Submitted by FERNANDO CHRISTIANO GABRIEL MORELLI null (fcgmorelli@yahoo.com.br) on 2016-02-12T17:49:35Z No. of bitstreams: 1 Fernando - Tese versão final Repositório UNESP.pdf: 1900802 bytes, checksum: a8c9044a1f5a46e203d3432398fc0a07 (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-02-15T11:32:07Z (GMT) No. of bitstreams: 1 morelli_fcg_dr_araca.pdf: 1900802 bytes, checksum: a8c9044a1f5a46e203d3432398fc0a07 (MD5) / Made available in DSpace on 2016-02-15T11:32:07Z (GMT). No. of bitstreams: 1 morelli_fcg_dr_araca.pdf: 1900802 bytes, checksum: a8c9044a1f5a46e203d3432398fc0a07 (MD5) Previous issue date: 2016-02-01 / Erosões e úlceras são achados comuns no abomaso e causam preocupação econômica nos mais variados sistemas de produção de gado. Muitos fatores podem predispor ao aparecimento de úlceras e acúmulo de gases no abomaso, incluindo alimentos grosseiros, estresse ambiental, deficiências de vitaminas e minerais e infecções bacterianas. Essas úlceras podem ser subclínicas, sendo descobertas nas necropsias ou após o abate do animal, ou levarem à redução da motilidade do órgão, prejudicando o fluxo do seu conteúdo e causando transtornos digestivos graves e até ao aparecimento de síndromes semelhantes à indigestão vagal. Existem informações a respeito da resposta do sistema imune na maior parte das mucosas do trato gastrintestinal de não-ruminantes e ruminantes, porém são raras a respeito do abomaso. Os objetivos desse estudo foram detectar os níveis de citocinas (IL-17A, IFN-γ, TNF-α, IL-10, IL-6, IL-4, IL-2) no conteúdo abomasal em bovinos de corte, determinar o perfil Th1 ou Th2 dessas citocinas em animais com úlceras de grau 1 e 2 na região cárdica abomasal e comparar esses valores com os níveis de citocinas de animais sem úlceras (controle), em amostras colhidas em abatedouro, para auxiliar na compreensão da fisiopatologia do processo inflamatório local. A avaliação macroscópica e a classificação das úlceras foi realizada por meio de exames visual e histológico em amostras de tecidos da parede da região cárdica abomasal ulcerada. Os níveis de citocinas produzidas do líquido abomasal dos animais com ou sem úlceras foram avaliados por citometria de fluxo (método Cytometric Bead Array). As citocinas citadas foram detectadas no líquido do abomaso dos bovinos. Não houve diferença na liberação das citocinas entre os grupos com úlceras e o grupo sem úlcera, indicando um equilíbrio entre perfis Th1 e Th2 da resposta inflamatória. / Erosions and ulcers are common findings in the abomasum and cause economic concern in several livestock production systems. Many factors may predispose to ulcers and bloat in the abomasum, including roughage, environmental stress, deficiencies of vitamins and minerals and bacterial infections. These ulcers may be subclinical and are found during necropsy or after slaughter, or lead to reduction of abomasal motility, hindering the flow of your content and causing serious digestive disorders and even the appearance of syndromes similar to vagal indigestion. There are some studies evaluating the immune system response in most of the mucous membranes of the gastrointestinal tract of non-ruminants and ruminants, but rarely related to the abomasum. The aims of this study were to investigate the levels of cytokines (IL-17A, IFN-γ, TNF-α, IL-10, IL-6, IL-4, IL-2) in the abomasal fluid of beef cattle, to determine the Th1 or Th2 profile of these cytokines in animals with types 1 or 2 ulcers located in the abomasal cardic region and to compare these levels with those of animals without ulcers (controls), in samples collected in an abbatoir, to help to the understand the pathophysiology of the local inflammatory process. Ulcers from the abomasal cardic region were macroscopicaly evaluated, then classified by histology. Cytokine levels in the abomasal fluid from animals with or without ulcers were evaluated by flow cytometry (Cytometric Bead Array). Cytokines were detected in the abomasum fluid of cattle. There was no difference in the release of cytokines between groups, indicating a balance between Th1 and Th2 profiles of the inflammatory response.

