Dissertations / Theses on the topic 'Venom allergy'

Ants from genus Myrmecia, subfamily Myrmeciinae, are aggressive stinging insects with medical importance in Australia, especially in the endemic areas of southeast Australia and Tasmania. Venoms of Myrmecia ants can cause allergic reactions with symptoms ranging from local to systemic. In some sensitized individuals of extreme severity, even death can occur. Two major allergens, Myr p 1 and Myr p 2 had been cloned and characterized from venom of one species, Myrmecia pilosula. The aim of this thesis was to study several aspects of the allergens from venoms of Myrmecia ants.

Orientador: Márcia Regina Brochetto Braga
Coorientador: Mário Sergio Palma
Banca: Henrique Ferreira
Banca: Ricardo de Lima Zollner
Banca: Rogilene Aparecida Prado
Resumo: A fosfolipase A1 é um dos principais alérgenos identificados no veneno do Polybia paulista (Hymenoptera: Vespidae), uma vespa social de elevada importância clínica no sudeste do Brasil. A produção recombinante deste alérgeno contribuirá com o desenvolvimento do diagnóstico molecular de alergia. Neste trabalho é descrita a produção recombinante da fosfolipase A1 de P. paulista (rPoly p 1) no sistema celular Escherichia coli. Elevados níveis da rPoly p 1 na forma insolúvel foram obtidos após expressão na bactéria. A otimização das condições de solubilização permitiu incrementar os níveis de recuperação do alérgeno recombinante. A rPoly p 1 foi purificada (99%) até homogeneidade mediante cromatografia de afinidade em coluna de Ni2+, mostrando valores de rendimento finais de 1.5 g/L de meio de cultura. A forma nativa do alérgeno (nPoly p 1) foi purificada mediante cromatografia de troca catiônica. A rPoly p 1 foi reconhecida pela IgE específica de soros de pacientes sensibilizados ao veneno de P. paulista. O uso da rPoly p 1 permite diferenciar a ocorrência de real dupla sensibilização ao veneno de vespa e formiga ou vespa e abelha da incidência de reatividade cruzada. Soros de pacientes com IgE específica ao veneno de abelha e formiga não reagiram com a rPoly p 1, enquanto que soros de camundongos sensibilizados com rPoly p 1 apresentaram reatividade cruzada exclusivamente com fosfolipases A1 (PLA1) de vespas Neotropicais ou de climas temperados. O alinhamento múltiplo do modelo... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Phospholipase A1 (PLA1) is one of the major allergens identified in the venom Polybia paulista (Hymenoptera: Vespidae), a clinically relevant social wasp from Brazil Southeast. The recombinant production of this allergen could result in the development of molecular diagnosis of allergy thus improving the outcomes of venom immunotherapy (IT). Here, we describe the heterologous production of the PLA1 from P. paulista venom in Escherichia coli. High levels of the insoluble recombinant allergen (rPoly p 1) were obtained after expression in the prokaryotic system. The downstream optimization of the solubilization process resulted in high levels of protein recovery. The rPoly p 1 was purified to homogeneity (99%) using an immobilized Ni2+ metal affinity chromatography while a single-step cation-exchange chromatography allowed the purification of native Poly p 1 (nPoly p 1) from the venom glands. Immunoblotting analyses showed the IgE-mediated recognition of the rPoly p 1 by sera from patients sensitized to P. paulista venom. The rPoly p 1 could allow the differentiation of true double sensitization to wasp/bee and wasp/ant venoms from cross-reactivity. The sera from patients with monosensitization to honey bee or fire ant venoms do not cross-reacted with the recombinant allergen. Meanwhile, the sera from rPoly p 1-sensitized mice cross-reacted with venoms of other clinically relevant wasps from Neotropical and temperate regions. The alignment of the 3-D model from rPoly p 1 with the PLA1s from some of these wasps suggested the presence of homologues epitopes as the molecular basis for the cross-reactivity. The presence of cross-reactive carbohydrates determinants (CCDs) in the venom of several Brazilian wasps, which is a major issue for understanding the incidence of cross-reactivity during diagnosis, was also analyzed. Overall, the results described .. (Complete abstract click electronic access below)
Doutor

Background: Myrmecia pilosula (the jack jumper ant, JJA) is the principal cause of ant venom anaphylaxis in Australia. Whereas honeybee and wasp venom allergy can be treated by venom immunotherapy (VIT), no such treatment is available for ant sting allergy. In addition, information on the natural history of JJA sting allergy is required to identify those most likely to benefit from immunotherapy. The main objectives of this research were to establish: (i) the prevalence, natural history and determinants of reaction severity for JJA allergy, and; (ii) the efficacy and tolerability of JJA VIT. Methods: A search of the Royal Hobart Hospital (RHH) forensic register, a random telephone survey, and a review of emergency department (ED) presentations were performed. Three hundred eighty-eight JJA allergic volunteers were assessed, including serum venom-specific IgE RAST, and then followed up for accidental stings over a 4-year period. Finally, a randomised double-blind, placebo-controlled, crossover trial of JJA VIT was performed. Laboratory parameters measured during the trial were; leukocyte stimulation index (SI), IL-4 production, IgE RAST, histamine release test (HRT), leukotriene release test (LRT) and basophil activation test (BAT). Intradermal venom skin testing (VST) was also performed at trial entry. Findings: The prevalence of JJA sting allergy was 2.7% in the Tasmanian population, compared to 1.4% for honeybee. People aged 35 or older had a greater risk of both sting allergy and hypotensive reactions. Four deaths were identified, all in adults with significant comorbidities. During follow-up, 79 (70%) of 113 accidental jack jumper stings caused systemic reactions. Only prior worst reaction severity predicted the severity of follow-up reactions, with the majority of people experiencing similar or less severe reactions when stung again. Sixty-eight otherwise healthy JJA allergic adult volunteers were enrolled in the clinical trial. Systemic reactions to therapy were recorded in 34% during VIT. Objectively defined systemic reactions to sting challenges arose in 1/35 after VIT (mild self-limiting urticaria only) versus 21/29 in the placebo group. Treatment with oxygen, intravenous adrenaline infusion and volume resuscitation was effective and well tolerated. Hypotension was always accompanied by a relative bradycardia, which was severe and treated with atropine in two patients. In the placebo group, only VST and HRT were predictive of sting challenge results. Although IgE RAST, leukocyte SI and IL-4 production, LRT and BAT all correlated well with VST, they did not predict sting challenge outcome. After successful VIT, venom-induced leukocyte IL-4 production tended to fall, whereas IgE RAST increased and a natural decline in HRT reactivity was reversed. Interpretation: VIT is highly effective in prevention of JJA sting anaphylaxis and is likely to be of most benefit to people with a history of severe systemic reactions, which usually occur in people aged over 35. Neurocardiogenic mechanisms &/or direct cardiac effects may be important factors in some anaphylaxis deaths. Systemic reactions to immunotherapy are common and require immediate access to resuscitation facilities. The HRT warrants further investigation as a test for selecting those most likely to benefit from VIT. None of the tests evaluated appear to be reliable markers of successful VIT.

