O envelhecimento é marcado por inúmeras alterações fisiológicas, dentre as quais encontramos um aumento da gordura corporal e alterações na resposta do sistema imunológico. Muito se sabe sobre a intrínseca ligação entre o aumento de gordura e as doenças de risco e fica a questão à cerca de sua influência sobre as respostas do sistema imunológico, já que estes dois fatores encontram-se alterados no envelhecimento. Portanto, o propósito deste estudo foi investigar a interação entre a quantidade de gordura corporal e as células do sistema imunológico de ratos envelhecidos. Para isso, num primeiro estudo, foram utilizados ratos ADULTOS e ENVELHECIDOS para a caracterização das alterações encontradas no processo de envelhecimento. Numa segunda etapa, os animais ENVELHECIDOS foram submetidos a dois protocolos de emagrecimento, a restrição calórica a 50%(RC) e o exercício aeróbio (EX) (natação), durante o período de quatro a seis semanas. Assim, foi possível avaliar alguns parâmetros do metabolismo de macrófagos e linfócitos em ratos envelhecidos com diferentes quantidades de gordura corporal. No primeiro estudo, os resultados comprovaram o aumento da gordura bem como algumas alterações no sistema imunológico de ratos envelhecidos, principalmente na capacidade funcional de macrófagos. Quanto ao segundo estudo, os resultados demonstram que ambos os protocolos foram eficientes em diminuir o peso corporal, bem como a gordura, sendo que para a RC os valores alcançados foram mais evidentes. Com relação ao sistema imunológico houve um aumento da resposta fagocitária e de produção de H2O2 por macrófagos e uma alteração da capacidade proliferativa de linfócitos, em ambos os protocolos. / Ageing is marked by several kinds of physiological changes, among then we find an increase of fat mass and a decline in several aspects of the immune system. It is well understood the intrinsical connexion between the increase in body fat and the risk of related diseases and the one question that remains is about the influence over the immune system response, since these two factors are altered on the ageing process. The purpose of this study was to investigate the relationship among fat content and immune cells of the aging rats. For this, in one first study, we used ADULTS and AGEING rats to determine the changes found in the ageing process. In the second time, the AGEING rats were submitted to two protocols for weight loss (slimming), 50% of caloric restriction (CR) and aerobic exercise (swimming) (EX), for four or six weeks. In this way, we can study some parameters of macrophages and lymphocytes metabolism in ageing rats with different body fat content. In the first study, the results confirmed the fat increase as well as some changes in the immune system of the ageing rats, especially in the macrophages functional capabilities. In the second study, the results showed that both protocols decreased body fat and weight, with the best results happening with CR protocol. The immune system had an increase on the phagocytic response and H2O2 production for macrophages and alterations of the proliferative capacities of the lymphocytes, in both protocols.

INTRODUÇÃO: A incidência de câncer da glândula tireoide é a que mais vem crescendo nas últimas décadas. Dentro desse grupo de diferentes neoplasias, o carcinoma papilífero, um dos carcinomas bem diferenciados, representa a maioria e tem prognóstico favorável, com sobrevida acima dos 90% em 5 anos. Embora sejam utilizadas diversas classificações de risco baseadas em diferentes fatores prognósticos, ainda não se consegue predizer quais pacientes terão maior chance de recorrência, metástases linfonodais e desfecho desfavorável, que se beneficiariam de um tratamento mais agressivo. Já foi demonstrado em diversos tipos de neoplasias que diferenças no perfil do infiltrado imune tumoral têm relação com prognóstico e resposta ao tratamento. Neste estudo caracterizou-se o microambiente imune