Orientador: Juliana Regina Peiró / Banca: Lina Maria Wehrle Gomide / Banca:Fernanda Bovino / Banca: José Paes de Oliveira Filho / Banca:Glenda Nicioli da Silva / Resumo: Erosões e úlceras são achados comuns no abomaso e causam preocupação econômica nos mais variados sistemas de produção de gado. Muitos fatores podem predispor ao aparecimento de úlceras e acúmulo de gases no abomaso, incluindo alimentos grosseiros, estresse ambiental, deficiências de vitaminas e minerais e infecções bacterianas. Essas úlceras podem ser subclínicas, sendo descobertas nas necropsias ou após o abate do animal, ou levarem à redução da motilidade do órgão, prejudicando o fluxo do seu conteúdo e causando transtornos digestivos graves e até ao aparecimento de síndromes semelhantes à indigestão vagal. Existem informações a respeito da resposta do sistema imune na maior parte das mucosas do trato gastrintestinal de não-ruminantes e ruminantes, porém são raras a respeito do abomaso. Os objetivos desse estudo foram detectar os níveis de citocinas (IL-17A, IFN-γ, TNF-α, IL-10, IL-6, IL-4, IL-2) no conteúdo abomasal em bovinos de corte, determinar o perfil Th1 ou Th2 dessas citocinas em animais com úlceras de grau 1 e 2 na região cárdica abomasal e comparar esses valores com os níveis de citocinas de animais sem úlceras (controle), em amostras colhidas em abatedouro, para auxiliar na compreensão da fisiopatologia do processo inflamatório local. A avaliação macroscópica e a classificação das úlceras foi realizada por meio de exames visual e histológico em amostras de tecidos da parede da região cárdica abomasal ulcerada. Os níveis de citocinas produzidas do líquido abomasal... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Erosions and ulcers are common findings in the abomasum and cause economic concern in several livestock production systems. Many factors may predispose to ulcers and bloat in the abomasum, including roughage, environmental stress, deficiencies of vitamins and minerals and bacterial infections. These ulcers may be subclinical and are found during necropsy or after slaughter, or lead to reduction of abomasal motility, hindering the flow of your content and causing serious digestive disorders and even the appearance of syndromes similar to vagal indigestion. There are some studies evaluating the immune system response in most of the mucous membranes of the gastrointestinal tract of non-ruminants and ruminants, but rarely related to the abomasum. The aims of this study were to investigate the levels of cytokines (IL-17A, IFN-γ, TNF-α, IL-10, IL-6, IL-4, IL-2) in the abomasal fluid of beef cattle, to determine the Th1 or Th2 profile of these cytokines in animals with types 1 or 2 ulcers located in the abomasal cardic region and to compare these levels with those of animals without ulcers (controls), in samples collected in an abbatoir, to help to the understand the pathophysiology of the local inflammatory process. Ulcers from the abomasal cardic region were macroscopicaly evaluated, then classified by histology. Cytokine levels in the abomasal fluid from animals with or without ulcers were evaluated by flow cytometry (Cytometric Bead Array). Cytokines were detected in the abomasu... (Complete abstract click electronic access below) / Doutor

A doença renal crônica é uma doença mediada pelo sistema imune e caracterizada por fibrose. Camundongos deficientes em TLR2, TLR4, MyD88 e NLRP3 se mostraram protegidos frente ao dano renal e à deposição de colágeno após serem submetidos à obstrução unilateral do ureter (UUO). Além disso, os camundongos protegidos exibiram menor produção de citocinas relacionadas com um perfil imune Th2 e apresentaram menor acúmulo de macrófagos do subtipo M2. Inicialmente, creditamos aos macrófagos M2 o papel de macrófagos formadores de fibrose uma vez que tal subpopulação é encontrada em maior número aos sete dias após a UUO em animais WT, porém, vimos que os personagens centrais no desenvolvimento da fibrose são macrófagos M1, encontrados no início da lesão renal. Também vimos que o ácido úrico é a molécula capaz de induzir a troca de fenótipo de M1 para M2 ao longo da UUO, além de ser capaz de ativar a via do inflamassoma. O ácido úrico solúvel é liberado em um contexto de hipóxia e ativa o complexo do inflamassoma NLRP3 por mecanismos diferentes, mas complementares. / Chronic kidney disease is an immune mediated disease characterized by fibrosis development. The damaged tissue releases molecules such as soluble uric acid resulting from the degradation of extracellular matrix or dead cells, which activate TLR and NLR, leading to the translocation of MyD88 in many cell types. This modulation of the immune system interferes with the activation of different subtypes of macrophages and activity of CD4+ T cells, with the Th1/Th2 paradigm as a possible effector mechanism of fibrosis. TLR2, TLR4, MyD88, and NLRP3 deficient