Orientador: Ricardo de Lima Zollner
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: As reações alérgicas à ferroada de inseto resultam de resposta exacerbada do sistema imune, com produção de elevados níveis de anticorpos IgE alérgeno-específicos e padrão de citocinas Th2, envolvidas na diferenciação de linfócitos B específicos para aquele antígeno em células produtoras de IgE e recrutamento de células efetoras da resposta alérgica. Neste contexto, granulocitos são células efetoras importantes na fase tardia da resposta alérgica e estão envolvidos na patogênese de diferentes doenças. Eosinófilos e neutrófilos, especificamente, modulam a resposta imune por meio de diferentes mecanismos, como a secreçao de citocinas, quimiocinas e mediadores lipídicos. A IgE desempenha papel central na patogênese das doenças alérgicas, interagindo com dois receptores de membranas: alta afinidade FcsRI e baixa afinidade FcsRII (CD23). A ligação da IgE ao seu receptor em mastocitos e basófilos promove a liberação de mediadores inflamatórios, dentre eles, a histamina. A histamina além de induzir os sintomas agudos da reação alérgica, sustenta a reação inflamatória até a fase crônica, sendo estes efeitos mediados através da ativação de diferentes receptores (H1, H2, H3 e H4). Os fatores liberadores de histamina (HRF), particularmente, HRF-dependentes de IgE, induzem a liberação de histamina na fase tardia da resposta alérgica, permitindo a perpetuação dos eventos inflamatórios crônicos. Muitos estudos demonstram a eficácia da imunoterapia específica na dessensibilização e no desenvolvimento de tolerância em indivíduos com quadros graves de hipersensibilidade à ferroada de insetos, sobretudo da classe Hymenoptera. Com base nestas informações, foram objetivos do presente trabalho avaliar os efeitos modulatórios da imunoterapia sobre a expressão gênica dos receptores de histamina (H1, H2 e H4), HRF- IgE dependente e de fatores apoptóticos (Bcl-2 e BID) por RT-PCR, além da expressão gênica, através da técnica de PCR em tempo real de fatores de transcrição envolvidos na diferenciação de granulocitos como PU.1, C/EBPa, C/EBPpe GATA-1, receptores de alta (FcsRla e FcsRly) e baixa afinidade de IgE (CD23), cuja detecção protéica foi realizada por imunofluorescência e citometria de fluxo, respectivamente. Além disso, foram avaliados os níveis séricos de IgE específica, secreçao de RANTES e IL-8 nos sobrenadantes das culturas celulares e quantificação de granulocitos apoptóticos através da técnica de TÚNEL. Os granulocitos foram isolados de pacientes submetidos à imunoterapia específica ao veneno de abelha, em diferentes períodos do tratamento (Pré, 1, 3, 6, 12, 18 e 24 meses), após injeção subcutânea, e submetidas à cultura por 72 horas, com estimulo de 1 ng/mL veneno de abelha. Indivíduos não alérgicos foram estudados como grupo controle. De maneira geral, a imunoterapia específica ao veneno de abelha foi capaz de modular os elementos analisados, reduzindo significativamente a expressão dos mesmos ao final de 24 meses de tratamento. Não verificamos, apenas, modulação no número de granulocitos apoptóticos ao longo da imunoterapia. Nossos resultados inéditos fornecem informações adicionais sobre os efeitos da imunoterapia sobre granulocitos, reforçando as propriedades supressoras e tolerogênicas desta forma de tratamento
Abstract: Allergic reactions to insect stings results from a exacerbated response of the immune system, resulting in the production of high levels of allergen-specific IgE antibodies and Th2 cytokine pattern, which are involved in the differentiation process of B lymphocytes, specific for that antigen, into IgE producing cells and the recruitment of effector cells of allergic response. Eosinophils and neutrophils, specifically, modulate the immune response through different mechanisms, such as the secretion of cytokines, chemokines and lipid mediators. IgE plays a central role on allergic diseases pathogenesis, interacting with two membrane receptors: high affinity FcsRI and low affinity FcsRII (CD23). Biding of IgE with receptors on mast cells and eosinophils promotes the release of inflammatory mediators, among them, histamine. Histamine, besides inducing acute symptoms of allergic reaction, supports inflammatory response until its chronic stage; these effects are mediated through the activation of distinct receptors (H1, H2, H3 and H4). Histamine releasing factors (HRF), particularly, IgE dependent HRF, induce histamine release during the late phase of allergic response, allowing the perpetuation of chronic inflammatory events. In this context, many studies have demonstrated the efficacy of specific immunotherapy on desensitization and tolerance development in subjects with severe hypersensivity to insect stings, especially Hymenoptera. Based on all these information, the aim of the present study were to evaluate the modulating effects of immunotherapy on gene expression of histamine receptors (H1, H2 and H4), IgE dependent HRF and apoptotic factors (Bcl-2 and BID), through RT-PCR; in addition to gene expression, through real time PCR, transcriptional factors involved at granulocytes differentiation as of PU.1, C/EBPa, C/EBPp and GATA-1, and protein expression of high (FcsRIa e FcsRly)and low affinity (CD23) IgE receptors, assessed by immunofluorescence and flow cytometry, respectively. Serum levels of specific IgE were also assessed, along with RANTES and IL-8 secretion in cell culture supernatant and quantification of apoptotic granulocytes through TUNEL technique. Granulocytes were isolated from patients undergoing bee venom specific immunotherapy in different periods of treatment (Pre, 1, 3, 6, 12, 18 and 24 months), after subcutaneous injection, and cultured for 72 hours, with bee venom 1ng/ml_. Non allergic subjects were studied as control group. Overall, bee venom specific immunotherapy was able to modulate the analyzed elements, significantly reducing their expression at the end of 24 months of treatment. Modulation on the number of apoptotic granulocytes were not observed during immunotherapy. Our results provide additional information about the effects of immunotherapy over granulocytes, reinforcing the suppressor and tolerogenic properties of this treatment
Doutorado
Ciencias Basicas
Doutor em Clínica Médica

