Among sheep producers, the parasitic nematode Haemonchus contortus is a major animal health concern. Caribbean hair sheep are more resistant than conventional wool breeds to this blood-feeding, abomasal parasite. Our objective was to determine differences in the immune response associated with parasite-resistant hair and susceptible wool lambs infected with 10,000 H. contortus and in uninfected controls. Animals were sacrificed and abomasum and lymph node tissues were collected at 3 or 27 days post-infection (PI), and for controls on day 17, 27, or 38 relative to d 0 of infected animals. Blood and fecal samples were collected throughout the study. Lower fecal egg counts, higher packed cell volumes, and heavier lymph nodes of infected hair compared to wool lambs, suggests hair lambs have increased parasite resistance. Greater tissue infiltration of eosinophils (P < 0.05) was observed in hair compared to wool sheep by 3 days PI, with no breed differences in globule leukocytes. Total serum IgA and IgE were greater in control hair versus wool sheep (P < 0.05). After 3, 5, and 21 of infection, total serum IgA (P< 0.05), total lymph node IgE (P < 0.01), but not total serum IgE were greater in hair sheep compared to wool sheep. Gene expression was measured between hair and wool lambs for abomasal and lymph node tissues using bovine cDNA microarrays and real-time RT-PCR. Microarray analysis revealed cell survival, endosome function, gut motility, and anti-coagulation pathways are important in abomasal and lymph node tissues during H. contortus infection. Immune genes, including IL-4, IL-4 Ra, IL-12 Rb1, and IL-12 Rb2, are also highly represented in abomasal or lymph node tissue of infected animals. Eleven genes were evaluated using real-time RT-PCR and included TH1 and TH2 cytokines, cytokine receptors, and IgE. Parasite infection leads to increased expression of IL-13 and IgE in both tissues and breeds when compared to control animals. Breed comparison of gene expression shows resistant hair sheep produce a stronger modified TH2-type immune response during infection. Differential cell infiltration, antibody production, and regulation of TH2 cytokines between breeds may be partially responsible for differences in parasite resistance. / Ph. D.

Objetivo: O objetivo deste estudo foi analisar a relação e a predominância dos padrões de resposta imune Th1 e Th2 em pacientes com endometriose. Pacientes e Métodos: Entre Fevereiro de 2004 e Abril de 2005 foram avaliadas 98 pacientes divididas em dois grupos de acordo com a presença (Grupo A) ou ausência de endometriose (Grupo B), confirmada histologicamente. Foram coletados sangue periférico e fluido peritoneal de todas as pacientes para a dosagem de interleucinas (IL) 2, 4 e 10, fator de necrose tumoral-alfa (TNF-alfa) e interferon-gama (IFN-gama) por citometria de fluxo. Além da presença da endometriose, foram analisadas a fase do ciclo menstrual, o quadro clinico, o estadiamento, o local de acometimento e a classificação histológica da moléstia. Resultados: Observou-se elevação estatisticamente significante nas concentrações de IFN-gama (mediana de 1,5pg/ml no Grupo A e de 0,4pg/ml no Grupo B, p=0,03) e de IL-10 (mediana de 38,6pg/ml no Grupo A e de 15,7pg/ml no Grupo B, p=0,03) do fluido peritoneal das pacientes com endometriose em relação àquelas sem a doença. As pacientes com endometriose apresentaram alteração estatisticamente significativa na relação das concentrações de IL-4/IFN-gama (p<0,001), IL-4/IL-2 (p=0,006), IL-10/IFN-gama (p < 0,001) e IL-10/IL-2 (p<0,001) do fluido peritoneal, com concentrações mais elevadas da IL-4 e da IL-10, o que reflete o predomínio da resposta Th2 sobre a Th1. Conclusão: Os resultados obtidos permitem concluir que, neste estudo, observou-se elevação de citocinas relativas à resposta imune Th2, denotando haver um predomínio deste padrão de resposta em pacientes com endometriose. / Objective: The objective of this study was to analyze the relation and the predominance of the immune response patterns Th1 and Th2 in patients with endometriosis. Patients and Methods: Between February 2004 and April 2005, 98 patients were evaluated and divided into two groups, according to the presence (Group A) or absence of endometriosis (Group B), confirmed by histology. Peripheral blood and peritoneal fluid were collected from all patients to obtain the concentrations of interleukines (IL) 2, 4 and 10, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) using flow cytometry. Besides the presence of endometriosis, we analyzed phase of menstrual cycle, clinical complaints, classification, site and histological differentiation of the disease. Results: We observed higher concentrations of IFN-gamma (median of 1.5pg/ml in Group A and 0.4pg/ml in Group B, p = 0.03) and IL-10 (median of 38.6pg/ml in Group A and 15.7pg/ml in Group B, p = 0.03) in peritoneal fluid of patients with endometriosis in relation to those without the disease. Patients with endometriosis presented a significant alteration in IL-4/IFN-gamma (p < 0.001), IL-4/IL-2 (p = 0.006), IL-10/IFN-gamma (p < 0.001) and IL-10/IL-2 (p<0.001) ratio concentrations of peritoneal fluid, with IL-4 and IL-10 predominance, reflecting a Th2 response predominance over the Th1. Conclusion: The results allow concluding that, in this study, it was observed a cytokine elevation related to Th2 immune response, indicating a predominance of this pattern of response in patients with endometriosis.

