In fact, changes in allergen conformation can modulate its identification by the specific antibody produced by immune system in allergic reactions. Structural modifications in allergens could be induced by conventional thermal treatments as well as by non-thermal technologies, namely High Hydrostatic Pressure (HHP), Pulsed Electric Fields (PEF), Pulsed Light (PL) and -radiations. Non-thermal technologies have been widely used in the last years for food preservation, having the advantage of increasing the shelf-life and freshness of the raw food products. These technologies are able to affect the food nutritional and organoleptic properties only slightly thanks to the use of a non-thermal stress to treat foodstuffs. Among them High Hydrostatic Pressure technology has been successfully used in food pasteurization, but also in processes involving the sol-gel transition such as the production of jams, jellies and dairy products. The ability of High Pressure to determine structural changes in foods was studied in order to assess if proteins unfolding and/or aggregation and gelation can be induced and if the treatment affects the functional properties and digestibility of proteins. These effects were studied on particular proteins, namely the allergens, for which unfolding and structural modification have been proven. However, the effectiveness of the High Pressure processing on the reduction of immunoresponse reduction was not clearly assessed so far. The objective of this PhD thesis was the study of the modifications induced by High Pressure Process on allergenic proteins and the possibility of obtaining hypoallergenic peptides by means of a combined High Hydrostatic Pressure hydrolysis. In particular, the effect of the HHP on the allergens structural modification was investigated in a wide range of operating conditions, including both gelling and ungelling conditions. Rheological behavior and functional properties of HHP processed allergens was also determined. [edited by Author]
XIII n.s.

Pollen grains are ubiquitous triggers of allergic asthma and seasonal rhinitis. Proteolytic enzymes in pollen as well as other sources are capable of disrupting airway epithelial integrity in vivo and in vitro. This provides a plausible mechanism for the initiation of sensitisation of the respiratory immune system to inhaled pollen allergens, comparable to that suggested for Group 1 allergens from house dust mites and cat dander, which are known to possess intrinsic proteolytic activity. This thesis explores the relationship between pollen allergens and proteolytic enzymes. It describes the different strategies used for the characterisation, purification and identification of immunogenic and proteolytic proteins in the complex mixtures of pollen diffusates. The peptidases in the diffusates of Kentucky blue grass, ryegrass and Bermuda grass pollens were characterised by a sensitive fluorescence assay and gelatin zymography. Among these, Bermuda grass pollen demonstrated the presence of a serine peptidase at Mr ~30,000 Da, which corresponded to an intense band by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense) group 1 allergen, Phl p 1. Purification of this enzyme from Bermuda grass was complicated by the low levels of the enzyme present in the diffusate, as well as by its autohydrolysis. Partial purification of the serine peptidase activity by affinity chromatography using Concanavalin A Sepharose demonstrated that the diffusate contained a trypsin-like peptidase, detected by the fluorescent assay, in addition to the ~30,000 Da serine endopeptidase, detected on gelatin zymography. Proteomic analysis of the ~30,000 Da protein using one- and two-dimensional electrophoresis and mass spectrometry identified it as the major pollen allergen of Bermuda grass, Cyn d 1. The studies reported here provide, for the first time, evidence that a pollen allergen may possess intrinsic proteolytic activity. This activity may play a role in the initiation of airway inflammation and allergic sensitisation.

Lipid transfer proteins (LTPs) are a class of low molecular weight hydrophobic conserved proteins comprising four intramolecular disulphide bonds making the structure very resistant to proteolysis and harsh food processing conditions. These proteins are identified as strong allergens sensitizing through the gut and share epitopes with LTPs from closely related species. Peach LTP, Pru p 3 is the primary sensitizer in the Mediterranean area being the most frequent food allergen. Wheat LTP, Tri a 14 is a relatively weak allergen with a very low prevalence. The study here compares the structural properties of these proteins and their resistance to various digestive and processing processes. Ligand binding experiments showed that Pru p 3 binds to ligands more strongly than Tri a 14. The gastroduodenal digestion of these LTPs revealed that both are stable to gastric digestion and while Pru p 3 is susceptible to duodenal digestion, Tri a 14 digestion is negligible. Ligand binding did not affect the digestibility of Pru p 3 but improved the duodenal digestibility of Tri a 14. The IgE binding studies using sera from peach allergic individuals confirmed that both Pru p 3 and its digestion fragments in the presence and absence of ligand were IgE reactive. Model processing conditions were employed to treat these LTPs. It was found that heat treatment destroys the secondary structure of Pru p 3 at 121°C and slightly affects that of Tri a 14. Heat treatment also increased the susceptibility of Pru p 3 to gastric digestion while Tri a 14 was less affected. The IgE binding studies showed that heat treatment of Pru p 3 appeared to reduce its IgE recognition while its digestion fragments lost all of their IgE reactivity. To investigate the effect of the food matrix on the digestibility of these LTPs, peach peel containing Pru p 3 and wheat flour containing Tri a 14 were digested under simulated conditions. It was found that they were resistant to proteolysis in their native matrices. Effect of heat treatment to the food matrix again confirmed that both of these proteins were more stable to heat in the matrix and were less digestible. In conclusion, this study shows that there are factors in food matrices which enhance structural stability of LTPs to both processing and digestion. Thus factors such as the effect of food matrix and effect of processing should be taken into account in assessing the allergenic risk posed by foods and not simply rely on data from purified proteins.

Orientadores: Maria Helena Andrade Santana, Ricardo de Lima Zollner
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica
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Resumo: Os lipossomas têm sido utilizados como imunoadjuvantes antigênicos, em estudos que enfatizam a sua atuação no sistema imunológico. Extratos peptídicos ou protéicos de várias fontes foram encapsulados em lipossomas ou associados à sua superficie, e aplicados em imunoterapias e vacinas. Apesar disso, poucos são os estudos voltados para a performance do processo de preparação desses lipossomas, eficiência da associação proteína/lipídio e estabilidade das vesículas. Este trabalho trata da avaliação do encapsulamento das proteínas: Albumina de Soro Bovino (BSA), Mioglobina (Mio) e Cito cromo C (Cit C), simulando extratos alergênicos provenientes de fungos e ácaros, no que se refere à faixa de peso molecular das suas proteínas. Os lipossomas foram preparados com os lipídios L-a-distearoilfosfatidi1colina (DSPC) e Colesterol (Col) na razão molar 70:30 respectivamente. As proteínas foram encapsuladas individualmente e em forma de misturas de composições molares BSA:Mio:Cit C de 20:40:40, 40:20:40 e 40:40:20, respectivamente. Além disso, a mistura de proteínas de composição 40:20:40 (BSA:Mio:Cit C) foi também associada covalentemente à superficie das vesículas. Os lipossomas foram caracterizados pelo teor de fósforo e diâmetro médio. O desempenho do encapsulamento foi analisado através dos perfis e eficiências de encapsulamento das proteínas individuais e em misturas, além da estabilidade das vesículas em tensoativo penta etileno glicol mono-n-dodecil éter (C12Es), e de plasma humano. Para as misturas, analisou-se a exclusão de proteínas durante o encapsulamento e a associação à superficie dos lipossomas. Os resultados indicam que a eficiência de encapsulamento depende mais das interações proteína:lipídio que do tamanho das proteínas. As eficiências de encapsulamento variaram entre 0,03% e 3,55% para as proteínas individuais e entre 1,04% e 3,94% para as misturas. Não houve alteração na estabilidade dos lipossomas em C12Es com a presença das proteínas. Em plasma, essas vesículas permaneceram estáveis por aproximadamente 20 horas. Na associação à superficie dos lipossomas, predominou a presença de BSA em relação às outras proteínas
Abstract: Liposomes has been studied as antigenic immunoadjuvants with emphasis on their performance in the imunological system. Peptides or proteins of various sources were encapsulated in liposomes or associated to their surface, and applied in immunotherapy and vaccines. In spite of this, there are few studies about the performance of preparation process ofthese liposomes, efficacy ofthe association proteinllipid and the stability ofvesic1es. This work concems with the evaluation of the encapsulation of the proteins Bovine Serum Albumin (BSA), Myoglobin (Myo) and Cythochrome C (Cyt C), simulating allergen extracts from molds and mites, related to the range of molecular weight of their proteins. Liposomes were prepared with lipid L-a-disteraoylphosphatidylcholine (DSPC) and Cholesterol (Chol) at molar ratio 70:30 respectively. The proteins were encapsulated individualyand in mixtures with molar composition BSA:Myo:Cyt C of20:40:40, 40:20:40 and 40:40:20 respectively. Furthermore, the mixture 40:20:40 (BSA:Myo:Cyt C) was also covalent1y associated to the surface of vesic1es. Liposomes were characterized by their phosphate contents and mean diameter. The performance of the protein encapsulation was evaluated through the profiles and efficiency of the encapsulation and throught the stability of vesic1es in the presence of the surfactant penta ethylene glycol mono-n-dodecyl ether (C12ES) and human plasma. For the mixtures, the exclusion of proteins duringentrapment or association to the liposome surface was also evaluated. The experimental results indicate that the efficacy of encapsulation is more dependent of the interactions proteinllipid than the size of the proteins. The efficiencies of encapsulation changed between 0.03% and 3,55% for individual proteins and were between 1,04% and 3,94% for the mixtures. The presence ofproteins does not altered the stability of liposomes in C12ES . In human plasma the vesic1es remained with stability about 20 hours. For the mixtures, the presence ofthe BSA protein predominated in the vesicles
Mestrado
Desenvolvimento de Processos Biotecnologicos
Mestre em Engenharia Química