Orientador: Ricardo de Lima Zollner
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: As reações alérgicas à ferroada de inseto resultam de resposta exacerbada do sistema imune, com produção de elevados níveis de anticorpos IgE alérgeno-específicos e padrão de citocinas Th2. A diferenciação de linfócitos Th2 e a secreção de citocinas por estas células são reguladas pelo fator de transcrição GATA-3. A ligação da IgE ao seu receptor de alta afinidade (Fc?RI) em mastócitos e basófilos, promove a liberação de mediadores inflamatórios. Muitos estudos demonstram a eficácia da imunoterapia específica na dessensibilização e no desenvolvimento de tolerância em indivíduos com quadros graves de hipersensibilidade à ferroada de insetos, sobretudo da classe Hymenoptera. A histamina é o principal mediador liberado durante a resposta alérgica; através da ativação de diferentes receptores (HR1, HR2, HR3 ou HR4) as células do sistema imune podem ser tanto inibidas como estimuladas. Via HR1, a histamina estimula principalmente linfócitos Th1, enquanto inibe linfócitos Th2 via HR2. O receptor 4 desempenha papel central na diferenciação de linfócitos Th2 e também é capaz de modular a produção de citocinas. A liberação de histamina é regulada por fatores de liberação de histamina (HRF), sendo que os HRF dependentes de IgE induzem a liberação de histamina na fase tardia das reações alérgicas. Considerando-se todas essas informações, o objetivo do presente trabalho foi avaliar os efeitos modulatórios da imunoterapia na expressão gênica dos receptores de histamina (HR1, HR2 e HR4), fatores de liberação de histamina (HRF) e cadeia ? do receptor de alta afinidade de IgE, além da expressão protéica do fator de transcrição GATA-3, em células linfomononucleares de pacientes alérgicos ao veneno de abelha. As células foram isoladas de pacientes submetidos à imunoterapia, em diferentes períodos do tratamento (Pré, 1, 3, 6, 12, 18 e 24 meses), após injeção subcutânea, e submetidas à cultura por 72 horas, com estimulo de veneno de abelha a 1 ng/ml. Indivíduos não alérgicos foram estudados como grupo controle. Com objetivo de avaliar a expressão gênica foram realizadas extração de RNA, seguida de síntese de cDNA e PCR, para avaliação da expressão protéica, utilizou-se a técnica de imunofluorescência. A expressão gênica de HR1 e HR4, assim como de HRF e da cadeia gama do Fc?RI foi significativamente reduzida ao final do período analisado. O receptor 2 de histamina não apresentou alterações bem definidas após 24 meses de imunoterapia. O fator de transcrição GATA-3 apresentou diminuição significativa na expressão a nível protéico. Nossos resultados demonstram que a imunoterapia específica ao veneno de abelha foi capaz de modular elementos envolvidos na resposta imune
Abstract: Allergic disease is an abnormal response from the immune system, with high levels of allergen specific IgE antibodies and Th2 pattern of cytokines. Development of Th2 cells and the production of cytokines are regulated by transcription factor GATA-3. Binding of IgE to its high affinity receptor (Fc?RI) in mast cells and basophils induces inflammatory mediators' release. Many studies have shown the efficacy of specific immunotherapy. Histamine is an important mediator in allergy, through activation of distinct histamine receptors (HR1, HR2, HR3 or HR4) in the immune system; cells can be either stimulated or inhibited. Histamine can stimulate, by HR1 receptor, especially Th1 response, and inhibit particularly Th2 cells through HR2 activation. HR4 plays a central role in Th2 polarization and also modulates cytokine profile production. IgE-dependent histamine releasing factors (IgE-HRF) induce histamine release in late phase reaction. In regard of this information, the aim of this study was to evaluate the modulating effects of specific immunotherapy in gene expression of histamine receptors (HR1, HR2 and HR4), histamine releasing factor (HRF), in patients with allergy and the gama chain of Fc?RI, and also GATA-3 protein levels. Bee venom allergic subjects underwent specific bee venom immunotherapy (VIT) in different stages of treatment (Pre, 3, 6, 12, 18 and 24 months) were studied. Peripheral blood mononuclear cells were isolated after subcutaneous venom injection and submitted to culture for 24, 48 and 72 hours stimulated with 1ng/ml of bee venom. In parallel healthy subjects were studied as well. Total RNA extraction, followed by cDNA synthesis and PCR were used to evaluate gene expression; GATA-3 protein expression was analyzed by immunofluorescence assay. Data from all time of cell culture - 24, 48 and 72 hours - were grouped and analyzed. Gene expression from HR1 and HR4 and also HRF and ? chain of Fc?RI were significantly reduced at the end of 24 months of immunotherapy. Histamine receptor 2 didn't show well established alterations. For transcription factor GATA-3 significant decrease at protein level was observed. Together, our results indicate that bee venom specific immunotherapy was able to modulate some of the elements involved in the immune response
Mestrado
Ciencias Basicas
Mestre em Clinica Medica