mice are protected against renal damage and collagen deposition after being submitted to unilateral ureteral obstruction (UUO), when compared to wild type animals. Moreover, protected mice exhibited less production of Th2 related cytokines and reduced accumulation of M2 macrophages. Initially, we hypothesized M2 macrophages are responsible for fibrosis formation since this subset is found in greater numbers seven days after UUO in WT mice, however, we observed the central characters on the development of fibrosis are M1 macrophages found in the onset of renal injury. These data were confirmed by the injection of Stat6 KO M1 macrophages into Rag deficient mice previously depleted of macrophages and subjected to UUO, in which we observed higher proteinuria and increased collagen deposition. We also observed that uric acid is able to induce the exchange of phenotype from M1 to M2 along the UUO, besides being able to activate the inflammasome pathway. The soluble uric acid is released in the context of hypoxia and activates the NLRP3 inflammasome complex by different, but complementary mechanisms. Therefore, the renal damage releases soluble uric acid, which signals via innate immune receptors, and the damage brings as a consequence the deposition of proteins in the renal interstitium, culminating in fibrosis.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-20T19:05:22Z No. of bitstreams: 1 alessasinsingerbrugiolo.pdf: 7024795 bytes, checksum: 9af11871ea610d9074a4267774a8aea9 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-26T20:25:08Z (GMT) No. of bitstreams: 1 alessasinsingerbrugiolo.pdf: 7024795 bytes, checksum: 9af11871ea610d9074a4267774a8aea9 (MD5) / Made available in DSpace on 2016-09-26T20:25:08Z (GMT). No. of bitstreams: 1 alessasinsingerbrugiolo.pdf: 7024795 bytes, checksum: 9af11871ea610d9074a4267774a8aea9 (MD5) Previous issue date: 2010-03-11 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A asma é uma doença caracterizada por obstrução intermitente das vias aéreas e inflamação crônica e afeta cerca de 300 milhões de pessoas em todo o mundo. É considerada um grave problema de saúde, trazendo consigo um grande encargo econômico. A resposta imunológica na asma é predominantemente de linfócitos Th2, com altos níveis de IgE total e alérgeno-específica e eosinofilia brônquica. O tratamento da asma 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. Neste trabalho, foi investigado o efeito do extrato aquoso de Echinodorus grandiflorus, planta utilizada pela população por suas propriedades diurética e anti-inflamatória, no modelo de alergia pulmonar no qual camundongos BALB/c foram sensibilizados e desafiados com OVA. O tratamento com o extrato por via oral reduziu marcadamente o número de neutrófilos, linfócitos, macrófagos e eosinófilos no lavado bronco-alveolar (LBA), a atividade da peroxidase eosinofílica (EPO) no tecido pulmonar, os níveis de IgE específica para OVA no soro, os níveis de CCL11 e a expressão gênica de IL-4 e IL-13 no tecido pulmonar e a expressão de CD25 em células do LBA. Entretanto, o tratamento não alterou o infiltrado inflamatório e o número de eosinófilos no tecido pulmonar, os níveis das quimiocinas CCL2 e CCL5, das citocinas IL-4, IL-13, IL-10 e IL-17A e a expressão gênica de Foxp3 no tecido pulmonar e a expressão de CD28 e CD152 nas células do LBA. Esses resultados sugerem que o extrato aquoso de E. grandiflorus é capaz de modular alguns aspectos da inflamação pulmonar alérgica e pode ser útil no tratamento da asma. / Asthma is a disease characterized by intermittent obstruction of the airways and chronic inflammation and affects about 300 million people around the world. It is considered a serious health problem, bringing a large economic burden. 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. In this study we investigated the effect of aqueous extract of Echinodorus grandiflorus, plant used in folk medicine for its diuretic and anti-inflammatory properties, in the model of lung allergy in which BALB/c mice were sensitized and challenged with OVA. Oral treatment with the extract markedly reduced the number of neutrophils, lymphocytes, macrophages and eosinophils in bronchoalveolar lavage (BAL), the activity of eosinophil peroxidase (EPO) in lung tissue, levels of OVA-specific IgE in serum, levels of CCL11 in lung tissue, the gene expression of IL-4 and IL-13 in lung tissue and the expression of CD25 on BAL cells. However, the treatment did not alter the inflammatory infiltrate and the number of eosinophils in lung tissue, levels of chemokines CCL2 and CCL5, levels of cytokines IL-4, IL-13, IL-10 and IL-17A and gene expression of Foxp3 in the lung tissue and the expression of CD28 and CD152 on BAL cells. These results suggest that the aqueous extract of E. grandiflorus is able to modulate some aspects of allergic pulmonary inflammation and may be useful in the treatment of asthma.