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
This work reports the biochemical characterization, cytotoxic and allergenic effects of a lipid transfer protein isolated from M. citrifolia seeds (McLTP1), with trypsin and alpha-amylase inhibition properties. McLTP1 was purified with a procedure involving trichloroacetic acid precipitation and gel filtration chromatography. This protein showed significant inhibitory activities against trypsin (767,10 Â 8,36 TIU/mgP), chymotrypsin (25,36 Â 0,86 IU/mgP), papain (65,419 Â 0,152 IU/mgP) and alpha-amylase (24,40%). Atomic force microscopy displayed that McLTP1 oligomerized in tetramers showing a central channel. Fluorescence and CD assays revealed that the McLTP1 structure is highly stable, regardless of pH and temperature levels. In vitro, McLTP1 presented a selective cytotoxic effect to human ovarian cancer cells (OVCAR-8; IC50 of 16,6 μg/mL) and demonstrated hemolytic effect against fresh rabbit red blood cels. Similarly to other non-specific lipid transfer protein reported, McLTP1 showed allergenic properties in mice, being considered as a true food allergen since it was able to sensitize the animals via the gastrointestinal tract.
Morinda citrifolia L. à uma espÃcie nativa do Sudeste da Ãsia intensamente investigada em funÃÃo de suas propriedades terapÃuticas reportadas hà mais de 2.000 anos. Recentemente, uma proteÃna transferidora de lipÃdeos denominada McLTP1 (UniProt Accession Number: C0HJH5) foi isolada de sementes de noni pelo nosso grupo de pesquisa. McLTP1 à uma proteÃna termoestÃvel de massa molecular 9,4 kDa, resistente à proteÃlise e dotada de atividades moduladoras da inflamaÃÃo e da dor pela via oral, promissoras e inÃditas para esse grupo de molÃculas. Este trabalho objetivou caracterizar bioquimicamente McLTP1, bem como avaliar o seu potencial alergÃnico em camundongos, como etapas bÃsicas para o seu uso racional e seguro do ponto de vista farmacolÃgico. Em adiÃÃo, as propriedades terapÃuticas de McLTP1 foram tambÃm ampliadas, atravÃs da investigaÃÃo de seu efeito citotÃxico em diferentes linhagens de cÃlulas tumorais. A proteÃna em estudo foi isolada utilizando o protocolo jà estabelecido, envolvendo as etapas de precipitaÃÃo seletiva de proteÃnas do extrato total das sementes de noni com Ãcido tricloroacÃtico 2,5% e cromatografia de exclusÃo molecular. O ensaio de alergenicidade in vivo foi conduzido apÃs prÃvia aprovaÃÃo pelo Comità de Ãtica para Uso de Animais da Universidade Federal do Cearà e utilizou fÃmeas nulÃparas com massa corporal entre 25 e 30 g. McLTP1 apresentou in vitro atividades inibitÃrias de tripsina (767,10  8,36 UIT/mgP), quimotripsina (25,36  0,86 UI/mgP), papaÃna (65,419  0,152 UI/mgP) e alfa-amilase (24,40%). A atividade inibitÃria de tripsina de McLTP1 foi reduzida significativamente em temperaturas superiores a 37 ÂC, apresentando atividade residual de apenas 5,91% quando aquecida a 100 ÂC por 30 min. Essa atividade foi tambÃm influenciada pelo pH, sendo de apenas 30,13% e 39,05% quando a proteÃna foi incubada em tampÃes de pH 3,0 e 12,0. O padrÃo de oligomerizaÃÃo de McLTP1 demonstrou a formaÃÃo de agregados dimÃricos/tetramÃricos delimitando um canal central de diÃmetro de 4,4 nm. As anÃlises espectroscÃpicas mostraram que McLTP1 apresenta espectro de CD similar Ãquele apresentado por outras proteÃnas transferidoras de lipÃdeos e caracterÃstico de proteÃnas ricas em alfa-hÃlice. Espectro de CD de McLTP1 nÃo mostrou alteraÃÃes significativas em diferentes temperaturas e pHs, corroborando com os dados de estabilidade obtidos anteriormente. Diferentemente, em condiÃÃes redutoras (DTT 1 mM) o espectro de CD mostrou alteraÃÃo na estrutura secundÃria da proteÃna e os mÃnimos e mÃximos de elipticidade molar foram tambÃm alterados na presenÃa de micelas iÃnicas de SDS (10 mM). McLTP1 apresentou atividade citotÃxica seletiva contra cÃlulas de cÃncer de ovÃrio (Ovcar-8; CI50: 16,6 μg/mL), nÃo sendo citotÃxica para as cÃlulas tumorais de cÃlon humano (HCT-116), leucemia humano (HL-60) e glioblastoma humano (SF-295) testadas. McLTP1 foi capaz de promover hemÃlise significativa em hemÃcias de coelho a partir da concentraÃÃo de 0,005 mgP/mL. McLTP1 apresentou potencial efeito alergÃnico in silico e em camundongos imunizados pela via oral, induzindo a sÃntese de anticorpos IgG e IgG1. Tal como descrito na literatura para outras LTPs, anticorpos anti-McLTP1 produzidos em coelho foram tambÃm capazes de reconhecer proteÃnas presentes em extratos de Rosaceae, Cucurbitaceae e na polpa do fruto de noni. Os dados obtidos permitiram caracterizar parcialmente a proteÃna em estudo, bem como avaliar o seu potencial imunogÃnico apÃs administraÃÃo oral. Novos testes serÃo conduzidos objetivando avaliar a importÃncia clÃnica dessas respostas, uma vez que testes de toxicidade demonstraram que McLTP1 nÃo foi capaz de promover reaÃÃes adversas em camundongos, mesmo apÃs administraÃÃo da dose de 8 mg/kg por 28 dias.