Die Wespengiftallergie stellt eine typische allergische Sofortreaktion dar. Für diese IgE-vermittelten, pathologischen Immunreaktionen ist die spezifische Immuntherapie (IT) die einzige zur Zeit zur Verfügung stehende kausale Therapie. Die Wirkmechanismen sind trotz intensiver Bemühungen weiterhin nicht vollständig aufgeklärt. Als wichtigste These wird zur Zeit eine Verlagerung des pathologischen, TH2-dominierten Zytokinmilieus in Richtung "normales" TH1-Milieu diskutiert. Es wurde auch eine reduzierte Mediatorfreisetzung von Effektorzellen, eine verminderte Leukozytenproliferation, eine verminderte Endorganantwort und charakteristische Ig-Titer-Veränderungen mit initialem Anstieg und längerfristigem Abfall des sIgE und Anstieg des sIgG4 beschrieben. In der vorliegenden Arbeit wurde der Einfluß der IT auf periphere B-Zellen hinsichtlich ihrer Ig-Produktion und ihres Phänotyps untersucht. 15 Patienten mit systemischen Reaktionen nach Wespenstich, Nachweis von spezifischem IgE und positivem Hauttest, bei denen eine Schnell-Immuntherapie eingeleitet wurde, wurden vor Beginn der Therapie (Tag 1), am Tag ihrer Entlassung (Tag 6), also einen Tag, nachdem die Erhaltungsdosis von 100 µg erreicht wurde, und vor der 2. ambulanten Allergeninjektion am 26. Tag untersucht. Die Expression von CD5, CD23, CD32, CD40, CD54, CD86, CD95, HLA-I-ABC und HLA-II-DR wurde auf peripheren mononukleären Blutzellen durchflußzytometrisch bestimmt. Anti-CD19 FITC wurde als spezifischer B-Zellmarker benutzt. Die Serum-Titer des Gesamt-IgE, Wespengift-spezifischen IgE und Wespengift-spezifischen IgG4 wurden mittels ELISA bestimmt. Zur statistischen Auswertung wurde der Wilcoxontest für nicht-parametrische, verbundene Daten benutzt. Die Expression von CD54, CD5, CD32 und HLA-II-DR wurde durch die IT signifikant und die von CD23 tendentiell modifiziert. So war die Expression dieser Moleküle auf der Oberfläche peripherer B-Zellen am Tag 6 im Vergleich zum Ausgangswert vom Tag 1 reduziert. Am 26. Tag wurden wieder Werte auf der Höhe der Ausgangswerte vom Tag 1 gemessen. Dagegen veränderte sich die Expression von CD40, CD86, CD95 und HLA-I-ABC während der untersuchten Zeitpunkte nicht. Die Ig-Titer veränderten sich in der für die IT charakteristischen Weise. So stieg nach 3 Wochen der Gesamt-IgE-, sIgE- und sIgG4-Titer hochsignifikant an. Die Expression der untersuchten Oberflächenmoleküle ist als Indikator für Veränderungen der Aktivationslage und des funktionellen Status der Zellen während der IT zu interpretieren. So spricht die Reduktion der Expression von CD32, CD54 und HLA-II für eine verminderte Aktivierungslage der peripheren B-Zellen. Ferner deutet die Reduktion von CD5 und CD32 auf eine Anergie der B-Zellen hin. Durch die reduzierte Expression von CD23 und CD54 könnte die T-B-Zell-Interaktion verschlechtert werden, die für die Effektorfunktionen beider Zellen bedeutsam ist.Einen wesentlichen Beitrag zur Wirksamkeit der IT könnte auch die verminderte Expression des HLA-II leisten, da HLA-II für die Ag-Präsentation essentiell ist. In dieser Arbeit wurde gezeigt, daß die spezifische Immuntherapie einen Einfluß nicht nur auf die Ig-Produktion der B-Zellen hat, sondern auch auf deren Phänotyp. Dies könnte Hinweise auf bisher nicht bekannte Mechanismen bieten, die an der Wirksamkeit der IT beteiligt sind.
Wasp-venom allergy is a typical IgE-mediated allergic reaction. Specific immunotherapy (IT) is the only currently available causal therapy for IgE-mediated allergies. The mechanisms responsible for the efficacy of IT are still not fully understood. So far, the main focus of research has been on changes of T-helper cell (TH) cytokine production with a shift from TH2 to TH1 cytokines. Reduced mediator secretion from effector cells of allergic reactions, decreased leukocyte proliferation, lowered responsiveness of end organs and changes in immunoglobulin levels have been reported as well. The purpose of this study was to investigate the influence of IT on phenotype and Ig-production of B-lymphocytes. 15 venom allergic patients with a history of systemic reactions after a wasp sting and venom-specific skin test reactivity as well as serum IgE were investigated before VIT (day 1), one day after reaching maintenance dose of 100 µg (day 6) during inpatient rush VIT, and again on day 26 during continued outpatient maintenance therapy. Changes in the serum levels of total IgE, allergen-specific IgE (sIgE) and sIgG4 were measured by ELISA. Expression of CD5, CD23, CD32, CD40, CD54, CD86, CD95, HLA-I-ABC and HLA-II-DR on double labeled B cells was studied by flow cytometry of peripheral blood mononuclear cells. On day 6, cell surface expression of CD54, CD5, CD32 and HLA-II-DR was decreased significantly in intensity and numbers of positive cells, compared to day 1, while on day 26, expression of these molecules approached again baseline levels. Furthermore, a trend to decreased CD23 was noted on day 6. No changes were observed for CD40, CD86, CD95 and HLA-I-ABC. Levels of total IgE, sIgE and sIgG4 showed a significant increase after 26 days of VIT. These data show that initiation of rush VIT has profound effects on B-cell phenotype and Ig-production. Reduced expression of surface molecules can be interpreted as a reduction of activation status of B-cells as well as reduced ability to present antigen and to costimulate other leukocytes. B cells may thus be additional direct or indirect targets of high dose antigen therapy and contribute to the efficacy of IT.

This study describes the identification and characterization of the Schistosoma mansoni venom allergen-like (SmVAL) proteins, the SCP/TAPS gene family found within the human parasite S. mansoni. 28 SmVALs with complete SCP/TAPS domains were identified within this parasitic trematode and comparison of their predicted protein features and gene structures indicated the presence of two distinct subfamilies (group 1 and group 2). Identification of SCP/TAPS containing proteins in other platyhelminth species demonstrated that both group 1 and group 2 proteins are present throughout the phylum with several SmVAL orthologs detected in both S. japonicum and S. haematobium. While detailed protein feature and phylogenetic analysis across multiple eukaryotic species found SCP/TAPS superfamily members to be present in representative species from four of the six eukaryotic supergroups (Opisthokonta, Amoebozoa, Archaeplastida and Chromalveolata), group 2 proteins were only detected within the Kingdom Animalia. SmVAL quantitative lifecycle expression profiling throughout parasite development demonstrated distinct transcription patterns, including transcripts specifically associated with life-stages involved in definitive host invasion, transcripts restricted to life-stages involved in the invasion of the intermediate host and transcripts ubiquitously expressed. Thorough analysis of SmVAL6 transcript diversity demonstrated a high degree of alternative splicing, including patterns that were statistically significant and developmentally regulated. In summary, this study provides a comprehensive analysis of the SmVAL gene family and presents a framework for understanding diversity within the SCP/TAPS superfamily across phyla. Additionally, the determination of SmVAL gene expression suggests SCP/TAPS proteins are an important protein family associated with schistosome developmental maturation and could, therefore, be viewed as anti-schistosome vaccine and/or drug targets.

