Endotoxemia is responsible for severe illness in horses. Individuals can become unresponsive to the endotoxin molecule after an initial exposure; this phenomenon has been called developing a state of "endotoxin tolerance" (ET). ET has been induced in horses in vivo; however, cytokine expression associated with ET has not been investigated. The purpose of this study was to develop and validate a method for inducing ET in equine peripheral blood mononuclear cells (PBMCs) in vitro, and to describe the cytokine profile which is associated with the ET. Blood was collected from 6 healthy horses and PBMCs were isolated. ET was induced by culturing cells with three concentrations of endotoxin given to induce ET, and evaluated after a second dose of endotoxin given to challenge the cells. The relative mRNA expression of IL-10 and IL-12 was measured by use of quantitative PCR. ET was induced in all cells (n=6) exposed to the 2-step endotoxin challenge. In PBMCs treated with 1.0 ng/ml of endotoxin followed by challenge with 10 ng/ml of endotoxin, the relative mRNA expression of IL-10 in tolerized cells was not different from positive control cells. In contrast, the relative mRNA expression of IL-12 in tolerized cells was decreased by 15-fold after the second endotoxin challenge compared with positive control cells. This experiment demonstrated a reliable method for the ex vivo induction of ET in equine PBMCs. A marked suppression of IL-12 production is associated with ET. The production of IL-10 was not altered in ET in our model. / Master of Science

Der Interleukin- 12 (IL-12)-Signalweg spielt eine wichtige Rolle bei der Alzheimer-Krankheit (AD)-bedingten Neuroinflammation, und es hat sich gezeigt, dass seine Hemmung die Pathologie und die kognitiven Defekte in einem AD-Mausmodell abschwächt. Es ist jedoch unklar, wie die IL-12-Ablation die Aβ-Belastung reduziert und kognitive Defekte umkehrt und welche Zelltypen an dieser neuroinflammatorischen Kaskade beteiligt sind. Um diese Fragestellung zu untersuchen, führten wir snRNA-seq-Methoden durch und analysierte ich die zelltypspezifische Antwort auf Il12b im Kontext von AD anhand von gealterten Wildtyp- (WT) und APPPS1-Mäusen, die entweder eine fehlende oder vorhandene IL-12-Signalgebung aufwiesen. Insbesondere fokussiere ich mich insbesondere auf Veränderungen der zellulären Zusammensetzung und der Genexpressionsprofile in einzelnen Zelltypen. Wie erwartet nahm der Anteil der Mikroglia in beiden AD-Mausmodellen zu. Interessanterweise beobachteten wir in den Mäusen, denen die IL-12-Signalisierung fehlte, eine Erholung der Anzahl der Oligodendrozyten. Bemerkenswert ist, dass die genetische Ausschaltung des IL-12-Signalwegs den APPPS1-bedingten Verlust von Myelin-produzierenden Oligodendrozyten, sowie den Verlust von Myelinschichten, als auch die Veränderungen der neuronalen Homöostase rückgängig machte. Darüber hinaus ergaben Trajektorieninferenz und Analyse der Expressionsniveaus von Transkriptionsfaktoren der Oligodendrozytenabstammung eine zeitspezifische Genexpressionsdynamik hinsichtlich der Myelinisierung. Die genetische Ablation der IL-12-Signalübertragung hatte jedoch keinen Einfluss auf das entzündliche Genexpressionsprofil der AD-spezifischen krankheitsassoziierten Mikroglia. Zusammenfassend konnte ich zeigen, dass der IL-12-Signalweg den AD-Phänotyp verursacht, welcher die Oligodendrozyten und Neuronen beeinträchtigt, und dass die Veränderung der Myelinisierung der Hauptphänotyp der IL-12-bedingten Neuroinflammation in einem AD-Mausmodell ist. / The interleukin-12 (IL-12) signaling pathway plays an important role in Alzheimer’s disease (AD)-related neuroinflammation and its inhibition has been shown to attenuate pathology and cognitive defects in an AD mouse models. However, it is unclear how IL-12 ablation reduces Aβ burden and reverses cognitive defects as well as which cell types are involved in this neuroinflammatory cascade. To address this question, we performed snRNA-seq methods and I scrutinized the cell type-specific response to Il12b in an AD specific context from aged wild type (WT) and APPPS1 adult mice, which were lacking or harboring the IL-12 gene signaling. In particular, I focused on changes in cellular composition and gene expression profiles in individual cell types. As expected, the proportion of microglia increased in both AD mice models, but, interestingly, we observed a rescue in the number of mature oligodendrocytes in the mice lacking IL-12 signaling. Notably, genetic ablation of IL-12 signaling reversed the APPPS1-driven loss of myelin sheets as well as alterations in neuronal homeostasis. Furthermore, trajectory inference and analysis of expression levels of transcription factors of oligodendrocytes lineage revealed time-specific gene expression dynamics regarding myelination. However, genetic ablation of IL-12 signaling did not affect the inflammatory gene expression profile of the AD-specific disease-associated microglia. In summary, the results demonstrate how IL-12 signaling alters the AD phenotype, affecting oligodendrocytes and neurons and myelination alteration which is the main phenotypes of IL-12 related neuroinflammation in an AD mice model.

