Diabetes tipo 1 (DM1) e uma doenca autoimune que se inicia devido a defeitos na tolerancia imunologica a auto-antigenos, resultando na destruicao autoimune das celulas pancreaticas em individuos geneticamente suscetiveis. Os receptores NOD-like (NLRs) sao receptores intracelulares responsaveis pelo reconhecimento de padroes moleculares associados a patogenos (PAMPs) e padroes moleculares associados ao dano (DAMPs). Estudos recentes tem demonstrado que os receptores NOD1 e NOD2 desempenham um importante papel na ativacao da imunidade inata contra patogenos e na regulacao da imunidade adaptativa, uma vez que sua ativacao leva a producao de citocinas relacionadas a diferenciacao de linfocitos T auxiliares produtores de IL-17 (Th17). Porem, a importancia desses receptores no DM1 ainda e incerto. Nesse sentido, investigamos o papel dos receptores NOD1 e NOD2 na patogenese do DM1, com enfoque na diferenciacao de linfocitos Treg/Th17/Th1 e na plasticidade desses subtipos celulares. Nossos resultados mostram que camundongos deficientes de NOD2, mas nao NOD1 ou RIP2, sao resistentes ao DM1, como comprovado por menor incidencia, hiperglicemia, diminuicao do infiltrado inflamatorio e normalizacao dos niveis de insulina quando comparado aos controles. Foi observado tambem que animais NOD2-/- tiveram uma reducao da populacao de linfocitos Th17, Tc17, Th1 e T citotoxicos nos linfonodos pancreaticos, o que correlaciona com a inibicao da producao de IL-23p19 e IFN- no pancreas. Em paralelo, foi evidenciado o aumento do numero de celulas T reguladoras, macrofagos do perfil M2 nos linfonodos pancreaticos e elevada producao de IL-10 no pancreas de animais NOD2-/-. Alem disso, foi observado que animais NOD2-/- apresentaram uma menor populacao de linfocitos T duplo-positivos (Foxp3+RORt+ e IL-17+IFN+). Posteriormente, foi detectado menor producao de IL- 1, IL-6, IL-23p19 e IL-12p40 por celulas dendriticas de animais deficientes de NOD2. De forma interessante, foi observada a translocacao de bacterias para os linfonodos pancreaticos de animais diabeticos. Adicionalmente, animais tratados com antibioticos tornaram-se resistentes ao DM1, o que nos fornece indicios da contribuicao da microbiota intestinal na inducao da doenca. Por fim, comprovamos alta expressao genica de NOD2 nos linfonodos pancreaticos e no pancreas na fase inicial (pre-diabetica) em outro modelo de DM1, utilizando camundongos NOD (nonobese diabetic mice). Portanto, nossos dados indicam que a ativacao do receptor NOD2 por componentes bacterianos da microbiota intestinal induz a producao de citocinas pro-inflamatorias com subsequente diferenciacao/conversao de linfocitos do perfil Th17/Th1 e progressao do DM1. Dessa forma, estes dados apontam o bloqueio do receptor NOD2 como uma potencial terapia imunomoduladora para o DM1 em humanos. / Type 1 diabetes is an autoimmune disease that precipitates due to defects in the self tolerance to auto- antigens, resulting in the autoimmune destruction of the pancreatic cells in genetically susceptible individuals. NOD-like (NLRs) receptors are intracellular receptors responsible for the recognition of pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). Recent studies have shown a role of NOD1 and NOD2 receptors in the innate immune response against pathogens and in the adaptive immune response, since its activation leads to the generation of cytokines related to the differentiation of IL-17-producing T helper cells (Th17). However, the role of these receptors in T1D remains elusive. Therefore, we investigated the role of NOD1 and NOD2 receptors in the pathogenesis of T1D, focusing on the differentiation of Treg/Th1/Th17 lymphocytes and in the plasticity of these subtypes. Our data demonstrate that NOD2-/- mice, but not NOD1-/- or RIP2-/-, are resistant to T1D, as shown by the lower incidence, hyperglycemia, less insulitis and normal insulin production when compared to wild type mice. It was also observed that NOD2-/- mice have a reduction in the Th17, Tc17, Th1 and cytotoxic T lymphocyte population within the pancreatic lymph nodes (PLNs), which correlates with the inhibition of IL-23p19 and IFN production in the pancreas. In parallel, there was an increase in Treg cells, M2 macrophages in the PLNs and IL-10 production in the pancreatic tissue of NOD2-/- mice. Also, NOD2-/- mice presented a downregulation of Foxp3+RORt+ and IL-17+IFN+ double-positive T cells. Later, it was shown that IL-1, IL-6, IL-23p19 and IL-12p40 production was downregulated in mice deficient to the NOD2 receptor. Interestingly, we observed a bacterial translocation to the pancreatic lymph nodes in diabetic mice, what could be triggering NOD2 activation, thus contributing to T1D development. As expected, mice pre-treated with antibiotics failed to become diabetic, suggesting a possible role of the gut microbiota in the development of the disease. Lastly, we observed a higher relative expression of NOD2 in the PLNs and pancreas of pre-diabetic mice, using another mouse model of the disease, the nonobese diabetic (NOD) mouse. Collectively, our data suggest that components from the gut microbiota are capable of translocating to the PLNs, thus triggering the activation of NOD2, which in turn induces the production of proinflammatory cytokines related to the differentiation of Th1/Th17 cells, thus contributing to T1D development in a mouse model of the disease. Therefore, the blockade of NOD2 appears as an interesting therapeutical target in the treatment of type 1 diabetes in humans.