L'immunothérapie spécifique (ou désensibilisation) aux venins d'hyménoptères est un traitement qui permet de prévenir la récidive d’une anaphylaxie chez les patients allergiques au venin de guêpe ou d’abeille. Une augmentation très rapide des doses est souvent utilisée lors de la phase initiale de ce traitement dont la bonne tolérance n’est pas bien expliquée. Le but de ce travail était de décrire les changements précoces du système immunitaire pendant l’initiation de l’immunothérapie aux venins d’hyménoptères pouvant expliquer cette bonne tolérance. Nous avons inclus 29 patients traités pour une allergie au venin d’hyménoptères avec une initiation de traitement par « ultra-rush » en 3h30. Des prélèvements sanguins ont été pratiqués avant le début du traitement, à 1h30 et juste avant la dernière injection de venin. L’évolution de la tryptase sanguine a été analysée. L'activation des polynucléaires basophiles ainsi que l'expression FcεRI à leur surface ont été analysées par intensité moyenne de fluorescence par cytométrie en flux. Pour évaluer l'évolution de la réactivité des polynucléaires basophiles, un test d'activation des basophiles (TAB) a été réalisé à chaque temps. L’évolution des populations lymphocytaires T et myéloïdes a été également analysée par cytométrie en flux. Nous avons montré une diminution significative de la tryptase sérique pendant l’ultra-rush, de même qu’une diminution significative de l’activation des polynucléaires basophiles et une diminution de l’expression de FcεRI à leur surface. Etonnamment, le TAB a montré une réponse in vitro des basophiles significativement plus élevée à l'extrait de venin à la fin de « l'ultra-rush » par rapport à avant le début du traitement. Nous avons également montré une augmentation significative des cellules dendritiques et une diminution significative des lymphocytes « Natural Killer » (NK) dans le sang. Concernant les populations lymphocytaires T, nous avons montré une ugmentation significative des populations lymphocytaires T dans le sang, sauf pour les Lymphocytes T CD4+et CD8+ naïfs. En conclusion, l’augmentation des doses de venin par « ultra-rush » est bien tolérée grâce à une inhibition des polynucléaires basophiles impliquant une diminution de l’expression de FcεRI à leur surface. L'ultra-rush entraîne également des modifications précoces dans la réponse immunitaire innée et adaptative
Hymenoptera venom immunotherapy (VIT) is a treatment that prevents sting inducing anaphylaxis in allergic patient. Fast-up dosing schedule are often used at the initial phase of VIT. This fast dosing schedule well tolerated, but the mechanisms behind this good tolerance have not yet been elucidated, as well as its consequences on the rest of the immune systems. The aim of this study is to describe early immune system change during initial phase of VIT We included 29 patients undergoing VIT by 3h30 ultra-rush up dosing phase. Blood puncture was performed before the beginning of the treatment, at 1h30 and just before the last venom injection. Blood tryptase evolution was measured. Basophils phenotype and FcεRI surface expression were analyzed by flow cytometry at each step of the ultra-rush. To assess basophils responsiveness evolution, basophils activation test (BAT) was also perform. Myeloid and T lymphocytes population’s evolution were analyzed by flow cytometry. We have shown a significantly lower concentration of blood tryptase at the end of ultra-rush, and a significantly lower basophils activation and FcεRI expression. Surprisingly, BAT has shown a significantly higher in vitro response to venom extract at the end of ultra-rush. We also found significantly increase in blood dendritic cells concentration and lower blood Natural Killer (NK) Cells. We observed higher lymphocytes population in blood except for naïve CD4+ and CD8+ T cells. In conclusion, ultra-rush fast up dosing is well tolerated thanks to a basophils inhibition involving lower FcεRI surface expression. Ultra-rush also leads to early change in innate and adaptive immune response

A esquistossomose é uma doença causada por trematódeos do gênero Schistosoma. Dentre os genes identificados no transcriptoma do parasita, membros da família gênica SmVAL (Schistosoma mansoni Venom Allergen-Like) foram apontados como candidatos vacinais. SmVALs foram identificadas em secreções de cercárias e esquistossômulos cultivados in vitro, os transcritos SmVAL4 e 24 foram localizados nas glândulas acetabulares de germ ball e a proteína nativa SmVAL4 foi identificada em extrato de cercárias, indicando funções durante a penetração da pele. Já os transcritos SmVAL13 e 14 foram localizados na glândula esofágica anterior de vermes adultos, sugerindo papéis no processo de alimentação sanguínea. A imunização com as proteínas rSmVAL4, 6, 7, 13, 14 e 18 coadministradas não protegeu camundongos contra o desafio experimental, porém, observou-se uma diminuição do número de fêmeas e do número de ovos no grupo imunizado. A investigação de funções para as proteínas secretadas mostrou que a rSmVAL18 interage com plasminogênio in vitro favorecendo a invasão do hospedeiro.
Schistosomiasis is a disease caused by trematodes of the genus Schistosoma. Among the genes identified in the parasite transcriptome, members of SmVAL (Schistosoma mansoni Venom Allergen-Like) gene family were proposed as vaccine candidates. SmVALs were identified in cercariae and schistosomule secretions in vitro, the SmVAL4 and 24 transcripts were located to the germ ball acetabular glands and SmVAL4 native protein was identified in cercariae extract, indicating functions in skin penetration. On the other hand, SmVAL13 and 14 transcripts were located to the anterior esophageal gland of adult worms, suggesting roles in the blood feeding processes. Immunization with rSmVAL4, 6, 7, 13, 14 and 18 proteins co-administered did not protected mice against experimental challenge, however, there was a decrease in the number of females and the number of eggs in the immunized group. The investigation of functions for secreted proteins showed that rSmVAL18 interacts with plasminogen in vitro thus favoring the host invasion.

Yes
Hymenoptera venom allergy is a potentially life‐threatening allergic reaction following a honeybee, vespid, or ant sting. Systemic‐allergic sting reactions have been reported in up to 7.5% of adults and up to 3.4% of children. They can be mild and restricted to the skin or moderate to severe with a risk of life‐threatening anaphylaxis. Patients should carry an emergency kit containing an adrenaline autoinjector, H1‐antihistamines, and corticosteroids depending on the severity of their previous sting reaction(s). The only treatment to prevent further systemic sting reactions is venom immunotherapy. This guideline has been prepared by the European Academy of Allergy and Clinical Immunology's (EAACI) Taskforce on Venom Immunotherapy as part of the EAACI Guidelines on Allergen Immunotherapy initiative. The guideline aims to provide evidence‐based recommendations for the use of venom immunotherapy, has been informed by a formal systematic review and meta‐analysis and produced using the Appraisal of Guidelines for Research and Evaluation (AGREE II) approach. The process included representation from a range of stakeholders. Venom immunotherapy is indicated in venom‐allergic children and adults to prevent further moderate‐to‐severe systemic sting reactions. Venom immunotherapy is also recommended in adults with only generalized skin reactions as it results in significant improvements in quality of life compared to carrying an adrenaline autoinjector. This guideline aims to give practical advice on performing venom immunotherapy. Key sections cover general considerations before initiating venom immunotherapy, evidence‐based clinical recommendations, risk factors for adverse events and for relapse of systemic sting reaction, and a summary of gaps in the evidence.
European Union's Seventh Framework Programme FP7. Grant Number: 601763

No
Background The European Academy of Allergy and Clinical Immunology (EAACI) is in the process of developing the EAACI Guidelines on Allergen Immunotherapy (AIT) for the management of insect venom allergy. To inform this process, we sought to assess the effectiveness, cost‐effectiveness and safety of AIT in the management of insect venom allergy. Methods We undertook a systematic review, which involved searching 15 international biomedical databases for published and unpublished evidence. Studies were independently screened and critically appraised using established instruments. Data were descriptively summarized and, where possible, meta‐analysed. Results Our searches identified a total of 16 950 potentially eligible studies; of which, 17 satisfied our inclusion criteria. The available evidence was limited both in volume and in quality, but suggested that venom immunotherapy (VIT) could substantially reduce the risk of subsequent severe systemic sting reactions (OR = 0.08, 95% CI 0.03–0.26); meta‐analysis showed that it also improved disease‐specific quality of life (risk difference = 1.41, 95% CI 1.04–1.79). Adverse effects were experienced in both the build‐up and maintenance phases, but most were mild with no fatalities being reported. The very limited evidence found on modelling cost‐effectiveness suggested that VIT was likely to be cost‐effective in those at high risk of repeated systemic sting reactions and/or impaired quality of life. Conclusions The limited available evidence suggested that VIT is effective in reducing severe subsequent systemic sting reactions and in improving disease‐specific quality of life. VIT proved to be safe and no fatalities were recorded in the studies included in this review. The cost‐effectiveness of VIT needs to be established.
EAACI and Grant agreement no: 601763.