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
ABSTRACT: Two experiments were conduct to evaluate the effect of using soy protein concentrate (SPC) in pre-starter piglet diets over performance, intestinal histomofometria and blood parameters. In the first trial 72 piglets weaned at 21 days of age were used and 54 for the second trial. Both were conducted as a randomized blocks design with six replications and three piglets per unit. The first trial had four diets (0% SPC, 3% SPC, 6% SPC and 9% SPC), which evaluated different levels of SPC inclusion in diets, and the second trial, which evaluated the replacement of spray dried blood plasma (SDP) by SPC, had three diets (0.0%-SPC + 5.0-SDP, 2.5%-SPC + 2.5%-SDP and 5.0%-SPC + 0.0%-SDP). In both trials, diets and water were offered ad libitum in pre-starter l (21-32 days old) and pre-starter ll (33-42 days old) phases, but during the starter phase (43-66 days old) all piglets received a single diet. At 32 days of age, blood was collected from one animal per experimental unit and then these animals were euthanized to collect samples of the small intestine. Linear effect was observed In the first trial, over feed conversion (FC) as the level of CPS in diet was increased in the period between 21 and 32 days of age. Linear effect was also observed on the FC in the period of 33-42 days old, however in a reverse form from the previous period, wherein the increase of the SPC level in diets resulted in the increase of FC. In the total period of this trial (21-66 days old), it was found a quadratic effect of SPC on piglets FC. In the second trial, the replacement of SDP by CPS caused effect on performance variables, except for feed conversion (FC) during 21-32 days of piglets age. The average daily feed intake (ADFI) was higher for pigs fed 2.5%-SPC + 2.5%-SDP and the average daily gain (ADG) and the final body weight (FBW) of the piglets in this period was higher for 2.5%-SPC + 2.5%-SDP compared to 5.0%-SPC + 0.0%-SDP. In the period of 21-42 days old, it was observed best results of ADFI and FBW for piglets fed diet containing 2.5%-SPC + 2.5%-SDP compared to piglets fed 5.0%-SPC + 0.0%-SDP. The ADG showed better results for diets containing 0.0%-SPC + 5,0-SDP and 2.5%-SPC + 2.5%-SDP and FC was higher for the diet with 0.0%-SPC + 5.0%-SDP compared to the diet with 5.0%-SPC + 0.0%-SDP. During total period of the experiment (21-66 days old) ADG and ADFI was influenced by substitution of SDP for SPC, wherein the best results was presented by piglets fed 2.5%-SPC + 2.5%-SDP compared to those fed 5.0%-SPC + 0.0%-SDP. In both trials, no effect was observed upon histomorphometric variables (villus height, crypt depth and villus: crypt). Regarding the blood variables (leukocytes, eosinophils and lymphocytes), no effect of SPC at 32 days of age the piglets was observed in the first trial. But in the second trial, total leukocyte count was higher in animals fed 5.0%-SPC + 0.0%-SDP compared to ones fed 0.0%-SPC + 5.0%-SDP and lymphocyte count was lower in piglets receiving 0.0%-SPC + 5.0%-SDP. In the first trial, the use of SPC in diets of post-weaning piglets during pre-starter period (21-42 days old) did not influence performance from 21 to 66 days of life, or intestinal morphology and leukocyte, lymphocytes and eosinophils count in these animals. In the second, the combined use of SPC and SDP in the diets of pigs between 21 and 42 days of age reduces the activation of the immune system and improves productive performance.
Foram conduzidos dois experimentos com objetivo de avaliar o efeito da utilização de Concentrado proteico de soja (CPS) em dietas pré-inicias de leitões sobre o desempenho, histomofometria intestinal e alguns parâmetros sanguíneos. Foram utilizados 72 leitões machos castrados desmamados aos 21 dias de idade no primeiro experimento e 54 no segundo. Ambos foram delineados em blocos ao acaso com seis repetições e três leitões por unidade experimental, tendo o primeiro quatro tratamentos (0% CPS, 3% CPS, 6% CPS e 9% CPS), o qual avaliou diferentes níveis de inclusão de CPS nas dietas, e o segundo, para avaliar a substituição de plasma sanguíneo (SDP) por CPS, três (0,0%-CPS + 5,0-SDP, 2,5%-CPS + 2,5%-SDP e 5,0%-CPS + 0,0%-SDP). Em ambos as rações experimentais e água foram fornecidas à vontade nas fases pré-inicial l (21 a 32 dias de idade) e pré-inicial ll (33 a 42 dias de idade), sendo que na fase inicial (43 a 66 dias de idade) todos os leitões receberam uma única dieta. Aos 32 dias de idade foi coletado sangue de um animal por unidade experimental e em seguida estes animais foram eutanasiados para coleta de amostras de intestino delgado. No primeiro experimento, foi observado efeito linear decrescente sobre a conversão alimentar (CA) à medida que aumentou o nível de CPS na dieta no período entre 21 e 32 dias de idade. Efeito linear também foi observado sobre a CA no período entre 33 e 42 dias de idade dos leitões, entretanto de forma inversa ao período anterior, ou seja, à medida que se aumentou o nível de CPS na dieta a CA aumentou. Foi observado no período total (21 a 66 dias de idade) do primeiro experimento efeito quadrático do CPS sobre a CA dos leitões. No segundo experimento a substituição de SDP por CPS causou efeito sobre as variáveis de desempenho no período entre 21 e 32 dias de idade, exceto sobre a CA. O CDR foi superior para os leitões alimentados com 2,5%-CPS + 2,5%-SDP e o GDP e o peso final (PF) dos animais neste período foi superior com 2,5%-CPS + 2,5%-SDP comparado a 5,0%-CPS + 0,0%-SDP. No período entre 21 e 42 dias de idade foi observado melhores resultados de CDR e PF para a dieta com 2,5%-CPS + 2,5%-SDP comparado a 5,0%-CPS + 0,0%-SDP. O GDP apresentou melhores resultados para as dietas com 0,0%-CPS + 5,0-SDP e 2,5%-CPS + 2,5%-SDP e a CA foi superior para a dieta com 0,0%-CPS + 5,0%-SDP diante da dieta com 5,0%-CPS + 0,0%-SDP. No período total do experimento (21 a 66 dias de idade) foi observado efeito da substituição dos SDP por CPS sobre o GDP e o CDR, sendo que os animais alimentados com 2,5%-CPS + 2,5%-SDP apresentaram resultados superiores aos alimentados com 5,0%-CPS + 0,0%-SDP. Emambos os experimentos nenhum efeito foi observado sobre as variáveis histomorfométricas (altura de vilosidade, profundidade de cripta e relação vilo:cripta). Em relação às variáveis sanguíneas (leucócitos, linfócitos e eosinófilos) do primeiro experimento, não foi observado nenhum efeito do CPS aos 32 dias de idade dos leitões. No segundo experimento, a contagem total de leucócitos foi superior para os animais alimentados com 5,0%-CPS + 0,0%-SDP sobre os alimentados com 0,0%-CPS + 5,0%-SDP e a contagem de linfócitos foi menor nos leitões que receberam 0,0%-CPS + 5,0%-SDP. No primeiro experimento, a utilização de CPS em dieta de leitões pós-desmame no período préinicial (21 a 42 dias de idade) não influenciou o desempenho produtivo dos leitões no período entre 21 e 66 dias de vida, nem a histomorfometria intestinal e a contagem de leucócitos, linfócitos e eosinófilos desses animais. No segundo, a utilização conjunta de CPS e SDP nas rações reduz a ativação do sistema imune e melhora o desempenho produtivo.

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.

