Klinische, epidemiologische und experimentelle Daten deuten darauf hin, dass Autoimmunkrankheiten wie z.B. rheumatoide Arthritis, multiple Sklerose oder Typ I Diabetes durch Infektionen ausgelöst oder verschlimmert werden können. Bis heute ist jedoch nicht bekannt, welche molekularen und zellulären Mechanismen den Zusammenhang zwischen Infektion und Autoimmunität vermitteln. Eine Hypothese, die diesen Zusammenhang zu erklären versucht, ist die Hypothese der molekularen Mimikry. Dieser Hypothese zufolge sind kreuzreaktive Lymphozyten, die sowohl Selbst- als auch Fremdantigene erkennen, für die Induktion von Autoimmunität verantwortlich. Die Hypothese der molekularen Mimikry erklärt die Kreuzreaktivität von Lymphozyten durch Sequenzhomologie oder Identität von Selbst- und Fremdantigenen. Wir haben diese Hypothese an zwei Modellen, der chronischen Lyme Arthritis und einem Maus Modell der multiplen Sklerose, getestet und dabei festgestellt, dass Kreuzreaktivität von Lymphozyten weitaus häufiger ist als bis vor kurzem noch vermutet wurde. Wir konnten weiterhin zeigen, dass nicht die Primärstruktur sondern definierbare strukturelle Motive die Ursache für die Kreuzerkennung von Selbst- und Fremdpeptiden sind, und das Kreuzreaktivität in den seltensten Fällen von pathogenetischer Relevanz ist. Die Vorstellung, immunologische Kreuzreaktivität zwischen einem definierten mikrobiellen Antigen und einem definierten Selbstantigen sei für die Pathogenese von Autoimmunkrankheiten verantwortlich, ist also zu einfach. Der zweite Schwerpunkt dieser Arbeit ist die immunologische Analyse eines von uns charakterisierten Th2-spezifisch exprimierten Moleküls, T1/ST2. Wir konnten zeigen, dass T1/ST2 auf Th2, nicht jedoch Th1-Zellen exprimiert wird; dass die Expression von T1/ST2 ex vivo die Lokalisation aktueller Th2-Antworten widerspiegelt; und dass T1/ST2 von funktioneller Bedeutung für die Th2 Zellen ist: Kreuzvernetzung des T1/ST2 Moleküls durch einen T1/ST2-spezifischen monoklonalen Antikörper induziert Proliferation und die Produktion von Typ 2 Zytokinen. In vivo läßt sich durch Applikation des löslichen Antikörpers gegen T1/ST2 die pathogene Th2-Immunantwort im Mausmodell von Asthma modulieren. T1/ST2 ist also ein Kandidat für die gezielte immunmodulatorische Therapie Th2-dominierter Erkrankungen wie Asthma und Allergie. / Clinical, epidemiological, and experimental data suggest that infections can sometimes trigger or exacerbate autoimmune diseases such as multiple sclerosis, type I diabetes, or rheumatoid arthritis. To date, the molecular and cellular mechanisms leading from infection to autoimmunity have not been defined. The molecular mimicry hypothesis proposes that crossreactive lymphocytes that recognize both self- and microbial antigens are key factors in the pathogenesis of autoimmunity. According to the molecular mimicry hypothesis, sequence identity or marked sequence similarity between self- and microbial antigens is the cause of such crossreactivity. We have examined the molecular mimicry hypothesis systematically in two different models: treatment-resistant Lyme arthritis and experimental autoimmune encephalitis (EAE). The major findings were: i) crossreactivity at the level of peptide recognition by T cells is far more frequent than previously expected; ii) structural criteria rather than sequence similarity determine cross-recognition; iii) immunoregulatory mechanisms normally prevent pathogenic effects mediated by crossreactive lymphocytes. Thus, the idea that crossrecognition of a defined microbial peptide and a particular self-peptide would explain autoimmunity is most likely too simple. The other major topic of this work was the immunological analysis of T1/ST2, a Th2-specific molecule that we characterized. Here, we could show that T1/ST2 is expressed on Th2 but not Th1 cells. Furthermore, T1/ST2 expression can be used to identify sites of ongoing Th2 reactions directly ex vivo. Most importantly, T1/ST2 is important for Th2 effector functions: crosslinking of T1/ST2 via a T1/ST2-specific monoclonal antibody induces proliferation and type 2-cytokine production. In vivo, administration of the soluble antibody against T1/ST2 ameliorates the immunological parameters of bronchial hyperreactivity in a murine model of asthma. Thus, T1/ST2 is a candidate target for therapeutic immunomodulation of diseases such as allergy and asthma.