Introdução: O Papilomavírus Humano está muito bem associado com diversos tipos de cânceres humanos, como câncer anogenital e oral. Alguns estudos demonstram que o aparecimento de lesões e a progressão para o câncer estão relacionados ao tipo de resposta imune do hospedeiro. Deste modo, evidências indicam que a resposta imune do hospedeiro tem um papel muito importante para o curso da infecção pelo HPV. Objetivo: Avaliar a resposta imune específica in vitro ao Papilomavírus Humano (HPV) em homens com lesões causadas por HPV e sem lesão por HPV. Material e Métodos: Foram recrutados 31 pacientes e 11 voluntários, que formaram 4 grupos de estudo; sendo 12 pacientes no Grupo A (HIV +/ HPV +); 09 pacientes no Grupo B (HIV-/HPV+); 10 pacientes no Grupo C (HIV+/ HPV-); e 11 indivíduos saudáveis no Grupo D (HIV-/HPV-). Foram realizados ensaios de cultura celular para mensurar a resposta celular específica \"in vitro\" do tipo Th1/Th2/Th17 (INF-y, IL-2, TNFalfa, IL-4, IL-10 e IL-17) sob o estímulo da vacina quadrivalente do HPV (HPV 6, 11, 16 e 18) e à proteína E7 de HPV-16. Resultados: O grupo coinfectado (HIV +/ HPV+) apresentou níveis mais elevados de citocinas, principalmente do perfil Th2, comparando-se com os dados dos demais grupos de estudo. O grupo coinfectado apresentou níveis elevados de IL-6 e IL-10 (Perfil Th2) em relação ao grupo controle (HIV-/HPV-), com significância estatística (p < 0.0001 e p < 0.0001, respectivamente). Conclusão: Foi demonstrada uma elevada produção de citocinas no grupo HPV+/HIV+, sugerindo uma forte imunomodulação pela coinfecção HIV/HPV. Entretanto, novos estudos devem ser realizados para comprovar estes dados. Além de apresentar um perfil essencialmente Th2 do grupo coinfectado, principalmente pelos níveis elevados de IL-6 e IL-10 apresentados, sugerindo que estas duas citocinas possam servir como biomarcadores para persistência viral, uma vez que, os pacientes soropositivos para HIV apresentam maior persistência de HPV, e monitorar a progressão para lesões mais graves / Introduction: Human Papillomavirus is associated with different types of human cancers, such as anogenital and oral cancer. Some studies show that the appearance of lesions and progression to cancer are related to the type of host immune response. Thus, evidence indicates that the host immune response has a role key in the course of HPV infection. Objective: To evaluate the specific immune response in vitro to HPV in men with lesions caused by HPV and without injury caused by HPV. Methods: We recruited 31 patients and 11 volunteers, who formed four groups, with 12 patients in Group A (HIV+/HPV+); 09 patients in Group B (HIV-/HPV+); 10 patients in Group C (HIV+/HPV-) and 11 healthy subjects in Group D (HIV-/HPV-). Cells culture assay was performed to measure the specific immune response \"in vitro\" Th1/Th2/Th17 (IFN-y, IL-2, TNF-alfa, IL-4, IL-10 and IL-17) under the stimulation of quadrivalent HPV vaccine (HPV 6, 11, 16 and 18) and the E7 protein of HPV-16. Results: The coinfected group (HIV+/HPV+) had higher levels of cytokines, especially Th2 profile, compared with data from the other study groups. The coinfected group showed high levels of IL-6 and IL-10 (Th2 profile) compared to the control Group (HIV- /HPV-), with statistical significance (p < 0.0001 and p < 0.0001, respectively). Conclusion: This study demonstrated a high production of cytokines in the coinfected group, suggesting a strong immunomodulation by coinfection HIV/HPV. However, further studies should be conducted to confirm these data. In addition to presenting essentially a Th2 profile, especially by high levels of IL-6 and IL-10 presented, suggesting that these two cytokines may serve as biomarkers for viral persistence, since HIV seropositive patients have a higher persistence of HPV, and monitor the progression to more serious injuries

Submitted by Wendell Amoedo (wendell.amoedo@hotmail.com) on 2018-11-26T14:35:47Z No. of bitstreams: 1 dissertação.kerollen.runa.pinto2017.pdf: 1354749 bytes, checksum: 47321c0e5d660481ffc33d9d3204b2f0 (MD5) / Submitted by Wendell Amoedo (wendell.amoedo@hotmail.com) on 2018-11-28T13:01:00Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertação.kerollen.runa.pinto2017.pdf: 1354749 bytes, checksum: 47321c0e5d660481ffc33d9d3204b2f0 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-11-28T13:20:12Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertação.kerollen.runa.pinto2017.pdf: 1354749 bytes, checksum: 47321c0e5d660481ffc33d9d3204b2f0 (MD5) / Made available in DSpace on 2018-11-28T13:20:12Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertação.kerollen.runa.pinto2017.pdf: 1354749 bytes, checksum: 47321c0e5d660481ffc33d9d3204b2f0 (MD5) Previous issue date: 2017-02-27 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / Enterokinase (EC 3.4.21.9) is a heterodimer serine protease, a natural activator of trypsinogen, capable of cleaving specifically the sequence Asp-Asp-Asp-Asp-Lys. Due to the high specificity of the recognition site, it became a great tool of biotechnological interest. It is usually used to remove affinity tags in vitro, of recombinant proteins. In this work, the molecular cloning strategy resulted in the