A malária permanece um sério problema de saúde em países subdesenvolvidos. O estágio sanguíneo da infecção é responsável por todos os sintomas associados com a malária. Recentemente, tem sido mostrado que receptores imunes inatos são capazes de detectar sinais de dano, tais como a adenosina trifosfato ATP. O receptor P2X7 detecta altas concentrações de ATP extracelular. Ao avaliarmos a parasitemia e os parâmetros clínicos da doença em camundongos C57BL/6 e P2X7-/-, observamos uma semelhança em ambos os grupos até o dia 7 p.i., mas após este período os camundongos P2X7-/- tiveram dificuldade de controlar a parasitemia e restaurar os parâmetros clínicos. O ineficiente controle da parasitemia durante o período agudo e crônico em camundongos P2X7-/- foi associado com a baixa produção de IFNγ. Além disso, o receptor P2X7 aumenta a expressão de T-bet em células Th1 e controla o número de células Tfh. Este estudo mostra que o equilíbrio mediado pelo receptor P2X7 entre os fatores de transcrição Bcl-6 e T-bet ajusta a imunidade celular e humoral na malária. / Malaria remains a serious healthcare problem in developing countries. The blood stage of infection is responsible for all symptoms associated with malaria. Recently, it has been shown that innate immune receptors are able to detect signals as adenosine triphosphate (ATP). P2X7 receptor detects high levels of extracellular ATP. Evaluating the parasitemia and clinical parameters in C57BL/6 (B6) and P2X7-/- mice, we observed a similarity in both groups to day 7 p.i., but after this period the P2X7-/- mice had difficulty in controlling the parasitemia and restoring the clinical parameters. The inefficient parasite control in acutely and chronically infected P2X7-/- mice was associated with low production of IFNγ. Furthermore, P2X7 receptor increases the expression of T-bet in Th1 cells and controls the Tfh cell number. This study provides a new insight into immunology by showing that the balance between T-bet and Bcl-6 transcriptional factors tunes the cellular and humoral immunity in malaria.

0 objetivo deste trabalho foi definir se a carga parasitaria na fase aguda da doenga de Chagas experimental afeta a parasitemia, a patologia e a resposta imune na fase cr6nica. Para obtengelo de diferentes cargas parasitoirias na fase aguda, camundongos A/J foram infectados corn 103 OU 105 formas tripomastigotas de T. cruzi e analisados urn ano depois. Os animais cr6nicos infectados corn 105 formas tripomastigotas apresentaram maior nivel de parasitemia residual, maior intensidade de inflamagclo no coragtio e no moscuio esquel6tico e maior grau de ativa95o do sistema imune do que os animais infectados corn 103 formas. Em reiagclo aos parametros imuno16gicos analisados, observou-se nos animais infectados corn 105 formas: i) expansio das populag6es B220-CD5- e CD8\'; ii) freq0@ncia maior de blastos nas populag6es linfocit@rias B220\', CD8\' e CD4\'; iii) mudanga acentuada nas c61ulas CD4+ para o fen6tipo CD4+CD45RBI-ow, indicando urn aumento das c61ulas efetoras elou de mem6ria; iv) freqGC=ncias elevadas de blastos CD4+CD45RB Hig\' e CD4+CD45RB Low; vi) nomero superior de c61ulas secretoras de lg principaimente IgG2a; v) niveis superiores de anticorpos IgG2a e IgGl especificos e vii) maior produgclo de IFN-Y e de IL-4. Estes resultados indicam que a carga parasitaria na fase aguda da infecggto influencia a ativagclo do sistema imune e o desenvolvimento da patologia na fase cr6nica da doenga de Chagas. / The objective of this project is to evaluate if the parasite load in the acute phase experimental Chagas\' disease affects the parasitemias, the pathology and the immune response in the chronic phase. To obtain low- and high-parasite loads in the acute phase of the disease, AlJ mice were infected with 103 or 105 T. cruzi trypomastigotes of the Y strain, and treated on day 6 with Benzonidazol. One year later, chronic mice were screened for subpatent parasitemias, tissue pathology and immune response. Mice infected with the high parasite inoculum showed higher levels of chronic parasitemias, heart and striated muscle inflammation and activation of the immune system when compared to mice infected with the low¬dose inoculum. Concerning the activation of the immune system, the main findings in high-dose infected mice were: i) increased numbers of splenocytes, with preferential expansion of CD8+ and B220-CDS- cells, many of them bearing a macrophage phenotype; ii) higher frequencies of B (B220+), CD4+ and CD8+ large lymphocytes; iii) a shift of CD4+ cells towards a CD4SRBLow phenotype; iv) increased frequencies of both CD4SRBLow and CD4SRBHigh large CD4+ cells; v) augmented numbers of total Ig-secreting cells, with predominance of IgG2a¬producing cells, and; vi) increased production of IFN-y and IL-4. In addition, these mice presented lower IgM and higher IgG2a and IgG1 parasite-specific serum antibody levels. Our results indicate that the parasite load at the acute phase of T. cruzi infection influences the activation of the immune system and development of Chagas pathology at the late chronic phase of the disease.