Submitted by PPG Pediatria e Sa?de da Crian?a (pediatria-pg@pucrs.br) on 2018-02-16T19:08:17Z No. of bitstreams: 1 Tese Vers?o Final Tiago Fazolo 18_01_2018.pdf: 3827531 bytes, checksum: b081e8333d16cb49ba00d0df25dff485 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-02-22T17:20:04Z (GMT) No. of bitstreams: 1 Tese Vers?o Final Tiago Fazolo 18_01_2018.pdf: 3827531 bytes, checksum: b081e8333d16cb49ba00d0df25dff485 (MD5) / Made available in DSpace on 2018-02-22T17:35:15Z (GMT). No. of bitstreams: 1 Tese Vers?o Final Tiago Fazolo 18_01_2018.pdf: 3827531 bytes, checksum: b081e8333d16cb49ba00d0df25dff485 (MD5) Previous issue date: 2017-03-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Respiratory syncytial virus (RSV) is the most common etiologic agent in severe lower respiratory tract infections (LRTI) in children. RSV-associated LRTI is the main cause of bronchiolitis, pneumonia and exacerbation of asthma. This infection is responsible for the high rates of hospitalizations related to respiratory diseases worldwide, especially in children younger than 2 years. Currently, annual mortality rate due to RSV infections is worrying worldwide and is estimated at approximately two hundred thousand cases. The treatment strategies to RSV infections are limited. Ribavirin is an approved drug for use in RSV infections, but its use is limited due to adverse side-effects and risks posed to health professionals who handle it. Palivizumab is a monoclonal antibody which targets RSV F glycoprotein and its use is only indicated as a prophylactic measure. This treatment is already accepted in several countries for groups of high risk children (premature children, with chronic lung disease and with congenital heart disease). However, palivizumab has a high cost for public health and is not available in all countries. The development of an effective RSV vaccine to generate a long-lasting immunological memory response that prevents infection may be the best alternative because it will reduce high public health expenditures with antiviral drugs and monoclonal antibodies. The first attempt in the search for a vaccine against RSV was in the 1960s. This vaccine produced high levels of serum antibodies but could not protect against infection. Children who were vaccinated developed a more serious disease when later infected with the same virus. To date, there is no licensed vaccine for RSV, so the search for effective vaccines is an important focus of research. Natural RSV infections do not induce lasting protective memory, and multiple reinfections can occur lifetime. Nasal secretions from infected infants presented a small number of regulatory CD4 T cells (Treg) in peripheral blood, an increase in interleukin 4 (IL-4) production and T helper type 2 (Th2) response. Treg cells are important for controlling an exacerbated increase in immune responses. A reduction of the Tregs caused by the RSV infection generates an exacerbation of the pulmonary disease due to a Th2 response. The M209-223 RSV peptide was identified to increase IFN-? production by peptide-specific CD4 T cells after challenge with the virus. The treatment with this peptide also induced an increase in pulmonary Treg frequency in infected mice. Recently, it has also been shown that Tregs aid in the development of a T CD8+ effector response, which is crucial for the control of RSV viral load. Our hypothesis is that the RSV M209-223 peptide impacts in the differentiation of CD4 T cells, increasing the population of specific Treg, reducing lung inflammation and modulating the anti-RSV immune response. This peptide in animal model induces the differentiation of specific Treg. Our findings suggest that vaccination with M209-223 peptide results in the differentiation of specific CD4 T cells into conventional effectors and Treg cells. Vaccination with this peptide decreased the expansion of a Th2 response in animals infected with