construction of the pDMK06, capable of programming the regulated expression of a heterologous gene ETK-Trx in E. coli. Through the cleavage process with restriction enzymes NdeI and BamHI, it was possible to obtain the coding sequence of the fusion protein between enterokinase and thioredoxin (ETK-Trx) of approximately 1259 bp from pENTK plasmid. Then, this sequence was subcloned at NdeI and BamHI sites of the expression vector pDM02, originating the recombinant plasmid pDMK06. This vector contains the TH2 promoter, which is efficiently regulated by Lac operator/repressor. E. coli JM110 cells transformed with the recombinant plasmid showed smaller growth in a solid medium when the expression of the heterologous protein was induced by IPTG in comparison with the control; however, this effect was not detected in the liquid medium. Furthermore, the E. coli cells morphology was analyzed through optical microscopy containing the recombinant plasmid pDMK06, when it was observed, all through the growth time, modifications on cell morphology, characterized by the formation of filaments in those induced with IPTG, in comparison with the control. For expression analysis of the recombinant protein ETKTrx, polyacrylamide gel electrophoresis SDS-PAGE was performed with the samples that grew with IPTG induction for eight hours. The results showed that the protein ETK-Trx is about 47 kDa with a high level of expression at the insoluble fraction, probably as an inclusion corpuscle. The high levels of expression of ETK-Trx protein occurred in a perfectly regulated way, showing the functionality of the pDM02 plasmid expression/regulation system. / A enteroquinase (EC 3.4.21.9) é uma serino protease heterodimérica, ativadora natural do tripsinogênio, capaz de clivar especificamente a sequência Asp-Asp-Asp- Asp-Lys. Devido à alta especificidade do sítio de reconhecimento tornou-se uma ferramenta de grande interesse biotecnológico. É comumente utilizada para a remoção in vitro de marcas de afinidade, como etiquetas de fusão (tags) de proteínas recombinantes. No presente trabalho, a estratégia de clonagem molecular resultou na construção do plasmídeo pDMK06, que é capaz de programar a expressão heteróloga regulada do gene ETK-Trx em E.coli. Por meio do processo de clivagem com as enzimas de restrição NdeI e BamHI foi possível obter a sequência codificadora da proteína de fusão entre enteroquinase e tiorredoxina (ETK-Trx) de aproximadamente 1259 pb partir do plasmídeo pENTK, a seguir essa sequência foi subclonada nos sítios de NdeI e BamHI do vetor de expressão pDM02, originando o plasmídeo recombinante pDMK06. Esse vetor contém o promotor TH2 que é regulado eficientemente pelo sistema operador/repressor Lac. Células de E.coliJM110 transformadas com o plasmídeo recombinante mostram menor crescimento em meio sólido quando a expressão da proteína heteróloga era induzida por IPTG em relação ao controle, porém esse efeito não foi detectado em meio líquido. Além disto foi analisado por microscopia ótica a morfologia das células de E.colicom o plasmídeo recombinante pDMK06, onde observou-se no decorrer do tempo de crescimento e indução, alterações na morfologia celular caracterizada de filamentação das células induzidas com IPTG quando comparadas com o controle. Para análise da expressão da proteína recombinante ETK-Trx utilizou-se a eletroforese em gel de poliacrilamida SDS-PAGE das amostras referentes a 8 horas de crescimento e indução com IPTG. Os resultados obtidos apresentaram a proteína ETK-Trx com tamanho aproximado de 47kDa com alto nível de expressão na fração insolúvel, provavelmente em forma de corpúsculo de inclusão. A expressão em altos níveis das proteínas ETK-Trx ocorreu de forma perfeitamente regulada mostrando a funcionalidade do sistema de expressão/regulação do plasmídeo pDM02.

Objetivo: O objetivo deste estudo foi analisar a relação e a predominância dos padrões de resposta imune Th1 e Th2 em pacientes com endometriose. Pacientes e Métodos: Entre Fevereiro de 2004 e Abril de 2005 foram avaliadas 98 pacientes divididas em dois grupos de acordo com a presença (Grupo A) ou ausência de endometriose (Grupo B), confirmada histologicamente. Foram coletados sangue periférico e fluido peritoneal de todas as pacientes para a dosagem de interleucinas (IL) 2, 4 e 10, fator de necrose tumoral-alfa (TNF-alfa) e interferon-gama (IFN-gama) por citometria de fluxo. Além da presença da endometriose, foram analisadas a fase do ciclo menstrual, o quadro clinico, o estadiamento, o local de acometimento e a classificação histológica da moléstia. Resultados: Observou-se elevação estatisticamente significante nas concentrações de IFN-gama (mediana de 1,5pg/ml no Grupo A e de 0,4pg/ml no Grupo B, p=0,03) e de IL-10 (mediana de 38,6pg/ml no Grupo A e de 15,7pg/ml no Grupo B, p=0,03) do fluido peritoneal das pacientes com endometriose em relação àquelas sem a doença. As pacientes com endometriose apresentaram alteração estatisticamente significativa na relação das concentrações de IL-4/IFN-gama (p<0,001), IL-4/IL-2 (p=0,006), IL-10/IFN-gama (p < 0,001) e IL-10/IL-2 (p<0,001) do fluido peritoneal, com concentrações mais elevadas da IL-4 e da IL-10, o que reflete o predomínio da resposta