Myrmecia pilosula (Jack Jumper) is an endemic Australian ant whose sting is a frequent cause of insect allergy in parts of South-Eastern and South-Western Australia, causing severe anaphylaxis in approximately 3% of the population. The venom of Myrmecia pilosula ant contains IgE-binding components frequently responsible for the severe anaphylactic reactions in humans. A treatment modality based on purified M. pilosula ant venom extract has been developed by members of the Tasmanian Jack Jumper Allergy Program. The treatment, known as Jack Jumper Ant Venom Immunotherapy (JJA VIT), was proven to reduce the risk of severe anaphylaxis in sensitized patients and improve patients’ Quality of Life. However, the current treatment is associated with frequent adverse reactions and long treatment duration. As the principal Pharmacist and Quality Manager responsible for the manufacture of JJA VIT products, it is my primary interest to continuously improve the quality, safety and efficacy of this important life-saving treatment, which is uniquely Australian. My review of allergic reactions to the venom of stinging ants (as detailed in Chapter 1 and Chapter 2) illustrated the burden and impact of M. pilosula venom allergy in Australia and highlights aspects of M. pilosula venom and JJA VIT that warrant further scientific investigations, which consequently shaped the objectives and research questions of this PhD thesis. This research has two general objectives that were aimed to advance this treatment modality, and I have performed several interconnected studies to answer my research questions (Chapter 3). My first research objective was to improve the quality of the JJA VIT produced. In Chapter 4 I explored the intrinsic and extrinsic factors that could influence batch-to-batch consistency and quality of pharmaceutical grade Jack Jumper ant venom (JJAV) extracts in the form of Active Pharmaceutical Ingredients, particularly with respect to their IgE-binding components and activities. In this analysis, I found that components of the venom with molecular weight of >20 kDa are significantly affected by elevated temperature above 40°C. Notably, these venom components are capable of binding to IgE and they were of unknown identity, and their identities are revealed in Chapter 5. I analysed the proteome and allergenome of JJAV separated using a combination of various gel electrophoresis and liquid chromatography techniques. To help divulge the identity of novel JJAV components capable of binding IgE, I employed a tandem Mass Spectrometry technique. From this study, I identified 17 novel JJAV proteins, including two glycoproteins, and confirmed the presence of four known Myr p and pilosulin peptides in JJAV. Most of the newly identified IgE-binding proteins were enzymes, including phospholipase A2, hyaluronidase, arginine kinase, and dipeptidyl peptidase IV. My second research objective was to improve the safety and efficacy of JJA VIT. For this purpose, I analysed the response of subjects undergoing JJA VIT with respect to their IgE-binding recognition to JJAV components pre-treatment and I correlated this information with treatment tolerability and efficacy. I subsequently linked this clinical data with the various JJAV components identified via tandem Mass Spectrometry and report my results in Chapter 5. In this study, I established correlations between recognition of certain IgE-binding bands with JJAV-specific IgE titre by ImmunoCAP, intradermal test threshold, and treatment-related issues. Finally, driven by the relative difficulty in obtaining pharmaceutical grade JJAV extracts and the recent increase in demand to treat patients with JJAV allergy within Tasmania and interstate, I explored the safety and efficacy of treatment with low-dose JJA VIT using Advax™ adjuvant. In order to enable a clinical trial using this novel combination product, I performed fundamental pharmaceutical and immunological studies. In Chapter 6 I report the physicochemical and microbiological stability and murine immunogenicity of low-dose JJA VIT in combination with Advax adjuvant. I observed that JJA VIT formulated with Advax is both physicochemically and microbiologically stable for at least 2 days when stored at 4 and 25°C, with a trend for an increase in allergenic potency observed beyond 2 days of storage. Importantly, JJA VIT formulated with Advax significantly increased the production of JJAV-specific IgG, consistent with a JJAV antigen-sparing effect of the adjuvant, which supports the use of Advax adjuvant with JJA VIT in future clinical trials. Overall, my PhD project has advanced our knowledge on the pharmaceutical and immunological aspects of JJA VIT and provides a robust platform to enhance the quality, safety and efficacy of this life-saving treatment modality.

The aim of my thesis is the introduction of the systemic mastocytosis and discussion about results of allergen immunotherapy in patients with systemic mastocytosis and its preventive effects against recurrent anaphylactic reactions. Patients with systemic mastocytosis are more prone to severe systemic anaphylactic reactions after Hymenoptera stings than in the general insect venom allergic population patients without elevated basal tryptase. This severe reaction can result in the death of the patient, it is important to prevent it prophylactically. The medication of choice in insect venom allergic patients is hyposensibilization therapy - allergen immunotherapy, which uses venom allergens of causal Hymenoptera (honey bee, yellow jacket). The thesis aims to summarize the results obtained so far about the appropriateness of this treatment in patients with systemic mastocytosis, side-effects during VIT, optimal dosing schedule and duration of treatment in these patients.

