O HDL-c é um fator de risco cardiovascular negativo e sua concentração plasmática apresenta relação inversa com a incidência de eventos cardiovasculares. Entretanto, as evidências relativas ao grupo de indivíduos com níveis de HDL-c acima do percentil 95 da população geral ainda são escassas e o impacto da hiperalfalipoproteinemia (HALP) sobre o risco cardiovascular continua representando motivo de controvérsia na literatura médica. Alguns estudos em populações específicas associam a HALP a aumento do risco cardiovascular. Ao mesmo tempo, outros estudos identificaram populações de indivíduos hipoalfalipoproteinêmicos com marcada longevidade. Assim, demonstrou-se aparente dissociação entre níveis de HDL-c e risco cardiovascular em determinadas populações, reconduzível a aspectos disfuncionais da HDL. O objetivo do presente estudo foi verificar o papel da HALP na determinação do risco cardiovascular; comparar a prevalência de doença cardiovascular subclínica, avaliada por meio da quantificação ultrassonográfica da Espessura Íntimo-Medial Carotídea (EIMC), entre portadores de HDL-c >= 90mg/dL (grupo HALP) e portadores de concentrações de HDL-c atualmente consideradas normais (entre 40 e 50mg/dL para os homens e entre 50 e 60mg/dL para as mulheres); e avaliar características e função da HDL em portadores de HALP por meio do estudo de sua composição, de sua capacidade de efluxo de colesterol, e de sua atividade anti-inflamatória e antioxidante, correlacionando estas características com a presença de doença cardiovascular subclínica avaliada por meio da determinação da EIMC, da Velocidade de Onda de Pulso (VOP) e da presença de Calcificação Arterial Coronariana (CAC) avaliada pela TCMD. Para responder estas perguntas, o presente estudo foi articulado em dois braços: Braço 1: Análise da coorte do estudo ELSA com o objetivo de determinar a prevalência de HALP em uma população geral; definir o perfil demográfico, antropométrico e metabólico dos portadores de HALP; e comparar a prevalência de doença vascular subclínica deste grupo com controles da mesma coorte com níveis normais de HDL-colesterol. Braço 2: Recrutamento de 80 voluntários hígidos e portadores de HALP para avaliação da correlação entre presença de doença vascular subclínica, e aspectos estruturais e funcionais da HDL. Em seus dois braços, o estudo levou a quatro conclusões principais: 1) Níveis marcadamente elevados de HDL-c estão associados a menor espessura íntimo-medial carotídea quando comparados a níveis de HDL-c considerados normais pelas diretrizes vigentes. Embora portadores do fenótipo HALP apresentem, como grupo, um perfil metabólico mais favorável que o encontrado em indivíduos com HDL-c normal, a associação entre EIMC e HALP foi independente dos fatores de risco tradicionais, indicando que a menor prevalência destes últimos em portadores de HDL-c marcadamente elevado justifica apenas parcialmente a menor prevalência de doença vascular subclínica neste grupo; 2) Embora a HALP se apresente como um fenótipo ateroprotetor, há indivíduos com níveis marcadamente elevados de HDL-c que evoluem com doença cardiovascular, clínica ou subclínica. Neste contexto, nossos resultados indicam correlação entre os três métodos avaliados para estudar doença vascular subclínica em portadores de HALP: EIMC, VOP e CAC; 3) Os fatores de risco tradicionais continuam exercendo seu peso na determinação do risco cardiovascular em portadores de HALP. Idade, tabagismo, hipertensão arterial, hipertrigliceridemia e altos níveis de LDL-c apresentaram associação estatisticamente significativa com a presença de doença vascular subclínica no grupo estudado; 4) A avaliação da composição e da função da HDL em portadores de HALP pode permitir identificar indivíduos especificamente mais suscetíveis à aterosclerose. Nossos resultados indicam que, em particular, a atividade anti-inflamatória da HDL, avaliada pela capacidade de inibição da produção de IL-6; o efluxo de colesterol e a capacidade de transferência de triglicérides apresentaram associação independente com menor espessura íntimo-medial carotídea em portadores de HALP, enquanto níveis mais altos de Apo A-IV se associaram a maior grau de doença cardiovascular subclínica / HDL-c is a negative cardiovascular risk factor and its plasma concentration is inversely related to the incidence of cardiovascular events. However, evidence of benefit among subjects with HDL-c levels above the 95th percentile of the general population is still scarce and the impact of hyperalphalipoproteinemia (HALP) on cardiovascular risk continues to represent matter of debate in the medical literature. Some studies with specific populations indicated an increased cardiovascular risk associated with HALP. In addition, other reports identified groups of patients with marked hypoalphalipoproteinemia and longevity. Hence, there could be a dissociation between HDL-c levels and cardiovascular risk in certain populations, possibly due to dysfunctional HDL particles. The aim of this study was to investigate the role of HALP phenotype in determining cardiovascular risk; to compare the prevalence of subclinical cardiovascular disease, assessed by ultrasound measurement of Carotid Intima-Media Thickness (CIMT) among patients with HDL-c >= 90mg/dL (HALP group) and patients with HDL-c currently considered normal (40-50mg/dL for men and 50-60mg/dL for women); and to evaluate HDL functionality in patients with HALP through the study of its composition, its cholesterol efflux capacity, and its anti-inflammatory and antioxidant activity; correlating those characteristics with the presence of subclinical cardiovascular disease assessed by CIMT, Pulse Wave Velocity (PWV) and Coronary Artery Calcification (CAC). To answer these questions, the present study was articulated into two arms: Arm 1: ELSA-Brasil study cohort analysis in order to assess HALP prevalence in a general population, defining demographic, anthropometric and metabolic profile of HALP individuals; and comparing the prevalence of subclinical vascular disease among HALP subjects with controls with normal HDL-c. Arm 2: Recruitment of 80 healthy volunteers with HALP to study the correlation between subclinical vascular disease and HDL functionality in this group. Our study led to four main conclusions: 1) markedly elevated HDL-c is associated with lower CIMT compared to the control group with normal HDL-c levels. Although individuals with HALP display a more favorable metabolic profile than subjects with normal HDL-c, the association between CIMT and HALP was independent of traditional risk factors, indicating that the lower prevalence of subclinical vascular disease in this group is only partially justified by the lower prevalence of cardiovascular risk factors; 2) Although HALP can be regarded as an atheroprotective phenotype, there are individuals with markedly elevated levels of HDL-c who develop cardiovascular disease. Our results indicate good correlation of the three methods here adopted to study subclinical vascular disease among HALP patients: CIMT, VOP and CAC; 3) Traditional risk factors continue to exert their weight in determining cardiovascular risk in patients with HALP. Age, smoking, hypertension, hypertriglyceridemia and high levels of LDL-c were significantly associated with the presence of subclinical vascular disease among HALP individuals; 4) the assessment of the HDL composition and functionality in patients with HALP may allow to identify individuals specifically more susceptible to atherosclerosis. Our results indicate that, in particular, cholesterol efflux capacity, the anti-inflammatory activity of HDL, and triglyceride transfer capacity were independently associated with lower CIMT in HALP individuals, while higher levels of Apo A-IV were associated with a greater burden of subclinical cardiovascular disease

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Multiple Sclerosis (MS) is a Central Nervous System disease, mediated by the Immune System, whose symptoms occur in episodes of relapses. Interferon-beta (IFN-β) is considered a safe treatment for the reduction of relapses, but its mechanisms of action have not yet been clear. Studies have shown involvement of tumor necrosis factor alpha (TNF-α) and of Interleukin-10 (IL-10) in the immunopathogenesis of MS. The role of IL-32, a proinflammatory cytokine has role on several chronic inflammatory diseases, was not elucidated in MS. The effect of IFN-β on these cytokines and disease severity, as measured by the Expanded Disability Status Scale (EDSS), has not yet been established. Objective. The objective of the present study was to evaluate TNF-α, IL-10 and IL-32γ concentrations in the peripheral blood and gene expression of patients with IFN-β. Methods. The sample were patients of the Department of Neurology of the Clinics Hospital of the Federal University, and healthy individuals. Blood collection, blood culture with lipopolysaccharide (LPS), Toll 4 receptor agonist (TLR4), and PAM3Cys, TLR2 agonist, and the quantification of cytokines by real-time polymerase chain reaction were performed. Mann Whitney tests were used for statistical analysis of unpaired data, Wilcoxon for paired samples and Spearman's correlation test, adopting significance level p <0.05. Results. Of