Produzida por macrófagos e células dendríticas, a IL-27 é uma citocina heterodimérica capaz de induzir células Tr1 produtoras de IL-10 e consequentemente regular linfócitos Th1, Th2 e Th17, dependendo da doença envolvida. Partindo-se do pressuposto de que a infecção causada por Trypanosoma cruzi normalmente induz miocardite refletida pela migração intensa de linfócitos Th1 para o tecido cardíaco, nós analisamos o papel regulador da IL-27 nesta condição inflamatória. Nós inicialmente verificamos que a IL-27 foi prontamente induzida in vitro em células infectadas com T. cruzi. Para gerar miocardite intensa coordenada por linfócitos Th1, nós polarizamos linfócitos T naïves para o padrão Th1 na ausência de moléculas relacionadas ao perfil Th17 (camundongos IL-17R-/-, IL-23-/- e IL-6-/-). Como esperado, a inflamação cardíaca intensa e o dano tecidual foram observados na ausência das moléculas do padrão Th17, o que contribuiu para a morte prematura dos animais IL-17R-/-, IL-23-/- e IL-6-/-, precisa e notoriamente pela indução da migração excessiva de linfócitos Th1 para o tecido cardíaco via CXCL-9 e CXCL-10. Para explorar os mecanismos pelos quais a IL-27 controla a miocardite induzida pelo T. cruzi, nós encontramos um recrutamento substancial de macrófagos produtores de IL-27 para o tecido cardíaco, o qual foi mediado pelas quimiocinas CCL3 e CCL4 na ausência de moléculas do padrão Th17. Para determinar quais os receptores necessários para a produção de IL-27, nós observamos que macrófagos derivados da medula óssea de camundongos deficientes de TLR4-/-, TLR9-/- e NLRP3-/- aboliram completamente a produção desta citocina após a infecção in vitro com T. cruzi, enquanto o receptor TLR2 foi dispensável. Nós também verificamos que macrófagos produtores de IL-27 suprimiram linfócitos Th1 através da indução de células Tr1 produtoras de IL-10 após a infecção com T. cruzi. Em seguida, nós avaliamos se a IL-27 foi correlacionada com a proteção cardíaca durante a doença de Chagas. Nós observamos níveis séricos elevados de IL-27 tanto em pacientes com a forma clínica indeterminada ou cardíaca leve, enquanto pacientes com cardiomiopatia moderada ou grave produziram níveis reduzidos de IL-27. Neste estudo, nós descrevemos um novo mecanismo regulador desempenhado por macrófagos produtores de IL-27 no controle da miocardite induzida por T. cruzi. Macrófagos produtores de IL-27 podem suprimir processos inflamatórios desencadeados por linfócitos Th1, os principais vilões na doença de Chagas. / IL-27 is a heterodimeric cytokine produced by macrophages and dendritic cells known to induce IL-10-producing Tr1 cells and to regulate Th1, Th2, and Th17 lymphocytes, depending on the underlying disease. Because the infection caused by Trypanosoma cruzi normally induces myocarditis mirrored by an outstanding migration of Th1 cells to the heart tissue, we analyzed the regulatory role of IL-27 in this inflammatory condition. We firstly verified that IL-27 was promptly induced by in vitro T. cruzi-infected spleen cells. To generate a robust myocarditis coordinated by Th1 lymphocytes, we polarized lymphocytes to a Th1 pattern by infecting mice in the absence of Th17-related molecules (IL-17R-/-, IL-23-/-, and IL-6-/- mice). As expected, an impressive cardiac inflammation and damage was observed in the absence of Th17-related molecules, leading IL-17R-/-, IL-23-/-, and IL-6-/- mice to the premature death, precisely and notably by inducing an exuberant Th1 migration to the heart tissue via CXCL9 and CXCL10 chemokines. To explore the mechanisms by which IL-27 controls T. cruzi-induced myocarditis, we found a striking recruitment of IL-27-producing macrophages to the heart tissue mediated by increased levels of CCL3 and CCL4 chemokines in the absence of Th17-associated molecules. To gain further insights into the receptors required to IL-27 production, we observed that bone marrow-derived macrophages from TLR4-/-, TLR9-/-, and NLRP3-/- mice completely abolished IL-27 production after in vitro T. cruzi infection, while TLR2 was dispensable. We also verified that IL-27-producing macrophages supressed Th1 lymphocytes by inducing IL-10-producing Tr1 cells after T. cruzi infection. We next assessed whether IL-27 was correlated to cardiac protection during Chagas Disease. We observed augmented serum levels of IL-27 in either patients with indeterminate (asymptomatic) form or mild cardiac form, whereas patients with moderate or severe cardiomyopathy were poor producers of IL-27. Here, we described a novel regulatory mechanism developed by IL-27-producing macrophages in the control of T. cruzi-induced myocarditis. IL-27-producing macrophages can suppress inflammatory processes caused by Th1 lymphocytes, the bona fide culprits of Chagas Disease.

La Néphropathie à IgA est la glomérulonéphrite primitive la plus fréquente, responsable d’une évolution vers l’insuffisance rénale terminale dans 10 à 30% des cas après 20 ans d’évolution. Les déterminants de cette maladie sont nombreux, impliquant de multiples acteurs de l’inflammation, qu’ils soient cellulaires ou humoraux. La physiopathologie générale de la maladie est actuellement considérée comme se déroulant en quatre « coups », (i) la production d’IgA1 polymériques présentant un déficit de galactosylation de la région charnière, (ii) l’existence d’un élément circulant capable de complexer ces IgA1 anormales, pouvant être une IgG anti-glycane ou la portion soluble du récepteur de type I aux IgA (sCD89), (iii) la constitution de complexes immuns circulants et (iv) le dépôt glomérulaire de ces complexes, générant des lésions inflammatoires puis cicatricielles responsables de l’évolution vers la maladie rénale chronique. La médiation inflammatoire est impliquée à différents niveaux comprenant entre autres le rôle de l’infiltration de macrophages dans le tissu rénal, l’orchestration de cette réponse inflammatoire glomérulaire par les sous-populations de lymphocytes T et l’importance de l’activation du complément sur le