Il est maintenant acquis que le système immunitaire occupe une place importante dans l’évolution les cancers (Hanahan et al., 2011). La compréhension actuelle de la réponse immunitaire adaptative en fait une cible de choix dans ce contexte. Il est apparu que les lymphocytes T CD4+, acteurs majeurs de la composante adaptative du système immunitaire, présentent des actions distinctes sur le contrôle de la croissance tumorale. Ainsi, les lymphocytes Th2 et Tfh, tous deux activateurs des lymphocytes B dans des conditions de lutte contre des infections pathogéniques, présentent des rôles ambivalents dans un contexte de cancer. En effet, de nombreuses études montrent que la présence de Th2 est corrélée à une progression de la maladie (notamment via l’action de l’IL-4 qu’ils sécrètent) (Koller et al., 2010 ; Roca et al., 2012) alors que les Tfh, seraient plutôt associés à un bon pronostic pour les patients (Gu-Trantien et al., 2013, 2017).Nos investigations actuelles nous ont permis de mettre en avant une caractéristique nouvelle de la biologie des lymphocytes Tfh. En effet, les Tfh expriment l’Hemathopoietic Prostaglandin D2 synthase (HPGDS). Cette enzyme de la voie de biosynthèse des eicosanoïdes est responsable de la production de Prostaglandine D2 (PGD2). Plusieurs travaux montrent que les cellules Th2 expriment le récepteur CRTH2, spécifique de la PGD2. Cette molécule agit sur ces cellules comme chemoattractant et permet également une augmentation de leur production cytokinique. Ainsi, nous posons l’hypothèse d’une communication potentielle entre lymphocytes Tfh et Th2 via la PGD2. Le projet présenté ici est alors axé sur la compréhension des mécanismes moléculaires et cellulaires sous-jacent à cette communication au sein des deux sous-types ainsi que sur son impact dans un contexte de cancer. Ce projet ayant également pour but de mettre en avant la PGD2 comme nouvelle cible thérapeutique dans le cancer. / It is now accepted that the immune system plays a critical role in cancers evolution (Hanahan et al., 2011). In this context, current understanding of the adaptive immune response made it a prime target. T CD4 cells, the main players of the adaptive immune system component, are known to possess distinct roles in the control of tumour growth. Thereby, Th2 and Tfh cells, both known to activate B cells in pathogenic infections, present antagonistic roles in cancer. Indeed, numerous studies demonstrate that Th2 cells are correlated with disease progression (especially via IL-4 secretion) (Koller et al., 2010 ; Roca et al., 2012), whereas Tfh cells are associated with a good prognosis for the patients (Gu-Trantien et al., 2013, 2017) despite the actual limited amount of available data.Our current researches highlighted a new property of the biology of Tfh cells. We found that Tfh cells are able to express the Hemathopoietic Prostaglandin D2 synthase (HPGDS), an eicosanoid pathway enzyme involved in Prostaglandin D2 (PGD2) production. Moreover, different studies revealed that Th2 cells expressed CRTH2, the specific PGD2 receptor. PGD2 is known as a chemoattractant molecule for Th2 cells and lead to the increase of their cytokine secretion. We hypothesized that Tfh communicate with Th2 cells via PGD2 signalling. The present project is focused on the understanding of the underlying molecular and cellular mechanisms involved in this cross-talk and their impact in cancer. The last aim of this work is to favor the development of PGD2 as a new cancer therapeutic target.

No presente trabalho investigamos a relevância dos mediadores lipídicos (Leucotrienos) gerados pela enzima 5-Lipoxigenase (5-LO) na susceptibilidade ou resistência de camundongos ao Melanoma experimental com células tumorais B16-F0, utilizando como modelo camundongos produtores de leucotrienos (129_WT) e camundongos geneticamente deficientes \"knockout\" de 5-LO (129_5-LO KO). Primeiramente, verificamos que leucócitos peritoneais provenientes de animais WT implantados com melanoma B16-F0, apresentam aumento da expressão do gene para 5-LO (Alox5). Nossos resultados mostram que animais 5-LO KO, deficientes de 5-LO são mais eficientes no controle da progressão do tumor e apresentam significativo aumento na sobrevivência, quando comparados a animais WT, produtores de 5-LO. A nossa análise do perfil imunológico em células esplênicas indicam que a maior eficiência dos camundongos 5-LO KO no controle do crescimento de células tumorais B16-F0 estariam associados à presença numérica aumentada de neutrófilos (Gr-1+), células apresentadoras de antígeno (I-Ab+) majoritariamente CD19+CD80+ e esplenócitos capacitados para produção de altos níveis de citocinas pró-inflamatórias/efetoras como a IL-6, TNF?, IFN-? e baixos níveis de citocinas regulatórias como IL-10, 15 dias pós-implantação do tumor; a rápida geração da resposta imune polarizada para produção elevada de citocinas Th1 (IFN-?), mas não, citocinas Th2 (IL-10) e presença de maiores números de linfócitos T CD4+ e CD8+ efetoras, expressando o fenótipo CD44high ou CD44highCD62Llow. Ainda, verificamos que a deficiência genética da 5-LO ou a inibição da 5-LO pelo MK886 em células LAK, aumenta significativamente sua atividade citotóxica em células do melanoma B16-F0. Nossos resultados em conjunto, indicam que leucotrienos gerados pela enzima 5-LO, modulam negativamente a geração de resposta imune protetora em camundongos para o Melanoma B16-F0. / In the present work we examine the contribution of 5-lipoxigenase-derived lipid mediators during experimental melanoma (B16-F0) in 5-LO gene knockout (KO) mice and wild-type (WT) mice. The 5-LO KO mice presented delayed tumor growth, lesser tumor volume and delayed mortality. The greater resistance of 5-LO KO mice correlated with the following: High splenic Gr-1+ leukocytes counts, High and dominant presence of splenic IAb+CD19+CD80+ antigen-presenting cells counts and capacity of spleen cell to produce high levels of IL-6, TNF-?, IFN-? and lower levels of IL-10 early after tumor cells implantation; rapid T-cell polarization to secret high quantities of Th1 type cytokine IFN-? and low quantities of Th2 type cytokine IL-10; rapid generation and greater numbers of CD4+ and CD8+ activated T cells expressing CD45RB or CD44 markers; and also CD4+ and CD8+ CD44high or CD44highCD62Llow effector T cells. Herein, IL-2 induced splenic LAK cells from 5-LO KO mice, compared with splenic LAK cells from WT mice, were more efficient at killing B16-F0 melanoma cells. The increased B16-F0 melanoma cells killing activity were also found by treatment of splenic LAK cells from WT mice with a 5-LO activity inhibitor, MK886. Our findings suggest that 5-LO deficiency altered antigen-presenting cells profile, IFN-? and IL-10 production during skin cancer disease favoring the generation of protective immune responses and also provide evidence that 5-LO-derived LTs negatively affect the host survival during experimental B16-F0 melanoma.