the 30 MS patients, 19 were treated with IFN-β and 11, untreated, with a mean age of 40.52 and 42 years, respectively, and female prevalence. TNF-α did not differ between groups but it was less produced after stimulation with Pam3Cys in treated patients compared to controls and untreated patients. IL- 10 concentrations were higher in cultures with LPS in patients treated compared to healthy controls. The mean EDSS of patients treated with IFN-β and untreated did not differ, and the correlation between and TNF-α and IL-10 concentrations produced in blood cultures and EDSS was not significant in the patients. There was a significant correlation between TNF-α concentrations and disease time in untreated patients in non-stimulated cultures and those with TLR2 agonist stimulus. Gene expression of IL-32γ was higher in IFN-β treated patients compared to controls. The gene expression of cytokines correlated positively and significantly in patients and controls and the IL-10 expression was correlated negative e significantly with the disease time in untreated patients. Conclusions. IFN-β reduced patients' response to Pam3Cys. IL-10 was higher in treated patients relative to controls. The correlations were not conclusive about the possible association between these cytokines and the clinic parameters of the disease. IL-32γ was higher in patients treated with IFN-β than in healthy subjects. / Introdução. A Esclerose Múltipla (EM) é uma doença do Sistema Nervoso Central, mediada pelo Sistema Imune,cujos sintomas ocorremem episódios de surtos.OInterferon-beta (IFN-β) é considerado um tratamento seguro para redução dos surtos, masseus mecanismos de ação ainda não foram bemesclarecidos. Estudos mostraram participaçãodo fator de necrose tumoral alfa (TNF-α) e da Interleucina-10 (IL-10) na imunopatogênese da EM. O papel da IL-32, citocina pró-inflamatória atuante emvárias doenças inflamatórias crônicas, não foi elucidado na EM. O efeito do IFN-β nestas citocinas e na gravidade da doença, medida pela Escala do Estado de Incapacidade Expandida (EDSS), ainda não foi estabelecido. Objetivo.O objetivo do presente estudo foi avaliar as concentrações de TNF-α, IL- 10 e IL-32γ no sangue periférico de pacientes com EM tratados com IFN-β e não tratados. Métodos.Foram recrutados portadores da doença, no Serviço de Neurologia do Hospital das Clínicas da Universidade Federal,e indivíduos sadios. Foi feita a coleta de sangue, hemoculturas com lipopolissacarídeo (LPS), agonista do receptor do tipo Toll 4 (TLR4), e PAM3Cys, agonista de TLR2, e a quantificação gênica das citocinas por reação em cadeia da polimerase em tempo real. Foram utilizados os testes Mann Whitney, para análise estatísticados dados não pareados, Wilcoxon para as amostras pareadas e o teste de correlação de Spearman, adotando nível de significância p<0,05. Resultados. Dos 30 pacientes com EM, 19 eram tratados com IFN-β e 11, não tratados, com idade média de 40,52 e 42 anos, anos, respectivamente, e prevalência do sexo feminino. As concentrações de IL-10 foram mais elevadasnas culturas dos pacientes tratados com IFN-β estimuladas com LPS comparado aos controles sadios.TNF-α não diferiu entre os grupos, mas foi menos produzido após estimulação com Pam3Cys nos pacientes tratados comparado aos controles e aos pacientes não tratados. O EDSS médio dos pacientes tratados com IFN-β e dos não tratados não diferiu, e a correlação entre e as concentrações de TNFα e IL-10 produzidas nas hemoculturas e o EDSS não foi significante nos pacientes. Houve correlação significante entre as concentrações de TNF-α e o tempo de doença nos pacientes não tratados nas culturas não estimuladas e naquelas com estímulo de agonista de TLR2. A expressão gênica de IL-32γ foi mais elevada nos pacientes tratados com IFN-β comparado aos controles. As expressões gênicas das citocinas se correlacionaram positiva e significantemente nos pacientes e controles e a expressão de IL-10 se correlacionou negativa e significantemente com o tempo de doença nos pacientes não tratados. Conclusões. O IFN-β reduziu a resposta dos pacientes ao Pam3Cys. IL-10 foi mais elevada nos pacientes tratados em relação aos controles. As correlações não foram conclusivas sobre a possível associação entre essas citocinas e os parâmetros clínicos da doença. IL-32γ foi mais elevada nos pacientes tratados com IFN-β em relação às pessoas sadias.

A desnutrição é uma condição nutricional que ainda constitui um grande problema de saúde publica e acomete grande parte da população mundial. Atualmente, sabe-se que vários aspectos da resposta imunológica podem estar alterados na decorrência da desnutrição, dentre eles alterações funcionais como redução da migração celular, fagocitose, atividade bactericida, bem como alterações na produção de espécies reativas do oxigênio e nitrogênio, além de comprometimento da expressão de importantes receptores de reconhecimento de patógenos e alteração na produção de citocinas pró-inflamatórias, dentre elas o TNF-&#945;. O TNF-&#945; é uma citocina, produzida principalmente por macrófagos, que participa de uma ampla gama de atividades biológicas, incluindo os processos de inflamação, crescimento, diferenciação e apoptose, essa citocina exerce seus efeitos através da ligação a dois receptores distintos, TNFR-I e TNFR-II. A ativação via receptor TNFR-I, no entanto, é responsável pela maioria dos efeitos do TNF-&#945;, e desencadeia uma série de eventos intracelulares que resultam, principalmente, na ativação do fator de transcrição NF&#954;B. Considerando a relevância dessa via de sinalização mediada por TNF-&#945;, avaliamos em um modelo de desnutrição protéica a via de sinalização de ativação do fator de transcrição NF&#954;B mediada pelo TNF-&#945; via TNFR-I. Sendo assim, utilizamos camundongos Balb/c, machos adultos, submetidos à desnutrição protéica, após perda de aproximadamente 20% do peso corpóreo, foram coletadas macrófagos peritoneais e cultivados ou não com TNF-&#945;, o sobrenadante foi utilizado para avaliação da produção de citocinas (IL-1&#945;, IL-1&#946;, IL-6, IL-12, IL-10) e as células utilizadas para avaliação da expressão do TNFR-I, bem como a expressão de proteínas da via de sinalização (TRADD, TRAF, RIP, IKK, IKB&#945;, p IKB&#945;, NF&#954;B e p NF&#954;B). Os resultados obtidos mostraram que os animais desnutridos apresentaram anemia, leucopenia, diminuição da celularidade medular e da cavidade peritoneal. Os resultados demonstraram redução da expressão de TNFR-I, além de diminuição da expressão das porções fosforiladas de IKB&#945; e NF&#954;B, associado a uma diminuição na produção de IL-1&#946; e IL-12. Nesse trabalho compreendemos aspectos relacionados a resposta imune inata e nosso dados permitem inferir que o estado nutricional interfere no estado de ativação de macrófagos e na capacidade de resposta dessas células. / Malnutrition is a nutritional condition that consists in a major public health issue and affects a considerable part of the global population. Currently, It is known that many aspects of the immunological response can be altered due to the malnutrition impairment, among them functional changes, for instance, cell migration reduction, phagocytosis, bactericidal response, as well as changes in the reactive oxygen and nitrogen species production, besides, substantially cell receptors expression impaired, responsible for the recognition of pathogens and changes in the production of proinflammatory cytokines, such as TNF-&#945;. This cytokine is primarily produced by macrophages, and it is associated to a wide range of biological activities, including the inflammation processes, growing, differentiation, and apoptosis. The TNF-&#945; act through the linkage to distinct receptors, TNFR-I and TNFR-II. The TNFR-I receptor activation, however, is responsible for most TNF-&#945; effects, and triggers a series of intracellular events that mainly result in the activation of the NF&#954;B transcription factor. Regarding the relevance of this signaling pathway mediated by the TNF-&#945;, we evaluated through a protein malnutrition model the signaling pathway for the NF&#954;B transcription factor mediated by the TNF-&#945; through TNFR-I. Adult male Balb/c mice, were submitted to a protein malnutrition and after a loss of nearly 20% body weight, peritoneal macrophages were collected and cultivated, or not with TNF-&#945;. The supernatant was collected for cytokine production evaluation (IL-1&#945;, IL-1&#946;, IL-6, IL-12, and IL-10) and the cell for TNFR-I expression evaluation, as well as the proteins of the signaling pathway (TRADD, TRAF, RIP, IKK, IKB&#945;, p IKB&#945;, NF&#954;B, and p NF&#954;B). The compiled results highlights that the malnourished animals present signs of anemia, leukopenia, and decrease of bone marrow cellularity and peritoneal cavity. The results presented reduction of the TNFR-I expression, in addition to the phosphorylated portions of the IKB&#945; and the NF&#954;B expression, associated to a decrease in the production of IL-1&#946; and IL-12. In this essay we perceive the aspects related to the innate immune response, and the outcome data allowed us to conclude that the nutritional status interferes on the macrophages activation and the response capabilities of these cells.