Th2 sobre a Th1. Conclusão: Os resultados obtidos permitem concluir que, neste estudo, observou-se elevação de citocinas relativas à resposta imune Th2, denotando haver um predomínio deste padrão de resposta em pacientes com endometriose. / Objective: The objective of this study was to analyze the relation and the predominance of the immune response patterns Th1 and Th2 in patients with endometriosis. Patients and Methods: Between February 2004 and April 2005, 98 patients were evaluated and divided into two groups, according to the presence (Group A) or absence of endometriosis (Group B), confirmed by histology. Peripheral blood and peritoneal fluid were collected from all patients to obtain the concentrations of interleukines (IL) 2, 4 and 10, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) using flow cytometry. Besides the presence of endometriosis, we analyzed phase of menstrual cycle, clinical complaints, classification, site and histological differentiation of the disease. Results: We observed higher concentrations of IFN-gamma (median of 1.5pg/ml in Group A and 0.4pg/ml in Group B, p = 0.03) and IL-10 (median of 38.6pg/ml in Group A and 15.7pg/ml in Group B, p = 0.03) in peritoneal fluid of patients with endometriosis in relation to those without the disease. Patients with endometriosis presented a significant alteration in IL-4/IFN-gamma (p < 0.001), IL-4/IL-2 (p = 0.006), IL-10/IFN-gamma (p < 0.001) and IL-10/IL-2 (p<0.001) ratio concentrations of peritoneal fluid, with IL-4 and IL-10 predominance, reflecting a Th2 response predominance over the Th1. Conclusion: The results allow concluding that, in this study, it was observed a cytokine elevation related to Th2 immune response, indicating a predominance of this pattern of response in patients with endometriosis.

Introdução: O Papilomavírus Humano está muito bem associado com diversos tipos de cânceres humanos, como câncer anogenital e oral. Alguns estudos demonstram que o aparecimento de lesões e a progressão para o câncer estão relacionados ao tipo de resposta imune do hospedeiro. Deste modo, evidências indicam que a resposta imune do hospedeiro tem um papel muito importante para o curso da infecção pelo HPV. Objetivo: Avaliar a resposta imune específica in vitro ao Papilomavírus Humano (HPV) em homens com lesões causadas por HPV e sem lesão por HPV. Material e Métodos: Foram recrutados 31 pacientes e 11 voluntários, que formaram 4 grupos de estudo; sendo 12 pacientes no Grupo A (HIV +/ HPV +); 09 pacientes no Grupo B (HIV-/HPV+); 10 pacientes no Grupo C (HIV+/ HPV-); e 11 indivíduos saudáveis no Grupo D (HIV-/HPV-). Foram realizados ensaios de cultura celular para mensurar a resposta celular específica \"in vitro\" do tipo Th1/Th2/Th17 (INF-y, IL-2, TNFalfa, IL-4, IL-10 e IL-17) sob o estímulo da vacina quadrivalente do HPV (HPV 6, 11, 16 e 18) e à proteína E7 de HPV-16. Resultados: O grupo coinfectado (HIV +/ HPV+) apresentou níveis mais elevados de citocinas, principalmente do perfil Th2, comparando-se com os dados dos demais grupos de estudo. O grupo coinfectado apresentou níveis elevados de IL-6 e IL-10 (Perfil Th2) em relação ao grupo controle (HIV-/HPV-), com significância estatística (p < 0.0001 e p < 0.0001, respectivamente). Conclusão: Foi demonstrada uma elevada produção de citocinas no grupo HPV+/HIV+, sugerindo uma forte imunomodulação pela coinfecção HIV/HPV. Entretanto, novos estudos devem ser realizados para comprovar estes dados. Além de apresentar um perfil essencialmente Th2 do grupo coinfectado, principalmente pelos níveis elevados de IL-6 e IL-10 apresentados, sugerindo que estas duas citocinas possam servir como biomarcadores para persistência viral, uma vez que, os pacientes soropositivos para HIV apresentam maior persistência de HPV, e monitorar a progressão para lesões mais graves / Introduction: Human Papillomavirus is associated with different types of human cancers, such as anogenital and oral cancer. Some studies show that the appearance of lesions and progression to cancer are related to the type of host immune response. Thus, evidence indicates that the host immune response has a role key in the course of HPV infection. Objective: To evaluate the specific immune response in vitro to HPV in men with lesions caused by HPV and without injury caused by HPV. Methods: We recruited 31 patients and 11 volunteers, who formed four groups, with 12 patients in Group A (HIV+/HPV+); 09 patients in Group B (HIV-/HPV+); 10 patients in Group C (HIV+/HPV-) and 11 healthy subjects in Group D (HIV-/HPV-). Cells culture assay was performed to measure the specific immune response \"in vitro\" Th1/Th2/Th17 (IFN-y, IL-2, TNF-alfa, IL-4, IL-10 and IL-17) under the stimulation of quadrivalent HPV vaccine (HPV 6, 11, 16 and 18) and the E7 protein of HPV-16. Results: The coinfected group (HIV+/HPV+) had higher levels of cytokines, especially Th2 profile, compared with data from the other study groups. The coinfected group showed high levels of IL-6 and IL-10 (Th2 profile) compared to the control Group (HIV- /HPV-), with statistical significance (p < 0.0001 and p < 0.0001, respectively). Conclusion: This study demonstrated a high production of cytokines in the coinfected group, suggesting a strong immunomodulation by coinfection HIV/HPV. However, further studies should be conducted to confirm these data. In addition to presenting essentially a Th2 profile, especially by high levels of IL-6 and IL-10 presented, suggesting that these two cytokines may serve as biomarkers for viral persistence, since HIV seropositive patients have a higher persistence of HPV, and monitor the progression to more serious injuries