RSV, protecting both the infection site and systemically. We believe that this approach could be an important component in vaccination strategies against this virus. / O v?rus sincicial respirat?rio (VSR) ? o agente etiol?gico mais comum nas infec??es graves do trato respirat?rio inferior (TRI) em crian?as. As infec??es do TRI associada com o VSR s?o a principal causa de bronquiolite, pneumonia e exacerba??o da asma. As TRI causadas pelo VSR s?o respons?veis pelas altas taxas das hospitaliza??es relacionadas ?s doen?as respirat?rias em todo o mundo, principalmente em crian?as menores de dois anos. Atualmente a taxa de mortalidade anual mundial devido ?s infec??es pelo VSR ? preocupante e ? estimada em aproximadamente duzentas mil crian?as. As estrat?gias de tratamento contra o VSR utilizadas s?o limitadas. A ribavirina ? um f?rmaco aprovado no uso para infec??es pelo VSR, por?m sua utiliza??o ? limitada devido aos efeitos secund?rios adversos e aos riscos que representam para os profissionais da sa?de que o manipulam. O palivizumabe ? um anticorpo monoclonal dirigido contra a glicoprote?na F do v?rus e sua utiliza??o ? apenas como medida profil?tica. Este tratamento j? ? aceito em v?rios pa?ses nos grupos de crian?as de alto risco (crian?as prematuras, com doen?a pulmonar cr?nica e com cardiopatia cong?nita). Entretanto o palivizumabe tem um alto custo para sa?de p?blica, n?o sendo disponibilizado em todos os pa?ses. O desenvolvimento de uma vacina eficaz contra o VSR pode ser a melhor alternativa, pois ao gerar resposta de mem?ria duradoura que previne a infec??o e reduz, desta forma, os altos gastos com a sa?de p?blica, com os f?rmacos antivirais e com os anticorpos monoclonais. A primeira tentativa na busca de uma vacina contra o VSR foi na d?cada de 60. A vacina produzida estimulou n?veis moderadamente elevados de anticorpos no soro, mas n?o conseguiu proteger contra ? infec??o. As crian?as que foram vacinadas desenvolveram uma doen?a mais grave quando mais tarde infectados com o v?rus. At? o presente momento n?o existe nenhuma vacina licenciada para o VSR. Desta forma, a busca de vacinas eficazes constitui um importante foco de pesquisa em todo mundo. As infec??es naturais pelo VSR n?o induzem mem?ria protetora duradoura, ocorrendo m?ltiplas reinfec??es ao longo da vida. Em crian?as infectadas, observou-se um n?mero reduzido de c?lulas T CD4+ regulat?rias (Treg) no sangue perif?rico, um aumento na produ??o de interleucina 4 (IL-4) e uma resposta T helper do tipo 2 (Th2) nas secre??es nasais. As c?lulas Treg s?o importantes para controlar um aumento exagerado da resposta imunol?gica. Por este fato acredita-se que quando h? uma redu??o das Tregs causada pela infec??o do VSR ocorre uma exacerba??o da doen?a pulmonar devido uma resposta Th2. Foi identificado que o pept?deo M209-223 do VSR aumenta a produ??o de IFN-? nas c?lulas T CD4+ ap?s o desafio com VSR. O tratamento com este mesmo pept?deo tamb?m apresentou um aumento na frequencia de c?lulas Treg ap?s infec??o prim?ria pelo VSR. Recentemente tamb?m foi demonstrado que as Tregs auxiliam no desenvolvimento de uma resposta efetora T CD8+, que ? crucial para o controle da carga viral do VSR. Nossa hip?tese ? que o pept?deo M209-223 do VSR influencia na diferencia??o das c?lulas T CD4+, aumentando a popula??o de c?lulas T efetoras e regulat?rias espec?ficas, reduzindo a inflama??o pulmonar e modulando a resposta imune. Os nossos resultados sugerem que a vacina??o com pept?deo M209-223 resulta na diferencia??o de c?lulas T CD4+ espec?ficas em efetoras convencionais, que produzem mais IFN-? e em c?lulas Treg. A vacina??o com este pept?deo diminuiu a expans?o de uma resposta Th2 nos animais infectados com o VSR, protegendo da inflama??o exacerbada tanto no local da infec??o como sistemicamente. Acreditamos que esta abordagem pode constituir um componente importante nas estrat?gias de vacina??o contra este v?rus.