Das kolorektale Karzinom (KRK) zählt zu den immunogenen Tumoren und zeichnet sich durch eine ausgeprägte Infiltration von verschiedenen Immunzell-Populationen aus. Dabei scheinen insbesondere CD8+ T-Lymphozyten und CD4+ T-Helfer-Zellen Typ 1 das Tumorwachstum zu beeinflussen und spielen somit eine zunehmende Rolle als prognostische Marker. Dementsprechend ergaben sich mehrere Hinweise, dass eine hohe Frequenz dieser beiden T-Zell-Populationen in KRK-Geweben mit einer erhöhten Überlebensrate assoziiert ist. Diese neuen Erkenntnisse könnten zukünftig in die Klassifikation des KRKs einfließen und therapeutische Entscheidungen beeinflussen. Im Gegensatz zu Tumor-infiltrierenden T-Zellen ist jedoch über die Frequenz und die Eigenschaften von nativen humanen dendritischen Zellen (DCs) in Kolonkarzinom-Geweben und deren mögliche Rolle in der immunologischen Abwehr von Tumoren nur sehr wenig bekannt. Als professionelle antigenpräsentierende Zellen spielen DCs eine Schlüsselrolle bei der Induktion und Aufrechterhaltung einer Tumor-gerichteten Immunantwort und können dadurch die Tumorentwicklung wesentlich beeinflussen. Daher wurden im Rahmen dieser Dissertation erstmalig Frequenz, Verteilung, Reifestatus und Zytokinexpression von 6-sulfo LacNAc+ (slan) DCs in Kolonkarzinom-Geweben sowie in korrespondierenden tumorfreien Kolon-Geweben untersucht. SlanDCs stellen eine große Subpopulation von humanen Blut-DCs dar, die nach Aktivierung hohe Konzentrationen von verschiedenen proinflammatorischen Zytokinen sezernieren. Darüber hinaus sind sie effizient in der Lage, die antitumoralen Eigenschaften von CD8+ T-Lymphozyten und CD4+ T-Helfer-Zellen sowie von Natürlichen Killer-Zellen zu fördern. Ausgehend von diesen Eigenschaften könnten slanDCs einen Beitrag zur Immunabwehr des Kolonkarzinoms leisten und somit das Tumorwachstum beeinflussen. Im Rahmen dieser Arbeit wurde zunächst mit Hilfe immunhistochemischer Färbungen der Nachweis von slanDCs in Kolonkarzinom-Geweben erbracht. In diesem Zusammenhang konnte eine höhere Frequenz von slanDCs in Kolonkarzinom-Geweben (Mittelwert: 16,69 slanDCs/mm2, n=38) im Vergleich zu den korrespondierenden tumorfreien Geweben (Mittelwert: 9,25 slanDCs/mm2, n=38) detektiert werden. Des Weiteren wurde eine höhere Dichte von infiltrierenden slanDCs in Kolonkarzinom-Gewebeproben (Mittelwert: 18,85 slanDCs/mm2, n=20) im Vergleich zu plasmazytoiden DCs (Mittelwert: 4,86 pDCs/mm2, n=20), welche eine andere Subpopulation von humanen DCs im Blut repräsentieren, nachgewiesen. Ausgehend von diesen Erkenntnissen erfolgten verschiedene Immunfluoreszenzfärbungen zur Untersuchung des Reifestatus und der Zytokinexpression der Kolonkarzinom-infiltrierenden slanDCs. Dabei konnten in allen zehn untersuchten Tumorgeweben CD83-exprimierende slanDCs detektiert werden (Mittelwert: 46,7 % CD83+ slanDCs), was auf einen reifen Phänotyp dieser DCs hinweist. Zudem erfolgte der Nachweis einer Interleukin (IL)-23-Expression in variabler Ausprägung durch infiltrierende slanDCs in zehn von elf analysierten Kolonkarzinom-Geweben (Mittelwert: 33,8 % IL-23+ slanDCs). Dabei stellte sich heraus, dass slanDCs einen wesentlichen Anteil der IL-23-exprimierenden Zellen in einigen untersuchten Gewebeproben darstellen. Eine Expression von Tumornekrosefaktor durch Kolonkarzinom-infiltrierende slanDCs wurde hingegen nur in einer geringen Frequenz detektiert. Weitere Untersuchungen identifizierten slanDCs als neue zelluläre Komponente der T-Zell-Zone von tertiären lymphoiden Strukturen (TLS) der Tumorumgebung des Kolonkarzinoms. Darüber hinaus wies ein deutlicher Anteil der dort lokalisierten slanDCs einen reifen Phänotyp oder eine Expression von IL-23 auf. Ausgehend von diesen neuen Ergebnissen könnten die infiltrierenden slanDCs an der Modulation einer adaptiven Immunantwort in der T-Zell-Zone Kolonkarzinom-assoziierter TLS beteiligt sein und einen Einfluss auf das Tumorwachstum ausüben. Weiterhin könnte die Expression des proinflammatorischen Zytokins IL-23 durch slanDCs im Tumor-umgebenden Stroma und in den TLS eine Induktion IL-17-produzierender Zellen fördern und damit auf eine Beteiligung der slanDCs an einem entzündungsbedingten Fortschreiten der Tumorerkrankung über die IL-23/IL-17-Achse hindeuten. Insgesamt leisten die gewonnenen Erkenntnisse einen Beitrag zum Verständnis der Rolle von humanen nativen DCs im Kolonkarzinom und könnten die Entwicklung neuer immuntherapeutischer Strategien in der Behandlung dieser Tumorerkrankung fördern. / Colorectal cancer as an immunogenic tumor is characterized by a marked infiltration of different immune cell populations. Especially CD8+ T-lymphocytes and CD4+ T helper cells type 1 seem to influence tumor growth and therefore play an increasing role as prognostic markers. Thus, it has been shown that high densities of these T cell subsets are associated with improved survival of colorectal cancer patients. These new insights could become part of the classification of colorectal cancer and influence therapeutic decisions. Despite these studies, little is known about the frequency and properties of native human dendritic cells (DCs) in colon cancer tissues and their potential role in antitumor immunity. DCs as professional antigen-presenting cells are critical for the induction and maintenance of antitumor immunity and can essentially influence tumor progression. Thus, the frequency, distribution, maturation, and cytokine expression of 6-sulfo LacNAc+ (slan) DCs in colon cancer tissues as well as in corresponding tumor-free colon specimens were investigated. SlanDCs represent a subset of human blood DCs that secrete large amounts of proinflammatory cytokines upon activation. Furthermore slanDCs are able to efficiently activate CD4+ T cells, tumor-reactive CD8 + T cells, and natural killer cells. Due to these functional properties, slanDCs may contribute to antitumor immunity and may influence tumor growth. Within this doctoral thesis the presence of slanDCs in primary colon cancer samples was immunohistochemically verified. In this context, a higher frequency of slanDCs in colon cancer tissues (mean: 16,69 slanDCs/mm2, n=38) in comparison to the corresponding tumor-free specimens (mean: 9,25 slanDCs/mm2, n=38) could be detected. Moreover, higher frequencies of infiltrating slanDCs in colon cancer tissues (mean: 18,85 slanDCs/mm2, n=20) were detectable compared to plasmacytoid DCs (mean: 4,86 pDCs/mm2, n=20), representing another human blood DC-subset. Based on these results, various immunofluorescence stainings were performed to investigate maturation and cytokine expression of the infiltrating slanDCs. SlanDCs expressing the maturation marker CD83 were detected in all 10 analyzed colon cancer tissues (mean: 46,7% CD83+ slanDCs). In addition, IL-23-expressing slanDCs were present at varying percentages in 10 of 11 evaluated colon cancer samples (mean: 33,8% IL-23+ slanDCs). Interestingly, in several tissues slanDCs represented a marked proportion of all IL-23-expressing cells. However, slanDCs expressing tumor necrosis factor could only be detected in low frequencies in the analyzed colon cancer specimens. Further studies revealed that slanDCs are a novel component of the T-cell zone of colon cancer-associated tertiary lymphoid structures (TLS). A proportion of these TLS-associated slanDCs displays a mature phenotype or express IL-23. These novel findings indicate that slanDCs may modulate adaptive immune responses in the T-cell zone of colon cancer-associated TLS and may contribute to the regulation of tumor progression. Furthermore the IL-23-expressing slanDCs in the tumor-surrounding stroma and the TLS may promote the generation of IL-17-producing cells and may participate in inflammation-related cancer progression mediated by the IL-23/IL-17 axis. These novel observations can help to decipher the role of human native DCs in colon cancer and may have implications for the design of therapeutic strategies against this tumor entity.