do carcinoma papilífero através de marcadores imuno-histoquímicos de células inflamatórias e relacionou-se este perfil de infiltração com fatores prognósticos clínico-patológicos e com sobrevida livre de recorrência. MÉTODOS: Foram incluídos 151 casos selecionados com base em um banco de dados que incluiu todos os pacientes submetidos a tratamento cirúrgico para câncer de tireoide no A.C.Camargo Cancer Center entre 2008 e 2010. Casos com tireoidite significante foram excluídos. Estes tumores selecionados foram então submetidos a reação imuno-histoquímica com marcadores de células inflamatórias e foram analisados por dois patologistas experientes. As características clínicas e patológicas foram avaliadas, assim como as recorrências e sobrevida, relacionando-as com as leituras de células marcadas. As análises de sobrevida global e livre de doença foram realizadas pelo método de Kaplan-Meier, com comparação de curvas de sobrevida pelo teste de Logrank. RESULTADOS: Cento e cinquenta e um pacientes foram incluídos, sendo 130 (86,1%) mulheres e 21 (13.9%) homens. Multifocalidade foi encontrada em 41 (27.2%), extensão extratireoidiana em 43 (28.5%) e metástase linfonodal em 36 (23.8%) casos. Apenas dois pacientes apresentaram metástase a distância. O tempo de seguimento médio foi 65,1 meses e observou-se nove (6%) pacientes com recorrências de neoplasia. Os pacientes com tumores com metástase linfonodal e/ou extensão extratireoidiana apresentaram maior risco de recorrência. Dos marcadores analisados, uma maior densidade de CD8 (que marca linfócitos citotóxicos) na área peritumoral esteve associada a uma tendência a melhor sobrevida livre de recorrência: 97,1% versus 87,5% (p=0,057), além de significativos menores índices de multifocalidade tumoral e de metástase linfonodal. A maior infiltração de linfócitos T CD8+ no tumor também se relacionou com menor ocorrência de metástase linfonodal nesta amostra (18,4% versus 38,1%, p=0,011). Além disso, a densidade desta marcação, tanto no interior da lesão neoplásica como em área peritumoral foi significativamente maior nos casos de carcinomas papilíferos restritos à tireoide, ou seja, sem extensão extratireoidiana e sem metástases. Os demais marcadores analisados não apresentaram relação significativa e consistente com recorrência ou outros fatores prognósticos. CONCLUSÕES: Nas neoplasias malignas de tireoide, o microambiente imune parece ter uma relação com características patológicas de agressividade. Este estudo mostrou que em carcinoma papilífero de tireoide quando não associado à tireoidite significativa, a densidade do infiltrado tumoral e peritumoral por linfócitos T CD8+ está inversamente relacionada com chance de disseminação metastática linfonodal e, provavelmente, com recidiva da doença, sendo, portanto, um marcador de melhor prognóstico. Este dado sugere que estes linfócitos exercem efeito antitumoral no carcinoma papilífero de tireoide, corroborando a importância da resposta imune na evolução desta neoplasia / INTRODUCTION: Within the last few decades thyroid cancer has the fastest rising incidence rate among all malignancies. In this group of different neoplasms, the papillary carcinoma, one of the well-differentiated carcinomas, represents the great majority and has favorable prognosis, with overall survival rates above 90% in five years. Although several risk classifications based on different prognostic features have been used, they are not accurate to predict which patients will have higher chance of recurrence, lymphatic metastasis and worse outcome, benefiting from more aggressive treatment. It has been described in several kinds of malignancies that tumor related immune infiltration has relation with prognosis and response to treatment. In this study, we characterize the immune microenvironment in papillary thyroid carcinomas, using immunohistochemical markers to inflammatory cells, and relate it with clinical and pathological prognostic