L’inflammasome NLRP3 est un complexe multiprotéique responsable notamment de la production d’IL-1β, une cytokine inflammatoire. Les effets délétères de l’inflammasome NLRP3 ont été démontrés dans de nombreuses maladies dont le cancer. Ce travail se concentre sur les effets de NLRP3 dans le contexte du cancer.Dans un premier projet, j’ai étudié l’activation de l’inflammasome NLRP3 dans les MSDC après un traitement par chimiothérapie. Deux chimiothérapies, le 5-Fluorouracile et la Gemcitabine, sont capables d’éliminer de façon spécifique les MDSC, population de cellules immunosuppressives dont la taille augmente en cas de cancer. J’ai découvert que le 5-Fluorouracile et la Gemcitabine activaient l’inflammasome NLRP3 dans les MDSC. En effet, le 5-Fluorouracile et la Gemcitabine provoquent la perméabilisation du lysosome des MDSC, permettant la sortie de la cathepsine B, protéine lysosomale, dans le cytoplasme où elle interagit directement avec NLRP3. Cette interaction active l’inflammasome NLRP3 et la production d’IL-1β. Cette IL-1β est responsable du développement d’une nouvelle population immunosuppressive, les Th17.J’ai ensuite étudié le rôle de NLRP3 dans la différenciation des lymphocytes T CD4 Th2. Dans ces cellules, le rôle de NLRP3 s’effectue indépendamment du reste du complexe multiprotéique qui forme l’inflammasome. Après avoir été induit par la cascade de signalisation de l’IL-2, NLRP3 interagit avec IRF4 (interferon regulatory factor) et agit comme un facteur de transcription sur le promoteur du gène de l’IL-4. L’absence de NLRP3 a pour conséquence une production moins importante d’IL-4 par les Th2 qui sont alors moins fonctionnels / The inflammasome NLRP3 (NOD like receptor pyd containing 3) is a multiprotein complex notably responsible for IL-1β (interleukine-1β) production, an inflammatory cytokine. Negative effects have been observed in various diseases including cancer. My thesis focuses on the effects of NLRP3 in cancer.In my first project, I studied the NLRP3 inflammasome activation in MDSC (myeloïd derived suppressor cells) after a chemotherapy treatment. Two chemotherapies, 5-Fluorouracil and Gemcitabine, are selectively able to kill MDSC, an immunosuppressive population growing during cancer evolution. MDSC’s death restores anti-tumor immunity for a while but another immunosuppressive population is established by MDSC produced IL-1β before their disappearance. I discovered that 5-Fluorouracil and Gemcitabine trigger NLRP3 inflammasome activation in MDSC. 5-Fluorouracil and Gemcitabine induce lysosomal permeabilisation, allowing for Cathepsin B release into the cytoplasm where it directly interacts with NLRP3. That interaction activates the inflammasome and induces IL-1β production which is responsible for the development of another immunosuppressive population, called Th17 cells.I then studied the role of NLRP3 during Th2 differentiation. Here, NLRP3 actions are done independently of the other inflammasome forming proteins. After being induced by IL-2 signalization pathway, NLRP3 interacts with IRF4 (interferon regulatory factor 4) and acts as a transcription factor on the IL-4 promoter gene. Lack of NLRP3 leads to a smaller IL-4 production by Th2 cells which are consequently less functional

<p>Linker for Activation of T cells, LAT, is a transmembrane adaptor protein that is vital for integrating TCR-mediated signals that modulate T cell development, activation, and proliferation. Upon engagement of the T cell receptor, LAT is phosphorylated and associates with Grb2, Gads, and PLC&#947;1 through its four distal tyrosine residues. Mutation of tyrosine 136 abolishes LAT binding to PLC&#947;1. This results in impaired TCR-mediated calcium mobilization and Erk activation. LATY136F knock-in mice have a severe but incomplete block in T cell development. Yet, CD4+ &#945;&#946; T cells undergo uncontrolled expansion in the periphery, resulting in a severe autoimmune syndrome characterized by Th2 skewing and resultant B cell autoreactivity. Here, we further studied the role of LAT-PLC&#947;1 signaling in T cell lineage commitment, cytokine production, and autoimmunity.