Le proteine della farina di frumento sono considerate gli allergeni responsabili del 60-70% dei sintomi respiratori che si manifestano nella malattia professionale denominata “asma dei panificatori”; inoltre il frumento è tra i 6 principali allergeni alimentari coinvolti in reazioni di ipersensibilità IgE-specifiche. Nel caso dell’allergia alimentare al frumento le manifestazioni variano da sintomi respiratori, gastrointestinali e cutanei fino a gravi reazioni sistemiche. Nonostante questa rilevanza allergologica, a livello molecolare la conoscenza degli allergeni del frumento è piuttosto scarsa. Una conseguenza di questa limitata conoscenza è che la diagnosi di malattie, quali l’asma del panificatore, può essere una difficile impresa. Si presuppone tuttavia che sia gli epitopi sequenziali che quelli conformazionali possano essere responsabili di queste reazioni allergiche. Lo Skin prick test (SPT) gioca un ruolo importante nella diagnosi allergologica ma i pazienti con asma del panificatore risultano positivi solo lievemente (dal 5 al 15%). Questa scarsa significatività dello SPT potrebbe essere una conseguenza della bassa solubilità di molte proteine del frumento così come un effetto di differenze nella standardizzazione degli estratti analizzati. OBIETTIVO Lo scopo di questo studio era quello di realizzare una caratterizzazione degli allergeni del frumento coinvolti nell’allergia alimentare e inalatoria. In particolare, l’obiettivo di questo studio era di esaminare la variabilità dei profili proteici riconosciuti dalle IgE di panificatori italiani sensibilizzati verso la farina di frumento e di identificare gli allergeni più frequentemente riconosciuti. Inoltre abbiamo voluto i) indagare quali epitopi fossero coinvolti nelle diverse forme di allergia al frumento ii) esaminare il reale contenuto allergenico negli estratti per lo SPT usati nella diagnosi dell’allergia al frumento mettendo in relazione la loro composizione con i risultati diagnostici prodotti. È stata sviluppata come strategia un’analisi proteomica del frumento seguita da IgE blotting cioè un approccio “allergonomico”. METODI per caratterizzare gli allergeni del frumento le proteine di farina di frumento, monocultivar Bolero, solubili in soluzione salina, sono state separate in elettroforesi monodimensionale (1-DE) e bidimensionale (2-DE) in condizioni ridotte e non ridotte. Inoltre, sono stati analizzati gli estratti di farina e di farina integrale usati per lo SPT. Le proteine IgE-specifiche sono state rivelate attraverso immunoblotting usando i sieri di 43 pazienti con allergia inalatoria e 9 con allergia alimentare al frumento. Dopo digestione triptica, sono state analizzate attraverso nano HPLC–ESI–MS/MS i peptidi di alcune proteine IgE-specifiche, frequentemente immunorivelate dalle IgE dopo elettroforesi bidimensionale. RISULTATI Gli immunoblot ottenuti con 43 sieri diversi utilizzati singolarmente presentavano una notevole eterogeneità. Sempre con questa metodica inoltre, sono stati realizzati profili di estratti in condizioni ridotte e non ridotte evidenziando il coinvolgimento di ponti disolfuro nella stabilizzazione dei determinanti. Infine è stato dimostrato che gli estratti usati per lo SPT sono solo parzialmente rappresentativi del reale contenuto allergenico del frumento e differenti lotti di questi estratti mostravano notevoli diversità. Si è inoltre osservato che la maggior parte degli allergeni non sono singoli spot proteici ma erano rappresentati da più isoforme proteiche con peso molecolare simile ma un punto isoelettrico diverso. Cinque dei principali spot proteici IgE-specifici sono stati identificati per mezzo della tecnica nano HPLC–ESI–MS/MS. In questo studio, abbiamo identificato un allergene inalatorio del frumento già riportato in letteratura [monomeric alpha-amylase inhibitor, 0.28]. Inoltre abbiamo identificato due allergeni inalatori del frumento, raramente descritti in letteratura [triosephosphate-isomerase e thioredoxin peroxidase], e abbiamo scoperto due nuovi allergeni mai riportati come tali [glucose and ribitol dehydrogenase homolog – barley e una heat shock protein]. Nel caso dell’allergia alimentare al frumento, sorprendentemente, non ci sono IgE-specifiche per le proteine del frumento in 8 casi su 9 (88%). CONCLUSIONI. La rilevanza clinica dei due nuovi allergeni identificati dovrà essere ulteriormente investigata ma certamente i nostri risultati potrebbero contribuire ad aumentare la specificità dei saggi diagnostici. L’IgE-blotting non ha rilevato allergeni alimentari perciò, nel prossimo futuro, sarà necessario sviluppare un approccio alternativo per lo studio di queste patologie.
Wheat flour proteins are allergens involved in 60% to 70% of workplace-related respiratory-symptoms of bakers, furthermore wheat belongs to the six major food allergens inducing IgE-mediated hypersensitivity-reactions. In the case of wheat food-allergy the manifestations range from cutaneous, gastrointestinal, and respiratory symptoms to severe systematic reactions. Despite this allergological relevance the knowledge of wheat allergens at molecular level is scanty. It is nevertheless assumed that conformational and sequential epitopes might be both responsible for wheat allergic reactions. As a consequence of this limited knowledge the diagnosis of wheat allergy is sometime a difficult task. Skin prick tests (SPTs) play an important role in the allergological diagnosis but baker’s asthmatic patients result positives in a limited (from 5 to 15%) range. This scarce SPT predictability may be a consequence of the low solubility of numerous wheat proteins as well as an effect of differences in test-extracts standardization. OBJECTIVE The aim of the present study was to achieve a more detailed and comprehensive characterization of the wheat allergens involved in food allergy and baker’s asthma. In particular, the aim of the study was to investigate the variability of IgE antibody patterns of wheat flour-sensitized Italian bakers and to identify the most frequently recognized allergens. Furthermore i) we tried to verify whether different epitopes are involved in the different forms of wheat allergy ii) we tested the real allergen content in SPT used in wheat allergy diagnosis in relationship to their performance in clinical practice. The employed strategy was the proteomic analysis of wheat followed by IgE blotting i.e. an “allergenomic” approach. METHODS To characterize wheat allergens, water/salt-soluble wheat flour proteins from the monocultivar Bolero were separated by using mono dimensional (1-DE) and 2-dimensional (2-DE) gel electrophoresis under reducing and non-reducing conditions. Furthermore, using 1-DE SPT-solutions, wholemeal wheat flour and flour extracts were separated. IgE-binding proteins were detected by immunoblotting using the sera of 43 patients with inhalant allergy and 9 with food allergy to wheat. After tryptic digestion, the peptides of some IgE-binding proteins, frequently recognized by IgE on 2-DE, were analyzed by nano HPLC–ESI–MS/MS. RESULTS The IgE immunoblots obtained with 43 different sera exhibited a remarkable inter-individual heterogeneity. Furthermore, the immunodetected profiles were very different under reducing and non-reducing condition. The analyzed SPT-solutions demonstrate to be only partially representative of the real allergenic content of wheat and different batch of these extracts show remarkable differences. It was also demonstrated that the majority of the allergens were not single polypeptide spots, but consisted of up to ten isoforms of similar molecular mass but with different isoelectric points. Five of the predominant IgE-binding protein spots were identified by using nano HPLC–ESI–MS/MS. In this study, we identified one already reported wheat inhalant allergen [monomeric alpha-amylase inhibitor, 0.28] by a database search. Moreover we identified two wheat inhalant allergens, rarely described in literature [triosephosphate-isomerase and thioredoxin peroxidase], and we found out two never reported wheat inhalant allergens [glucose and ribitol dehydrogenase homolog – barley and a heat shock protein]. In the case of food allergy, surprisingly, no specific IgEs for wheat proteins were detected in 8 out of 9 cases (88%). CONCLUSIONS. The clinical relevance of the identified 2 new allergens will be further investigated in the near future but certainly our findings could contribute to increase the specificity of diagnostic assays. IgE-blotting did not reveal food allergens for this reason, in the future, an alternative approach to allergomic must be developed.