features and with recurrence free survival rates. METHODS: The 151 included cases were selected from a database that included all patients who underwent surgical treatment for thyroid cancer at A.C.Camargo Cancer Center between the years 2008 and 2010. Tumor with significant thyroiditis were excluded. The selected tumors were submitted to immunohistochemical reactions with markers of inflammatory cells and analysis in complete slides by two experienced pathologists. Clinical and pathological features were evaluated, as well as recurrence and survival, relating them with the reading of marked cells. Survival analysis were made using Kaplan-Meier method, comparing survival curves with the Logrank test. RESULTS: One hundred and fifty one patients were included, of which 130 (86.1%) were females and 21 (13.9%) males. Multifocal disease was found in 41 cases (27.2%), extrathyroidal extension in 43 (28.5%) and lymph node metastasis in 36 (23.8%). Only two patients had distant metastasis. The mean follow-up time was 65.1 months and we observed nine (6%) tumor recurrences. Tumors with lymph node metastasis and/or extrathyroidal extension showed significantly higher recurrence rates. Of the analyzed markers, the cases with a higher density of CD8 (which marks cytotoxic T lymphocytes) in peritumoral areas presented a trend to better recurrence-free survival: 97.1% versus 87.5% (p=0.057), in addition to lower rates of mutifocal tumors and lymph node metastasis. A higher infiltration rate of CD8+ T lymphocytes in the tumor also correlated with less risk of lymph node metastasis in this sample (18.4% versus 38.1%, p=0.011). Besides that, the density of this marking both in the tumor and in peritumoral areas, was significantly higher in the papillary carcinomas limited to the thyroid gland (without extrathyroidal extension or metastasis). The other markers analyzed did not presented significant or consistent relation with recurrence or other prognostic factors. CONCLUSIONS: In thyroid cancer, the immune microenvironment seems to relate with pathological features of aggressiveness. This study showed that in papillary thyroid carcinomas without significant thyroiditis, the density of tumoral and peritumoral infiltration by CD8+ T lymphocytes is inversely related with lymph node metastasis rate and probably with recurrence, being therefore a marker of better prognosis. These data suggest that these lymphocytes play an anti-tumoral role in papillary thyroid carcinoma, supporting the implication of immune response in the progression of this neoplasm

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-04-26T14:09:15Z No. of bitstreams: 1 lucianomazzoccoli.pdf: 1325202 bytes, checksum: 7054f92647dc1e9d86442d0be8eec899 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-12T15:47:04Z (GMT) No. of bitstreams: 1 lucianomazzoccoli.pdf: 1325202 bytes, checksum: 7054f92647dc1e9d86442d0be8eec899 (MD5) / Made available in DSpace on 2017-05-12T15:47:04Z (GMT). No. of bitstreams: 1 lucianomazzoccoli.pdf: 1325202 bytes, checksum: 7054f92647dc1e9d86442d0be8eec899 (MD5) Previous issue date: 2009-04-29 / A talidomida é utilizada no tratamento de diversas doenças incluindo o eritema nodoso leproso (ENL), uma complicação inflamatória da Hanseníase. Contudo, apresenta atividade teratogênica severa e novos análogos podem ser usados no tratamento da doença sem apresentar este efeito colateral. Uma série de compostos diamínicos contendo duas subunidades ftalimídicas abertas foram escolhidos como análogos da talidomida. Nossos resultados mostram que compostos contendo dois grupos ftalimídicos podem ser facilmente obtidos em elevada quantidade através da condensação de anidrido ftálico ou anidrido 3-nitroftálico com diferentes diaminas comercialmente disponíveis. O efeito destes compostos na produção de TNF-α, IL-12, IL-10 e NO, e na expressão de CD80 e CD86 em células J774A.1 estimuladas com LPS/IFN-γ foi investigado. O nível de mRNA