L’utilisation d’agents anti-TNF a grandement amélioré la prise en charge de certaines maladies inflammatoires chroniques comme la polyarthrite rhumatoïde (PR) ou les maladies inflammatoires chroniques de l’intestin (MICI). Cependant, le quart des patients environ ne vont pas répondre au traitement ou présenteront une perte de réponse secondaire. Des marqueurs prédictifs de réponse sont nécessaires afin limiter les effets secondaires et les coûts inutiles en ciblant les patients qui pourraient être améliorés par les anti-TNF. Ces travaux de recherche se sont dans un premier temps concentrés sur l’importance de l’immunogénicité de ces traitements. Des anticorps anti-médicaments (ADAs) étaient bien associés à un taux bas d’anti-TNF avec des conséquences cliniques en termes de perte de réponse clinique et d’absence de cicatrisation muqueuse dans les MICI. Des seuils cliniques d’interprétation des tests biologiques pour la détection du médicament et de ses anticorps ont pu être définis et correspondent à 4.9 μg/ml pour l’infliximab et 200 ng/ml pour les ADAs. Ces résultats obtenus par ELISA sont bien corrélés avec les tests fonctionnels réalisés en parallèle et confirment l’intérêt de cette technique dans ce dépistage. Les ADAs étaient diminués par traitement immunosuppresseur concomitant. Ensuite, la persistance d’une infection chronique mise en évidence par des anticorps anti-bactériens a été évaluée en tant que marqueur prédictif de réponse aux anti-TNF. Aucun résultat statistiquement significatif n’a pu être relevé sur ces premières données, que ça soit pour les anticorps dirigés contre la flore intestinale pour les MICI ou contre le microbiote oral dans la PR. Seul un taux élevé de MMP-3 à l’initiation de l’infliximab chez les patients PR prédisait d’une bonne réponse clinique selon les critères de l’EULAR par la suite / The use of anti-TNF agents has greatly improved the management of chronic inflammatory diseases such as rheumatoid arthritis (RA), or chronic inflammatory bowel disease (IBD). However, about a quarter of patients will not respond to treatment or will present a secondary loss of response. Predictive biomarkers of response are needed to reduce side effects and unnecessary costs by targeting patients that could be improved by anti-TNF. This research work was initially focused on the importance of immunogenicity of these treatments. Anti-drug antibodies (ADAs) were well associated with low levels of anti-TNF with clinical consequences in terms of loss of clinical response and absence of mucosal healing in IBD. Clinical thresholds for drug and ADAs have been defined and correspond to 4.9 μg/ml for infliximab and 200 ng/ml for ADAS. These results obtained by ELISA correlate well with functional tests done in parallel, and confirm the value of this technique for screening. The ADAs were decreased with concomitant immunosuppressive therapy. Then, the persistence of chronic infection as evidenced by anti-bacterial antibody was evaluated as a predictive marker for response to anti-TNF. No statistically significant results could be raised on these first data, for antibodies against the intestinal flora in IBD or against the oral microbiota in RA. Only high levels of MMP-3 at the initiation of infliximab in RA patients predicted a good clinical response according to the EULAR criteria

O objetivo desse trabalho foi estudar os mecanismos que alteram a produção de melatonina na glândula pineal durante o processo de caquexia associada ao câncer e o papel de citocinas neste contexto. Os resultados mostraram um aumento da produção de melatonina no grupo inoculado com o Tumor de Walker 256 (GT) junto com uma maior atividade e expressão gênica da enzima AA-NAT, principal enzima reguladora da síntese de melatonina. O estudo in vitro mostrou que a glândula do GT produziu menos melatonina que a glândula do grupo controle (GC) após estimulação com noradrenalina (NOR). Além disso, o TNF-<font face=\"symbol\">a foi capaz de modular a síntese de melatonina em cultura, promovendo um efeito estimulatório 2h após o inicio da estimulação com NOR, e um inibitório 4h após essa estimulação. Dessa forma, os resultados indicam que produtos na circulação do rato do GT estão envolvidos na modulação encontrada in vivo e que o TNF<font face=\"symbol\">a- é um forte candidato a participar dessa modulação promovida pelo estabelecimento da síndrome da caquexia sobre a produção de melatonina na glândula pineal. / The purpose of this work was to investigate the mechanisms that modify melatonin synthesis in pineal gland during cancer related cachexia and the involvement of cytokines in this context. The results showed an increment of melatonin production in the group inoculated with Walker 256 Tumor (GT) together with a higher activity and gene expression of AA-NAT enzyme, main enzyme that regulates melatonin synthesis. The in vitro study showed that glands from GT produced less melatonin than glands of the control group (GC) after noradrenalin (NOR) stimulation. Besides, TNF-<font face=\"symbol\">a was capable to modulate melatonin synthesis in pineal gland culture, promoting a stimulatory effect 2 hours after NOR stimulation and an inhibitory effect 4 hours after this stimulation. Therefore, the results indicate that products from tumor bearing rat\'s circulation are probably involved in the modulation found in vivo and that TNF-<font face=\"symbol\">a is a strong candidate to participate in cachexia-related modulation of melatonin production in pineal gland.

Background: Horses are much predisposed and susceptible to excessive and acute inflammatory responses that cause the recruitment and stimulation of polymorphnuclear granulocytes (PMN) together with peripheral blood mononuclear cells (PBMC) and the release of cytokines. The aim of the study is to develop easy, quick, cheap and reproducible methods for measuring tumor necrosis factor alpha (TNF-α) and interleukin-1 receptor antagonist (IL-1Ra) in the equine whole blood cultures ex-vivo time- and concentration dependently. Results: Horse whole blood diluted to 10, 20 and 50 % was stimulated with lipopolysaccharide (LPS), PCPwL (a combination of phytohemagglutinin E, concanavalin A and pokeweed mitogen) or equine recombinant TNF-α (erTNF-α). TNF-α and IL-1Ra were analyzed in culture supernatants, which were collected at different time points using specific enzyme-linked immunosorbent assays (ELISA). Both cytokines could be detected optimal in stimulated 20 % whole blood cultures. TNF-α and IL-1Ra releases were time-dependent but the kinetic was different between them. PCPwL-induced TNF-α and IL-1Ra release was enhanced continuously over 24–48 h, respectively. Similarly, LPS-stimulated TNF-α was at maximum at time points between 8–12 h and started to decrease thereafter, whereas IL-1Ra peaked later between 12–24 h and rather continued to accumulate over 48 h. The equine recombinant TNF-α could induce also the IL-1Ra release. Conclusions: Our results demonstrate that similar to PCPwL, LPS stimulated TNF-α and IL-1Ra production time-dependently in whole blood cultures, suggesting the suitability of whole blood cultures to assess the release of a variety of cytokines in health and diseases of horse.