博士
國立中興大學
食品暨應用生物科技學系所
99
Rice is the staple food in Asia, it plays an important role in providing energy to general population within the region. Rice 14-16 kDa allergenic proteins, a salt-soluble protein fraction, were the products of homologous genes with similar structures. The 14-16 kDa allergenic proteins have been reported as human salivary α-amylase inhibitor, barley trypsin inhibitor and storage protein of grate asako. It was demonstrated that these proteins were the major allergens which caused allergic reactions with consumption of cereals. It was reported that the 14-16, 26, 33 and 56 kDa proteins in rice could react with IgE of rice allergic patients in which the 14-16 kDa proteins, especially, were found to be recognized by IgE antibodies from 90-95% of rice allergic patients. Therefore, 14-16 kDa allergens are thought to be the main allergy proteins in rice. Environmental pollution and dietary factors on human allergenicity has drawn much attention lately. More information in this regard is needed. To develop an allergen-specific antibody is the first step for food allergen detection. It is difficult to obtain the specific antibodies against rice 14-16 kDa allergens. The objectives of this study were to utilize the molecular transgenic technology to amplify rice 14-16 kDa allergen genes and transfer the genes into the expression vector followed by expressing the target recombinant protein in Escherichia coli expression host, to purify target recombinant protein, and to study the use of purified protein on detection of rice 14-16 kDa allergens. pET-29a(+)-RA17 Expression vector of rice was constructed by using the RT-PCR method to amplify the cDNA of RA17 (one of rice 14-16 kDa allergens) from rice mRNA. Recombinant RA17 (rRA17) was expressed in Escherichia coli expression host as a histidine-tag fusion protein and was purified to its homogeneity. The polyclonal antibody was then prepared from the rRA17 protein to be further used as a tool to analyze the content of 14-16 kDa allergens in phytase-transgenic rice (GR) and non-transgenic rice (NR) (wild-type, Oryza sative L. cv. Tainung 67). Result of western blotting showed that the content of major allergenic proteins in GR was 96.3% of that in NR, which were 1.66 ± 0.08 mg/g and 1.73 ± 0.09 mg/g for GR and NR, respectively. It appeared that transferring phytase gene into rice plant did not affect the content of rice major allergenic proteins significantly. The 14-16 kDa allergens of rice was purified and was further analyzed and characterized. Salt-souble proteins of rice were first extracted with 1M NaCl solution followed by precipitation with 90% saturated ammonium sulfate solution. The precipitate was dialyzed and then subjected to DEAE-Sephadex ion-exchange chromatography. Four fractions (I-IV) were obtained. Each fraction was further purified by Sephadex G-50 gel-filtration. The purified fractions were identified as RA14/RA14B (FDEAE-Peak I), RA14C/RAG2 (FDEAE-Peak II), similar to RA17 precursor (FDEAE-Peak III ) and RA14/RA14B (FDEAE-Peak IV) by LC-MS-MS analysis. In a simulated gastric fluid (SGF) test, both RA14/RA14B and RA14C/RAG2 were fully digested in 5 min whereas similar to RA17 precursor was stable up to 60 min. In contrast, both RA14/RA14B and RA14C/RAG2 were relatively stable when subjected to a simulated intestinal fluid (SIF) test. Besides, RA14/RA14B, RA14C/RAG2 and similar to RA17 precursor remained to be stable when heated at 100°C for 60 min. Contents of 14-16 kDa allergens in 9 studied rice samples were as follows: 2.36 ± 0.12 mg/g (Oryza sative L. cv. Taikeng 2), 2.16 ± 0.13 mg/g (Oryza sative L. cv. Taikeng 4), 1.91 ± 0.11 mg/g (Oryza sative L. cv. Taikeng 9), 2.26 ± 0.10 mg/g (Oryza sative L. cv. Taoyuan 3), 2.61 ± 0.07 mg/g (Oryza sative L. cv. Tainan 11), 1.81 ± 0.11 mg/g (Oryza sative L. cv. Kaohsiung 139), 2.26 ± 0.09 mg/g (Oryza sative L. cv. Kaohsiung 145), 2.12 ± 0.10 mg/g (Oryza sative L. cv. Taisen 22), 1.95 ± 0.09 mg/g (Imported Thailand Indica rice), 1.69 ± 0.08 mg/g (Sihu long waxy rice), 1.67 ± 0.12 mg/g (Xinhua long waxy rice), 1.73 ± 0.11 mg/g (Sihu round waxy rice) and 1.80 ± 0.10 mg/g (Siluo round waxy rice). Data indicated that there was no correlation between contents of 14-16 kDa allergens and growing areas of rices. Furthermore, the 14-16 kDa allergen contents were different in some japonica and indica rice strains. In SGF test, the 14-16 kDa allergens in tested rices could be completely digested in 2 min, and all fragments after SGF digesting showed no allergic activity when subjected to Western blotting.

碩士
逢甲大學
資訊工程學系
106
Allergy is the abnormal reaction of the body when exposed to some substances that cause the immune system overreacts by producing Immunoglobulin E antibodies and following upon symptoms include fever, allergy with food, atopic dermatitis as well as anaphylaxis. In recent years, the number of allergenic reactions increases significantly depending much on the developing of modified proteins in foods, therapeutics and biopharmaceuticals and allergenic protein (allergen) prediction becomes an urgent problem. Although, there are many previous methods developing for allergy protein prediction, more and more precise and sophisticated method is still necessary, especially on discriminating of allergens and allergen-like non-allergens. To overcome this problem, we developed a new approach which combines Support Vector Machine (SVM) and Genetic Algorithm (GA) processing on the n-peptide composition properties characteristic to improve the accuracy performance of allergen protein identification. In our experiment, we not only improved the prediction ability but also obtained the knowledge of which protein properties store the allergenic information. Although the sensitive value is slightly lower than previous method (78.4%), our results emphasize the accuracy performance 96.6%, Matthew’s correlation coefficient MCC 0.805 and precision 87% when applying on the independent dataset of 1384 protein sequences. Our approach predicts very well allergen-like non allergen sequences and it becomes a promised tool for allergen non-allergen protein prediction in recent.

碩士
國立交通大學
生物科技學系
106
Allergy is a common disease for modern people, and is difficult to prevent and treat. Due to the increase of allergens in the modern environment, people suffering allergy become more and more. Hypersensitivity to allergy can be prevented by combining allergen specific tests and immunotherapy, but the types of allergens that have been tested are very few. Therefore, we aim to purify allergenic proteins in large scale through the E.coli expression system in order to understand the mechanism of allergen induced by immune responses and for applyication in biomedical detection. We selected 18 allergenic proteins, which are divided into five types: the food type (Ara h 1, Ara h 2, Gly m 5 and Pen a 1), the pollen type (Phl p 1, Phl p 5, Amb a 1, Art v 1, Par j 2, Cry j 1, Cry j 2, Cup s 1, Jun a 2 and Pla a 1), the rubber type (Hev b 5 and Hev b 6), the venom type (Ves v 1) and the microorganism type (Asp f 1). The allergenic genes were cloned into pET22b or pET28a plasmids and then expressed through the E. coli expression system. These proteins were further purified in a homogenous state by column chromatography and used on antibody synthesizion for application in biomedical detection. In addition, we will try to determine the crystal structures of these allergenic proteins to understand the allergen induced immune responses.

Cardiovascular diseases (CVD) and their risk factors remain the leading cause of morbidity and mortality in North America. Food-derived bioactive peptides (BAP) have been shown to play a role in regulating physiological pathways of CVD risk factors including hypertension, diabetes, and chronic inflammation. Common sources of BAP include dairy and plant proteins. In addition to being an alternative protein source, it is now accepted that edible insect proteins can also confer health benefits beyond nutrition. However, as with any novel protein source, allergenicity remains a major concern surrounding edible insect consumption.

This dissertation aimed to: 1) Evaluate the bioactive potential of peptides from an edible cricket species and; 2) determine the effects of BAP production methods on immunoreactivity. First, peptide-rich extracts were generated from farmed food-grade crickets via enzymatic hydrolysis techniques with the commercial protease Alcalase™. To measure the in vitro bioavailability, cricket peptides were also subject to simulated gastrointestinal digestion (SGD). Peptides and their digests were tested for relevant bioactivities and active groups were further fractionated by chromatographic methods to identify the major peptides responsible for the bioactivity. When tested for in vitro antihypertensive and anti-glycemic properties, cricket peptides were found to inhibit the activities of angiotensin converting enzyme, dipeptidyl peptidase-4, α-amylase, and α-glucosidase. The anti-inflammatory potential was expounded using RAW-264.7 macrophages and human umbilical vein endothelial cells (HUVEC). Cricket peptides (after SGD) effectively lowered NF-κB, MCP-1, and IL-6 production in cells without affecting their viability. Proteomic analyses identified 18 sequences from the enriched cationic peptide fraction that showed the highest activity. Three novel peptides were identified via molecular docking, as potent ACE-inhibitors and binding was similar to that of the commercial drug captopril. Key binding characteristics included interaction with hydrophobic amino acids (Phe, Pro, Leu) near the C-terminal position and coordination with Zn (II) ions near the ACE active site.