</p><p>First, we investigated the importance of the LAT-PLC&#947;1 interaction in &#947;&#948; T cells by crossing LATY136F mice with TCR&#946;-deficient mice. Our data showed that the LATY136F mutation had no major effect on the homeostasis of epithelial &#947;&#948; T cells, which could be found in the skin and small intestine. Interestingly, a population of CD4+ &#947;&#948; T cells in the spleen and lymph nodes underwent continuous expansion and produced elevated amounts of IL4, resulting in an autoimmune syndrome similar to that caused by &#945;&#946; T cells in LATY136F mice. Development of these hyperproliferative &#947;&#948; T cells was not dependent on expression of MHC class II or CD4, and their proliferation could be partially suppressed by regulatory T cells. Our data indicated that a unique subset of CD4+ &#947;&#948; T cells could hyperproliferate in LATY136F mice and suggested that LAT-PLC&#947;1 signaling may function differently in various subsets of &#947;&#948; T cells. </p><p>In addition to examining &#947;&#948; and &#945;&#946; T cell development, we also were interested in further exploring the role of LAT in cytokine production. While our previous data have demonstrated that T cells in LATY136F mice are Th2 skewed, producing large amounts of IL4, we investigated other cytokines that may be important for autoimmunity and found that these CD4+ &#945;&#946; T cells could also produce the proinflammatory cytokine IL6. Analysis of whole cell lysates from CD4+ &#945;&#946; LATY136F T cells demonstrated that NF&#954;B, AKT, and p38 were constitutively phosphorylated, and inhibition of these pathways resulted in reduced IL6 production. By crossing LATY136F mice with IL6 deficient mice, we demonstrated that early T cell survival was diminished in the absence of IL6. We further showed that this reduced CD4+ T cell pool was not due to further blocks in development, or an increase in FoxP3+ regulatory T cells. Finally, we demonstrated that over time, CD4+ T cells do hyperproliferate, yet B cell class switching and autoreactivity remains low. Our data uncovered a novel role for LAT-PLC&#947;1 signaling in regulating IL6 production by T cells during autoimmunity. </p><p>Finally, we wanted to further examine IL4 production and T helper cell differentiation in LATY136F mice. We examined IL4 production using KN2 reporter mice, where huCD2 marks T cells that have recently produced IL4 protein. We demonstrated that only a small proportion of the LATY136F T cells were actively secreting IL4. This subset of T cells were Tfh cells that expressed BCL6 and localized to B cell-rich germinal centers within the spleen. Most studies to date have examined Tfh cells in infection models, and have demonstrated that Tfh cells have very low expression of GATA3. Our results revealed in a spontaneous T cell-mediated autoimmune model system, that Tfh cells express both high levels of BCL6 and GATA3. Additionally, using an inducible deletion system, where normal development occurs, we showed that Tfh cells differentiation is the result of aberrant LAT signaling, rather than autoreactive TCRs with high affinity for self-peptide-MHC. LATY136F Tfh cells did require B cells for their development. Together, these results displayed a novel role for tonic LAT-PLC&#947;1 signaling in modulating Tfh cell differentiation and BCL6 expression.</p> / Dissertation

Type 2 (Th2) immune responses are required for immune defence against helminths, but can also have pathogenic effects in allergic conditions. This thesis examined two factors which may influence Th2 immunity at a cellular and molecular level: cross-talk between Natural Killer (NK) cells and dendritic cells (DCs) and the cell surface organisation of DCs. Cross-talk between NK cells and DCs is well-established to impact Th1 responses against tumours and infection; however the influence of this interaction during Th2 inflammation is unknown. To investigate this, human monocyte-derived DCs were stimulated in vitro with different pathogen-associated molecules; LPS or Poly(I:C) which polarise a Th1 response, or soluble egg antigen (SEA) from the helminth worm Schistosoma mansoni, a potent Th2-inducing antigen. These cells were then combined with autologous NK cells. Confocal microscopy showed polarisation of the NK cell microtubule organising centre (MTOC) and accumulation of LFA-1 at contacts between NK cells and immature or Th2-polarising DCs, but not Th1-polarising DCs, indicative of the assembly of an activating immune synapse. NK cells did not lyse DCs treated with LPS or Poly(I:C), but degranulated to and lysed both immature DCs and Th2 polarising DCs. Antibody blockade of NK cell activating receptors NKp30 and DNAM-1 prevented this lysis. Furthermore, depletion of NK cells in mice which were then transferred with Th2 polarising DCs led to an enhanced Th2 recall response. Thus, these data indicate a previously unrecognised role of NK cell cytotoxicity in restricting the pool of DCs involved in Th2 immune responses. Secondly, this thesis investigated the nanoscale organisation of MHC-II on the surface of Th1 and Th2 polarising DCs using ground state depletion super-resolution microscopy. MHC-II was relatively homogenously distributed across the membrane with no significant changes in clustering between immature, Th1 and Th2 polarising DCs. In contrast, imaging CD74, which can mediate internalisation of MHC-II, revealed increased expression and a more homogenous distribution of this receptor on the surface of Th2-polarising DCs compared to Th1-polarising DCs. These data suggest that changes in the clustering of CD74 could modulate MHC-II surface expression during Th2 responses. Overall, the results in this thesis indicate that both molecular and cellular level modulation of DC function contribute to the development of Th2 responses.