Introdução. A paracoccidioidomicose (PCM) é uma micose sistêmica crônica, endêmica na América Latina, principalmente Brasil, sendo a oitava causa de morte entre as doenças infecciosas crônicas recorrentes. A PCM infecção é caracterizada por uma resposta Th1, a forma aguda por um perfil misto da resposta Th2/Th9, enquanto na forma crônica caracteriza-se pelo perfil Th17/Th22. A ocorrência e gravidade da PCM humana podem também estar associadas a fatores genéticos como os polimorfismos dos genes de citocinas. Objetivos. 1. Descrever a frequência dos polimorfismos de (SNPs) IFNG +874 T/A, IL12B 3\' UTR +1188 A/C e IL12RB1 11014 A/G no éxon 7 em pacientes e controles; 2. Investigar a associação entre esses polimorfismos e as diferentes formas clínicas da micose; 3. Verificar se há associação entre esses polimorfismos e a secreção das citocinas IFN-y, IL-12p40 e IL-12p70. Materiais e Métodos. 143 pacientes com PCM foram incluídos (40 com a forma aguda, 100 com a forma crônica multifocal e 17 unifocal). Critérios de inclusão: ter doença ativa (DA) comprovada por exame micológico ou histopatológico positivo ou presença de anticorpos anti-Paracoccidioides brasiliensis (>= 1/32 por contraimunoeletroforese) ou ter doença curada/tratada (CT) quando comprovada anteriormente pelos critérios de DA e atualmente com títulos de anticorpos estáveis e <= 4 em dois períodos com intervalo >= 6 meses. Analisaram-se os SNPs IFNG pela técnica de PCR-ARMS (\"Polymerase Chain Reaction - Amplification Refractory Mutational System\"), IL12B e IL12RB1 por RFLP (\"PCR-Restriction Fragment Lenght Polymorphism\"). Para a dosagem de citocinas foram utilizadas as técnicas de ELISA (n=29) e CBA (\"Cytometric Bead Array\"; n= 18), sendo considerados estatisticamente significantes, os valores de p < 0,05 para os testes de x2 e o teste de Kruskal-Wallis, com pós-teste de Dunn. Resultados. O genótipo AA do SNP IL12RB1 foi mais frequente na forma crônica multifocal e o genótipo AG, na forma unifocal masculina (p= 0,048). À análise desta forma clínica entre ambos os sexos, o genótipo AG foi também mais frequente no sexo masculino (p= 0,009). Segundo a etnia, foi demonstrada diferença estatisticamente significante nas frequências dos genótipos e alelos dos SNPs IFNG e IL12RB1 (p < 0,05). Em relação às formas clínicas da PCM, houve similaridade nas frequências dos genótipos e alelos dos SNPs estudados. Quanto aos níveis das citocinas, para os SNPs IFNG, IL12B e IL12RB1, maiores níveis de secreção de citocinas, frente a PHA, foram registrados nos grupos CT e CO em relação ao DA, sugerindo relação com a evolução da doença e com a imunossupressão já descrita na doença ativa. Conclusão. Não houve associação entre os SNPs IFNG, IL12B e IL12RB1 e as diferentes formas da doença quando todos os pacientes foram analisados; no sexo masculino, sugere-se que o genótipo AA esteja associado à doença crônica mais disseminada (IL12RB1). Houve diferença significante entre as etnias nos SNPs IFNG e IL12RB1, sugerindo-se a ampliação do número de pacientes em determinadas etnias e na forma clínica unifocal para melhor compreensão dessas associações / Introduction. Paracoccidioidomycosis (PCM) is a systemic chronic mycosis, endemic in Latin America, mainly in Brazil where it is the eighth cause of death among chronic recurrent infectious diseases. PCM infection is characterized by the Th1 immune response, the acute form, by a mixed Th2/Th9 profile, while the chronic form is characterized by Th17/Th22 profile. The occurrence and severity of human PCM can also be associated with genetic factors such as polymorphisms on genes of cytokines. Objectives. 1. To describe the frequencies of the single nucleotide polymorphisms (SNPs) IFNG +874 T/A, IL12B 3\'UTR +1188 A/C and IL12RB1 11014 A/G on exon 7, on patients with PCM and non-PCM controls; 2. To investigate the association between those SNPs and the different clinical forms of PCM. 3. To verify the possible association between those SNPs and the secretion of the cytokines IFN-?, IL-12p40 and IL12p70. Materials and Methods. 