Einleitung Zytokine bewirken maßgeblich die Kommunikation und Koordination der zellulären und humoralen Effektorsysteme der angeborenen und erworbenen Immunität. Die Immunzellen stellen selbst die Hauptproduzenten der Zytokine dar. Pro- und anti-inflammatorische Zytokine nehmen nicht nur innerhalb der Zellkommunikation ablaufender Immun- und Entzündungsreaktionen eine Schlüsselrolle ein, sondern sind somit ebenso am Ablauf von Pathogenesen zahlreicher Erkrankungen beim Pferd beteiligt. Trotz verschiedener Studien anhand unterschiedlicher Modelle existiert keine einheitliche Datenlage zu validierten, vergleichbaren in vivo-nahen Zellkultursystemen, die es erlauben die Kinetiken equiner Zytokine als Grundlage zur weiterführenden Erforschung von Zytokinwechselwirkungen sowie zur Testung potentieller Arzneimittel abzubilden. Aktuell werden insbesondere Glukokortikoide weiterhin aufgrund ihrer anti-inflammatorischen und immunmodulatorischen Eigenschaften häufig, aber in Bezug auf Zytokine unspezifisch zur Therapie equiner Erkrankungen eingesetzt. Ziele der Untersuchung Das Ziel der vorliegenden Studie bestand darin, eine einfache, schnelle, günstige und reproduzierbare Methodik zur ex vivo-Messung von Zytokinen (Tumornekrosefaktor-alpha [TNF-α], Interleukin-1 Rezeptor-Antagonist [IL-1Ra] und Interferon gamma [IFN-γ]) und deren zeit- und konzentrationsabhängige Freisetzung in der equinen Vollblutzellkultur zu entwickeln. Anhand dessen sollte weiterführend der Effekt der Glukokortikoide Dexamethason (DEX) und Hydrocortison (HC) auf die Produktion von TNF-α, IL-1Ra und IFN-γ im equinen Vollblut untersucht werden. Zurzeit sind Zytokine, ihre Freisetzung sowie das Eintreten ihrer vermittelten Effekte Gegenstand der gegenwärtigen Forschung, mit dem Ziel spezifische Wechselwirkungen aufzuzeigen und somit zielgerichtete Therapeutika etablieren zu können. Insbesondere Pferde, die eine Anfälligkeit gegenüber mit Sepsis einhergehenden Erkrankungen oder für equines Asthma aufweisen, könnten davon profitieren. Material und Methoden Hierfür wurde Pferdevollblut, in den Verdünnungen zu 10%, 20% und 50% eingesetzt und mit Lipopolysaccharid (LPS), einer Kombination aus Phytohämagglutinin, Concanavalin A, Pokeweed-Mitogen und LPS (PCPwL) oder equinem rekombinantem TNF-a (erTNF-α) zur Zytokinfreisetzung stimuliert. Zur Erstellung der Zytokinkinetiken wurden Zellkulturüberstände zu verschiedenen Zeitpunkten gesammelt und die Konzentrationen von TNF-α, IL-1Ra und IFN-γ mit spezifischen enzyme-linked immunosorbent assays (ELISA) analysiert. In weiterführenden Versuchen wurden in der etablierten equinen Vollblutzellkultur DEX und HC in Konzentrationen von 10-12 - 10-5 M eingesetzt, um die LPS- oder PCPwL- induzierte Zytokinfreisetzung zu modulieren. Statistische Analysen erfolgten über die Berechnungen der Mittelwerte mit den dazugehörigen Standardfehlern. Signifikanzen wurden über ein- und zweifaktorielle ANOVA bestimmt. Ergebnisse Die durchgeführten Untersuchungen ergaben, dass die optimale Detektion der Zytokine in equinen Vollblutzellkulturen mit einem Blutanteil von 20% durchgeführt werden kann. TNF-α, IL-1Ra und IFN-γ wurden zeitabhängig freigesetzt und zeigten unterschiedliche Freisetzungskinetiken. Die PCPwL- induzierte TNF-α- und IL-1Ra-Freisetzung stiegen jeweils kontinuierlich über 24 - 48 Stunden an. In ähnlicher Weise erreichte die LPS- stimulierte TNF-α-Konzentration ein Maximum zu Zeitpunkten zwischen 8 - 12 Stunden und begann daraufhin abzufallen, wohingegen die Konzentration von IL-1Ra 24 Stunden später gipfelte und vielmehr fortgeführt über 48 Stunden hinaus akkumulierte. Equines rekombinantes TNF-α konnte ebenso die IL-1Ra-Freisetzung induzieren. Die PCPwL-induzierte IFN-γ-Freisetzung begann zeitversetzt und verlief kontinuierlich ansteigend über 48 - 72 Stunden. In weiterführenden konzentrationsabhängigen Untersuchungen konnte anhand der equinen Vollblutzellkultur eine stärkere Suppression der LPS-induzierten TNF-α- und IL-1Ra-Produktion sowie der PCPwL-induzierten IFN-γ-Produktion durch DEX als durch HC nachgewiesen werden. DEX hemmte die Zytokinfreisetzung mit einer mittleren inhibitorischen Konzentration (IC50) von 0,09 μM (TNF-α), 0,453 μM (IL-1Ra) und 0,001 μM (IFN-γ), während HC IC50 Werte von 1,45 μM (TNF-α), 2,96 μM (IL-1Ra) und 0,09 μM (IFN-γ) aufwies. Schlussfolgerungen Schlussfolgernd kann zusammengefasst werden, dass sich das Model der equinen Vollblutzellkultur hervorragend eignet, um nach erfolgreicher Mitogenstimulation den zeitabhängigen Freisetzungsverlauf von Zytokinen evaluieren zu können. Somit bietet das Model der equinen Vollblutzellkultur durch die Vorteile einer einfachen, günstigen Durchführung im in vivo-nahen, physiologischen Milieu, die Möglichkeit den Zytokinstatus gesunder sowie kranker Pferde zu beurteilen und stellt seinen Nutzen und die Verlässlichkeit unter Beweis potentielle Arzneimittel und immunologische Zusammenhänge des Pferdes untersuchen zu können.:INHALTSVERZEICHNIS I ABBILDUNGSVERZEICHNIS III TABELLENVERZEICHNIS III ABKÜRZUNGSVERZEICHNIS IV 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 3 2.1 Allgemeine wissenschaftliche Hintergründe 3 2.1.1 Das Blut und das Immunsystem des Pferdes 3 2.1.1.1 Zusammensetzung des equinen Blutes 3 2.1.1.2 Allgemeiner Aufbau des Immunsystems 4 2.1.2 Zytokine und Entzündungsreaktionen - Mediation der Immunantwort durch Zytokine und Chemokine 14 2.1.2.1 Pro-inflammatorische Zytokine: Tumornekrosefaktor-α und Interferon-γ 17 2.1.2.2 Anti-inflammatorische Zytokine: Interleukin-1 Rezeptor-Antagonist 19 2.2 Therapeutische Beeinflussung der Zytokin- und Mediator-Freisetzung 21 2.2.1 Inhibition der Zytokinfreisetzung durch Glukokortikoide 21 2.2.2 Inhibition der Zytokinfreisetzung durch weitere Pharmaka und Substanzen 23 2.2.2.1 NSAID 23 2.2.2.2 Small molecules und Anti-Zytokinantikörper 24 2.3 Equine Zellkulturmodelle zur Zytokindetektion 25 2.3.1 Stimulation der Zytokinfreisetzung 26 2.3.2 Vollblutzellkultursysteme und Systeme mit isolierten Zellen 27 2.4 Fragestellung der Dissertation 30 3 PUBLIKATIONEN 31 3.1 Freisetzungskinetik von TNF-α und IL-1Ra im equinen Vollblut 32 3.2 Modulation der Freisetzung von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur durch Glukokortikoide 40 4 DISKUSSION 45 4.1 Zytokinfreisetzung im equinen Vollblut 46 4.2 Der Einfluss von Glukokortikoiden auf die Zytokinfreisetzung 52 4.3 Schlussfolgerungen 56 4.4 Ausblick 56 5 ZUSAMMENFASSUNG 58 6 SUMMARY 60 7 LITERATURVERZEICHNIS 62 8 ANHANG 72 8.1 Freisetzungskinetik von IFN-γ 72 8.2 Konzentration von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur 72 9 DANKSAGUNG 74 / Introduction The communication and coordination between the cellular and humoral effector compartments of the innate and adaptive immunity were mainly accomplished by cytokines. Immunocompetent cells themselves represent the main source for cytokines. Pro- and anti-inflammatory cytokines not only play a pivotal role within the cell signaling of expiring immune- and inflammatory reactions but also take part in the pathogenesis of several equine diseases. Despite various studies based on different experimental setups no uniform availability of data about validated, comparable in vivo cell culture systems exists which enables the description of the kinetically time course of cytokines as foundation of further investigations of cytokine interactions as well as the testing of potential drugs. These days especially glucocorticoids are still frequently used for treatment of equine diseases because of their anti-inflammatory and immunomodulatory, but with respect to