para TNF-α, IL-12, IL-10 e iNOS em J774A.1 foi analisado por RT-PCR em tempo real. Células do sangue periférico humano (PBMC) foram usadas para avaliar a produção de TNF-α, IL-6, IFN-γ , CXCL9 e CXCL10. A produção de citocinas foi avaliada por ELISA, a produção de NO pelo método de Griess, e a expressão de CD80, CD86, CXCL9 e CXCL10 por citometria de fluxo. As células J774A.1 foram incubadas com diferentes concentrações dos compostos (entre 1 a 880 μM) e após 1h foram estimuladas com LPS (1 μg/ml) e IFN-γ (0,4 ng/ml) por 18h. PBMC humano foi incubado de forma similar e estimulado com LPS (2 μg/ml). Três compostos inibiram de forma elevada a produção de TNF-α , IL-12 e NO, conquanto que aumentaram a produção de IL-10. Além disto, inibiram a expressão de CD80, mas não de CD86. Os resultados analisados por RT-PCR em tempo real indicaram que esses compostos atuaram em eventos pós-transcricionais. Os compostos N, N'-Di-(2-carboxi-benzoil)-1,3-propanodiamina, N, N'-Di-(2-carboxi-3-nitro-benzoil)-1,2-etilenodiamina e N, N'-Di-(2-carboxi-3-nitro-benzoil)-1,6-hexanodiamina inibiram mais a produção de TNF-α do que a talidomida (IC50 de 55 μM, 5 μM, 6,1 μM e 220 μM, respectivamente) e também tiveram efeito inibitório na produção de IFN-γ, IL-6, CXCL9 e CXCL10. Os compostos não tiveram efeito na viabilidade celular avaliada por azul de Trypan e por MTT. Este trabalho mostra a síntese e caracterização de novos análogos da talidomida, obtidos em bom rendimento utilizando metodologia simples. Nossos resultados sugerem que a potencial atividade anti-inflamatória e imunorregulatória destes compostos diamínicos apresentam potencial aplicação no tratamento de ENL e outras doenças. / Thalidomide is used to treat various diseases including erythema nodosum leprosum (ENL), an inflammatory complication of leprosy. However, it has severe teratogenic activity and novel thalidomide analogues might be used to treat the disease without this severe side effect. A series of diamine compounds containing two hydrolyzed phthalimido structures were chosen as analogues of thalidomide. Our results show that compounds bearing two phthalimido units could easily be obtained in high yield by condensation of phthalic or 3-nitrophthalic anhydride with the different diamines comercially available. The effects of these compounds on production of TNF-γ, IL-12, IL-10 and NO, and on expression of CD80 and CD86 in LPS/IFN-γ stimulated J774A.1 cells were investigated. The level of TNF-, IL-12, IL-10 and iNOS mRNA in J774A.1 was analyzed by real time RT-PCR. Human peripheral blood (PBMC) was used to evaluate TNF-γ, IFN-α, CXCL9 and CXCL10 production. Cytokine production was evaluated by ELISA, NO production by the Griess assay, and CD80, CD86, CXCL9 and CXCL10 expression by cytometry. J774A.1 cells were incubated with different concentrations of the compounds (between 1 and 880 μM) and after 1h stimulated with LPS (1 μg/ml) and IFN-γ (0,4 ng/ml) for 18h. Human PBMC were similarly incubated with the compounds and stimulated by LPS (2 μg/ml). Three compounds greatly inhibited TNF-α, IL-12 and NO production while enhancing IL-10. In addition, CD80 expression was inhibited, but not CD86. The result analyzed by real time RT-PCR indicates that these compounds act in the blocking of post-transcriptional events. The compounds N, N'-Di-(2-carboxy-benzoyl)-1,3-propanediamine, N, N'-Di-(2-carboxy-3-nitro-benzoyl)-1,2-ethylenediamine e N, N'-Di-(2-carboxy-3-nitro-benzoyl)-1,6-hexanediamine inhibited TNF-α production by PBMC greater then thalidomide (IC50 de 55 μM, 5 μM, 6.1 μM and 220 μM, respectively) and also had a inhibitory effect on IFN-γ , IL-6, CXCL9 and CXCL10 production. The compounds had no effect on cell viability, evaluated by Trypan blue exclusion and MTT assay. This work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. Our results suggest that the potential anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating ENL and other diseases.