143 patients with PCM were included (40 with acute form, 100 with multifocal chronic form and 17 unifocal). Inclusion criteria: active disease (DA) proved by fungal identification on direct microscopy/histopathology or culture, or presence of antibodies antiParacoccidioides brasiliensis ( >= 1/32 by counterimmunoelectrophoresis) or cured/treated disease (CT) when previously proved by criteria of DA and present stable antibodies titles =6 months in between. The SNP IFNG was analyzed by PCR-ARMS (Polymerase Chain Reaction - Amplification Refractory Mutational System) and the SNPs IL12B and IL12RB1 by PCR-RFLP (PCR-Restriction Fragment Length Polymorphism). The levels of cytokines were detected by ELISA (n= 29) and CBA (Cytometric Bead Array; n= 18) and values of p < 0.05 for ?2 test and Kruskal-Wallis\' test, with Dunn\'s post-test were considered statistically significant. Results. The AA genotype of SNP IL12RB1 was the most frequent in the multifocal chronic form while the AG was more frequent in men with the unifocal chronic form of PCM (p = 0.048). On this clinical form in the comparison between genres, the AG genotype was also more frequent in men (p= 0.009). On ethnicity, it was demonstrated statistical difference between the frequencies of genotypes and alleles of SNPs IFNG and IL12RB1 (p < 0.05). In the comparison between the clinical forms of PCM, the frequencies of genotypes and alleles of the evaluated SNPs were similar. On the levels of cytokines, for SNPs IFNG, IL12B and IL12RB1, increased levels of cytokines were observed with PHA on the CT and CO groups compared with DA, suggesting a connection with the evolution of the disease and the previously described immunosuppression during active disease. Conclusion. There was no association between the SNPs IFNG, IL12B and IL12RB1 and the different forms of PCM when all patients were analyzed; among men, it is suggested that the AA genotype of IL12RB1 is associated with a more disseminated chronic disease. There was a significant difference between the ethnicities on SNPs IFNG and IL12RB1, being the latter also associated with the chronic form in men. The increase in the number of patients in certain ethnic groups and in the unifocal clinical form of PCM might help the better understanding of these associations

Introdução. A paracoccidioidomicose (PCM) é uma micose sistêmica crônica, endêmica na América Latina, principalmente Brasil, sendo a oitava causa de morte entre as doenças infecciosas crônicas recorrentes. A PCM infecção é caracterizada por uma resposta Th1, a forma aguda por um perfil misto da resposta Th2/Th9, enquanto na forma crônica caracteriza-se pelo perfil Th17/Th22. A ocorrência e gravidade da PCM humana podem também estar associadas a fatores genéticos como os polimorfismos dos genes de citocinas. Objetivos. 1. Descrever a frequência dos polimorfismos de (SNPs) IFNG +874 T/A, IL12B 3\' UTR +1188 A/C e IL12RB1 11014 A/G no éxon 7 em pacientes e controles; 2. Investigar a associação entre esses polimorfismos e as diferentes formas clínicas da micose; 3. Verificar se há associação entre esses polimorfismos e a secreção das citocinas IFN-y, IL-12p40 e IL-12p70. Materiais e Métodos. 143 pacientes com PCM foram incluídos (40 com a forma aguda, 100 com a forma crônica multifocal e 17 unifocal). Critérios de inclusão: ter doença ativa (DA) comprovada por exame micológico ou histopatológico positivo ou presença de anticorpos anti-Paracoccidioides brasiliensis (>= 1/32 por contraimunoeletroforese) ou ter doença curada/tratada (CT) quando comprovada anteriormente pelos critérios de DA e atualmente com títulos de anticorpos estáveis e <= 4 em dois períodos com intervalo >= 6 meses. Analisaram-se os SNPs IFNG pela técnica de PCR-ARMS (\"Polymerase Chain Reaction - Amplification Refractory Mutational System\"), IL12B e IL12RB1 por RFLP (\"PCR-Restriction Fragment Lenght Polymorphism\"). Para a dosagem de citocinas foram utilizadas as técnicas de ELISA (n=29) e CBA (\"Cytometric Bead Array\"; n= 18), sendo considerados estatisticamente significantes, os valores de p < 0,05 para os testes de x2 e o teste de Kruskal-Wallis, com pós-teste de Dunn. Resultados. O genótipo AA do SNP IL12RB1 foi mais frequente na forma crônica multifocal e o genótipo AG, na forma unifocal masculina (p= 0,048). À análise desta forma clínica entre ambos os sexos, o genótipo AG foi também mais frequente no sexo masculino (p= 0,009). Segundo a etnia, foi demonstrada diferença estatisticamente significante nas frequências dos genótipos e alelos dos SNPs IFNG e IL12RB1 (p < 0,05). Em relação às formas clínicas da PCM, houve similaridade nas frequências dos genótipos e alelos dos SNPs estudados. Quanto aos níveis das citocinas, para os SNPs IFNG, IL12B e IL12RB1, maiores níveis de secreção de citocinas, frente a PHA, foram registrados nos grupos CT e CO em relação ao DA, sugerindo relação com a evolução da doença e com a imunossupressão já descrita na doença ativa. Conclusão. Não houve associação entre os SNPs IFNG, IL12B e IL12RB1 e as diferentes formas da doença quando todos os