déterminisme des lésions glomérulaires inflammatoires médiées par les complexes immuns déposés. Au cours de ce travail de thèse, l’implication de ces différents acteurs a été explorée. Les macrophages expriment le récepteur de type I aux IgA (CD89, issu du gène FCAR), dont une mutation de la portion intracytoplasmique modifie in vitro la transduction du signal. La première étude a visé à évaluer sur une grande cohorte rétrospective l’impact de cette mutation sur le risque d’occurrence de la maladie ainsi que son impact pronostique. Le rôle des sous-populations T a été abordé au cours d’une seconde étude, suivant l’hypothèse que l’orientation pro inflammatoire au cours de la NIgA pourrait être liée à un déficit de régulation par une sous-population de lymphocytes T, les Tregs. Cette étude prospective a évalué la représentation de la sous-population CD4+CD25+CD127low et le profil d’expression génique de gènes prototypiques des sous-populations Th1, Tregs et Th17. Le rôle du complément comme médiateur inflammatoire à l’interface entre les complexes immuns à IgA1 et les cellules mésangiales a été exploré par une étude in vitro. La mutation 844 A->G de FCAR n’a pas été associée à un risque supplémentaire de développer la maladie et n’impactait pas le pronostic des patients. L’étude des lymphocytes T n’a pas montré de différence de quantité de cellulesCD4+CD25+CD127low entre patients et volontaires sains et ne suggérait qu’une tendance en faveur d’un déficit fonctionnel de régulation (plus faible expression des gènes FoxP3, IL10, TGFβ chez les patients). Enfin, des fragments issus de l’activation et de la protéolyse par le facteur I de C3 ont été mis en évidence par immunoblot et spectrométrie de masse au sein des complexes immuns générés artificiellement en présence de serum. Ces études suggèrent qu’une modification du rôle fonctionnel du CD89 n’impacte pas le devenir de la NIgA, ce qui est en défaveur d’un rôle critique de ce récepteur dans la pathogénie de la maladie. La tendance au déficit fonctionnel Tregs nécessite d’être confirmée au sein d’un effectif plus conséquent et présentant une forme plus active de la maladie, mais elle corrobore deux autres études comparables dans leur méthodologie. Enfin, le rôle du complément se situe à l’interface entre les complexes immuns et les effecteurs inflammatoires glomérulaires tels que les cellules mésangiales / IgA Nephropathy (IgAN) is the most common primary glomerulonephritis, leading to end stage renal failure in 10 to 30% of cases after 20 years. This disease is determined by numerous inflammatory players, including cells and molecules. The pathogeny of the disease is likely to be driven by a 4 « hits » model, (i) increased systemic production of aberrantly O galactosylated polymeric IgA1, (ii) the existence of circulating abnormal IgA1 binding element, which could be either an anti-glycan IgG or the soluble fragment of the main IgA receptor (sCD89), (iii) the formation of circulating immune complexes, and (iv) the glomerular deposition of these complexes, that accounts for a variable local inflammation leading to scarring processes and finally to the chronic kidney disease. Inflammatory mechanisms operate at several levels, including the macrophage cells infiltration in the kidney tissue, the orchestration of the immune response by T-cells subsets, including regulatory T-cells, and the role of complement activation to induce the glomerular inflammatory response from the immune complexes deposition. In the present work, we aimed to explore the implication of these inflammation response players. Macrophages express the type I IgA receptor (CD89, downstream from its gene FCAR), whose function can be affected in vitro by a common mutation of its intracytoplasmic portion. A first study evaluated the impact of this mutation on the risk to develop the disease as well as on the global prognosis. A second study evaluated the role of T-cell subsets during IgAN, following the hypothesis that the pro-inflammatory balance of the disease could be a consequence of a defect in the immune regulation by the Tregs. This prospective study aimed to assessing the frequency of CD4+CD25+CD127low cells in peripheral blood and the characteristic gene expression profile from Th1, Th17 and Tregs subsets. The role of complement as an inflammatory player at the interface between IgA1 containing immune complexes and mesangial cells was explored by an in vitro study. The single nucleotide polymorphism 844 A->G of FCAR had no impact neither on disease risk of occurrence neither on the renal survival. The T-cell subsets study failed to demonstrate any difference in the proportion of CD4+CD25+CD127low cells and only suggested a defect in functional activity of Tregs, according to a lower expression of FoxP3, IL10, TGFβ genes. The third in vitro study demonstrated by immunoblot and mass spectrometry the presence of C3 breakdown products accompanying IgA1 based engineered immune complexes formed in presence of normal immunoglobulin depleted serum. The lack of effect of the mutation of FCAR on the IgAN prognosis is not in favour to a critical role of this receptor on the pathogeny of the disease. The trend in the functional defect of Tregs subset needs to be confirmed in a larger study, including patients with a more severe form of their disease. This result is however consistent with two other studies displaying a similar methodology. The role of complement is confirmed to be a key player, as it is likely to act at the interface between the IgAN particular immune complexes and mesangial cells

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