Experiências vivenciadas durante o período pré-natal são determinantes para a saúde do feto. A ocorrência de infecções maternas e a consequente ativação do sistema imune da mãe ocasionam uma série de alterações estruturais e funcionais no cérebro da prole, podendo predispor o indivíduo a transtornos psiquiátricos na vida pós-natal, como esquizofrenia e autismo. No entanto, estudos que investigam as alterações imunes na prole ainda são escassos na literatura. Dessa forma, o objetivo do presente estudo foi avaliar, na prole, o impacto da ativação imune materna sobre a atividade imune periférica, a resposta imune-inflamatória no sistema nervoso central (SNC), e sobre o desenvolvimento da encefalomielite autoimune experimental (EAE), o modelo murino de Esclerose Múltipla. Camundongos fêmeas prenhes receberam uma administração de salina ou lipopolissacarídeo (LPS) ao final da gestação (dia gestacional 17) e, quando adulta, a prole foi submetida a 3 experimentos principais, analisando: (1) produção de citocinas, atividade de células da periferia e desenvolvimento da hipersensibilidade do tipo tardia; (2) produção de mediadores inflamatórios por células residentes do SNC e; (3) desenvolvimento dos sintomas clínicos e da resposta imune no decorrer da EAE. Nossos resultados mostraram que a ativação imune materna provocou na prole alterações imunes periféricas, como aumento da produção de Interleucina(IL)- 12 e exacerbação da resposta de hipersensibilidade do tipo tardia; potencialização da produção das citocinas IL-1β e IL-6 em cultura primária de células residentes do SNC e; piora na severidade dos sintomas clínicos causados pela EAE, que coincide com aumento do infiltrado de linfócitos e macrófagos no SNC e ativação imuneinflamatória das células da glia. Tomados em seu conjunto, os dados do presente trabalho sugerem que condições inflamatórias durante a gestação, particularmente durante o final da gestação, podem predispor o feto a distúrbios autoimunes e neurodegenerativos na vida adulta. / Prenatal period experiences are crucial for the fetal health. The occurrence of maternal infections and subsequent maternal immune system activation cause a number of structural and functional changes in the brain of the offspring that may predispose individuals to psychiatric disorders in post-natal life, such as schizophrenia and autism. However, studies investigating offspring´s immune alterations are still scarce in the literature. The aim of this study was to evaluate, in mice offspring taken from LPS-treated dams, the impact of maternal immune activation on peripheral immune cell activity, central nervous system (CNS) inflammatory response, and development of experimental autoimmune encephalomyelitis (EAE), the murine model of multiple sclerosis. Pregnant female mice received a dose of either saline or lipopolysaccharide (LPS) during late gestation (gestational day 17), and offspring were used in three experiments to analyze: (1) cytokine production and activity by peripheral immune cells and development of delayed type hypersensitivity, (2) production of inflammatory mediators by resident CNS cells and, (3) development of clinical symptoms and immune response during the course of EAE. Our results showed that maternal immune activation resulted in immune alterations in the offspring, such as increased peripheral production of interleukin (IL) -12 and exacerbated response of delayedtype hypersensitivity; enhancement of IL-1β and IL-6 productions in primary CNS resident cells culture and; increased severity of EAE clinical symptoms, which is positively correlated with the increased lymphocytes and macrophages infiltration within the CNS and also with the immune-inflammatory activation of glial cells. Taken together, the data from this study suggest that inflammatory conditions during pregnancy, especially during the late pregnancy, may predispose the fetus to autoimmune and neurodegenerative disorders in adulthood.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-17T14:40:56Z No. of bitstreams: 1 anaclaudiacarvalhogouveia.pdf: 2060044 bytes, checksum: a2757483182ff953fdffbf86f346380b (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-06-28T14:35:33Z (GMT) No. of bitstreams: 1 anaclaudiacarvalhogouveia.pdf: 2060044 bytes, checksum: a2757483182ff953fdffbf86f346380b (MD5) / Made available in DSpace on 2016-06-28T14:35:33Z (GMT). No. of bitstreams: 1 anaclaudiacarvalhogouveia.pdf: 2060044 bytes, checksum: a2757483182ff953fdffbf86f346380b (MD5) Previous issue date: 2012-08-30 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A asma alérgica é uma doença inflamatória crônica das vias aéreas, caracterizada por uma resposta de hipersensibilidade imediata, obstrução brônquica, inflamação pulmonar e níveis elevados de IgE. A doença é mediada principalmente por uma resposta imunológica alérgeno-específica tipo