Immunological reactivity was measured by IgE-binding from shrimp-allergenic patient sera to antigens present within cricket peptides. Our studies demonstrate that immunoreactivity was impacted by enzymatic hydrolysis, depending on the conditions and heating source used. Tropomyosin (a major shrimp allergen) was extracted from both untreated crickets and protein hydrolysates, and verified as the major reactive protein. Tropomyosin reactivity decreased (under both partial and extensive hydrolysis) or retained (under conditions which prevented epitope cleavage). However, using microwave-assisted enzymatic hydrolysis was effective at decreasing tropomyosin reactivity in all immunoassays tested (IgG and IgE). Proteomic and immunoinformatic analyses revealed prominent allergen binding regions of cricket tropomyosin available for cleavage during enzymatic hydrolysis. Conserved antigen regions showed greater homology with other crustacean species, but not with other well studied allergenic insect proteins (i.e., cockroach). Lastly, LC-MS/MS and FT-Raman spectrometry suggests that reactivity was affected due to distinct epitope cleavage within the protein instead of decreased antigen extractability/solubility.

The findings of this dissertation support that edible cricket proteins are a potential source of bioactive peptides for functional food or nutraceutical development. Additionally, using protein extraction methods such as microwave-assisted hydrolysis seems a promising tool for minimizing the immunoreactivity of the allergen present in this edible cricket species.

碩士
國立中興大學
食品科學系
93
Soybean meals have been used extensively as protein sources in animal feedstuffs because of their high protein content (around 44%). However, the thermo-stable anti-nutritional factors, i.e., allergenic proteins (mainly β-conglycinin and glycinin) and flatulence-producing factors (mainly stachyose and raffinose) that commonly exist in these soybean meals will affect the growth and health of animals and have limited their use in young animal diets. The purpose of this study was to investigate the factors affecting the solid-state fermentation condition of soybean meal by koji-mold Aspergillus oryzae in order to reduce anti-nutritional factors and improve its nutritional value. Pre-treatment of raw material before fermentation was investigated. The water absorption ability was good for soybean meal. The moisture content reached equilibrium when ratio of soybean meal to water was at 1: 2.5, and which was independent of soaking time. Cooking methods had no effect on moisture content and could be omitted. The optimum fermentation conditions performed in Erlenmeyer flasks (capacity 250 ml) were established. The optimal ratio of soybean meals to water was 1:1 (moisture content 58.82%), and the fermentation was performed at 25℃ for 6~7 days with inoculum size at 5% (v/w). Peak activities of α-galactosidase and acid protease obtained were 4.52 and 109.85 U/g, respectively. The optimum solid-state fermentation conditions by tray method were established. The optimal ratio of soybean meals to boiled water was 2:1 (moisture content 41.09%), and the fermentation was performed at 30℃ under 90% RH for 30~35 hr with inoculum size at 1% (v/w). Maximumα-galactosidase activity and acid protease activity obtained were 4.83 and 43.05 U/g, respectively. The oligosaccharides (stachyose and raffinose) of soybean meal were removed after fermented for 42 hr. Theβ-conglycinin of allergenic protein was also removed and the amount of glycinin decreased. Moreover, crude protein content and the amino nitrogen content of soybean meal increased after fermentation for 30~35 hr, and the maximum values obtained were 51.43% and 0.453%, respectively. The optimum temperature and pH for both α-glactosidase and acid protease activity from crude enzyme solution of a fermented soybean meal (24 hr) were determined. The optimum temperature of α-galactosidase and acid protease activity were 47℃and 52℃, respectively. The optimum pH of α-galactosidase and acid protease activity were 4.5 and 2.6 U/g, respectively. These two enzymes were stable in the temperature range of 37-52℃. The optimum conditions of limited enzymatic hydrolysis of 24 h fermentation were determined. The ratio of fermented soybean meal to water was 1:2, and the reaction was carried out at pH 3.0 and 42℃ for 1 hr. Under this condition, all stachyose and part of raffinose in the fermented soybean meal were removed. The β-conglycinin of allergenic protein was degraded and the amount of glycinin also decreased considerably. It is concluded that solid-state fermentation of soybean meal by Aspergillus oryzae produced high levels of α-galactosidase and acid protease, and thereafter increased crude protein and amino nitrogen content. Contents of both flatulence-producing factors and allergenic proteins were largely removed. Nutritional value of soybean meals by such solid-state fermentation will be considerably upgraded.

碩士
國立中興大學
食品暨應用生物科技學系
94
Abstract Soybean meals have been used extensively as one of major plant protein sources from plant origins in animal feedstuffs because of their high protein content (around 44%). However, anti-nutritional factors that commonly exist in these soybean meals have limited their nutritive values and utilization. Fermented soybean meals by Aspergillus oryzae removed most of thermo stable anti-nutritional factors such as allergenic proteins (mainly β-conglycinin and glycinin) and flatulence-causing factors (mainly stachyose and raffinose). However, in order to reduce most allergenic proteins in a greater extent before koji-sporulation, and to improve flavors of koji-fermented soybean meals, a two-stage solid state fermentation using combination of a fungal strain A. oryzae and a lactic acid bacterium Lactobacillus casei were used in this study. Effects of inoculum types, in spore or in mycelial form, on the removal of allergenic proteins and oligosaccharides were also examined. Finally, proximate analysis of fermented soybean meal was performed, and the optimal fermentation mode was suggested. Three kinds of fermentation mode were examined in the two-stage solid state fermentation. These included the combination of treating soybean meals with A. oryzae first, followed by L. casei (A-L); the one with L. casei first, followed by Asp. oryzae (L-A), and the third one with two strains being used simultaneously (A/L). Results showed β-conglycinin, acidic polypeptides subunits of glycinin, stachyose and raffinose of soybean meals were hydrolyzed by A-L 48 hr-fermentation. The 2nd stage fermentation exerted by lactic acid bacteria contributed the acidic environment to meals and facilitated the enzymatic hydrolysis by 1st stage koji. Alternatively, use of mycelial Asp. oryzae as inoculum for the initial 20 h-fermentation, followed by increasing moisture content and temperature of meals and maintained for 4 h. The results revealed that their hydrolysis effect on removal of allergenic proteins and oligosaccharides was almost similar to those by A-L mode for 48 h fermentation (use spores as inoculum). The fermentation conditions of soybean meals by A-L mode with spores or mycelial inoculum were used in the 15 kg scale fermentation. Results showed it is not facile to control the temperature of fermented meals when the tray method was used, due to the active fungal growth during fermentation. Higher temperatures of meals hurt the Asp. oryzae deeply, and led to lower enzyme production or less stability of enzymes. Although use of Asp. oryzae in spores form was common for a large scale or mass production, using mycelial Asp. oryzae as inoculum has some advantages over spore ones, yet the hydrolysis rate was the same. The removal of these thermo stable anti-nutritional factors in the 15-kg scale of soybean meals fermentation was not effective as expected, but most of allergenic proteins and flatulence-causing oligosaccharides were degraded, and the amino nitrogen content of soybean meals was also increased after fermentation.