Die Prävalenz einer Insektengiftallergie beträgt 2,8 %. Am häufigsten sind Honigbienen (Apis melliferi) und Faltenwespen (Vespula vulgaris, Vespula germanica) Auslöser einer Insektengiftallergie in Deutschland. Sie ist eine allergische Typ-I-Reaktion und durch eine Immunglobulin-E-vermittelte (IgE) Immunreaktion charakterisiert. IgE führt zur Sensibilisierung der an einer Immunreaktion beteiligten Mastzellen und basophilen Granulozyten. Durch den Zweitkontakt erfolgt die Aktivierung jener mit Degranulation von Histamin, Serinproteasen, Prostaglandinen, Leukotrienen und Zytokinen. Dies spiegelt sich in einer allergischen Reaktion mit Vasodilatation, Tachykardie, Hypotonie, Bronchokonstriktion, Pruritus, Schmerzen, Erythem oder Flush, Nausea, Vomitus und Diarrhoe wieder. Mittels Hauttests wie Prick- und Intrakutantest kann eine IgE-vermittelte Sensibilisierung auf das Insektengiftallergen nachgewiesen werden. Es werden die Serumparameter Gesamt-IgE und spezifisches IgE auf native und rekombinante Allergene des Insektengiftes gemessen. Der Basophile Aktivierungstest (BAT) kann ergänzt werden. Hierbei wird die immunologische Quantifizierung der Rezeptorenaktivität auf basophilen Granulozyten mittels Detektion der membranständigen Aktivierungsmarker CD63 und CD203c vor und nach Antigenexposition gemessen. CD63 befindet sich intragranulär gespeichert in ruhenden basophilen Granulozyten, Mastzellen, Makrophagen und Monozyten. In vitro konnte eine verstärkte Expression von CD63 v.a. allergen-induziert und FcεRI-vermittelt beobachtet werden. CD203c ist ein hochspezifischer Marker für die basophile Differenzierungslinie. Nach Allergenstimulation wird eine rasche Expression von CD203c beobachtet, welche FcεRI-vermittelt ist. Am Ausmaß der Expression von CD63 und CD203c kann die allergene Eigenschaft abgeleitet und bei Kreuzreaktivität i.R. der in-vitro-Diagnostik das auslösende Allergen identifiziert werden. Die spezifische Immuntherapie (SIT) ist die einzige kausale Therapie einer Insektengiftallergie. Pathophysiologisch wird eine immunmodulatorische Wirkung angenommen. Die Stichprovokation schätzt die Therapieeffektivität einer SIT auf Grund von Mangel an validen laborchemischen Kriterien ein. Bei Asymptomatik oder lokaler allergischer Reaktion hat das Ergebnis einen hohen prädiktiven Wert für die Verträglichkeit weiterer Stiche. Bei ausbleibender systemischer Reaktion nach 3 bis 5 Jahren SIT kann jene beendet werden, wenn SIT oder Stichprovokation ohne Nebenwirkungen vertragen wurden. Ein Feldstich ist der Stichprovokation ebenbürtig, wenn das allergieauslösende Insekt sicher identifiziert wurde. Nach SIT kommt es bei bis zu 15% der Patienten zum Verlust des Schutzes nach 5-10 Jahren (Epidemiology of Insect Venom Sensitivity | JAMA | The JAMA Network, 2017). Die Leitlinie empfiehlt daher ein Notfallset dauerhaft mitzuführen. Vor einer Stichprovokation muss eine 109 Risiko-Nutzen-Abwägung bei relevanten Nebenerkrankungen oder vorbekannter Mastozytose erfolgen. Da die Aktivität des Insektes sowie die Giftzusammensetzung in Abhängigkeit der Jahreszeit variieren, geht die Stichprovokation mit einer eingeschränkten Beurteilbarkeit einher. Auf Grund von erhöhter Angst und Arbeitsausfall für bis zu 3 Tage wird eine Provokation häufig nicht durchgeführt. Daher besteht die Notwendigkeit nach Messmethoden, welche die Therapieeffektivität einer SIT sicher beurteilen. Ziel der vorliegenden Arbeit ist den BAT bezüglich seines Nutzens als Monitoringinstrument während einer SIT zu prüfen und die Therapieeffektivität jener beurteilen zu können. Es wurden 50 Probanden während einer SIT begleitet (6 Kinder mit 4x Bienengift- und 2x Wespengiftallergie, 44 Erwachsene mit 4x Bienengift- und 40x Wespengiftallergie). Die Probanden erklärten sich zu Blutentnahmen vor der SIT und alle 6 Monate bis 3 Jahre während der SIT einverstanden. Hieraus erfolgte die Bestimmung der Serumparameter mittels des ImmunoCAP® 250 der Firma Thermo Fisher Scientific/ Phadia. Mit Hilfe des Flow CAST® Kit (FK-CCR) von der Firma Bühlmann Laboratories AG wurde die Expression der Oberflächenmarker basophiler Granulozyten CD63 und CD203c im BAT gemessen. Anhand des a2-Wertes wurde der relative Anteil an aktivierten basophilen Granulozyten [%] auf die Stimulation mit der Allergenkonzentration von 56,8 ng/ml (c2) bestimmt. Der kalkulierte c50-Wert definiert die mindest notwendige Konzentration an Allergen, um eine Aktivierung von 50% aller in der Testprobe vorliegenden basophilen Granulozyten zu induzieren. Klinische Daten wurden in halbjährlichen Visiten telefonisch, vor Ort, mittels Fragebogen und durch Einsicht in Patientenakten erhoben. Probanden wurden nach Stichprovokation oder Feldstich während SIT und Studie zu Verträglichkeit und Klinik befragt. 90,9% der Erwachsenen und 100% der Kinder boten eine allergische Reaktion Stadium II oder III nach Ring & Messmer. Anaphylaktische Reaktionen wurden nicht beschrieben oder beobachtet. Es waren 40 Erwachsene mit einer Wespengift-SIT zu verzeichnen. Für CD203c sind jene im BAT alle Responder, für CD63 waren 4 Probanden Nonresponder. Für CD63 zeigten sich für 60% steigende und für etwa 30% konstante c50-Werte i.V. der SIT. Dies spiegelt sich in fallenden a2-Werten bei etwa 60% aller Probanden dieser Gruppe wieder. Für CD203c konnten für 60-75% der Probanden steigende sowie für 20% konstante c50-Werte kalkuliert werden. Nach einem Jahr SIT boten 20%, nach zwei Jahren 32,5% und nach drei Jahren SIT 47,5% aller Probanden eine geringere Aktivierung basophiler Granulozyten. In der vorliegenden Studie konnte für 4 Testkonzentrationen (c1 = 284 ng/ml, c2 = 56,8 ng/ml, c3 =11,4 ng/ml sowie c4 = 2,27 ng/m) in allen untersuchten Studiengruppen (Kinder mit Bienengift-SIT, Erwachsene mit Bienengift-SIT, Kinder mit Wespengift-SIT und Erwachsene mit Wespengift-SIT) fallende am-Werte über die Zeit der SIT ermittelt werden. 110 Dies korrelierte mit steigenden c50-Werten über die Dauer der SIT. Die Routinetest-konzentration des Flow CAST® Kit (56,8 ng/ml) gibt hierbei die beste Diskriminierung wieder. Alle 40 Erwachsenen mit einer Wespengiftallergie beschrieben eine mildere klinische Symptomatik unter SIT. Für alle 21 Probanden, welche an einer Stichprovokation teilnahmen oder einen Feldstich erlitten, bestätigte sich dies im BAT. Anhand von Serologie oder Kinetikmessungen lässt sich in der vorliegenden Studie keine Aussage zur Immunmodulation einer SIT und ihrer Therapieeffektivität treffen. Der BAT hingegen ist ein mögliches valides Messverfahren, um die Effektivität einer SIT zu prüfen. Hierfür eignen sich bei guter Korrelation zu Klinik und Stichprovokation/ Feldstich der a2-Wert sowie der kalkulierte c50-Wert. Das Ergebnis eines BAT ist reproduzierbar. Es können Arbeitsausfall und ein erhöhtes Angstempfinden vermieden werden. Ein Routineeinsatz des BAT kann anhand der Studie noch nicht abgeleitet werden. Hausmann et. al konnten jedoch 2014 den c50-Wert als valides Monitoringinstrument der Effektivität bei guter Korrelation zur Stichprovokation belegen. Der BAT ist daher in Fällen interessant, in denen eine Stichprovokation nicht möglich ist (Kontraindikationen, Schwangerschaft, Patientenwunsch). Eine Langzeitwirkung der SIT könnte ggf. mit begleitenden BAT-Messungen geprüft werden. Damit ließe sich die Effektivität einer SIT zum Beispiel auch nach 10 oder 15 Jahren prüfen. Dann wäre eine Aussage darüber möglich, ob das Notfallset lebenslang indiziert ist. Weitere Studien sind notwendig, um die vorliegenden Ergebnisse zu stützen. Eine multizentrische Studie mit standardisiertem BAT und begleitender Stichprovokation ist hierfür Voraussetzung.:Abbildungsverzeichnis Tabellenverzeichnis Abkürzungsverzeichnis 1 Einleitung und Zielstellung 2 Hintergrund und Wissensstand 2.1. Insektengiftallergie 2.1.1. Terminologie der Allergie 2.1.2. Prävalenz der Insektengiftallergie 2.1.3. Hymenoptera - Arten, Taxonomie und Gifte 2.1.4. Immunologische Grundlagen der allergischen Reaktion 2.2. Diagnostik allergischer Reaktionen 2.2.1. Anamnese 2.2.2. Klinik 2.2.3. Hauttests 2.2.4. In-vitro-Allergiediagnostik 2.2.5. Zelluläre Testverfahren 2.3. Therapie 2.3.1. Allgemeine Maßnahmen 2.3.2. Notfalltherapie 2.3.3. Spezifische Immuntherapie (SIT) nach aktueller Leitlinie 2.3.4. Stichprovokation 3 Material und Methoden 3.1. Patientenkollektiv 3.2. Blutentnahmen im Rahmen der Studie während der SIT 3.3. Serologische Untersuchungen 3.4. Zelluläre Testverfahren 3.4.1. Basophiler Aktivierungstest (BAT) 3.4.2. Kinetikuntersuchungen 3.5. Stichprovokation und Feldstiche 3.6. Anamnestische Datenerhebung 3.7. Statistische Methoden 4 Ergebnisse 4.1. Studienpopulation 4.2. Stichereignisse 4.3. Aktivierung basophiler Granulozyten im BAT 4.3.1. Aktivierung basophiler Granulozyten im BAT - c50 und a2 4.3.2. Zeitlicher Verlauf der mittleren Aktivierung basophiler Granulozyten während SIT, Dosis-Wirkungs-Kurve 4.4. Verlauf der serologischen Messdaten 4.5. Kinetikuntersuchungen im BAT 4.6. Anamnese und Klinik 4.7. Korrelation BAT-Ergebnisse und Anamnese/ Klinik 4.8. Korrelation BAT-Ergebnisse und Stichprovokationen/ Feldstiche 5 Diskussion 5.1. Prävalenz der Insektengiftallergie und Verteilungsmuster der allergischen Reaktion nach Ring und Messmer 5.2. Diagnostik und Therapie einer Insektengiftallergie, Studienpopulation 5.3. State of the art - Lücken in Diagnostik und Therapie einer Insektengiftallergie 5.4. Alternative Methoden der Beurteilung der Effektivität einer SIT 5.5. Aktivierung basophiler Granulozyten im BAT 5.5.1 Aktivierung basophiler Granulozyten BAT - a2 und c50 5.5.2. Kinetik der Immunmodulation unter SIT 5.5.3. Interleukin-3 und Expressionskinetik der Oberflächenmarker CD63/ CD203c 5.5.4. Responder versus Nonresponder 5.5.5. Korrelation BAT-Ergebnisse und Anamnese/ Klinik 5.5.6. Korrelation BAT-Ergebnisse und Stichprovokation 5.6. Verlauf der serologischen Messdaten 5.7. Fehleranalysen 5.8. Ausblick in die zukünftige Forschung 6 Zusammenfassung 7 Literaturverzeichnis 8 Danksagung Anhang A Curriculum vitae Anhang B. Veröffentlichungen Anhang C. Anlage 1 - Eröffnung Promotionsverfahren Anhang D. Anlage 2 - Einhaltung gesetzlicher Vorgaben Anhang E. Anlage 3 - Eidesstattliche Erklärung Anhang F. Anlage 4 - Fragebogen Anhang G. Anlage 5 - Experimentelle Daten