As doenças cardiovasculares são a principal causa de mortalidade no mundo. A aterosclerose é a base fisiopatológica dessas doenças, sendo definida como um processo crônico-inflamatório multifatorial, resultando da interação de diferentes células como linfócitos, macrófagos, células endoteliais e células musculares lisas na parede arterial. A lipoproteína de baixa densidade eletronegativa [LDL(-)], uma subfração modificada da LDL nativa, desempenha um papel-chave na aterosclerose, uma vez que as modificações sofridas por esta partícula são capazes de induzir o acúmulo de ésteres de colesterol em macrófagos e a subsequente formação de células espumosas. O sistema imunológico é crucial no processo aterogênico e estratégias terapêuticas direcionadas à imunoregulação deste processo têm sido utilizadas como novas alternativas tanto na prevenção do desenvolvimento quanto da progressão desta doença. Dentre essas estratégias, destaca-se o uso de fragmentos de anticorpos como o scFv (do inglês, single chain fragment variable), que podem ainda estar conjugados a nanopartículas com o intuito de aumentar sua eficiência de ação no organismo. Diante do papel da LDL(-) na aterosclerose, este projeto objetivou avaliar os efeitos in vitro e in vivo de um sistema nanoestruturado contendo fragmentos scFv anti-LDL(-) derivatizados na superfície de nanocápsulas sobre macrófagos murinos e humanos primários e em camundongos knockout para o gene do receptor da LDL (Ldlr-/-) no desenvolvimento e na progressão dessa doença. Demonstrou-se que o tratamento de macrófagos com a formulação scFv anti-LDL(-)-MCMN-Zn diminuiu de forma significativa a captação de LDL(-), assim como a expressão de IL-1β (mRNA e proteína) e MCP-1 (mRNA). Foi demonstrada a internalização da nanoformulação pelos macrófagos via diferentes mecanismos de endocitose, demonstrando seu potencial uso como carreador de fármacos. In vivo, a nanoformulação diminuiu de forma significativa a área da lesão aterosclerótica em camundongos Ldlr-/- submetidos à avaliação pela técnica de tomografia por emissão de pósitrons (do inglês, PET), utilizando o radiotraçador 18F-FDG (18F-desoxiglicose), associada à tomografia computadorizada (CT) com agente de contraste iodado, além da análise morfométrica das lesões no arco aórtico. O conjunto dos resultados obtidos evidenciou a ação ateroprotetora da formulação scFv anti-LDL(-)-MCMN-Zn, reforçando seu potencial como estratégia terapêutica na aterosclerose. / Cardiovascular diseases are the leading cause of mortality worldwide. Atherosclerosis is the pathophysiological basis of these diseases, defined as a chronic inflammatory multifactorial process, resulting from the interaction of several cells such as lymphocytes macrophages, endothelial cells and smooth muscle cells within the arterial wall. The electronegative low-density lipoprotein [LDL(-)], a modified subfraction of native LDL, plays a key role in atherosclerosis, since its modifications are capable of inducing the accumulation of cholesteryl esters in macrophages and the subsequent foam cells formation. The immune system is crucial in atherogenic process and therapeutic strategies directed to the immunoregulation of this process have been used as a new alternative in the prevention of the development as well as the progression of this disease. Among these strategies, it is the use of antibody fragments such as scFv (single chain fragment variable), which may be also conjugated to nanoparticles in order to increase their efficiency in the body. Given the role of LDL(-) in atherosclerosis, the aim of this project was to evaluate the in vitro and in vivo effects of a nanostructured system containing scFv anti-LDL(-) fragments derivatized on the surface of nanocapsules on murine and human primary macrophages and in the development and progression of the disease in LDL receptor knockout mice (Ldlr-/-). It was demonstrated that the treatment of macrophages with scFv anti-LDL(-)-MCMN-Zn formulation significantly decreases the uptake of LDL(-) and the expression IL-1β (mRNA and protein) and MCP-1 (mRNA). Moreover, the internalization of the nanoformulation by macrophages through different endocytosis mechanisms was shown, demonstrating its potential use as a nanocarrier. In vivo, the nanoformulation decreased the area of atherosclerotic lesions in Ldlr-/- mice evaluated by positron emission tomography with 18F-FDG associated with computed tomography with iodinated contrast agent (PET/CT), besides the lesion morphometric analysis at the aortic arch Thus, these data provide evidence of the atheroprotection action of the ateroprotection action of the scFv anti-LDL(-)-MCMN-Zn formulation, suggesting its promising use as a therapeutic strategy for atherosclerosis.