Zielstellung: Ziel dieser kumulativen Dissertationsarbeit war die vergleichende Untersuchung der Wirtsantwort in verschiedenen Mauslinien nach Infektion mit Streptobacillus (S.) moniliformis und Rodentibacter (R.) pneumotropicus mit Anzeigeparametern für die Klinik, die Pathologie und die Immunantwort. Es sollten neue erregerspezifische enzyme-linked immunosorbent assays (ELISA) evaluiert und durch die Bestimmung der Immunglobulin G (IgG)-Subklassen mausstammspezifische Unterschiede in der Immunantwort aufgezeigt werden. Darüber hinaus waren Sentinelsysteme zu bewerten. Material und Methoden: Es wurden BALB/c und C57BL/6-Mäuse intranasal mit S. moniliformis- oder R. pneumotropicus infiziert und mit Kontaktsentinels vergesellschaftet. Zusätzlich wurde benutzte Einstreu der Infektionskäfige auf Käfige mit nichtinfizierten CD1-Mäusen (Einstreu-Sentinels) übertragen. Die Infektionsgruppen wurden über 4 Wochen, die Sentinels mindestens 7 Wochen alle 12 Stunden klinisch untersucht und die Verläufe dokumentiert. Am Ende der Experimente bzw. bei Erreichen spezifischer Abbruchkriterien wurden die Mäuse tierschutzgerecht euthanasiert und definierte Organproben für pathohistologische und bakteriologische Untersuchungen gewonnen. Die Erreger wurden dabei massenspektrometrisch sowie mittels polymerase chain reaction (PCR) differenziert und in Proben des Respirationstraktes quantitativ erfasst. Erregerspezifische Antikörper wurden in tracheonasaler Spülflüssigkeit und im Serum in eigens etablierten ELISA‘s auf Basis von Ganzzellextrakten bestimmt. Weiterhin erfolgte die Messung des Verhältnisses der IgG-Subtypen IgG1 und IgG2 im ELISA. Ergebnisse: Der Infektionsversuch mit S. moniliformis bestätigte mit einer Mortalität von 75% die bekannte hohe Infektionsanfälligkeit der C57BL/6-Mäuse im Gegensatz zu BALB/c, die keine Krankheitsanzeichen entwickelten. Die wichtigsten pathologischen Manifestationen waren eitrig-nekrotisierende Lymphadenitiden und Pneumonien in Verbindung mit der Reisolation des Infektionsstammes. Mithilfe des etablierten ELISA‘s gelang der Nachweis erregerspezifischer IgG-Antikörper im Serum der Tiere beider Linien. Bei den Kontaktsentinels konnte, bis auf eine Ausnahme, weder kulturell, noch serologisch eine Infektion nachgewiesen werden. Gleiches gilt für alle Einstreu-Sentinels. Molekularbiologisch wurde aber Erreger-DNA in den Lungen der Sentinels festgestellt. Die Infektion mit einem R. pneumotropicus Stamm, welcher genotypisch positiv für alle drei bekannten RTX-Toxine dieses Erregers war, führte zu einer unerwartet hohen Morbidität und Mortalität in beiden Mauslinien. In frühzeitig euthanasierten Tieren beider Linien konnten katarrhalisch-eitrige bis nekrotisierende Bronchopneumonien sowie eine Dissemination des Belastungsstammes in zahlreiche innere Organe nachgewiesen werden. In überlebenden Tieren beider Linien wurde eine deutliche Kolonisation respiratorischer Schleimhäute mit dem Belastungsstamm trotz z.T. hoher mukosaler IgA-Spiegel und Serokonversion im Blut nachgewiesen. Überlebende C57BL/6 Mäuse zeigten eine signifikant niedrigere Bakterienlast in inneren Organen als BALB/c Mäuse. In allen Kontaktsentinels, aber nicht in einem einzigen Einstreu-Sentinel, konnte kulturell und indirekt serologisch eine Infektion mit R. pneumotropicus nachgewiesen werden. Die Bestimmung der IgG-Subklassen in den Seren der C57BL/6-Mäuse beider Infektionsstudien ergab eine Verschiebung des Verhältnisses IgG2/IgG1 von unter 0,8 vor zu über 1,6 nach Infektion. Dies weist auf eine T-Helferzell (Th) 1-dominierte Immunantwort hin. BALB/c-Mäuse behielten dagegen ein Verhältnis unter 0,8 auch nach der Infektion bei, sodass auf eine Th2- Antwort zu schließen war. Schlussfolgerungen: Sowohl für S. moniliformis als auch für R. pneumotropicus konnten Tiermodelle mit diversen Anzeigeparametern etabliert werden, welche für Folgestudien zur Pathogenese oder Immunprophylaxe genutzt werden können. Für beide Erreger wurden neue sensitive und spezifische ELISA-Protokolle in die Diagnostik eingeführt. Kontaktsentinels, aber nicht Einstreu-Sentinels, sind gut geeignet, um R. pneumotropicus-Infektionen nachzuweisen. Die beobachtete stammspezifische Klinik, Pathologie und Immunantwort der C57BL/6-Mäuse nach experimenteller S. moniliformis-Infektion sprach für eine pathologische Th1-Immunantwort. Im Gegensatz dazu war im R. pneumotropicus – Infektionsversuch die Th1-Immunantwort der C57BL/6-Mäuse mit einer effektiveren Reduktion des Erregers in inneren Organen assoziiert. Die unerwartet hohe Morbidität und Mortalität im R. pneumotropicus –Infektionsversuch weist auf eine besonders hohe Virulenz des eingesetzten Stammes hin, sodass in dieser Arbeit erstmalig ein septikämischer Verlauf in Wildtyp-Mäusen nach intranasaler R. pneumotropicus-Infektion nachgewiesen werden konnte.:Inhaltsverzeichnis (I) Abkürzungsverzeichnis (III) 1 Einleitung (1) 2 Literatur (3) 2.1 Streptobacillus moniliformis (3) 2.1.1 Allgemeine Charakteristika (3) 2.1.2 Differenzierung von Streptobacillus spp.(4) 2.1.3 Serologische Methoden zum indirekten Nachweis einer Streptobacillus moniliformis - Infektion (5) 2.1.4 Epidemiologie der durch Streptobacillus moniliformis hervorgerufenen Zoonose (6) 2.1.5 Klinik und Pathologie der Streptobacillus moniliformis-Infektion bei Nagetieren (8) 2.1.6 Klinik und Pathologie der Streptobacillus moniliformis-Infektion in Menschen und anderen Nebenwirten (9) 2.1.7 Pathogenese und Virulenzfaktoren (10) 2.1.8 Prävalenz in Nagern (11) 2.1.9 Sanierung Streptobacillus moniliformis infizierter Nagetierbestände und Prävention (12) 2.2 Rodentibacter (R.) pneumotropicus und heylii (Pasteurella (P.) pneumotropica Biotyp Jawetz und Heyl) (14) 2.2.1 Allgemeine Charakteristika (14) 2.2.2 Ursprüngliche Einteilung in Biotypen und Reklassifikation zu Rodentibacter spp. (14) 2.2.3 Differenzierung von Rodentibacter spp. (15) 2.2.4 Serologische Methoden zum indirekten Nachweis einer Rodentibacter- Infektion (15) 2.2.5 Übertragung (15) 2.2.6 Klinik und Pathologien der Rodentibacter-Infektion in Nagern (16) 2.2.7 Pathogenese und Virulenzfaktoren (18) 2.2.8 Prävalenz (19) 2.2.9 Sanierung Rodentibacter pneumotropicus infizierter Nagetierbestände und Prävention (20) 2.3 Mäuse als Versuchstiere (22) 2.3.1 Inzucht-Stämme (22) 2.3.1.1 Merkmale, Verwendung und Historie der BALB/c-Wildtypmäuse (22) 2.3.1.2 Merkmale, Verwendung und Historie der C57BL/6-Wildtypmäuse (23) 2.3.1.3 Unterschiede der Immunreaktionen in C57BL/6- und der BALB/c- Mäusen (23) 2.3.2 Auszucht-Stämme (24) 2.3.2.1 Merkmale, Verwendung und Historie der CD1-Wildtypmäuse (24) 2.4 Gesundheitsmonitoring in Labortierhaltungen (25) 2.4.1 Empfehlungen der Federation of Laboratory Animal Science Associations (FELASA) (25) 2.4.2 Sentinelsysteme für das Gesundheitsmonitoring in Labormausbeständen (26) 3 Publikationen (28) 3.1 Fornefett J, Krause J, Klose K, Fingas F, Hassert R, Eisenberg T, Grunwald T, Müller U, Schrödl W, Baums CG. Comparative analysis of clinics, pathologies and immune responses in BALB/c and C57BL/6J mice infected with Streptobacillus moniliformis. Microbes and Infection. 