cytokines unspecific properties. Objectives of the investigations The aim of the study was to develop an easy, quick, cheap and reproducible ex vivo method for measuring cytokines (tumor necrosis factor alpha [TNF-α], interleukin-1 receptor antagonist [IL-1Ra] and interferon gamma [IFN-γ]) and their time- and concentration-dependent release in the equine whole blood cell culture. Whereby the impact of the glucocorticoids dexamethasone (DEX) and hydrocortisone (HC) on production of TNF-α, IL-1Ra and IFN-γ should be investigated subsequently. Currently, cytokines, their release and eventuation of their mediated effects are objects of actual research with the aim to reveal specific interactions and thus be able to establish purposeful therapeutic agents. This could be beneficial especially for horses which display a susceptibility to septic diseases or equine asthma. Material and Methods Therefore horse whole blood diluted to 10%, 20% and 50% was stimulated with lipopolysaccharide (LPS), a combination of phytohemagglutinin, concanavalin A, pokeweed mitogen and LPS (PCPwL) or equine recombinant TNF-α (erTNF-α). To generate cytokine kinetics TNF-α, IL-1Ra and IFN-γ were analyzed in culture supernatants, which were collected at different time points using specific enzyme-linked immunosorbent assays (ELISA). In further investigations within the equine whole blood cell culture DEX and HC were applied with concentrations between 10-12 and 10-5 M to modulate LPS- or PCPwL-induced cytokine release. Statistics were performed by calculation of means with associated standard errors. Statistical significances were assessed by one- and two-way analysis of variance. Results The evaluations revealed that cytokines could be detected optimal in whole blood cell cultures with 20% blood volume. TNF-α, IL-1Ra and IFN-γ were released time-dependently and differing kinetics were displayed. PCPwL-induced TNF-α and IL-1Ra release was enhanced continuously over 24 - 48 hours, respectively. Similarly, LPS-stimulated TNF-α was at maximum at time points between 8 - 12 hours and started to decrease thereafter, whereas IL-1Ra peaked 24 hours later and rather continued to accumulate beyond 48 hours. ErTNF-α could induce also the IL-1Ra release. PCPwL- induced IFN-γ release started time displaced and showed a continuously enhanced course over 48 - 72 hours. In subsequent investigations within equine whole blood cell culture, LPS-induced TNF-α and IL-1Ra as well as PCPwL-induced IFN-γ production were more potently suppressed concentration-dependently by DEX than by HC. DEX inhibited cytokine release with the inhibition concentration (IC50) 0.09 μM (TNF-α), 0.453 μM (IL-1Ra) and 0.001 μM (IFN-γ), whereas HC with IC50 values of 1.45 μM (TNF-α), 2.96 μM (IL-1Ra) and 0.09 μM (IFN-γ). Conclusion In conclusion our results could suggest the eminent suitability of equine whole blood cell culture to assess the release of a variety of cytokines following successful mitogen stimulation. Therefore the model of the equine whole blood cell culture provides, because of its advantages including simple and cheap performance in an in vivo close physiological ambient, the opportunity to evaluate the cytokine status of healthy and diseased horses. Furthermore it could give the proof of its benefit and reliability to evaluate potential equine drugs and immunological coherences of the horse.:INHALTSVERZEICHNIS I ABBILDUNGSVERZEICHNIS III TABELLENVERZEICHNIS III ABKÜRZUNGSVERZEICHNIS IV 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 3 2.1 Allgemeine wissenschaftliche Hintergründe 3 2.1.1 Das Blut und das Immunsystem des Pferdes 3 2.1.1.1 Zusammensetzung des equinen Blutes 3 2.1.1.2 Allgemeiner Aufbau des Immunsystems 4 2.1.2 Zytokine und Entzündungsreaktionen - Mediation der Immunantwort durch Zytokine und Chemokine 14 2.1.2.1 Pro-inflammatorische Zytokine: Tumornekrosefaktor-α und Interferon-γ 17 2.1.2.2 Anti-inflammatorische Zytokine: Interleukin-1 Rezeptor-Antagonist 19 2.2 Therapeutische Beeinflussung der Zytokin- und Mediator-Freisetzung 21 2.2.1 Inhibition der Zytokinfreisetzung durch Glukokortikoide 21 2.2.2 Inhibition der Zytokinfreisetzung durch weitere Pharmaka und Substanzen 23 2.2.2.1 NSAID 23 2.2.2.2 Small molecules und Anti-Zytokinantikörper 24 2.3 Equine Zellkulturmodelle zur Zytokindetektion 25 2.3.1 Stimulation der Zytokinfreisetzung 26 2.3.2 Vollblutzellkultursysteme und Systeme mit isolierten Zellen 27 2.4 Fragestellung der Dissertation 30 3 PUBLIKATIONEN 31 3.1 Freisetzungskinetik von TNF-α und IL-1Ra im equinen Vollblut 32 3.2 Modulation der Freisetzung von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur durch Glukokortikoide 40 4 DISKUSSION 45 4.1 Zytokinfreisetzung im equinen Vollblut 46 4.2 Der Einfluss von Glukokortikoiden auf die Zytokinfreisetzung 52 4.3 Schlussfolgerungen 56 4.4 Ausblick 56 5 ZUSAMMENFASSUNG 58 6 SUMMARY 60 7 LITERATURVERZEICHNIS 62 8 ANHANG 72 8.1 Freisetzungskinetik von IFN-γ 72 8.2 Konzentration von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur 72 9 DANKSAGUNG 74

This dissertation focuses on rationalised DNA design as a tool for the discovery and development of new therapeutic entities, as well as understanding the biological function of DNA beyond the storage of genetic information. The study is comprised of two main areas of study: (i) the use of DNA as a coding unit to illustrate the relationship between code-diversity and dynamics of self-assembly; and (ii) the use of DNA as an active unit that interacts and regulates a target protein. In the study of DNA as a coding unit in code-diversity and dynamics of self-assembly, we developed the DNA-Based Diversity Modelling and Analysis (DDMA) method. Using Polymerase Chain Reaction (PCR) and Real Time Polymerase Chain Reaction (RT-PCR), we studied the diversity and evolution of synthetic oligonucleotide populations. The manipulation of critical conditions, with monitoring and interpretation of their effects, lead to understanding how PCR amplification unfolding could reshape a population. This new take on an old technology has great value for the study of: (a) code-diversity, convenient in a DNA-based selection method, so semi-quantitation can evaluate a selection development and the population\'s behaviour can indicate the quality; (b) self-assembly dynamics, for the simulation of a real evolution, emulating a society where selective pressures direct the population's adaptation; and (c) development of high-entropy DNA structures, in order to understand how similar unspecific DNA structures are formed in certain pathologies, such as in auto-immune diseases. To explore DNA as an active unit in Tumour Necrosis Factor α (TNF-α) interaction and activity modulation, we investigate DNA's influence on its spatial conformation by physical environment regulation. Active TNF-α is a trimer and the protein-protein interactions between its monomers are a promising target for drug development. It has been hypothesised that TNF-α forms a very intricate network after its activation between its subunits and receptors, but the mechanism is still not completely clear. During our research, we estimate the non-specific DNA binding to TNF-α in the low micro-molar range. Cell toxicity assays confirm this interaction, where DNA consistently enhances TNF-α's cytotoxic effect. Further binding and structural studies lead to the same conclusion that DNA binds and interferes with TNF-α structure. From this protein-DNA interaction study, a new set of tools to regulate TNF-α's biological activity can be developed and its own biology can be unveiled.