As relações bidirecionais entre o Sistema Nervoso e o Sistema Imune são relevantes para a manutenção da homeostase do organismo. Estudos realizados em nosso laboratório mostraram que 14 dias de coabitação com um conspecífico doente (injetado com células do tumor de Ehrlich-TAE) produziu mudanças comportamentais, endócrinas e imunológicas. Este estudo analisa os efeitos da convivência com um animal portador de tumor de Ehrlich em camundongos OVA sensibilizados e desafiados sobre a resposta alérgica pulmonar. Pares de camundongos machos foram separados em três grupos: naïve, controle e experimental. Os animais do grupo naïve não foram manipulados sendo utilizados para a avaliação de parâmetros basais. Um animal de cada par dos grupos experimental e controle foi imunizado com OVA. No dia D(0), os animais imunizados receberam uma dose reforço de OVA. No dia D(0) os camundongos do grupo experimental que não foram manipulados foram inoculados com 5x106 células de tumor de Ehrlich; seus companheiros de gaiola moradia foram designados CAD (companheiro do animal doente). Os camundongos não perturbados de cada par do grupo controle foram tratados (i.p.) em D(0) com 0,9% de NaCl, sendo designados CAS (companheiro do animal saudável). O desafio intranasal com OVA foi realizado nos camundongos CAS e CAD nos dias D(12) e D(13); colheram-se o sangue e os tecidos no dia D(14). Em comparação com o grupo CAS, os camundongos do grupo CAD apresentaram 14 dias após a coabitação: (1) aumento do número de eosinófilos e neutrófilos no LBA, (2) diminuição na contagem de células da medula óssea, (3) aumento do níveis de IL-4 e IL-5 e diminuição de IL-10 e INF-ϒ no sobrenadante do LBA, (4) aumento dos níveis de IgG1-OVA, diminuição dos níveis de IgG2a-OVA e nenhuma alteração na IgE-OVA no sangue periférico, (5) aumento na expressão de ICAM-1, VCAM-1 e L-selectina em granulócitos do LBA, (6) diminuição da reatividade da traquéia à metacolina in vitro, (7) aumento da desgranulação de mastócitos, (8) nenhuma alteração nos níveis plasmáticos de corticosterona, (9) aumento dos níveis de adrenalina e noradrenalina plasmáticas, (10) diminuição no tempo de permanência e entradas nos braços abertos do labirinto em cruz elevado, (11) diminuição da expressão de IL-6 no PVN e (12) diminuição da expressão de C-fos no PFC. Estes resultados mostram que a convivência forçada com um animal portador de um tumor ascitico de Ehrlich exacerba a inflamação alérgica pulmonar de camundongos. Eles foram discutidos como decorrentes da estimulação do Sistema Nervoso Autônomo Simpático (SNS) pelo estresse psicológico gerado pela coabitação com o parceiro doente, via liberação de adrenalina e noradrenalina e consequente mudança no perfil de citocinas Th1/Th2 para uma resposta do tipo Th2. Esta alteração seria, provavelmente, um dos mecanismos responsáveis pelo aumento do recrutamento celular para as vias aéreas dos camundongos do grupo CAD. / The bidirectional relationship between the nervous system and the imune system is relevant for homeostatic organism maintenance. Studies from our laboratory showed that 14 days of cohabitation with a sick conspecific (injected with Ehrlich tumor cells-TAE) produced behavioral, endocrinological and immunological changes. This study analyzes the effects of cohabitation with an Ehrlich tumor-bearing animal on ovalbumin (OVA)-induced lung inflammatory response in mice. Pairs of male mice were separate into three groups: naïve, control and experimental. Animals of the naïve group were kept undisturbed being used for assessment of basal parameters. One animal of each experimental and control pair of mice was immunized with OVA. On D(0), these OVA-immunized animals received an OVA booster. At this day (D(0)) the experimental mice that were kept undisturbed were inoculated with 5x106 Ehrlich tumor cells; their immunized cage-mates were then referred as to CSP(companion of sick partner). The undisturbed mice of each control pair were i.p. treated on D(0) with 0.9% NaCl; their sensitized cage-mate were subsequently referred as CHP (companion of health partner). The intranasal OVA challenge was performed on CSP and CHP mice on D(12) and D(13); blood and tissue collection were performed on D (14). Fourteen days after cohabitation, in comparison to the CHP mice, the CSP mice displayed the following: (1) an increased number of eosinophils and neutrophils in the BAL, (2) a decreased bone marrow cell count, (3) increased levels of IL-4 and IL-5 and decreased levels of IL-10 and INF-ϒ in the BAL supernatant, (4) increased levels of IgG1-OVA, decreased levels of IgG2a-OVA and no changes in OVA-specific IgE in the peripheral blood, (5) increased expression of ICAM-1, VCAM-1 and L-selectin in the BAL granulocytes, (6) decreased tracheal reactivity to metacholine measured in vitro , (7) increased mast cell degranulation, (8) no changes in plasma corticosterone levels (9) increased levels of plasmatic adrenaline and noradrenaline, (10) decreased time and % of entries on open arms of elevated plus maze, (11) decreased expression of IL-6 on PVN and (12) decreased expression of C-fos on PFC. These results suggest that cohabitation with an Ehrlich tumor bearing mice exacerbates allergic lung inflammatory response in mice. Most probably, the changes observed in CSP mice are a consequence of the psychological stress induced by forced cohabitation with the sick partner. Strong involvement of the sympathetic nervous system through adrenaline and noradrenaline release and a shift of the Th1/Th2 cytokine profile toward a Th2 response were considered to be the mechanisms underlying the cell recruitment to the animal´s airways.