Allergic diseases are considered to be one of the epidemics of the century, affecting approximately one-third of the general population. Classically, among the allergenic sources able to induce IgE-mediated reactions, fungi have been one of the less favored areas of study. It has been demonstrated that sensitization to fungal aeroallergens, particularly from Alternaria alternata, represents an unequivocal risk factor for the development, persistence and severity of asthma. Thus, the better understanding and management of fungal allergy, namely by improvements in the actual assessment and diagnostic approaches are needed. Regarding the assessment of fungal exposure, actual methodologies are generally laborious and time-consuming making difficult to establish or exclude a fungal contamination and potential associations with allergic disease. In terms of diagnosis, the main difficulty arises from the high number of patients that are apparently sensitized to multiple fungi in which the identification of primary cause of sensitization is complex. Because A. alternata is one of the most abundant and potent source of airborne allergens, the panel of allergenic components produced by this fungal specie, seems to be a very relevant target of study. Among the several allergens described in this mold, Alt a 1 has been demonstrated to be the most important, sensitizing approximately 80% of A. alternata allergic patients. For this reason, Alt a 1 has been used as the diagnostic marker of genuine sensitization to A. alternata. However, given the complex A. alternata sensitization data, which shows a significant level of polysensitization, due to co-sensitization and/or cross-reactivity to several other phylogenetically related and non-related molds, other allergenic A. alternata components should be studied to explain the whole broad range of reported clinical observations. In the last years, componentresolved diagnosis (CRD) has been shown to be a valuable tool to elucidate clinical observations and to differentiate between cases of co-sensitization and cross-reactivity. Nevertheless, the actual available panel of A. alternata allergens appears to not be enough for achieving an accurate diagnosis and prognosis of sensitization to this mold. Considering the above mentioned facts, the major aims of this work were, on one hand to develop an approach to specifically detect Alternaria and related species by using the A. alternata major allergen, Alt a 1, as a specie-specific molecular marker. And, on other hand, to characterize two new cross-reactive A. alternata allergens belonging to the manganesedependent superoxide dismutase (MnSOD) and serine protease (SP) protein families and to study their role in A. alternata sensitization. The strategies used to accomplish both aims made use of phylogenetic relationships among fungal species that share A. alternata allergen homologues. First, investigating conservation of the genes encoding for Alt a 1 and its homologues which allowed the design of a set of primers in the conserved immunologically relevant Alt a 1 coding sequence region. This primer set, together with a pair of primers to amplify the complete Alt a 1 encoding gene, was applied in a polymerase chain reaction (PCR)-based system. This approach yielded two rapid, sensitive and specific methodologies with high potential to be applied both for the detection of Alt a 1 and Alt a 1 homologues and for specific identification of the existence of contamination by the very close taxonomically related species A. alternata and A. tenuissima. The investigation of conservation of the genes encoding for A. alternata protein homologues, was also employed to design primers in the invariant region of fungal MnSOD and SP nucleotide sequences available in public databases. The aforementioned primers along with rapid amplification of cDNA ends (RACE) and sequencing assays allowed for the isolation of the full-length cDNA encoding for A. alternata MnSOD and SP. Both proteins were then produced as recombinant proteins in E. coli and evaluated for IgE immunoreactivity using a comprehensive panel of sera from patients clinically labeled as to be sensitized to A. alternata. Immunoblotting analysis showed that IgE antibodies from A. alternata-sensitized patients bound to recombinant A. alternata MnSOD and SP with prevalence of 11.5% (n=61) and 10.2% (n=59), respectively. These results were reported to the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub- Committee, and both proteins, respectively named Alt a 14 and Alt a 15, are currently included in the official A. alternata allergen list. By performing immunoblotting inhibition assays it was demonstrated that Alt a 14 and Alt a 15 are able to mediate IgE cross-reactivity with similar homologues allergens from other important allergenic fungal species, such as A. fumigatus and C. lunata, respectively. Furthermore, evidence of reactivity of Alt a 14 to IgE of Allergic Bronchopulmonary Aspergillosis (ABPA)-diagnosed patients was found, thus indicating that sensitization to this A. alternata allergen could play important implications in the development of ABPA. On the other hand, sensitization to Alt a 15 was shown to be restricted to apparently poly-sensitized patients and to justify some cases of sensitization to A. alternata in which there is no evidence of Alt a 1 sensitization. A homologue to Alt a 14, together with a manitol desidrogenase (MtDH), of the edible mushroom A. bisporus were identified to induce an anaphylactic shock reaction in a patient who presented a previous history of respiratory allergic symptoms associated to mold aeroallergens. These results were useful in proving that although minor allergens, A. alternata cross-reacting proteins may be the primary cause of strong allergic reactions to various other allergenic sources, such as mushrooms. Overall, in this work we successfully developed a specific and sensitive PCR method based on the amplification of regions of the gene encoding for the allergenic Alt a 1 and Alt a 1 homologues which it is intended to be applied for environmental monitoring as well as for quality and biosecurity control of food stuffs. Moreover, cloning and characterization of Alt a 14 and Alt a 15 as minor allergens of A. alternata that can trigger cross-reactive IgE response with other important and prevalent allergenic fungal species were also accomplished. The availability of these allergens as recombinant molecules suitable for application in a molecule-based diagnostic approach to fungal allergy can improve the diagnostic process prognosis of clinical manifestations and potential cross-reactivities. Furthermore, this can guide to a more effective specific immunotherapy using a single or a few allergenic molecules. Hence, this work provided valuable findings that can contribute to improving the accuracy of assessment of allergen exposure, diagnosis and management of IgE-mediated fungal diseases.
As doenças alérgicas são consideradas uma das epidemias do século XXI, afectando aproximadamente um terço da população em geral. De entre as fontes alergénicas ambientais capazes de induzir reacções de hipersensibilidade de tipo I, os fungos têm sido uma das áreas de estudo menos favorecidas. No entanto, a sensibilização a aero-alergénios de origem fúngica tem sido apontada por vários autores como um factor de risco indiscutível para o desenvolvimento, persistência e severidade de doença alérgica respiratória, nomeadamente da asma. Estes dados sugerem que a realização de estudos visando progressos, nomeadamente, nos métodos de detecção de exposição ambiental, assim como nas metodologias de diagnóstico, são necessários e cruciais para a melhor compreensão, diagnóstico e tratamento das doenças alérgicas provocadas por espécies fúngicas. Os métodos de avaliação de contaminação ambiental por fungos de importância alergológica, tradicionalmente usados em estudos epidemiológicos e aerobiológicos, têm como base a detecção e identificação de espécies por observação microscópica de características morfológicas e contagem de esporos. Na sua generalidade, a execução destes métodos é bastante demorada, complexa e requer a presença de pessoal treinado com elevados conhecimentos na área da micologia. Desta forma, a definição ou exclusão de contaminação ambiental por uma determinada espécie fúngica e a associação de exposição com um padrão de sintomatologia alérgica tem sido bastante problemática. Em termos de diagnóstico, a principal dificuldade atual resulta do grande número de doentes que estão aparentemente sensibilizados a mais do que uma espécie fúngica. Nestes casos clínicos, o diagnóstico da causa primária de sensibilização é bastante complexo. Vários estudos epidemiológicos realizados, fundamentalmente na Europa, têm descrito Alternaria alternata como a fonte de aero-alergénios de origem fúngica mais importante e com maior alergenicidade. Tais relatos sugerem que o estudo do painel de alergénios produzidos por esta espécie pode ser um alvo de estudo que pode contribuir para o avanço significativo na área de conhecimento das alergias a fungos. Entre as várias proteínas alergénicas descritas até ao momento, Alt a 1, constitui o alergénio de A. alternata mais importante, sendo responsável pela sensibilização de, pelo menos, 80% dos doentes diagnosticados como alérgicos a Alternaria. Devido a esta elevada prevalência de sensibilização, este componente alergénico tem sido correntemente utilizado como um marcador de sensibilização genuína a A. alternata, sendo referido por muitos autores como um marcador de sensibilização à família das Pleosporaceae. Nos últimos anos, o diagnóstico molecular tem sido apresentado como uma ferramenta com resultados bastante promissores para definir o perfil de sensibilização individual de cada doente alérgico e sua associação com as manifestações clínicas observadas, assim como para discriminar entre casos de cosensibilização e reatividade cruzada. Nestes estudos, a sensibilização a Alt a 1 e aos outros alergénios descritos, parece não ser suficiente para justificar todo o espectro de sintomas clínicos observado em doentes sensibilizados a A. alternata. Tendo por base estes conhecimentos, estabeleceram-se como objectivos principais deste trabalho: por um lado, desenvolver uma tecnologia por detecção específica de A. alternata e espécies taxonomicamente relacionadas, utilizando Alt a 1 como um marcador molecular espécie-específico. E por outro lado, ampliar o painel alergénico de A. alternata, por caracterização de uma superóxido dismutase dependente de manganês (MnSOD) e uma protease de serina (SP) como dois novos alergénios de A alternata e estudar o seu papel no diagnóstico e prognóstico das doenças alérgicas causadas por fungos. As estratégias usadas para a concretização destes objectivos tiveram como princípio base o estudo das relações filogenéticas das espécies fúngicas que partilham proteínas homólogas aos alergénios de A. alternata. Assim, primeiramente, o estudo de conservação dos genes de expressão de Alt a 1 e homólogos possibilitou o desenho de um par de primers para o reconhecimento da região conservada e imunologicamente relevante, ou seja, da zona que codifica para os epítopos alergénicos da molécula de Alt a 1. Foram então realizados ensaios de PCR utilizando este par de primers e um segundo par de oligonucleótidos que permite a amplificação do gene completo de Alt a 1. Desta forma, foram desenvolvidas e optimizadas duas metodologias específicas, sensíveis e rápidas: a primeira com potencial para a detecção de Alt a 1 e homólogos, independentemente da fonte alergénica que o produz e uma segunda que permitiu detetar de forma restringida as espécies filogeneticamente próximas, A. alternata e A. tenuissima. O estudo de homologia e conservação de genes que codificam para proteínas homólogas às de A. alternata, foi também aplicado no desenho de primers degenerados para a região não variável das sequências nucleotídicas codificantes de MnSODs e SPs de origem fúngica disponíveis nas bases de dados. Os cDNA completos que codificam estas proteínas foram isolados pela realização de ensaios RACE-PCR; as respectivas moléculas recombinantes foram produzidas em Escherichia coli e purificadas a partir dos corpos de inclusão. A capacidade de reconhecimento de IgE existente em soros de uma população representativa de doentes sensibilizados a A. alternata foi analisada mediante immunoblotting. Desta análise verificaram-se prevalências de sensibilização às MnSOD e SP de A. alternata recombinantes de 11.5% (n=61) e 10.2% (n=59), respetivamente. Estes resultados foram comunicados ao World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-Committee e ambas proteínas foram registadas por esta entidade como alergénios clinicamente relevantes de A. alternata, oficialmente designados de Alt a 14 e Alt a 15. Pela realização de ensaios de inibição por ELISA pôde constatar-se que os alergénios recombinantes produzidos apresentavam características imunoquímicas de reatividade para a IgE, similares às respetivas moléculas nativas existentes num extracto crude de A. alternata. Adicionalmente, Alt a 14 e Alt a 15 demonstraram um alto potencial para mediar reações de reatividade cruzada entre A. alternata e outras espécies fúngicas de importância alergológica, nomeadamente Aspergillus fumigatus e Curvularia lunata. A associação de dados clínicos com as evidências de immunorreatividade e reatividade cruzada permitiu demonstrar que a sensibilização a Alt a 14 parece ser um indicador de desenvolvimento de aspergilose broncopulmonar alérgica (ABPA). Em relação a Alt a 15, a sensibilização a esta proteína alergénica pareceu ser restrita a doentes aparentemente sensibilizados a múltiplos fungos. Além disso, esta molécula parece justificar a sensibilização a A. alternata em alguns casos nos quais a sensibilização ao marcador de sensibilização genuína a Alternaria (Alt a 1) não foi verificada. Neste trabalho de investigação foi ainda apresentado um caso clínico de um doente com história clínica prévia de sintomas respiratórios associados a alergia diagnosticada a fungos ambientais, entre os quais a A. alternata, que recentemente sofreu um choque anafilático após a ingestão de cogumelos. Os resultados obtidos pela análise da reatividade da IgE sérica da doente identificaram uma MnSOD (homóloga da Alt a 14) e uma manitol desidrogenase (MtDH) existentes num extrato da espécie de cogumelo comestível Agaricus bisporus, como as moléculas responsáveis pelo desencadeamento da reação sistémica observada. Estas demonstrações sustentam a teoria de que uma anterior sensibilização a alergénios fúngicos ambientais de reatividade cruzada, tais como os que são apresentados neste trabalho, em particular Alt a 14, pode suscitar o desenvolvimento de reações sistémicas a alimentos que partilham alergénios homólogos aos sensibilizantes primários. Em suma, neste trabalho doutoral, foi desenvolvida uma técnica de PCR específica e bastante sensível para aplicação no controlo de contaminação por espécies fúngicas produtoras de Alt a 1. Além disso, dois novos alergénios de A. alternata, Alt a 14 e Alt a 15, foram caracterizados como alergénios minor capazes de mediar fenómenos de reatividade cruzada com várias espécies fúngicas, resultando em quadros clínicos de resposta alérgica complexos. A disponibilidade destes alergénios como moléculas recombinantes com aptidão para serem aplicadas em diagnóstico molecular pode constituir uma mais valia para melhorar a precisão do diagnóstico das doenças alérgicas provocadas por fungos, através da definição de perfis de sensibilização individuais. Pelas evidências clínicas demonstradas ao longo deste trabalho pode também concluir-se que a correta identificação de sensibilização a Alt a 14 e Alt a 15, pode orientar o alergologista e o doente para a tomada de medidas de prevenção de potenciais quadros clínicos que podem ocorrer, nomeadamente, em consequência de reatividade cruzada. Este trabalho fornece ferramentas e informações úteis e valiosas que podem contribuir para um avanço significativo na avaliação de exposição a alergénios de origem fúngica, assim como no diagnóstico, prognóstico e tratamento das doenças alérgicas provocadas por espécies fúngicas.