Das kolorektale Karzinom (KRK) zählt zu den immunogenen Tumoren und zeichnet sich durch eine ausgeprägte Infiltration von verschiedenen Immunzell-Populationen aus. Dabei scheinen insbesondere CD8+ T-Lymphozyten und CD4+ T-Helfer-Zellen Typ 1 das Tumorwachstum zu beeinflussen und spielen somit eine zunehmende Rolle als prognostische Marker. Dementsprechend ergaben sich mehrere Hinweise, dass eine hohe Frequenz dieser beiden T-Zell-Populationen in KRK-Geweben mit einer erhöhten Überlebensrate assoziiert ist. Diese neuen Erkenntnisse könnten zukünftig in die Klassifikation des KRKs einfließen und therapeutische Entscheidungen beeinflussen. Im Gegensatz zu Tumor-infiltrierenden T-Zellen ist jedoch über die Frequenz und die Eigenschaften von nativen humanen dendritischen Zellen (DCs) in Kolonkarzinom-Geweben und deren mögliche Rolle in der immunologischen Abwehr von Tumoren nur sehr wenig bekannt. Als professionelle antigenpräsentierende Zellen spielen DCs eine Schlüsselrolle bei der Induktion und Aufrechterhaltung einer Tumor-gerichteten Immunantwort und können dadurch die Tumorentwicklung wesentlich beeinflussen. Daher wurden im Rahmen dieser Dissertation erstmalig Frequenz, Verteilung, Reifestatus und Zytokinexpression von 6-sulfo LacNAc+ (slan) DCs in Kolonkarzinom-Geweben sowie in korrespondierenden tumorfreien Kolon-Geweben untersucht. SlanDCs stellen eine große Subpopulation von humanen Blut-DCs dar, die nach Aktivierung hohe Konzentrationen von verschiedenen proinflammatorischen Zytokinen sezernieren. Darüber hinaus sind sie effizient in der Lage, die antitumoralen Eigenschaften von CD8+ T-Lymphozyten und CD4+ T-Helfer-Zellen sowie von Natürlichen Killer-Zellen zu fördern. Ausgehend von diesen Eigenschaften könnten slanDCs einen Beitrag zur Immunabwehr des Kolonkarzinoms leisten und somit das Tumorwachstum beeinflussen. Im Rahmen dieser Arbeit wurde zunächst mit Hilfe immunhistochemischer Färbungen der Nachweis von slanDCs in Kolonkarzinom-Geweben erbracht. In diesem Zusammenhang konnte eine höhere Frequenz von slanDCs in Kolonkarzinom-Geweben (Mittelwert: 16,69 slanDCs/mm2, n=38) im Vergleich zu den korrespondierenden tumorfreien Geweben (Mittelwert: 9,25 slanDCs/mm2, n=38) detektiert werden. Des Weiteren wurde eine höhere Dichte von infiltrierenden slanDCs in Kolonkarzinom-Gewebeproben (Mittelwert: 18,85 slanDCs/mm2, n=20) im Vergleich zu plasmazytoiden DCs (Mittelwert: 4,86 pDCs/mm2, n=20), welche eine andere Subpopulation von humanen DCs im Blut repräsentieren, nachgewiesen. Ausgehend von diesen Erkenntnissen erfolgten verschiedene Immunfluoreszenzfärbungen zur Untersuchung des Reifestatus und der Zytokinexpression der Kolonkarzinom-infiltrierenden slanDCs. Dabei konnten in allen zehn untersuchten Tumorgeweben CD83-exprimierende slanDCs detektiert werden (Mittelwert: 46,7 % CD83+ slanDCs), was auf einen reifen Phänotyp dieser DCs hinweist. Zudem erfolgte der Nachweis einer Interleukin (IL)-23-Expression in variabler Ausprägung durch infiltrierende slanDCs in zehn von elf analysierten Kolonkarzinom-Geweben (Mittelwert: 33,8 % IL-23+ slanDCs). Dabei stellte sich heraus, dass slanDCs einen wesentlichen Anteil der IL-23-exprimierenden Zellen in einigen untersuchten Gewebeproben darstellen. Eine Expression von Tumornekrosefaktor durch Kolonkarzinom-infiltrierende slanDCs wurde hingegen nur in einer geringen Frequenz detektiert. Weitere Untersuchungen identifizierten slanDCs als neue zelluläre Komponente der T-Zell-Zone von tertiären lymphoiden Strukturen (TLS) der Tumorumgebung des Kolonkarzinoms. Darüber hinaus wies ein deutlicher Anteil der dort lokalisierten slanDCs einen reifen Phänotyp oder eine Expression von IL-23 auf. Ausgehend von diesen neuen Ergebnissen könnten die infiltrierenden slanDCs an der Modulation einer adaptiven Immunantwort in der T-Zell-Zone Kolonkarzinom-assoziierter TLS beteiligt sein und einen Einfluss auf das Tumorwachstum ausüben. Weiterhin könnte die Expression des proinflammatorischen Zytokins IL-23 durch slanDCs im Tumor-umgebenden Stroma und in den TLS eine Induktion IL-17-produzierender Zellen fördern und damit auf eine Beteiligung der slanDCs an einem entzündungsbedingten Fortschreiten der Tumorerkrankung über die IL-23/IL-17-Achse hindeuten. Insgesamt leisten die gewonnenen Erkenntnisse einen Beitrag zum Verständnis der Rolle von humanen nativen DCs im Kolonkarzinom und könnten die Entwicklung neuer immuntherapeutischer Strategien in der Behandlung dieser Tumorerkrankung fördern.:Abbildungsverzeichnis IV Tabellenverzeichnis VI Abkürzungsverzeichnis VII 1 Einleitung 1 1.1 Das humane Immunsystem 1 1.2 Dendritische Zellen 2 1.2.1 Phänotyp und Funktion dendritischer Zellen 3 1.2.2 Aktivierung von T-Lymphozyten durch dendritische Zellen 4 1.2.3 Subpopulationen humaner dendritischer Zellen 6 1.3 Interaktion von Immunsystem und Tumor 8 1.4 Infiltration von Tumoren durch Immunzellen 10 1.5 Kolonkarzinome 11 1.5.1 Entwicklung, Charakteristika und Klassifikation kolorektaler Karzinome 12 1.5.2 Therapieansätze des Kolonkarzinoms 14 1.6 Zielstellung 17 2 Material und Methoden 19 2.1 Material 19 2.1.1 Chemikalien und Reagenzien 19 2.1.2 Lösungen und Puffer 19 2.1.3 Testkitsysteme 19 2.1.4 Antikörper zur Detektion von Oberflächenmolekülen 20 2.1.5 Seren 20 2.1.6 Geräte 21 2.1.7 Sonstige Materialien 21 2.1.8 Gewebeproben 21 2.2 Methoden 23 2.2.1 Vorbereitung der Gewebeproben zur Immunmarkierung 23 2.2.2 Immunhistochemischer Nachweis von slanDCs und pDCs in Kolonkarzinom-Geweben und tumorfreien Kolon-Geweben 24 2.2.3 Untersuchung der Zytokinexpression von slanDCs in Kolonkarzinom-Geweben und tumorfreien Kolon-Geweben mit Fluoreszenzfärbungen 25 2.2.4 Nachweis der Expression von CD83 durch slanDCs in Kolonkarzinom-Geweben mit einer Fluoreszenzfärbung 26 2.2.5 Analyse einer Kolokalisation von slanDCs und CD3+ T-Lymphozyten in Kolonkarzinom-Geweben 26 2.2.6 Detektion von slanDCs in tertiären lymphoiden Strukturen von Kolonkarzinom-Geweben 26 2.2.7 Nachweis der Expression von CD83 und Interleukin-23 durch slanDCs in TLS von Kolonkarzinom-Geweben 28 2.2.8 Auswertung der immunhistochemischen Färbungen von slanDCs und pDCs in Kolonkarzinom-Geweben und in tumorfreien Kolon-Geweben 29 2.2.9 Statistik 30 3 Ergebnisse 31 3.1 Nachweis von slanDCs in Kolonkarzinom-Geweben 31 3.2 Assoziation der ermittelten Frequenz infiltrierender slanDCs mit der TNM-Klassifikation des Kolonkarzinoms 37 3.3 Nachweis von pDCs in Kolonkarzinom-Geweben 39 3.4 Expression von CD83 durch slanDCs in Kolonkarzinom-Geweben 42 3.5 Expression von Interleukin-23 durch slanDCs in Kolonkarzinom-Geweben 44 3.6 Expression von Tumornekrosefaktor durch slanDCs in Kolonkarzinom-Geweben 47 3.7 Kolokalisation von slanDCs und CD3+ T-Lymphozyten in Kolonkarzinom-Geweben 50 3.8 Detektion von slanDCs in tertiären lymphoiden Strukturen von Kolonkarzinom-Geweben 51 3.9 Expression von CD83 durch slanDCs in TLS von Kolonkarzinom-Geweben 54 3.10 Expression von IL-23 durch slanDCs in TLS von Kolonkarzinom-Geweben 56 4 Diskussion 57 4.1 DCs als zentrale Mediatoren einer Tumor-gerichteten Immunantwort 57 4.2 DCs als Komponente des Immunzellinfiltrats im Kolonkarzinom 58 4.2.1 Immunhistochemischer Nachweis von DCs in Kolonkarzinom-Geweben 58 4.2.2 Zielstrukturen eingesetzter Antikörper