Th2. Nas últimas décadas, a prevalência da asma alérgica tem aumentado significativamente, sobretudo nos países desenvolvidos. A Hipótese da Higiene atribui este aumento a uma menor exposição a determinados microrganismos durante a infância, quando o amadurecimento adequado do sistema imunológico requer estímulos que induzam respostas imunológicas de perfil Th1, fundamentais para o equilíbrio de respostas Th2 exacerbadas. Diversos trabalhos epidemiológicos parecem comprovar esta hipótese, evidenciando a existência de uma relação inversa entre o contato com microrganismos indutores de uma resposta Th1 e o desenvolvimento de asma alérgica. Paralelamente, estudos em modelos murinos constataram que o tratamento com Mycobacterium bovis BCG (BCG) reduz respostas Th2 alérgenoespecíficas. No entanto, os mecanismos pelos quais a micobactéria inibe o desenvolvimento da resposta alérgica são ainda pouco conhecidos. Este estudo avaliou o efeito da administração do BCG sobre a resposta imunológica ocorrida na alergia pulmonar em camundongos BALB/c previamente sensibilizados e desafiados com OVA. Vinte e quatro horas após o último desafio, o sangue e o lavado broncoalveolar foram coletados para análises de imunoglobulinas e contagem de células, respectivamente. Adicionalmente, os pulmões foram submetidos à análise histológica, avaliação da atividade de EPO e dosagens de citocinas e quimiocinas, assim como avaliação da expressão de CTLA-4, Foxp3 e IL-10 por citometria de fluxo. Os resultados obtidos indicam que o tratamento com BCG melhorou o processo alérgico através da redução dos principais parâmetros relacionados à resposta Th2, como o infiltrado eosinofílico pulmonar, a atividade de EPO, IL-4, IL-13, CCL11, além de IgE e IgG1 específicas anti-OVA. Por outro lado, a administração da micobactéria aumentou os níveis de IFN-γ, IL-10 e TGF-β, além das expressões de Foxp3 e CTLA-4 pelos linfócitos T CD4+. Paralelamente, houve um aumento na produção de IL-10 pelos linfócitos T CD8+. Esses dados sugerem que, além da indução de uma resposta imune Th1, a ação imunomoduladora do BCG está relacionada também à indução de mecanismos reguladores. / Atopic asthma is a chronic respiratory disease characterized by airway hyperresponsiveness, reversible airway obstruction, lung inflammation, and high levels of allergen-specific IgE, driven by allergen-specific Th2 cells. The increasing prevalence of allergic diseases, particularly in industrialized countries, has led to the hygiene hypothesis, which states that the newborn infant’s immune system is skewed toward Th2 responses and needs timely and appropriate environmental stimulus to create a balanced immune response. Supporting this hypothesis, epidemiological and experimental evidence has shown an inverse correlation between Th1-induced microbial infections and atopic asthma. Similarly, some animal studies have demonstrated that exposure to Mycobacterium tuberculosis or to environmental mycobacteria is able to protect against the development of allergic responses. However the exact mechanism underlying this inhibition still remains poorly understood. This study aimed to evaluate the ability of BCG to suppress an established allergic response in a mouse model of OVA-induced airway inflammation. To achieve this, OVA sensitized and challenged BALB/c mice were twice treated with BCG via nasal and 21 days after the first treatment, mice were rechallenged with OVA. Twenty-four hours after the last challenge, blood samples were collected to detect anti-OVA immunoglobulin isotypes, and bronchoalveolar lavage (BAL) was harvested for cell count. Additionally, lungs were collected for histological analysis, detection of EPO activity and measurement of cytokines and chemokines. The expression of CTLA-4, Foxp3 and IL-10 was also determined in lung tissue by flow cytometry. The data indicated that BCG treatment was able to inhibit an established allergic Th2-response by decreasing the allergen-induced eosinophilic inflammation, EPO activity, levels of IL-4, IL-13, CCL11 and serum levels of IgE and IgG1. Mycobacteria treatment increased lung levels of IFN-γ, IL-10 and TGF-β, and expressions of Foxp3 and CTLA-4 in CD4+T cells. Additionally, an increased production of IL-10 by CD8+ T cells was observed, even though no detectable changes in CD4+IL-10+ was noticed. Altogether, these results suggest that the mechanism underlying the down-regulatory effects of BCG on OVA-induced airway inflammation appear to be associated with the induction of both Th1 and T regulatory immune responses.