pacientes foram analisados; no sexo masculino, sugere-se que o genótipo AA esteja associado à doença crônica mais disseminada (IL12RB1). Houve diferença significante entre as etnias nos SNPs IFNG e IL12RB1, sugerindo-se a ampliação do número de pacientes em determinadas etnias e na forma clínica unifocal para melhor compreensão dessas associações / Introduction. Paracoccidioidomycosis (PCM) is a systemic chronic mycosis, endemic in Latin America, mainly in Brazil where it is the eighth cause of death among chronic recurrent infectious diseases. PCM infection is characterized by the Th1 immune response, the acute form, by a mixed Th2/Th9 profile, while the chronic form is characterized by Th17/Th22 profile. The occurrence and severity of human PCM can also be associated with genetic factors such as polymorphisms on genes of cytokines. Objectives. 1. To describe the frequencies of the single nucleotide polymorphisms (SNPs) IFNG +874 T/A, IL12B 3\'UTR +1188 A/C and IL12RB1 11014 A/G on exon 7, on patients with PCM and non-PCM controls; 2. To investigate the association between those SNPs and the different clinical forms of PCM. 3. To verify the possible association between those SNPs and the secretion of the cytokines IFN-?, IL-12p40 and IL12p70. Materials and Methods. 143 patients with PCM were included (40 with acute form, 100 with multifocal chronic form and 17 unifocal). Inclusion criteria: active disease (DA) proved by fungal identification on direct microscopy/histopathology or culture, or presence of antibodies antiParacoccidioides brasiliensis ( >= 1/32 by counterimmunoelectrophoresis) or cured/treated disease (CT) when previously proved by criteria of DA and present stable antibodies titles =6 months in between. The SNP IFNG was analyzed by PCR-ARMS (Polymerase Chain Reaction - Amplification Refractory Mutational System) and the SNPs IL12B and IL12RB1 by PCR-RFLP (PCR-Restriction Fragment Length Polymorphism). The levels of cytokines were detected by ELISA (n= 29) and CBA (Cytometric Bead Array; n= 18) and values of p < 0.05 for ?2 test and Kruskal-Wallis\' test, with Dunn\'s post-test were considered statistically significant. Results. The AA genotype of SNP IL12RB1 was the most frequent in the multifocal chronic form while the AG was more frequent in men with the unifocal chronic form of PCM (p = 0.048). On this clinical form in the comparison between genres, the AG genotype was also more frequent in men (p= 0.009). On ethnicity, it was demonstrated statistical difference between the frequencies of genotypes and alleles of SNPs IFNG and IL12RB1 (p < 0.05). In the comparison between the clinical forms of PCM, the frequencies of genotypes and alleles of the evaluated SNPs were similar. On the levels of cytokines, for SNPs IFNG, IL12B and IL12RB1, increased levels of cytokines were observed with PHA on the CT and CO groups compared with DA, suggesting a connection with the evolution of the disease and the previously described immunosuppression during active disease. Conclusion. There was no association between the SNPs IFNG, IL12B and IL12RB1 and the different forms of PCM when all patients were analyzed; among men, it is suggested that the AA genotype of IL12RB1 is associated with a more disseminated chronic disease. There was a significant difference between the ethnicities on SNPs IFNG and IL12RB1, being the latter also associated with the chronic form in men. The increase in the number of patients in certain ethnic groups and in the unifocal clinical form of PCM might help the better understanding of these associations

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives (norethisterone enanthate (NET-EN); norethisterone acetate (NET-A)), designed to mimic the actions of the endogenous hormone progesterone (Prog), are extensively used by women as contraceptives and in hormone replacement therapy (HRT). A number of reports have indicated that these synthetic progestins affect immune function in the female genital tract thereby increasing the risk of acquiring sexual transmitted infections. Despite these findings, very little is known about their mechanism of action at the cellular level, in particular their steroid receptor-mediated effects on cytokine gene expression. In the first part of this thesis, the effect of Prog, MPA and NET-A on the expression of the endogenous pro-inflammatory cytokine gene, interleukin (IL)-12p40, and anti-inflammatory cytokine gene, IL-10, was investigated in a human ectocervical epithelial cell line, Ect1/E6E7. Quantitative realtime PCR (qPCR) showed that all