in immunhistochemischen Färbungen von DCs 60 4.2.3 SlanDCs als DC-Subpopulation in Kolonkarzinom-Geweben 61 4.3 Phänotyp infiltrierender slanDCs im Kolonkarzinom 64 4.3.1 Expression von CD83 durch slanDCs 64 4.3.2 Expression von Interleukin-23 durch slanDCs 67 4.3.3 Expression von Tumornekrosefaktor durch slanDCs 69 4.4 SlanDCs als Komponente von tertiären lymphoiden Strukturen im Kolonkarzinom 71 5 Zusammenfassung 75 6 Summary 77 7 Literaturverzeichnis 79 Anlage 1 91 Anlage 2 92 Danksagung 93 / Colorectal cancer as an immunogenic tumor is characterized by a marked infiltration of different immune cell populations. Especially CD8+ T-lymphocytes and CD4+ T helper cells type 1 seem to influence tumor growth and therefore play an increasing role as prognostic markers. Thus, it has been shown that high densities of these T cell subsets are associated with improved survival of colorectal cancer patients. These new insights could become part of the classification of colorectal cancer and influence therapeutic decisions. Despite these studies, little is known about the frequency and properties of native human dendritic cells (DCs) in colon cancer tissues and their potential role in antitumor immunity. DCs as professional antigen-presenting cells are critical for the induction and maintenance of antitumor immunity and can essentially influence tumor progression. Thus, the frequency, distribution, maturation, and cytokine expression of 6-sulfo LacNAc+ (slan) DCs in colon cancer tissues as well as in corresponding tumor-free colon specimens were investigated. SlanDCs represent a subset of human blood DCs that secrete large amounts of proinflammatory cytokines upon activation. Furthermore slanDCs are able to efficiently activate CD4+ T cells, tumor-reactive CD8 + T cells, and natural killer cells. Due to these functional properties, slanDCs may contribute to antitumor immunity and may influence tumor growth. Within this doctoral thesis the presence of slanDCs in primary colon cancer samples was immunohistochemically verified. In this context, a higher frequency of slanDCs in colon cancer tissues (mean: 16,69 slanDCs/mm2, n=38) in comparison to the corresponding tumor-free specimens (mean: 9,25 slanDCs/mm2, n=38) could be detected. Moreover, higher frequencies of infiltrating slanDCs in colon cancer tissues (mean: 18,85 slanDCs/mm2, n=20) were detectable compared to plasmacytoid DCs (mean: 4,86 pDCs/mm2, n=20), representing another human blood DC-subset. Based on these results, various immunofluorescence stainings were performed to investigate maturation and cytokine expression of the infiltrating slanDCs. SlanDCs expressing the maturation marker CD83 were detected in all 10 analyzed colon cancer tissues (mean: 46,7% CD83+ slanDCs). In addition, IL-23-expressing slanDCs were present at varying percentages in 10 of 11 evaluated colon cancer samples (mean: 33,8% IL-23+ slanDCs). Interestingly, in several tissues slanDCs represented a marked proportion of all IL-23-expressing cells. However, slanDCs expressing tumor necrosis factor could only be detected in low frequencies in the analyzed colon cancer specimens. Further studies revealed that slanDCs are a novel component of the T-cell zone of colon cancer-associated tertiary lymphoid structures (TLS). A proportion of these TLS-associated slanDCs displays a mature phenotype or express IL-23. These novel findings indicate that slanDCs may modulate adaptive immune responses in the T-cell zone of colon cancer-associated TLS and may contribute to the regulation of tumor progression. Furthermore the IL-23-expressing slanDCs in the tumor-surrounding stroma and the TLS may promote the generation of IL-17-producing cells and may participate in inflammation-related cancer progression mediated by the IL-23/IL-17 axis. These novel observations can help to decipher the role of human native DCs in colon cancer and may have implications for the design of therapeutic strategies against this tumor entity.:Abbildungsverzeichnis IV Tabellenverzeichnis VI Abkürzungsverzeichnis VII 1 Einleitung 1 1.1 Das humane Immunsystem 1 1.2 Dendritische Zellen 2 1.2.1 Phänotyp und Funktion dendritischer Zellen 3 1.2.2 Aktivierung von T-Lymphozyten durch dendritische Zellen 4 1.2.3 Subpopulationen humaner dendritischer Zellen 6 1.3 Interaktion von Immunsystem und Tumor 8 1.4 Infiltration von Tumoren durch Immunzellen 10 1.5 Kolonkarzinome 11 1.5.1 Entwicklung, Charakteristika und Klassifikation kolorektaler Karzinome 12 1.5.2 Therapieansätze des Kolonkarzinoms 14 1.6 Zielstellung 17 2 Material und Methoden 19 2.1 Material 19 2.1.1 Chemikalien und Reagenzien 19 2.1.2 Lösungen und Puffer 19 2.1.3 Testkitsysteme 19 2.1.4 Antikörper zur Detektion von Oberflächenmolekülen 20 2.1.5 Seren 20 2.1.6 Geräte 21 2.1.7 Sonstige Materialien 21 2.1.8 Gewebeproben 21 2.2 Methoden 23 2.2.1 Vorbereitung der Gewebeproben zur Immunmarkierung 23 2.2.2 Immunhistochemischer Nachweis von slanDCs und pDCs in Kolonkarzinom-Geweben und tumorfreien Kolon-Geweben 24 2.2.3 Untersuchung der Zytokinexpression von slanDCs in Kolonkarzinom-Geweben und tumorfreien Kolon-Geweben mit Fluoreszenzfärbungen 25 2.2.4 Nachweis der Expression von CD83 durch slanDCs in Kolonkarzinom-Geweben mit einer Fluoreszenzfärbung 26 2.2.5 Analyse einer Kolokalisation von slanDCs und CD3+ T-Lymphozyten in Kolonkarzinom-Geweben 26 2.2.6 Detektion von slanDCs in tertiären lymphoiden Strukturen von Kolonkarzinom-Geweben 26 2.2.7 Nachweis der Expression von CD83 und Interleukin-23 durch slanDCs in TLS von Kolonkarzinom-Geweben 28 2.2.8 Auswertung der immunhistochemischen Färbungen von slanDCs und pDCs in Kolonkarzinom-Geweben und in tumorfreien Kolon-Geweben 29 2.2.9 Statistik 30 3 Ergebnisse 31 3.1 Nachweis von slanDCs in Kolonkarzinom-Geweben 31 3.2 Assoziation der ermittelten Frequenz infiltrierender slanDCs mit der TNM-Klassifikation des Kolonkarzinoms 37 3.3 Nachweis von pDCs in Kolonkarzinom-Geweben 39 3.4 Expression von CD83 durch slanDCs in Kolonkarzinom-Geweben 42 3.5 Expression von Interleukin-23 durch slanDCs in Kolonkarzinom-Geweben 44 3.6 Expression von Tumornekrosefaktor durch slanDCs in Kolonkarzinom-Geweben 47 3.7 Kolokalisation von slanDCs und CD3+ T-Lymphozyten in Kolonkarzinom-Geweben 50 3.8 Detektion von slanDCs in tertiären lymphoiden Strukturen von Kolonkarzinom-Geweben 51 3.9 Expression von CD83 durch slanDCs in TLS von Kolonkarzinom-Geweben 54 3.10 Expression von IL-23 durch slanDCs in TLS von Kolonkarzinom-Geweben 56 4 Diskussion 57 4.1 DCs als zentrale Mediatoren einer Tumor-gerichteten Immunantwort 57 4.2 DCs als Komponente des Immunzellinfiltrats im Kolonkarzinom 58 4.2.1 Immunhistochemischer Nachweis von DCs in Kolonkarzinom-Geweben 58 4.2.2 Zielstrukturen eingesetzter Antikörper in immunhistochemischen Färbungen von DCs 60 4.2.3 SlanDCs als DC-Subpopulation in Kolonkarzinom-Geweben 61 4.3 Phänotyp infiltrierender slanDCs im Kolonkarzinom 64 4.3.1 Expression von CD83 durch slanDCs 64 4.3.2 Expression von Interleukin-23 durch slanDCs 67 4.3.3 Expression von Tumornekrosefaktor durch slanDCs 69 4.4 SlanDCs als Komponente von tertiären lymphoiden Strukturen im Kolonkarzinom 71 5 Zusammenfassung 75 6 Summary 77 7 Literaturverzeichnis 79 Anlage 1 91 Anlage 2 92 Danksagung 93