2018;20(2):101-110 (28) 3.2 Fornefett J, Krause J, Klose K, Fingas F, Hassert R, Benga L, Grunwald T, Müller U, Schrödl W, Baums CG. Comparative analysis of humoral immune responses and pathologies of BALB/c and C57BL/6 wildtype mice experimentally infected with a highly virulent Rodentibacter pneumotropicus (Pasteurella pneumotropica) strain. BMC Microbiology. 2018;18(1):45 (39) 4 Übergreifende Diskussion (51) 5 Zusammenfassung (59) 6 Summary (61) 7 Literaturverzeichnis (63) 7.1 Fachliteratur (63) 7.2 Internet (76) 7.3 Gesetzestexte (78) Anhang (79) i Ergänzende Abbildungen (79) ii Ergänzendes Material zu 3.1 “Comparative analysis of clinics, pathologies and immune responses in BALB/c and C57BL/6J mice infected with Streptobacillus moniliformis” (81) iii Ergänzendes Material zu 3.2 “Comparative analysis of humoral immune responses and pathologies of BALB/c and C57BL/6 wildtype mice experimentally infected with a highly virulent Rodentibacter pneumotropicus (Pasteurella pneumotropica) strain” (83) Liste mit weiteren Veröffentlichungen Danksagung / Objective: Aim of this cumulative doctoral thesis was the comparative analysis of the host response in different mice strains infected with Streptobacillus (S.) moniliformis and Rodentibacter (R.) pneumotropicus with readout parameters for clinics, pathology and immune response. New pathogen specific enzyme linked immunosorbent assay’s (ELISA) were evaluated. Differentiation of immunoglobulin (Ig) G subclasses was conducted to reveal differences in the immune response between the two mice strains. Furthermore, sentinel systems were assessed. Materials and methods: BALB/c and C57BL/6 mice were infected intranasally with S. moniliformis or R. pneumotropicus and housed together with contact sentinels. Soiled bedding from infection cages was transmitted to cages with uninfected CD1 mice (bedding sentinels). Infection groups were observed for 4 weeks, sentinels for at least 7 weeks and the clinical course was documented. At the end of the experiments or when predefined termination criteria were reached, animals were humanely killed. Predefined organ samples were collected for pathohistological and bacteriological screenings. Pathogens were differentiated via mass spectrometry and via polymerase chain reaction (PCR). The specific bacterial load was quantified in samples of the respiratory tract. Pathogen-specific antibodies were detected in tracheonasal lavages and sera using newly established ELISAs based on whole cell extracts. Determination of the ratios of the IgG subtypes (IgG1 to IgG2) was conducted using ELISAs. Results: The S. moniliformis experiment confirmed the known high susceptibility of C57BL/6 mice with a mortality of 75%. This was in contrast to BALB/c, which developed no signs of illness. The major pathologies were purulent-necrotizing inflammations of the lymph nodes and the lung associated with detection of the challenge strain. Specific IgG-antibodies were detected in sera of both mice strains by the newly established ELISAs. In contact and bedding sentinels the infection was not detected by culture or indirectly by serology, except for one contact sentinel. However, pathogen DNA was detectable in the lungs of these animals via PCR. The infection with the R. pneumotropicus strain, which is genotypically positive for all 3 known RTX toxins of this pathogen, leaded to an unexpected high morbidity and mortality in both mice strains. In early losses a catharal-purulent to necrotizing bronchopneumonia as well as dissemination of the challenge strain in various inner organs was recorded. Efficient colonization of the respiratory mucosa through the challenge strain was detected in survivors of both lines despite high mucosal IgA levels and seroconversion in the blood. Surviving C57BL/6 mice showed a significant lower bacterial burden in inner organs than BALB/c. All contact sentinels were culturally and serologically positive for R. pneumotropicus infection in contrast to all bedding sentinels. Differentiation of IgG subclasses in sera of C57BL/6 mice of both experiments revealed a shift of the IgG2/IgG1 ratio from 0.8 prior to infection to 1.6 post infection suggesting a T helper (Th) 1-prone immune response. BALB/c mice remained under 0.8 after infection indicating a Th2-prone immune response. Conclusions: New animal models with various readout parameters were established for both S. moniliformis and R. pneumotropicus. These models can be used in future studies on pathogenesis and immunoprophylaxis. Sensitive und specific ELISA-protocols were established for both pathogens. Contact sentinels but not bedding sentinels are appropriate for detection of R. pneumotropicus-infections. The observed distinct clinic, pathology and immune response of C57BL/6 mice experimentally infected with S. moniliformis indicated on the one hand a pathological Th1 immune response. On the other hand, the Th1 response of C57BL/6 mice to R. pneumotropicus infection was associated with a more efficient clearance of the challenge strain in internal organs. The unprecedented high morbidity and mortality in the R. pneumotropicus experiment indicates high virulence of this strain. Accordingly, this work revealed for the first time septicaemia in wildtype mice after intranasal R. pneumotropicus-infection.:Inhaltsverzeichnis (I) Abkürzungsverzeichnis (III) 1 Einleitung (1) 2 Literatur (3) 2.1 Streptobacillus moniliformis (3) 2.1.1 Allgemeine Charakteristika (3) 2.1.2 Differenzierung von Streptobacillus spp.