博士
國立臺灣大學
生物化學暨分子生物學研究所
99
Part I: Although cockroaches are known to produce allergens that can cause IgE-mediated hypersensitivity reactions, including perennial rhinitis and asthma, the various cockroach allergens have not yet been fully studied. Many proteins from the German cockroach (B. germanica) show high IgE reactivity, but have never been comprehensively characterized. To identify these potential allergens, proteins was separated by 2-DE and IgE-binding proteins were analyzed by nanoLC-MS/MS or N-terminal sequencing analysis. Using a combination of proteomic techniques and bioinformatic allergen database analysis, we identified a total of ten new B. germanica IgE-binding proteins. Of these, aldolase, arginine kinase, enolase, Hsp70, triosephosphate isomerase (TIM), and vitellogenin have been reported as allergens in species other than B. germanica. Analysis of the FARRp allergen database indicated that arginine kinase, enolase, and TIM showed significant potential cross-reactivity with other related allergens. This study revealed that vitellogenin is an important novel B. germanica allergen. Personalized profiling and reactivity of IgE Abs against the panel of IgE-binding proteins varied between cockroach-allergic individuals. These findings make it possible to monitor the individual IgE reactivity profile of each patient and facilitate personalized immunotherapies for German cockroach allergy disorders. Part II: The German cockroach （B. germanica） is a well-known indoor aeroallergen, considered as one the causative agents of extrinsic bronchial asthma and frequently included in serum test panels for allergic diagnosis. Molecular characterization of allergens by recombinant DNA technology progressed rapidly during the last few years. The Bla g 9 protein, a novel allergen from B. germanica was identified by two-dimensional immunoblotting followed by the molecular cloning, and expression of this allergen as a 6 × His-tagged fusion protein in Escherichia coli. A purified Bla g 9 was obtained by anion exchange chromatography and had arginine kinase activity. It reacted with serum IgE from cockroach-allergic patients and shown high prevalence recognition. In ELISA inhibition assay, we observed that P. americana arginine kinase （Per a 9） inhibited the IgE-binding to B. germanica arginine kinase （Bla g 9）, which indicated the same IgE epitope between arginine kinase from different cockroach species. In order to identify the allergenic determinants of Bla g 9, recombinant （r）Bla g 9 and fragments were also generated, and IgE-binding epitopes were assessed by ELISA. Recombinant fragmented Bla g 9 proteins not containing 280-300 amino acid residues were significantly decrease the reactivity to sera from cockroach sensitized individuals, suggesting that this region contains the IgE-binding epitope. Despite strong IgE reactivity to rBla g 9, the pooled serum from 10 cockroach-sensitized patients did not show IgE reactivity to two synthetic peptides consisting of 15 residues covering 280-305 amino acids. These results suggest the possibility that Bla g 9 may have a conformational epitope in the C-terminal region.