Une forte production de la chimiokine CCL2 par les cellules malignes et les cellules stromales a été démontrée dans la plupart des cancers humains. Ainsi, l’axe chimiokinique CCR2/CCL2 est un important marqueur du développement des cancers ; ce même axe est associé à la récurrence de tumeurs après thérapie anticancéreuses. Les macrophages associés aux tumeurs (TAM) et les lymphocytes T régulateurs (Treg) ont des capacités immunosuppressives robustes et contribue à la croissance tumorale. Durant cette thèse, je me suis intéressé à la fonction de l’expression du récepteur de chimiokine CCR2 par ces cellules dans le contexte de thérapies anticancéreuses. Nous avons montré que le récepteur de chimiokines CCR2 contrôle la migration des Treg en contexte tumoral, chez l’homme et la souris, et que son expression par les Treg peut servir de biomarqueur de la réponse à la chimiothérapie. Notre étude indique une nouvelle fonction de CCR2 et définie un nouveau sous-type de Treg impliqué dans la régulation de l’immunité antitumorale. En parallèle, nous avons pu mettre en évidence que les métastases pulmonaire sont composées à la fois de macrophages résident du tissu et de macrophages recrutés via l’axe CCR2. La présence de macrophages résidents au sein des tumeurs pourrait contribuer à l’hétérogénéité des microenvironnements de diffèrent type de tumeurs. Le récepteur CCR2 est important pour le la phase de rechute après chimiothérapie, indiquant un rôle limité des macrophages résidents dans ce phénomène. De plus, nous avons montré que le VEGF joue un rôle direct dans la survie des TAM. Ainsi, la combinaison de la chimiothérapie avec un anticorps anti-VEGF cible simultanément les TAM résidents et recrutés et permet d’augmenter l’efficacité de la chimiothérapie. / Malignant and stromal cells are strong producer of the chemokine CCL2 in most human cancers. The chemokine axis CCR2/CCL2 is thus a key marker of cancer development, but is also associated with relapse following therapy. Tumour associated macrophages (TAM) and regulatory T cells (Treg) display robust immunosuppressive capacities and contribute to tumour growth. My thesis work focused on the function of the expression of the chemokine receptor CCR2 by these cell types in the context of anticancer therapies. We have shown that CCR2 controls the migration of Treg in tumoral context, in both human and mice, and that the expression of this receptor by Treg could serve as a biomarker of the response to chemotherapy. Our study indicate a novel function of CCR2, defining at the same time a new Treg subset implicated in the regulation of antitumor immunity.We have also demonstrated that pulmonary metastases are composed of both tissue resident and recruited macrophages. The presence of resident macrophages within tumours could contribute to the heterogeneity of the microenvironment of different tumour types. CCR2 is largely implicated in the relapse phase following chemotherapy, indicating a limited role for resident macrophages in this phenomenon. Meanwhile, we have demonstrated that VEGF plays a direct role in TAM survival. The combination of chemotherapy with an anti-VEGF antibody targets both resident and recruited TAM, thereby enhancing the efficacy of chemotherapy. Finally, we have shown that the CCR2/CCL2 axis is implicated in the response to radiotherapy by enhancing the recruitment of both Treg and TAM. This work provides evidences for a central role of the CCR2/CCL2 axis in mediating Treg and TAM co-localization in response to anticancer therapy, this axis could also contribute to establishment of immunosuppressive networks in tumours. Our results provide a better understanding of the immune mechanism implicated in resistance to anticancer therapies.

The adenylate cyclase toxin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates CR3-expressing phagocytes and catalyzes the uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by oxidative burst and opsonophagocytic mechanisms. Here we show that CyaA suppresses the production of bactericidal reactive oxygen and nitrogen species in neutrophils and macrophages, respectively. The inhibition of reactive oxygen species (ROS) production is most-likely achieved by the combined PKA-dependent inhibition of PLC and Epac-dependent dysregulation of NADPH oxidase assembly. Activation of PKA or Epac interfered with fMLP-induced ROS production and the inhibition of PKA partially reversed the CyaA-mediated inhibition of ROS production. CyaA/cAMP signaling then inhibited DAG formation, while the PIP3 formation was not influenced. These results suggest that cAMP produced by CyaA influences the composition of target membranes. We further show here that cAMP signaling through the PKA pathway activates the tyrosine phosphatase SHP-1 and suppresses the production of reactive nitrogen species (RNS) in macrophages. Selective activation of PKA interfered with LPS- induced iNOS expression...