Die von T-Helfer (Th) -Zellen produzierten Zytokine spielen eine entscheidende Rolle bei der Einleitung, der Aufrechterhaltung und der Regulation von Immunantworten. Bei der Untersuchung von Immunantworten hat sich eine vereinfachte Einteilung der Th-Zellen in zwei Klassen als hilfreich erwiesen: Th1 und Th2. Stabil differenziell exprimierte Oberflächenmoleküle werden benötigt, um lebende Th1- und Th2-Zellen identifizieren, auf Einzelzellebene charakterisieren und möglicherweise die von ihnen erzeugten Immunantworten modulieren zu können. Auf der Suche nach solchen Molekülen wurde in Zusammenarbeit mit der Firma Millennium Pharmaceuticals das Oberflächenmolekül TIM-3 entdeckt. Die Ergebnisse der vorliegenden Arbeit belegen, dass TIM-3 nicht nur von CD4+ Th-Zellen, sondern auch von CD8+ T-Zellen, gamma/delta-T-Zellen, sowie einigen Makrophagen und der Mehrheit der dendritischen Zellen in der Milz von Mäusen auf der Zelloberfläche exprimiert wird. Die Expression von TIM-3 auf Th-Zellen ist klar mit einem aktivierten Phänotyp assoziiert. TIM-3 wird unter polarisierenden Bedingungen in vitro im Vergleich zu Th2-Zellen bevorzugt, jedoch nicht ausschließlich von Th1-Zellen exprimiert. Erstmals wurde auf Einzelzellebene die Zytokinproduktion TIM-3 exprimierender Th-Zellen untersucht. Die Analyse von Th0-Zellen, welche unter nichtpolarisierenden Bedingungen in vitro hergestellt wurden, ergab keine bevorzugte Produktion von Th1-Zytokinen und keine verminderte Expression von Th2-Zytokinen durch TIM-3 exprimierende Th-Zellen. Aufgrund der in dieser Arbeit erhaltenen Ergebnisse erlaubt die Expression von TIM-3 allein daher nicht die Identifizierung von Th1-Zellen. Nach einer Infektion mit Toxoplasma gondii lag jedoch eine bevorzugte Assoziation zwischen der Expression von TIM-3 und der pathogenspezifischen Produktion von Interferon (IFN)-gamma, Interleukin (IL)-2 und Tumor Nekrose Faktor (TNF)-alpha vor. Somit korreliert die TIM-3 Expression auf Th-Zellen nur unter bestimmten Bedingungen mit einem Th1-Phänotyp. / The cytokines that are produced by T helper (Th) cells are decisive for the initiation, the maintenance and the regulation of immune responses. A simplified classification of Th cells has proven to be useful for the analysis of immune responses: Th1 and Th2. Stably and differentially expressed surface molecules are required for the identification of live Th1- and Th2-cells, their characterisation at the single cell level and the possible modulation of the immune responses that they induce. On the search for such molecules the surface molecule TIM-3 was discovered in collaboration with Millennium Pharmaceuticals. The present work shows that TIM-3 protein is not only expressed on the cell surface by CD4+ Th cells but also by CD8+ T cells and gamma/delta T cells as well as by some macrophages and the majority of the dendritic cells in the murine spleen. TIM-3 expression on Th cells is clearly associated with an activated phenotype. Under polarising conditions in vitro TIM-3 is expressed preferentially albeit not exclusively by Th1 cells compared to Th2 cells. For the first time, the cytokine production of TIM-3 expressing Th cells has been analysed at the single cell level. The analysis of Th0 cells, generated under non-polarising conditions in vitro showed no preferential production of Th1-cytokines and no diminished production of Th2-cytokines by TIM-3 expressing Th-cells. The results obtained in this work lead to the conclusion that expression of TIM-3 does not permit the identification of Th1-cells. However upon infection with Toxoplasma gondii a positive association between the expression of TIM-3 and the pathogen-specific production of Interferon (IFN)-gamma, Interleukin (IL)-2 and Tumor Necrosis Factor (TNF)-alpha was observed. Therefore the expression of TIM-3 on Th-cells only correlates under specific conditions with a Th1-phenotype.