Das Bestreben, den Aufbau, die Funktion sowie die Entwicklung des Immunsystems zu verstehen, steht schon lange Zeit im Zentrum des Interesses vieler Forschungsarbeiten, insbesondere um auf Grundlage der gewonnenen Erkenntnisse neue Behandlungsansätze für immunologisch relevante Krankheitsbilder zu entwickeln. Stillen könnte ein wichtiger Faktor sein, der bei der Entwicklung und Differenzierung von T-Zell-Subpopulationen und Zytokinmustern im Säuglings- und Kindesalter eine bedeutende Rolle spielt. Die Zielsetzung der hier vorgelegten Promotionsarbeit war es, den potentiellen Effekt des Faktors Stillen auf die Entwicklung, die Verteilung und die Differenzierung von Zell-populationen sowie die Expression von Zytokinen bei gesunden Kindern zu untersuchen. Dies geschah insbesondere im Hinblick auf einen möglicherweise vorhandenen Shift der relativen Verteilung der TH1- und TH2-Zytokinen, da in retrospektiven Kohortenstudien bereits gezeigt werden konnte, dass gestillte Kinder eine geringere Anfälligkeit gegenüber schwerwiegenden bakteriellen Infektionen (BACHRACH ET AL., 2003) sowie einer verminderten Inzidenz von Autoimmunerkrankungen (KOLETZKO ET AL., 1989; PISACANE ET AL., 1994) aufweisen. Die Studienkohorte bestand aus 196 gesunden Kindern im Alter zwischen 26 Tagen und 12 Jahren und 352 Tagen. Diese wurde in vier Altersgruppen unterteilt (<1, 1- <3, 3- <6 und 6-<13 Jahre) und mittels eines Fragebogens im Hinblick auf ein möglicherweise vorhandenes Bias bezüglich exogener Einflussfaktoren wie Impfungen, Nikotinexposition (FELESZKO ET AL., 2006) und allergische Erkrankungen in der Familie (HRDÝ ET AL., 2010), die in diesem Zusammenhang diskutiert werden, überprüft. Dabei zeigten sich keine signifikanten Unter-schiede zwischen den Gruppen. Alle immunologischen Parameter wurden in peripherem, heparinisiertem Blut ermittelt. Zunächst wurde mittels Durchflusszytometrie (FACS) eine Phänotypisierung, anhand von antikörpermarkierten Oberflächenantigenen der T-, B- und NK-Zellpopulationen („Immun-status“), durchgeführt. Des Weiteren wurden die mononuklearen Zellen des peripheren Blutes (PBMC) mittels PMA und Ionomycin stimuliert und die Zytokinsekretion durch Brefeldin blockiert. Durch FACS-Analyse wurde die nach 20-24 stündiger Anregung in der Kultur vorhandene intrazellulärer Zytokinexpression von IL2, IFNγ, TNFα, IL4, IL10, TGFβ und IL17 in den T-Zellpopulationen ermittelt. In einem zweiten Schritt wurde der Quotient aus IFNγ und IL4 berechnet, um das Verhältnis zwischen TH1 und TH2 zu analysieren. Das Datenmaterial zeigt die Entwicklung der T-Zell-Subpopulationen mit dem Alter. Junge Kinder zeigen eine durch regulatorische T-Zellen (Treg) bzw. TH0-Zellen vorherrschende TGFβ und IL2-Expression, während ältere Kinder das gesamte Repertoire an TH1 (IFNγ, TNFα) und TH2-Zytokinen (IL4), insbesondere durch T-Gedächtniszellen, exprimieren. Diese Ergebnisse bestätigten bereits zuvor beschriebene – vom Faktor Stillen unabhängige – altersabhängige Veränderungen der Zellpopulationen und der Zytokinexpression. Aus diesem Grund konnte von einer „normalen Verteilung“ der Studienkohorte ausgegangen werden. Zwischen gestillten und nicht-gestillten Kindern hat sich gezeigt, dass sich die Größe und das Verhältnis der übergeordneten Zellpopulationen (T-Helferzellen, zytotoxische T-Zellen) sowie die Reifung der T-Zellen (naive T-Zellen, T-Gedächtniszellen) bezogen auf das Alter nicht unterscheiden. Hingegen zeigt der Faktor Stillen in der Tat einen Einfluss auf die TH1/TH2-Balance. Bei gestillten Kindern lässt sich eine stärkere Gewichtung in Richtung der TH2-Zytokine feststellen (niedrigere IFNγ/IL4-Ratio), so dass davon auszugehen ist, dass Stillen einen so genannten TH2-Shift induziert. Dieses gegenüber nicht-gestillten Kindern „verschobene“ Gleichgewicht bleibt bis zur Altersgruppe der 6-13 Jährigen konstant. Gestillte Kinder haben zudem, im Vergleich zu Formula-ernährten Kindern, ein höheres Vermögen TH1-Zytokine wie TNFα und IFNγ zwischen dem dritten und sechsten Lebensjahr zu bilden. Diese Daten entsprechen nicht einem vorherrschenden TH2-Muster und einer Allergiedisposition, aber sie können die geringere Inzidenz von bakteriellen Infektionen im Vorschulalter (TH1-Antworten benötigt) und von TH1-vermittelten Autoimmunerkrankungen bei gestillten Kindern erklären. Aufgrund der Ergebnisse wird deutlich, dass Muttermilch einen bedeutenden Einfluss auf das Immunsystem hat. Insbesondere konnte gezeigt werden, dass der Einfluss der Muttermilchernährung über den eigentlichen Zeitraum des Stillens hinausreicht. Eine Prägung des Immunsystems durch die Muttermilch, während der frühen Kindheit, erscheint deshalb sehr wahrscheinlich. Diese Ergebnisse sollten selbstverständlich durch weiterreichende Studien, die eventuell vorhandene weitere Störgrößen wie genetische Prädispositionen oder auch Umwelttoxine einschließen, verifiziert werden. Zudem wäre es von Interesse welche in der Muttermilch enthaltene Stoffe zu diesem TH2-Shift beitragen oder ob es sich bei den Einflussfaktoren vielmehr um in der Muttermilch nicht enthaltene Stoffe handelt, die in Formula-Nahrung enthalten sind, wie zum Beispiel Kuhmilchantigene. Schlussendlich bleibt festzuhalten, dass Muttermilch erwiesene positive Vorteile mit sich bringt, wie geringere Infektionsraten (Atemwegsinfektionen, Otitis media) und eine Risiko-verminderung für bestimmte Erkrankungen (Diabetes mellitus Typ1, Morbus Crohn, Multiple Sklerose) im späteren Leben. Die Ergebnisse dieser Arbeit können bestätigen, dass das Stillen mit Muttermilch tatsächlich einen Einfluss auf die Entwicklung des individuellen Immunsystems hat, der auch nach dem Abstillen weiter anhält. Eine wichtige Rolle kann diese Erkenntnis bei der Beratung werdender Mütter spielen, gerade