(4) 2.1.3 Serologische Methoden zum indirekten Nachweis einer Streptobacillus moniliformis - Infektion (5) 2.1.4 Epidemiologie der durch Streptobacillus moniliformis hervorgerufenen Zoonose (6) 2.1.5 Klinik und Pathologie der Streptobacillus moniliformis-Infektion bei Nagetieren (8) 2.1.6 Klinik und Pathologie der Streptobacillus moniliformis-Infektion in Menschen und anderen Nebenwirten (9) 2.1.7 Pathogenese und Virulenzfaktoren (10) 2.1.8 Prävalenz in Nagern (11) 2.1.9 Sanierung Streptobacillus moniliformis infizierter Nagetierbestände und Prävention (12) 2.2 Rodentibacter (R.) pneumotropicus und heylii (Pasteurella (P.) pneumotropica Biotyp Jawetz und Heyl) (14) 2.2.1 Allgemeine Charakteristika (14) 2.2.2 Ursprüngliche Einteilung in Biotypen und Reklassifikation zu Rodentibacter spp. (14) 2.2.3 Differenzierung von Rodentibacter spp. (15) 2.2.4 Serologische Methoden zum indirekten Nachweis einer Rodentibacter- Infektion (15) 2.2.5 Übertragung (15) 2.2.6 Klinik und Pathologien der Rodentibacter-Infektion in Nagern (16) 2.2.7 Pathogenese und Virulenzfaktoren (18) 2.2.8 Prävalenz (19) 2.2.9 Sanierung Rodentibacter pneumotropicus infizierter Nagetierbestände und Prävention (20) 2.3 Mäuse als Versuchstiere (22) 2.3.1 Inzucht-Stämme (22) 2.3.1.1 Merkmale, Verwendung und Historie der BALB/c-Wildtypmäuse (22) 2.3.1.2 Merkmale, Verwendung und Historie der C57BL/6-Wildtypmäuse (23) 2.3.1.3 Unterschiede der Immunreaktionen in C57BL/6- und der BALB/c- Mäusen (23) 2.3.2 Auszucht-Stämme (24) 2.3.2.1 Merkmale, Verwendung und Historie der CD1-Wildtypmäuse (24) 2.4 Gesundheitsmonitoring in Labortierhaltungen (25) 2.4.1 Empfehlungen der Federation of Laboratory Animal Science Associations (FELASA) (25) 2.4.2 Sentinelsysteme für das Gesundheitsmonitoring in Labormausbeständen (26) 3 Publikationen (28) 3.1 Fornefett J, Krause J, Klose K, Fingas F, Hassert R, Eisenberg T, Grunwald T, Müller U, Schrödl W, Baums CG. Comparative analysis of clinics, pathologies and immune responses in BALB/c and C57BL/6J mice infected with Streptobacillus moniliformis. Microbes and Infection. 2018;20(2):101-110 (28) 3.2 Fornefett J, Krause J, Klose K, Fingas F, Hassert R, Benga L, Grunwald T, Müller U, Schrödl W, Baums CG. Comparative analysis of humoral immune responses and pathologies of BALB/c and C57BL/6 wildtype mice experimentally infected with a highly virulent Rodentibacter pneumotropicus (Pasteurella pneumotropica) strain. BMC Microbiology. 2018;18(1):45 (39) 4 Übergreifende Diskussion (51) 5 Zusammenfassung (59) 6 Summary (61) 7 Literaturverzeichnis (63) 7.1 Fachliteratur (63) 7.2 Internet (76) 7.3 Gesetzestexte (78) Anhang (79) i Ergänzende Abbildungen (79) ii Ergänzendes Material zu 3.1 “Comparative analysis of clinics, pathologies and immune responses in BALB/c and C57BL/6J mice infected with Streptobacillus moniliformis” (81) iii Ergänzendes Material zu 3.2 “Comparative analysis of humoral immune responses and pathologies of BALB/c and C57BL/6 wildtype mice experimentally infected with a highly virulent Rodentibacter pneumotropicus (Pasteurella pneumotropica) strain” (83) Liste mit weiteren Veröffentlichungen Danksagung

The socio-economic and geographical distribution of malaria overlaps with that of many parasitic helminths and in these areas co-infections are common. Co-infection with helminths can influence disease outcome causing either exacerbation or amelioration of malaria. Understanding the complex host-parasite interactions that lead to these different disease outcomes is important for the success of control programmes aimed at these parasites. The immune system has evolved diverse types of response (e.g. T-helper 1 (Th1) and T-helper 2 (Th2)) to efficiently combat infection with ‘microparasites’ and helminths respectively. When faced with co-infection however, the need for the host to multitask means it must manage these counter-regulatory responses. In this study a murine model of malaria-hookworm (Plasmodium chabaudi- Nippostrongylus brasiliensis) co-infection was utilised to investigate how changes in T-helper bias affect malaria disease outcome. Antibody isotypes were used as indicators of Th1/Th2 bias and revealed that helminth co-infection reduced the malaria-specific Th1 response. Counter-intuitively this resulted in ‘protection’ from malaria with co-infected mice having reduced peak P. chabaudi parasitaemia and suffering less severe anaemia. In addition to providing a measure of Th1/Th2 bias, analysis of antibody responses revealed the occurrence of cross-reactive antibodies. The potential for these crossreactive antibodies to influence disease outcome was investigated but in this murine model resource-mediated mechanisms of parasite regulation appear to be responsible for the ‘protection’ that co-infection affords. The question of why cross-reactive antibodies are produced has important immunological and ecological implications. Cross-reactive responses may arise through some physiological constraint on the immune mechanisms that usually result in antibody-specificity. However experiments designed to investigate if the specificity of antibodies is constrained by availability of antigen suggest that this is not the case in the model system used here. There is also the possibility that production of cross-reactive antibodies represents an evolutionary optimal strategy for a host faced with unpredictable exposure to a variety of parasites. However a major finding of this study indicates these two taxonomically distinct parasite species share antigens, which in itself is crucial to understanding host-parasite interactions in a co-infection setting. The main findings of this thesis are relevant to co-infection studies in general and the implications for both evolutionary and applied biology are discussed.