Die vorliegende Arbeit befaßt sich mit T-Helferzellen und ihren Interaktionen mit Gewebszellen, wie sie im gesunden Organismus und in Autoimmunerkrankungen auftreten. Es werden Fragen der Toleranzinduktion durch orale Gabe von Antigenen, speziell der oralen Verabreichung von Collagen II bei Patienten mit rheumatoider Arthritis diskutiert. Eine Immundeviation als Mittel, inflammatorische Th1-Zellantworten in anti-inflammatorische Th2-Zellantworten zu verwandeln, kann durch Eingriffe in die T-Zell-Signaltransduktion erreicht werden. Es werden neue Ansätze zu Mechanismen diskutiert, die das Immunprivileg des Zentralnervensystems gewährleisten. Die hirnresidenten Immunzellen, zu denen Mikrogliazellen und Astrozyten zählen, besitzen Eigenschaften, die eine Entzündung unwahrscheinlich machen. Sie müssen aktiviert werden, um Antigene präsentieren zu können. In organtypischen entorhinal-hippocampalen Schnittkulturen konnte gezeigt werden, dass Mikrogliazellen durch Th1-Zellen aktiviert, von Th2-Zellen hingegen deaktiviert werden. Die Möglichkeit, dass die Costimulation über CD80 oder CD86 differentielle Effekte auf den Charakter der Immunantwort hat, wird diskutiert. Der Einfluß von pro-inflammatorischen Zytokinen auf Mikrogliaaktivierung und den Erhalt von Nervenfasern wurde ebenfalls in Hirnschnittkulturen untersucht. Astrozyten sind wesentlicher Bestandteil der Blut-Hirn-Schranke. Diese kann jedoch von aktivierten T-Zellen überwunden werden. In dieser Arbeit wird gezeigt, dass Astrozyten über eine Expression von CD95L in aktivierten T-Zellen Apoptose induzieren können. Davon sind jedoch nicht alle T-Zellen betroffen. Andererseits wird eine T-Zellproliferation unterdrückt, indem T-Zellen unter Astrozyteneinfluß verstärkt CTLA-4 exprimieren, was einen Zellzyklusarrest zur Folge hat. Darüber hinaus ist eine verstärkte Produktion von Nervenwachstumfaktor (NGF; nerve growth factor) nach antigenspezifischer Interaktion von Astrozyten mit Th1- und Th2-Zellen als zusätzliches Mittel, eine Neuroinflammation einzudämmen, anzusehen. Die Arbeit stellt diese Ergebnisse in fünf Kapiteln dar, welche gleichzeitig eine Einführung in die als Anlagen enthaltenen zehn Publikationen geben. / This thesis deals with T helper cells and their interactions with tissue cells as they occur in the healthy organism and in autoimmune diseases. Questions of tolerance induction by oral application of antigens are discussed especially oral treatment with type II collagen in patients with rheumatoid arthritis. In order to transform inflammatory Th1 responses into anti-inflammatory Th2 responses, immune deviation can be reached by interference with T-cell signal transduction. New approaches towards the different ways that the immune privilege of the central nervous system is maintained are discussed. The resident immune cells, i.e. microglia and astrocytes possess properties that make inflammation unlikely. They have to be activated in order to present antigens. It has been shown in organotypic entorhinal-hippocampal slice cultures that Th1 cells activate whereas Th2 cells deactivate microglial cells. The possibility is discussed as to whether costimulation via CD80 or CD86 differentially influences the character of the immune response. The influence of pro-inflammatory cytokines on microglial activation and preservation of nerve fibers has also been studied in brain slice cultures. Astrocytes are an essential part of the blood-brain barrier, which can be crossed by activated T cells. The thesis shows that astrocytes can induce apoptosis in activated T cells via expression of CD95L. However, not all T cells are affected. T cell proliferation is suppressed by increased CTLA-4 expression in T cells under the influence of astrocytes, resulting in a cell cycle arrest. An additional mechanism of confining neuroinflammation is increased production of the nerve growth factor (NGF) following antigen-specific interaction of astrocytes and Th1 and Th2 cells, respectively. These results are presented in five chapters that also introduce the ten attached publications.