three ligands significantly upregulated the tumor necrosis factor alpha (TNF )-induced IL-12p40 gene expression, while IL-10 gene expression was downregulated. Moreover, by reducing the glucocorticoid receptor (GR) levels with siRNA, these effects were shown to be mediated by the GR. A more detailed investigation into the molecular mechanism of the progestogen-induced upregulation of IL-12p40 gene expression, using chromatin immunoprecipitation (ChIP), siRNA, co-immunoprecipitation and re-ChIP analyses, showed that the progestogen-bound GR is recruited to the CCAAT enhancer binding protein (C/EBP)- regulatory element of the IL-12p40 promoter, most likely via an interaction with the transcription factor C/EBP . Similar experiments for the progestogen-induced downregulation of IL-10 gene expression showed that the progestogen-bound GR is recruited to the signal transducer and activator of transcription (STAT)-3 regulatory element of the IL-10 promoter, most likely via an interaction with the transcription factor STAT-3. The second part of this study elucidated the influence of the HIV-1 accessory viral protein R (Vpr) on progestogen-induced regulation of IL-12p40, IL-12p35 and IL-10 in the Ect1/E6E7 cell line. Results showed that in these cells, the overexpression of Vpr significantly modulated the effects of Prog, MPA and NET-A on the mRNA expression of IL- 12p40 and IL-10, while only the NET-A effect was modulated on IL-12p35. Moreover, reducing the GR protein levels by siRNA suggested that the GR is required by Vpr to mediate its effects. Taken together, these results show that Prog, MPA and NET-A promote the pro-inflammatory milieu in the ectocervical environment, and that during HIV-1 infections, this milieu is modulated. Furthermore, the results suggest that the use of MPA or NET in vivo may cause chronic inflammation of the ectocervical environment, which may have important implications for ectocervical immune function, and hence susceptibility to infections such as HIV-1. / AFRIKAANSE OPSOMMING: Medroksieprogesteroon asetaat (MPA), noretisteroon (NET) en derivate daarvan noretisteroon enantaat (NET-EN); noretisteroon asetaat (NET-A), ontwerp om die funksies van die natuurlike hormone progesteroon (Prog) na te boots, word wêreldwyd deur vroue as voorbehoedmiddels sowel as vir hormoon vervangingsterapie (HVT) gebruik. Daar is verskeie aanduidings dat hierdie sintetiese progestiene die immuunfunksie in die vroulike geslagskanaal kan beïnvloed en ook die moontlike vatbaarheid van seksueel oordraagbare infeksies kan verhoog. Ten spyte hiervan, is baie min bekend oor hulle meganisme van werking op ‘n molekulêre vlak, veral in die besonder hul effek op sitokinien geenuitdrukking. Die effek van Prog, MPA en NET-A op die geenuitdrukking van ’n endogene pro-inflammatoriese sitokinien, interleukin (IL)-12, en ’n anti-inflammatoriese sitokinien, IL-10, asook die onderliggend meganisme van werking, in ’n menslike ektoservikale sellyn, Ect1/E6E7, is in die eerste deel van hierdie studie ondersoek. Kwantitatiewe “realtime” polimerisasie ketting reaksie (PKR) het getoon dat al drie die ligande die tumor nekrosis faktor alfa (TNF- )-geïnduseerde IL-12p40 geenuitdrukking opreguleer en IL-10 geenuitdrukking onderdruk. Verder is gevind dat induksie van IL-12p40 en inhibisie van IL-10 deur Prog, MPA en NET-A deur die glukokortikoïed reseptor (GR) gedryf word, aangesien volledige opheffing van die effekte op hierdie sitokinien gene waargeneem is wanneer die GR proteïen vlakke deur middel van kort inmengende ribonukleïensuur (siRNS) verminder is. 'n Meer beskrywende ondersoek in die molekulêre meganisme is uitgevoer deur gebruik te maak van chromatien immunopresipitasie (ChIP), siRNS, mede-immunopresipitasie en her-ChIP analises. Hierdie resultate het voorgestel dat die progestogeen (Prog en die sintetiese progestiene)-gebonde GR tot die CCAAT verbeterende bindings protein (C/EBP)- regulatoriese element van die IL-12p40 promotor betrek word en dat die transkripsie faktor C/EBP benodig word om transkripsie van die IL-12p40 geen te aktiveer. Met betrekking tot IL-10, het die resultate voorgestel dat die progestogeen-gebonde GR tot die sein transduksie en aktiveerder van transkripsie (STAT)-3 regulatoriese element van die IL-10 promotor betrek word en dat die transkripsie faktor STAT-3 benodig word om transkripsie van die IL-10 geen te onderdruk. Die tweede deel van die studie het die invloed van die MIV-1 aksesorale virale proteïen R (Vpr) op sitokinien geenuitdrukking, spesifiek die progestogeen-geïnduseerde regulering van