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-07-25T12:36:24Z No. of bitstreams: 1 alessasinsingerbrugiolo.pdf: 28268825 bytes, checksum: 19819230b54e983b1d5aa62f656ce02c (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-09T13:50:57Z (GMT) No. of bitstreams: 1 alessasinsingerbrugiolo.pdf: 28268825 bytes, checksum: 19819230b54e983b1d5aa62f656ce02c (MD5) / Made available in DSpace on 2017-08-09T13:50:57Z (GMT). No. of bitstreams: 1 alessasinsingerbrugiolo.pdf: 28268825 bytes, checksum: 19819230b54e983b1d5aa62f656ce02c (MD5) Previous issue date: 2016-03-11 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A asma é uma doença heterogênea, caracterizada por inflamação crônica das vias aéreas, definida pela história de sintomas respiratórios e associada a limitação variável do fluxo expiratório. Considerada um grave problema de saúde, estima-se que afete mais de 300 milhões de pessoas em todo o mundo. A resposta imunológica na asma envolve predominantemente linfócitos Th2, com elevados níveis de IgE total e alérgeno-específica e eosinofilia brônquica. O tratamento visa ao controle da doença e os medicamentos utilizados atualmente apresentam efeitos colaterais sistêmicos e, em geral, não são eficazes nos casos de asma de difícil controle, sendo crucial o desenvolvimento de novos tratamentos. Echinodorus grandiflorus é uma espécie vegetal conhecida como chapéu-de-couro muito utilizada por suas propriedades anti-inflamatórias. Em estudo anterior, o tratamento com o extrato aquoso dessa espécie reduziu a resposta imune patogênica Th2 no modelo de alergia pulmonar induzida por OVA. O ácido ferúlico é um ácido fenólico amplamente presente no reino vegetal e encontrado em E. grandiflorus que apresenta atividades antioxidante e anti-inflamatória. Neste trabalho, foram investigados os efeitos do extrato aquoso de E. grandiflorus, de suas frações metanólica e residual e do ácido ferúlico na modulação da resposta imune no modelo de alergia pulmonar. O extrato, as frações e o ácido ferúlico foram analisados por cromatografia líquida de alta eficiência. Para indução da alergia pulmonar camundongos fêmeas BALB/c foram sensibilizados intraperitonealmente com OVA e alumen nos dias 0 e 14. Nos dias 21, 23, 25, 27 e 29, os animais foram desafiados com OVA 1% por 20 minutos. Os tratamentos foram administrados por gavagem, entre os dias 21 e 29 do protocolo. Foram coletados soro, lavado broncoalveolar e pulmões. Inicialmente, a análise cromatográfica revelou dois ácidos fenólicos como componentes majoritários do extrato e das frações, possivelmente relacionados às suas atividades farmacológicas, entretanto, o ácido ferúlico não foi identificado no extrato e nas frações. Todos os tratamentos foram promissores na modulação da resposta patogênica Th2, com os melhores resultados obtidos com o extrato aquoso 25 mg/kg, fração metanólica 25 mg/kg, fração residual 100 mg/kg e ácido ferúlico 25 mg/kg. Foram observadas reduções do infiltrado inflamatório pulmonar, do número de células caliciformes produtoras de muco, do número total de células e de eosinófilos, linfócitos e neutrófilos no lavado bronco-alveolar, dos níveis de TSLP, IL-25, IL-33, IL-4, IL-5, IL-13, IFN-γ, CCL5, CCL11 e CCL20 no tecido pulmonar e níveis séricos de IgE e IgG1. Juntos, esses resultados indicam que o extrato aquoso de E. grandiflorus, suas frações metanólica e residual e o ácido ferúlico, reduzem a inflamação pulmonar alérgica, atuando principalmente na fase inicial da resposta imune, reduzindo a liberação de citocinas e quimiocinas pelas células epiteliais das vias aéreas. / Asthma is a heterogeneous disease characterized by chronic airway inflammation, defined by history of respiratory symptoms and associated with variable expiratory flow limitation. Regarded as a serious health problem affecting an estimated over 300 million people worldwide. The immune response in asthma is predominantly Th2 lymphocytes, with high levels of total and allergen-specific IgE and bronchial eosinophilia. Asthma treatment is aimed at controlling the disease and the drugs used currently have systemic side effects and generally are not effective in cases of asthma are difficult to control, and it is crucial the developing new treatments. Echinodorus grandiflorus is a plant species known as “chapéu-de-couro” widely used for their anti-inflammatory properties. In previous work, the treatment with the aqueous extract of this specie reduced the pathogenic Th2 response in the OVAinduced pulmonary allergy. The ferulic acid is a phenolic acid widely present in the plant kingdom and found in E. grandiflorus which has antioxidant and antiinflammatory activities. In this study, the effect of aqueous extract of E. grandiflorus, its methanol and residual fractions, and ferulic acid in modulating the immune response of pulmonary allergy model was investigated. The aqueous extract, fractions and ferulic acid were analyzed by high-performance liquid chromatography. For induction of pulmonary allergy female BALB/c mice were intraperitoneally sensitized with OVA and alum on days 0 and 14. On days 21, 23, 25, 27 and 29, animals were challenged with OVA 1% for 20 minutes. Treatments were