in Hinblick auf ein gegebenenfalls erhöhtes endogenes familiäres Risiko für beispielsweise Autoimmunerkrankungen. Folgearbeiten sind sicher wünschenswert, um den pathophysiologischen Hintergrund dieser beobachteten Daten besser zu verstehen. / The aspiration to understand the construction, the function as well as the development of the immune system is for a long time in the centre of the interest of many research projects, in particular to develop new attempts of treatment for immunological relevant clinical pictures on basis of the won knowledge. Breast-feeding could be an important factor which plays an important role by the development and differentiation of T-cell subsets and cytokine profiles in infancy and childhood. The objective of the doctorate work presented here was to examine the potential effect of the factor breast-feeding for the development, the distribution and the differentiation of cell populations as well as the expression of cytokines with healthy children. This happened in particular in view of a possibly available Shift of the relative distribution of the TH1 and TH2 cytokines, because in retrospective cohort studies could be already demonstrated, that breastfed children show a lower susceptibility for serious bacterial infections (BACHRACH ET AL.,2003) as well as a decreased incidence of autoimmune illnesses (KOLETZKO ET AL.,1989; PISACANE ET AL.,1994). The study cohort existed of 196 healthy children at the age between 26 days and 12 years and 352 days. This was divided into four age groups (<1, 1-<3, 3-<6 and 6-<13 years) and by means of a questionnaire in view of a possibly available Bias with regard to exogenous factors of influence like vaccinations, nicotine exposition (FELESZKO ET AL.,2006) and allergic illnesses in the family (HRDÝ ET AL.,2010) which are discussed in this connection, checks. Besides, no significant differences appeared between the groups. All immunological parametres were determined in peripheral, heparinised blood. First a phenotype determination, on the basis of antibody-marked surface antigens of the T-, B- and NK-cell populations ("immune status"), was carried out by means of flow cytometry (FACS). Besides the mononuclear cells of the peripheral blood (PBMC) were stimulated by means of PMA and Ionomycin and the cytokine secretion was blocked by Brefeldin. By FACS analysis the cytokin expression of IL2, IFN γ, TNF α, IL4, IL10, TGF β and IL17 in the T-cell subpopulations was determined. In the second step the quotient from IFN γ and IL4 was calculated to analyse the relation between TH1 and TH2. The data material shows the development of the T-cell subsets with the age. Young children show one by regulatory T-cells (Treg) or TH0-cells prevailing TGF β and IL2 expression, while older children can produce the whole repertoire of TH1-(IFN γ, TNF α) and TH2-cytokines (IL4), in particular by memory T-cells. These results already confirmed before described – from the factor breast-feeding independent – changes of the cell populations and the cytokine production dependent on age. That's why could be gone out from a „normal distribution“ of the study cohort. Between breastfed and non-breastfed infants has appeared that the size and the relation of the higher cell populations (T-helper cells, zytotoxic T-cells) as well as the maturation of the T- cells (naive T-cells, memory T-cells) did refer to the age make no distinction. Indeed, however, breastfeeding shows an influence on the TH1/TH2-balance. Breastfed children show a stronger weighting in the direction of the TH2-Zytokine (lower IFN γ/IL4-Ratio), so that is to be assumed from the fact that breast-feeding induces a so-called TH2-Shift. These towards non-breastfed children "postponed" balance remains up to the age group of the 6-13 year-old steady. Besides, breastfed children have a higher ability, in comparison to Formula-fed children, to produce TH1-Zytokine like TNF α and IFN γ between the third and sixth year of life. These data do not correspond to a prevailing TH2 pattern and an allergy arrangement, but they can explain the lower incidence of bacterial infections in the pre-school age (TH1-answers needed) and from TH1-provided auto immune illnesses with breastfed children. On account of the results becomes clear that breastmilk has an important influence on the immune system. In particular it could be shown that the influence of breastmilk stretches beyond the real period of the breast-feeding. Therefore, an imprinting of the immune system by breastmilk, during the early childhood, seems very likely. These results should be verified of course by the handing on studies which enclose, perhaps, available other sturgeon dimensions like genetic prearrangements or also environmental toxins. Besides, it would be of interest which ingredients of breastmilk to this TH2-Shift contribute or whether it concerns with the factors of influence rather materials not contained in the mother's milk which are included in Formula food, as for example cow's milk antigens. Finally remains to stick that mother's milk brings proved positive advantages with itself, how lower infection rates (breath way infections, Otitis media) and a risk decrease for certain illnesses (diabetes mellitus Typ1, Morbus Crohn, multiple sclerosis) in the later life. The results of this work can confirm that the breast-feeding with mother's milk really has an influence on the development of the individual immune system. An important role can play this knowledge with the consultation of mothers, just in view of an endogenous informal risk raised if necessary for example auto immune illnesses. Subsequent works are surely desirable to understand the pathophysiological background of these observed data better.