IL-12p40, IL-10 en IL-12p35, in die Ect1/E6E7 sellyn ondersoek. Resultate het getoon dat ooruitdrukking van Vpr in hierdie sellyn die effekte van Prog, MPA en NET-A op die mRNS uitdrukking van IL-12p40 en IL-10, en slegs die NET-A effek op IL-12p35, aansienlik moduleer. Vermindering van die GR proteïen vlakke deur middel van siRNS het getoon dat Vpr die GR benodig om hierdie veranderinge mee te bring. In samevatting, die resultate van hierdie proefskrif stel voor dat Prog, MPA en NET-A die pro-inflammatoriese milieu in die ektoservikale omgewing bevorder, en dat hierdie milieu gedurende MIV-1 infeksies verander. Verder, die resultate van hierdie studie impliseer dat die gebruik van MPA en NET in vivo nadelige lokale immuunonderdrukkende effekte mag hê wat kan lei tot kroniese inflammasie van die ektoservikale omgewing en ‘n moontlike verhoging in die vatbaarheid van infeksies soos MIV-1.

Orientador: Valéria Marçal Felix de Lima / Resumo: A Leishmaniose Visceral (LV) é causada nas Américas, pelo protozoário intracelular obrigatório Leishmania infantum e os cães domésticos são os principais reservatórios urbanos do parasita e em áreas endêmicas, o aumento da LV em humanos tem sido associado ao aumento da infecção canina. Os atuais medicamentos disponíveis para a Leishmaniose Canina (CanL) não são completamente eficientes e meses após o tratamento a maioria dos cães apresentam recidiva, indicando a necessidade de buscar formas alternativas de tratamento. Na CanL, cães desenvolvem uma resposta imune celular (Th1) ineficiente para combater o parasita e a estimulação das vias de citocinas em células de defesa com proteínas recombinantes, tem o potencial de se tornar parte de métodos imunoterapêuticos eficazes. Neste estudo, as citocinas recombinantes caninas (IL-12, IL-2, IL-15 e IL-7) e o receptor solúvel de IL-10R1 (sIL-10R1), com atividade antagonista, foram avaliadas pela primeira vez em combinações (IL-12/IL- 2, IL-12/IL-15, IL-12/sIL-10R1, IL-15/IL-7) ou isoladamente (sIL-10R1) quanto à capacidade imunomodulatória em células mononucleares do sangue periférico (sigla em inglês PBMC) de cães com leishmaniose. Todas as combinações de proteínas recombinantes testadas mostraram melhorar a resposta linfoproliferativa. Além disso, as combinações de IL-12/IL-2 e IL-12/IL-15 promoveram a diminuição na expressão da proteína “Programed Cell Death 1” (PD-1) nos linfócitos. Estas mesmas combinações de citocinas e IL-12/sI... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Visceral Leishmaniasis (LV) is caused in the Americas by the obligate intracellular protozoan Leishmania infantum and domestic dogs are the major urban reservoirs of the parasite and in endemic areas, and increase LV in humans has been associated with increased canine infection. The current medications available for Canine Leishmaniasis (CanL) are not completely effective and months after treatment most dogs present with relapse, indicating the necessity to looking for alternative forms of treatment. In CanL, dogs develop an ineffective cellular immune response (Th1) to combat the parasite. Then, the stimulation of cytokine pathways in defense cells with recombinant proteins, has the potential to become part of effective immunotherapeutic methods. In this study, the canine recombinant cytokines (IL-12, IL-2, IL-15 and IL-7) and the soluble receptor of IL-10R1 (sIL-10R1) with antagonistic activity, were evaluated for the first time in combinations IL-12/IL-2, IL-12/IL-15, IL12/sIL-10R1, IL-15/IL-7) or alone (sIL-10R1) for their immunomodulatory capacity in peripheral blood mononuclear cells (PBMC) from dogs with leishmaniasis. All combinations of recombinant proteins tested were shown to improve lymphoproliferative response. Further, combinations of IL-12/IL-2 and IL-12/IL-15 promoted decrease in programmed cell death protein 1 (PD-1) expression in lymphocytes. These same combinations of cytokines and IL-12/casIL-10R1 induced IFN-y production in PBMC. Furthermore, the combinat... (Complete abstract click electronic access below) / Mestre