administered by gavage, between days 21 and 29 of protocol. The serum, bronchoalveolar lavage fluid and lungs were collected. Chromatographic analysis revealed two phenolic acids as major components of extract and fractions that possibly are related to their pharmacological activities, however ferulic acid was not identified in the extract and fractions. The treatments were promising in the modulation of pathogenic Th2 response, with the best results obtained with the aqueous extract 25 mg/kg, methanolic fraction 25 mg/kg, residual fraction 100 mg/kg and ferulic acid 25 mg/kg. The treatments reduced the inflammatory infiltration in lung tissue, the number of goblet cells in lung tissue, the number of total cells, eosinophils, lymphocytes and neutrophils in bronchoalveolar lavage, the levels of TSLP, IL-25, IL33, IL-4, IL-5, IL-13, IFN-γ, CCL5, CCL11, and CCL20 in lung tissue, and the serum levels of IgE and IgG1. Taken together, these results indicate that the aqueous extract of E. grandiflorus, methanolic and residual fractions, and ferulic acid reduced the allergic pulmonary inflammation, act primarily in the early phase of the immune response, reducing the release of cytokines and chemokines by airways epithelial cells.

<p>Polyacryl starch microparticles have been developed as an oral vaccine adjuvant capable of inducing strong local and systemic immune responses in mice. In this thesis, the starch microparticles were studied in order to increase basic understanding of their function. In particular, the thesis addressed aspects of the uptake of the particles and their presentation to the immune system after different routes of administration, in correlation with the differentiation of the induced immune response.</p><p>When using human serum albumin as a model antigen conjugated to the microparticles, it was found that the route of administration and the use of different combinations of routes, parenteral or oral, affect the profile (Th1/Th2 balance) of the induced immune response. It was also found that oral boosters are needed for the development of a local s-IgA response. </p><p>Ligated mouse intestinal loops in combination with confocal laser-scanning microscopy demonstrated that the uptake of the particles by the intestinal mucosa takes place over the follicle-associated epithelium (FAE) that covers the Peyer’s patches. The particles are also taken up in the villus epithelium when conjugated with rCTB, a ligand to the GM1 receptor. This qualitative difference in uptake did not affect the induced immune response. Thus, the addition of rCTB to the microparticles did not improve or influence the profile of the immune response. Chronic stress, known to alter the barrier function of the FAE, increased the cellular response but did not affect the humoral immune response. </p><p>Despite positive results in rodents, the particles were not able to boost a humoral immune response in man when tested with diphtheria toxin-cross reacting material (CRM197). Possible methods of improving the adjuvant effect in man are discussed.</p>

Polyacryl starch microparticles have been developed as an oral vaccine adjuvant capable of inducing strong local and systemic immune responses in mice. In this thesis, the starch microparticles were studied in order to increase basic understanding of their function. In particular, the thesis addressed aspects of the uptake of the particles and their presentation to the immune system after different routes of administration, in correlation with the differentiation of the induced immune response. When using human serum albumin as a model antigen conjugated to the microparticles, it was found that the route of administration and the use of different combinations of routes, parenteral or oral, affect the profile (Th1/Th2 balance) of the induced immune response. It was also found that oral boosters are needed for the development of a local s-IgA response. Ligated mouse intestinal loops in combination with confocal laser-scanning microscopy demonstrated that the uptake of the particles by the intestinal mucosa takes place over the follicle-associated epithelium (FAE) that covers the Peyer’s patches. The particles are also taken up in the villus epithelium when conjugated with rCTB, a ligand to the GM1 receptor. This qualitative difference in uptake did not affect the induced immune response. Thus, the addition of rCTB to the microparticles did not improve or influence the profile of the immune response. Chronic stress, known to alter the barrier function of the FAE, increased the cellular response but did not affect the humoral immune response. Despite positive results in rodents, the particles were not able to boost a humoral immune response in man when tested with diphtheria toxin-cross reacting material (CRM197). Possible methods of improving the adjuvant effect in man are discussed.