Bacillus anthracis produces lethal toxin (LT) and edema toxin (ET) and they suppress the function of LPS-stimulated dendritic cells (DC). Because DCs respond differently to various microbial stimuli, we compared toxin effects in bone marrow DCs stimulated with either LPS or Legionella pneumophila (Lp). DCs were enriched with GM-CSF for 9 days, purified by positive selection, and treated with toxins for 6h; cells were then stimulated with either LPS or Lp-infection for 24h. DC cytokine production and maturation marker expression varied depending upon cell stimulus and the mouse strain used. LT but not ET was more toxic for cells from BALB/c than from C57BL/6 (B6) as measured by 7-AAD uptake; however, ET suppressed CD11c expression. LT suppressed IL-12, IL-6, and TNF-a in cells from BALB/c and B6 mice but increased IL-1ß in LPS-stimulated cultures. ET also suppressed IL-12 and TNF-a but increased IL-6 and IL-1ß in Lp-stimulated cells from B6. Regarding maturation marker expression, LT increased MHCII and CD86 while suppressing CD40 and CD80; ET, on the other hand, generally decreased marker expression across all groups. We conclude that the modulation of cytokine production by anthrax toxins is dependent on variables including the source of the DCs, the type of stimulus and cytokine measured, and the individual toxin tested. However, LT and ET enhancement or suppression of maturation marker expression is more related to the marker studied than the cell stimulus or cell source. Anthrax toxins are not uniformly suppressive of DC function but instead can increase function under defined conditions.

SerpinB2 (aka plasminogen activator type 2) is well described as an extracellular inhibitor of urokinase-type plasminogen activator (uPA). However, the majority of SerpinB2 is retained intracellularly, and many uPA-independent activities have been reported for SerpinB2 suggesting an alternate function. This thesis explores the role of SerpinB2 in epithelial tumour cell lines, highlights the problems associated with various expression systems and argues that SerpinB2 has no role in growth or apoptosis of tumour cells. A potential role for immune modulation and angiogenesis is suggested in in vivo models. Previous research using SerpinB2 transfected, clonally selected tumour cell lines suggested that SerpinB2 regulates the retinoblastoma tumour suppressor protein (Rb) by binding and protecting Rb from degradation. Despite the use of two techniques under numerous conditions and positive controls, no significant interaction between SerpinB2 and Rb was found. SerpinB2 was reported to bind Rb through a PENF homology motif located within the SerpinB2 C-D interhelical loop region. The PENF homology motif was postulated to represent the motif responsible for binding to the C-pocket of Rb. Epstein Barr Virus nuclear antigen 6 (EBNA6) is a known Rb binding protein, which contains two predicted PENF homology motifs. However, mutation of the two PENF homology motifs within EBNA6 did not reduce Rb binding. Furthermore, the SerpinB2 PENF homology motif is actually not well conserved between SerpinB2 proteins from multiple species, whereas other regions of the SerpinB2 C-D loop show a high level of conservation. These data do not support a role for SerpinB2 and the PENF homology motif in Rb binding. SerpinB2 has been proposed to have a role in regulating growth and apoptosis. To further investigate this proposed phenotype of SerpinB2, SerpinB2 was expressed in a range of epithelial tumour lines using transient transfection. No change in growth, apoptosis or Rb levels were found. After ≈2-3 month antibiotic selection for the SerpinB2-expressing plasmid, SerpinB2 protein was lost without the loss of the transgene, indicating selective pressure against long-term SerpinB2 protein expression. To further investigate long-term SerpinB2 expression adenovirus and lentivirus vectors were used. Infection of tumour cell lines with adenovirus vectors expressing SerpinB2 resulted in reduced cell growth, characterised by increased p53 (but not Rb) levels and G2 arrest or apoptosis. When SerpinB2 expressing lentivirus vectors were used to transduce the same tumour cell lines, high levels of long-term expression of functional SerpinB2 was achieved. However, SerpinB2-expressing cell lines showed no differences in growth, proliferation, Rb levels, or apoptosis induced by a range of agents. Growth and apoptosis observed with adenovirus SerpinB2 had all the characteristics of adenovirus-associated toxicity, which has been reported previously for specific proteins. These experiments highlighted the problems associated with SerpinB2 expression systems and suggest that SerpinB2 expression per se is not toxic nor has a role in regulating Rb, growth and apoptosis. Screening of a number of tumour cell lines identified the HPV16 transformed cervical cancer line as expressing high levels of SerpinB2. SerpinB2 was located both extracellularly and intracellularly with a cytoplasmic and nuclear distribution. A high molecular weight SerpinB2 species was identified in CaSki cells and was shown to be the N-linked glycosylated species. Sequencing showed the protein to be Type A SerpinB2 and the protein was shown to form an inhibitory complex with uPA. An abundant low molecular weight SerpinB2 species was also identified in CaSki cell supernatants and appeared to be a proteolytic fragment of SerpinB2. Treatment of CaSki with PMA, TNFα and IFNγ increased SerpinB2 levels. Lentiviral based shRNA failed to significantly down regulate SerpinB2 expression and increasing SerpinB2 levels with lentiviral expression did not change growth, apoptosis, Rb levels or E7 transcription. Lentiviral expression of SerpinB2 in (normally SerpinB2 negative) HPV16 transformed SiHa cells, also failed to show changes in Rb levels or E7 transcription. CaSki thus express wild-type and functional SerpinB2, but no evidence could found that SerpinB2 effects HPV16 E7 transcription or Rb levels. The data presented identifies CaSki as valuable source of biologically functional SerpinB2. SerpinB2 expression in breast cancer cells has been associated with positive prognosis. Tubo, a SerpinB2-negative murine breast carcinoma cell line, was transduced with lentivirus expressing SerpinB2 and grown subcutaneously in BALB/c mice. SerpinB2 expressing tumours appeared red and were larger than control tumours. Furthermore, SerpinB2 expressing tumours had a ≈2 fold higher density of blood vessels when compared to Tubo and Tubo expressing EGFP. Mice carrying tumours expressing SerpinB2 also showed reduced anti-tumour IgG2 responses. These data suggest that a role for SerpinB2 in regulating angiogenesis and antitumour immunity. In conclusion, this thesis challenges the notion that SerpinB2 regulates Rb, cell cycle, and apoptosis and suggests a potential role for SerpinB2 in tumour angiogenesis and immunity.