Die Impfung mittels Antigen-beladener dendritischer Zellen [DZ] ist mittlerweile eine gut etablierte Technik, die dann zum Einsatz kommt, wenn Standard-Impftechniken versagen, vor Krankheiten zu schützen beziehungsweise diese zu heilen. Die Effizienz dieser Technik konnte bereits für diverse Infektionskrankheiten und Krebserkrankungen in experimentellen Tiermodellen sowie am Menschen gezeigt werden. Hierbei ist die Möglichkeit zur wohldefinierten Manipulation und Antigenbeladung der DZ ein großer Vorteil gegenüber den konventionellen Ansätzen. Jedoch ist vor allem bei der Anwendung im klinischen Bereich die Präparation, Herstellung und Manipulation dieser autologen DZ mit einem erheblichen technischen, zeitlichen sowie finanziellen Aufwand verbunden. Hinsichtlich einer Präventivimpfung gegen eine pandemische Infektionskrankheit, die in hauptsächlich unterentwickelten Ländern vorkommt, wird dieser Aufwand sicherlich ein Hindernis darstellen. Daher muss für solche Fälle ein maßgeschneiderter Impfstoff entwickelt werden, der sich am Vorbild des effektiven DZ-basierten Impfstoffs orientiert. Für die Impfung gegen die Leishmania Parasiten besteht so ein DZ-basierter Impfstoff bereits. Dessen Wirkung, eine T-Zell Antwort vom Typ Th1 zu induzieren, wurde bereits in mehreren Veröffentlichungen demonstriert. Zusätzlich hat aber eine unserer Studien gezeigt, dass das typische Th1-bezogene Zytokin IL-12 zur Differenzierung naiver T-Zellen nicht von den injizierten DZ bereitgestellt werden muss, sondern von der geimpften Maus. Dies gab erste Hinweise auf eine stärkere Beteiligung des Wirts-Immunsystems als zuvor angenommen. Daher sollte hier vertieft der Mechanismus dieser DZ-basierten Impfung untersucht werden, wobei modifizierte Impfstoff-Ansätze zum Einsatz kommen sollten. Dabei wurden die Fragen nach der vom Impfstoff transportierten Information und dem Empfänger dieser Information berücksichtigt. Das aktuelle Paradigma zur DZ-basierten Impfung besagt, dass transferierte DZ im direkten Kontakt mittels dreier Signale T-Zellen stimulieren und aktivieren. Dafür müssen diese DZ mit dem entsprechenden Antigen beladen und aktiviert worden sein um das Antigen-Peptide mittels MHC Molekül im Kontext der Co-Stimulation präsentieren zu können. Jedoch zeigt diese Studie hier, dass weder eine Aktivierung der DZ noch die Präsentation des Antigens mittels passender MHC Moleküle notwendig ist für die Induktion einer protektiven Immunantwort gegen Leishmania Parasiten. Aufgeschlossene, mit Antigen beladene DZ müssen nicht vor dem Transfer mit CpG ODN aktiviert worden sein, um entsprechende Immunität zu verleihen. Ebenso hat der MHC Typ in diesem Falle auch keinen Einfluss auf die Effektivität des Impfstoffs. Da im Weiteren aufgeschlossene mit Leishmania-Antigen beladene Makrophagen nach Impfung die gleiche Wirkung erzielen, wie vorangegangene DZ-basierte Impfstoffe, können keine DZ spezifischen Mechanismen Schlüsselkomponenten der Induktion einer protektiven Immunität sein. Darüber hinaus konnte gezeigt werden, dass die DZ der geimpften Mäuse, eine maßgebliche Rolle bei der Verarbeitung transferierter Signale spielen. Suspensionen aufgeschlossener DZ stellen eine Kombination aus freigesetzten löslichen Molekülen sowie Membranvesikeln dar, die sich nach dem Aufschluss gebildet haben. Nach Auftrennung dieser beiden Fraktionen konnte gezeigt werden, dass ausschließlich die Membran-Fraktion nach Verimpfung eine geeignete Immunantwort zum Schutz vor Leishmania Parasiten induzieren kann. Als Vorteil dieser Aufreinigung erweist sich zudem die stabile Lagermöglichkeit bei -80°C. Somit ist klar gezeigt, dass die Immunität-verleihende Einheit dieser Impfstoffvarianten in der Membran-Fraktion liegt. Verfolgt man die Induktion Th1-zugehöriger Zytokine in in vivo Experimenten so ergibt sich im Falle der Gesamtsuspension aufgeschlossener, mit Leishmania-Antigen beladener DZ ein klares Bild. Diese Suspension erzeugt das volle Spektrum der DZ-basierten Impfung gegen Leishmania Parasiten. Es kann sowohl Produktion von IL-12 und IL-2 als auch eine antigenspezifische T-Zell Proliferation nach Stimulation von Splenozyten mit der entsprechenden Suspension verzeichnet werden. Außerdem produzieren Splenozyten von entsprechend geimpften Mäusen nach Stimulation mit Leishmania-Antigen erhebliche Mengen des entscheidenden Zytokins IFNγ. Obwohl jedoch die Verimpfung aufgereinigter Membranvesikel dieses Ansatzes im Tierversuch zu biologisch sowie statistisch signifikanten Ergebnissen führt, lassen sich die entsprechend Th1-bezogenen Zytokine im in vivo Ansatz nur in geringen Maße nachweisen. Ob dies jedoch für einen in vivo unbemerkten Aktivitätsverlust des Vakzins oder für andere lymphatische Organe als Ort der T-Zell Instruktion spricht, ist noch unbekannt und muss noch geklärt werden. / Dendritic cell-based vaccination is a well established technique for preventive and therapeutic instruction of the immune system where conservative vaccine formulations fail to cure or prevent diseases, respectively. Efficiency of this technique already was demonstrated in infectious