Parasitische Nematoden beeinflussen gezielt die Abwehrreaktionen ihres Wirtes. Dies wird besonders während der chronischen Infektionsphase durch eine herabregulierte T-Zell-Antwort auf Parasitenantigene und andere Stimuli ersichtlich. In dieser Arbeit wurde die T-Zell-Antwort gegen einen intestinalen Nematoden untersucht. Mäuse wurden mit dem Trichostrongyliden Heligmosomoides polygyrus infiziert und in der Folge Effektor- sowie regulatorische T-Zellen (Tregs) untersucht. Subpopulationen von CD4+ T-Zellen wurden aus chronisch infizierten Mäusen isoliert und in naive Empfänger transferiert, welche nachfolgend infiziert wurden. Dabei zeigte sich, dass der Transfer von CD4+ Effektor-T-Zellen zu einer verminderten Wurmlast in den Empfängertieren führte, diese Zellen also einen partiellen Schutz gegen die Primärinfektion vermitteln. Der gleichzeitige Transfer von Tregs beeinflusste diesen Effekt nicht. Tregs allein zeigten keinerlei Einfluss auf die Wurmlast der Empfänger. Die Protektion durch Transfer von Effektor-T-Zellen kann vermutlich auf eine kleine Antigen-spezifische Population von CD4+ Zellen zurückgeführt werden. Diese Zellen wurden durch die Expression von CD40-L (CD154) nach Restimulation mit Parasitenantigen in vitro charakterisiert und enthielten einen Großteil der Zytokinproduzenten unter den CD4+ Zellen. Während diese Effektorzellen ein deutliches Th2-Zytokinprofil durch Produktion von Interleukin-4 (IL-4) und IL-13 zeigten, reagierte eine Treg-Subpopulation mit der Sekretion hoher Mengen von IL-10 auf Antigenstimulation. Diese Tregs waren durch Expression des Integrins AlphaE (CD103)Beta7 sowie CD25 und Foxp3 charakterisiert und vermittelten in vitro die stärkste Suppression anderer T-Zellen, wenn sie aus chronisch infizierten Mäusen isoliert wurden. Durch Untersuchung der zellulären Zusammensetzung von mesenterialen Lymphknoten und Milz konnte gezeigt werden, dass die Frequenz solcher regulatorischer Zellen im Verlauf der Infektion dauerhaft und überproportional zunimmt. Im Gegensatz dazu wurde am Infektionsort nur eine vorübergehende Akkumulation von Tregs (Foxp3+) während der akuten Phase der Infektion nachgewiesen. Diese Ergebnisse zeigen den Einfluss einer intestinalen Nematodeninfektion auf die Aktivität von Tregs und das Potential parasitenspezifischer CD4+ Effektor-Zellen zur Vermittlung von Schutz gegen die Infektion. Ein weiteres Projekt dieser Arbeit wahr die Verabreichung eines immunmodulatorischen Parasitenproteins, des Filariencystatins Av17, in einem Mausmodell entzündlicher Darmerkrankungen. In Mäusen wurde eine kolitisartige Entzündung durch eine Chemikalie im Trinkwasser induziert. Die regelmäßige Verabreichung von rekombinant exprimiertem Cystatin verminderte die Entzündungsreaktion signifikant. Damit konnte in dieser Arbeit gezeigt werden. dass Entzündungsreaktionen, die nicht durch den Parasiten selbst hervorgerufen werden, durch die Applikation einer einzelnen Parasitenkomponente unterdrückt werden können. / Parasitic nematodes specifically modulate the immune response of their hosts. A cellular hyperreactivity, especially during the chronic phase of infection, is a distinct finding of such infections. The T cell response against an intestinal nematode was analyzed in this work. Mice were infected with the trichostrongylid Heligmosomoides polygyrus and surveyed for changes concerning effector and regulatory T cells (Tregs). Subpopulations of CD4+ T cells were isolated from chronically infected mice and adoptively transferred to naive recipients, which were subsequently infected. The Transfer of CD4+ effector cells conferred partial protection, seen as decreased worm burdens in recipients. This effect was unimpaired by simultaneous transfer of Tregs. The transfer of purified Tregs alone showed no effect on worm burdens. The protection by transfer of effector T cells was probably due to a small parasite-specific population, which was characterized by the expression of CD40-L (CD154) after antigen-restimulation. The CD154+ population contained high frequencies of cells reacting with production of the Th2 key cytokines interleukin-4 (IL-4) and IL-13. On the other hand, a subpopulation of Tregs secreted high amounts of IL-10 in response to the antigen. These Tregs were characterized by the expression of the integrin AlphaE (CD103)Beta7, as well as CD25 and Foxp3. They showed a peculiar strong suppressive efficacy on the proliferation of other T cells, especially when derived from chronically infected donors. Analyzing the cellular composition of mesenteric lymph nodes and spleens in response revealed a lasting and over-proportional increase in frequencies of these Tregs. In clear contrast, only a transient increase of Foxp3-expressing Tregs was detected at the site of infection during the acute phase. These results point out the changes Treg activity during an intestinal nematode infection and show the potential of CD4+ effector cells in mediating protection against infection. A second project of this work was the application of an immunomodulatory parasite protein, the filarial cystatin Av17, in a mouse model of inflammatory bowel disease. Mice developed an inflammatory response to a chemical applied in the drinking water. The repeated application of recombinantly expressed cystatin significantly diminished the inflammatory response. Hence, this work showed the potential of a single parasite component in suppressing inflammatory processes not caused by the parasite itself.