碩士
國立彰化師範大學
生物學系
98
Abstract 　　The ant is evolved from a Hymenoptera Vespoidea. Lay ovipositor, aculeate and surrounding tissue turn to venom gland for survived. The red imported fire ant (RIFA) and tropical fire ant (TFA) are exotic species in Taiwan, they are aggressive, have a painful and allergenic sting. RIFA is distributed in the north of Taiwan at present. TFA is distributed in the south of Taiwan. Leptogenys kitteli, Odontomachus monticola and Pachycondyla javana are native in Taiwan. Workers easy to respond defensively to nest disturbance. The poison protein in the venom composition is the main cause of the allergy. Sol i 1 and Phospholipase A1 are the same poison protein composition, and the amino acid sequence has already been determined. In this studies, ant's venom glands were dissected under the microscope and we finds five kinds of ants have a lot of the cells in the filamentous gland, convoluted gland and sting apparatus. And cell is distributed on the poison sac. surface, but the Odontomachus monticola does not this discovery. It’s to be aimed at Sol i 1 amino acid sequence to synthesize antigen, and make the polyclonal antibody from rabbit. Use Western blot analysis and Liquid Chromatography –Mass Spectrometry Analysis to investigate two fire ants species in Taiwan, with three Ponerinae ants comparing the poison protein and COI sequence Phylogenesis, and proved the antibody of Sol i 1 can binding forms the similar protein. Pachycondyla javana may to cross- react with RIFA and TFA phylogeny relationship between these Hymenoptera insects and RIFA is closer, the their antigen binding site of Phospholipase A1 would be more similar. The similar of these antigen binding site may inereaoe the probability of the cross reactivity. Analyse via the IV protein composition. There are 5 base of TFA protein Sol g 1 with Sol i 1 amino acid sequence. And the sequence coverage is 20 %, so it confirmed to be similar Phospholipase A1. Perhaps the Pachycondyla javana protein Pjav02 is similar Phospholipase A1, but because pressed for self-same amino acid sequence, so undeterminable. Sol g 1 and Pjav02 can be identified by Sol i 1 antibody, because both of them have the 50 % similar amino acid sequence of the Sol i 1 compose antigen sequence. Pass the research of this thesis so the Sol i 1 antibody can screened the Phospholipase A1 for other ants species.