diseases as well as for cancer in animal or human studies. Well controlled manipulation and antigen-loading of immature DC is most beneficial to this technique. But, time-consuming and cost-extensive procedures for preparation of DC precursors, expansion and stimulation of DC and inpatient administration are big disadvantages regarding vaccine development for pandemic infectious diseases that occur mainly in underdeveloped countries. Therefore vaccines are needed that are pathogen-tailored and able to induce equal immune responses as their DC-based vaccine models. For vaccination against Leishmania parasites such a DC-based vaccine is feasible and its efficacy to induce protective Th1-based immune responses was already demonstrated in several animal studies. But, one of our own studies indicated supportive activity of host cells exceeding the allocation of T cells to become activated by transferred DC. IL-12, an important cytokine for the induction of Th1-related immune responses, has to be produced by host cells. Therefore, the aim of this study was to investigate the mechanism of BMDC-based vaccination with regard to simplification of the vaccine formulation. Key questions that have been addressed are: Which cells process the information that is transferred by the injected DC and what are the key components of this information? Further more, it was looked at whether altered vaccine formulations are able to induce protective immunity and whether they share equal molecular mechanisms. The current paradigm of BMDC-based vaccination proposes direct interaction of transferred BMDC with host T cells. These BMDC have to be antigen-loaded for stimulation via antigen-peptide-MHC molecule-complexes and they have to be activated for proper co-stimulation of T cells. Here, this study demonstrates that neither activation for co-stimulation nor direct interaction with adequate MHC molecules is needed for the induction of protective immunity against infection with Leishmania-parasites. Disrupted antigen-loaded BMDC are able to induce protective immunity in BALB/c mice without pre-stimulation via CpG ODN. Beyond, if BMDC were used with a different MHC-background than recipient mice then the vaccine still would be efficient in terms of reduction of footpad swelling and parasite load in draining lymph nodes. Even more, DC-specific features are no key component that leads to protective immunity as vaccination with disrupted antigen-loaded MΦ shows equal properties than before mentioned vaccine formulations. Further more, it was found that host DC play a major role in transforming the incoming signal, received from transferred antigen-loaded DC, into Th1-related stimuli and Leishmania-antigen-specific T cell activation. Suspensions of disrupted antigen-loaded DC resemble a combination of laid off soluble molecules together with exosome-like vesicles that formed after disruption of membranes. Here it was shown that separation of the membranous and soluble fractions and subsequent transfer into BALB/c mice will lead to protection of these mice against infection with L. major promastigotes only if the membranous fraction is used as vaccine. More, this vaccine formulation takes advantage of easy storage at -80°C with no need of fresh production. This clearly demonstrates that the immunity-inducing principle of disrupted DC-based vaccination lies within the membrane enclosed fraction. On a molecular level, disrupted antigen-loaded DC induce Th1-related cytokines during vaccination and as response on pathogen encounter. In vivo assays revealed IL-12 production and antigen-specific T cell proliferation among splenocytes that were stimulated with disrupted antigen-loaded DC. Splenocytes of accordingly vaccinated mice produce tremendous amounts of IFNγ after stimulation with Leishmania parasites. In summary, disrupted antigen-loaded BMDC fulfil all characteristics of DC-based vaccination against Leishmania major. But, while purification of membranes of antigen-loaded DC and subsequent transfer to BALB/c mice leads to control of the disease in the animal model, only slight levels of Th1-related cytokines are seen in the in vivo assays. Whether this points towards a loss of vaccine activity on unseen levels or unknown sites where Th1-related immunity is induced by both, complete solution and purified membranes, still has to be determined.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Tübingen, Univ., Diss., 2006.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed on June 24, 2009). Includes bibliographical references (p. 88-101).