The host immune response to parasitic helminths is usually characterized by a Th2 phenotype. As the Jagged/Notch pathway has been implicated in driving Th2 development, it was hypothesized that host macrophages and dendritic cells (DCs) could detect helminth products and mount an appropriate response via this pathway. Schistosoma mansoni soluble egg antigen (SEA) rapidly up-regulated expression of the Notch ligand, Jagged 1, in both mouse and human macrophages, as well as in conventional mouse DCs. Other factors associated with Th cell development, including the Th1-promoting factor IL-12 p40, as well as another potential Th2-promoting factor, interleukin (IL)-33, were not transcriptionally responsive to SEA in these same cell types, thus indicating the selectivity of the response. Inducible gene expression was modified by the presence of the macrophage growth factor colony-stimulating factor (CSF)-1, which inhibited Jagged 1 induction by SEA and lipopolysaccharide (LPS), but enhanced LPS-induced IL-12p40 expression. Despite the observation that SEA upregulated Jagged 1 in both macrophages and DCs, only SEA-pulsed DCs promoted IL-4 production upon T-cell activation, suggesting that Jagged 1 induction alone is insufficient for instructing Th2 development. A recombinant form of the extracellular region of Jagged 1 did, however, enhance IFN-γ production in splenocytes, thus implying that the rapid induction of Jagged 1 in macrophages and DCs can regulate T cell responses. A potential role for SEA-induced Jagged 1 in autocrine responses in macrophages was also investigated through studies with recombinant extracellular Jagged 1, as well as ectopic expression of Jagged 1 in macrophages. A comparison of the responses initiated in macrophages by SEA and the bacterial endotoxin lipopolysaccharide (LPS) revealed common activation of extracellular signal regulated kinase-1/2 (ERK-1/2) and p38 phosphorylation. However, only LPS triggered IκB degradation, phosphorylation of c-Jun N-terminal kinase (JNK) and phosphorylation of Tyr701 of signal transducer and activator of transcription 1 (STAT1). SEA robustly activated signalling in HEK293 cells expressing either Toll-like receptor 2 (TLR2) or TLR4/MD2, as well as variably in cells expressing TLR3. Jagged 1 upregulation by SEA was not abrogated in TLR4 knockout macrophages, in contrast to the LPS response. Pharmacological inhibition of the ERK-1/2 pathway impaired both SEA- and LPS-inducible Jagged 1 expression in macrophages. In conclusion, the data within this thesis suggests that Jagged 1 is an ERK-dependent target of TLR signalling that has a macrophage-specific function in the response to SEA.

[Truncated abstract] The last few decades have witnessed an increase in the prevalence, morbidity and economic burden associated with asthma and allergic disease. This rising incidence cannot be completely explained by changes in genetic factors or by improvements in diagnostic procedures. Environmental factors, particularly those associated with a westernised lifestyle, are considered to be involved in this increase. In the late 1980’s Strachan was the first to link environmental factors with allergic disease, this theory became to be known as the ‘hygiene hypothesis’. This hypothesis links the “cleaner” more “healthy ” environment we now live in, with an increased risk of developing allergic disease. This effect is highlighted by studies linking farm and animal exposure (rich in microbial compounds) during early life with a decrease in allergic disease. Since then numerous studies have been undertaken to ascertain the factors present in the microbe rich environment, which elicit this protective effect. Many studies have revolved around endotoxin, however microbial components (mainly from Gram-positive bacteria) which signal through Toll-like receptor 2 (TLR2), have also shown that they can alter the allergic immune response. In mice models TLR2 has been shown to both exacerbate and inhibit allergic disease. The above research highlights the need for further studies into the effect of TLR2 ligands, and to define the mechanisms by which they exert their effects in human allergic disease. These mechanisms will be relevant to understanding the pathogenesis of allergy, but also might provide novel ways to treat allergy. The aims of the study outlined in this thesis were to determine whether in vitro exposure to TLR2 ligands could modify the established immune response to house dust mite allergen (HDM), and to examine the mechanisms by which this occurs. ... The addition of glucocorticoids to LTA enhanced the ability of this TLR2 ligand to inhibit IL-5 and IL-13 production by HDM-activated blood mononuclear cells. In conclusion, this study shows that TLR2 ligands have the ability to inhibit the Th2 response to mite allergen in previously sensitized individuals by an as yet unknown mechanism. However the findings described herein do provide an impetus for future studies designed to uncover novel mechanisms by which allergic responses can be ameliorated, and may open new treatment modalities.

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 100 pages. Includes Vita. Includes bibliographical references.