Adipose tissue (AT) expansion and remodeling that maintains healthy function relies on stromal preadipocytes capable of differentiating into new adipocytes (adipogenesis). During chronic positive energy balance, a relative deficit in adipogenesis, from either a decrease in preadipocyte number or their capacity to differentiate, leads to excessive adipocyte hypertrophy and AT dysfunction. AT contains macrophages whose number and activation state is dynamically regulated with changes in AT mass. This study aims to investigate the effect of macrophage-secreted factors on preadipocyte survival. To assess the effect of macrophage-secreted factors on preadipocytes, murine 3T3-L1 preadipocytes or human primary preadipocytes were incubated with macrophage-conditioned medium (MacCM), prepared from either murine (J774A.1, RAW264.7, bone marrow-derived) or human (THP-1, monocyte-derived) macrophage models, respectively. MacCM inhibited preadipocyte apoptosis and activated pro-survival signaling in both preadipocyte models. Inhibition of PDGFR, Akt, or ERK1/2 reduced the pro-survival effect of MacCM in 3T3-L1 preadipocytes. Inhibition of reactive oxygen species (ROS) generation, or enhancement of ROS clearance, reduced MacCM-dependent 3T3-L1 preadipocyte survival. Whereas anti-inflammatory activated macrophages retained the ability to prevent preadipocyte apoptosis, pro-inflammatory activated macrophages did not. TNF-α immunoneutralization restored the survival activity of pro-inflammatory MacCM on 3T3-L1 preadipocytes. These studies reveal a novel pro-survival effect of MacCM on preadipocytes, and identify signaling molecules (PDGF, Akt, ERK1/2, and ROS) that underlie this action. Macrophage activation was found to regulate the pro-survival activity of MacCM. These in vitro cell culture studies are consistent with a model in which the extent of preadipocyte apoptosis in vivo may determine preadipocyte number and the ability of AT to expand while maintaining healthy function during chronic positive energy balance.

No description available.

Inflammatory processes mark all stages of atherogenesis. One of the key regulators of inflammation is the transcription factor nuclear factor kappa B (Nf-kb). Nf-kb is the general name for a whole family of dimeric transcription factors. One can distinguish between a classical and an alternative pathway with Rela/p50 (Nf-kb1) and Relb/p52 (Nf-kb2) representing the terminal transcription factors, respectively. Classical Nf-kb1 signaling has been associated with atherosclerotic lesion development many times, mainly because of its regulation of many pro-inflammatory proteins with an established role in atherogenesis. Recent studies provided evidence of crosstalk between classical Nf-kb1 and alternative Nf-kb2 signaling, implicating a potential role for Nf-kb2 in atherogenesis. The aim of the present study was to investigate the influence of Nf-kb2 on atherosclerotic lesion development in a knockout mouse model. Nfkb2 knockout (Nfkb2-/-) mice were generated on two different atherosclerosis sensible backgrounds, the Apoe- and Ldlr- deficient background. Quantification of atherosclerotic lesion development showed, that Nfkb2-/- mice developed significantly more atherosclerosis at the brachiocephalic artery than wild type controls, indicating a protective effect of Nf-kb2 on atherogenesis. Further expression analyses in bone marrow-derived macrophages (BMDM) revealed highly significant upregulation of matrix metalloproteinase 9 (Mmp9) in Nfkb2-/- mice. Overexpression of Mmp9 was associated with enhanced macrophage migration across extracellular matrix in vitro and an inflammatory plaque phenotype with advanced, macrophage-rich lesions. Accordingly, increased Mmp9 expression in Nfkb2-/- macrophages might have contributed to enhanced lesion development in these mice.

Subklinische entzündliche Veränderungen des Endometriums können nur durch die histopathologische Untersuchung eines Bioptates diagnostiziert werden und sind von besonderer Bedeutung bei Fertilitätsstörungen. Die nicht-eitrige (NE) Endometritis ist gekennzeichnet durch eine Infiltration des Endometriums mit Lymphozyten, Plasmazellen und/oder Makrophagen. Die genaue Pathogenese ist unklar. Die immunhistologische Phänotypisierung dieser Zell-(sub-)populationen kann möglicherweise pathogenetische Hinweise geben. Der immunhistologische Nachweis equiner Immunzellen ist jedoch fast ausschließlich an Gefrierschnitten etabliert. Dies ist für die Routinediagnostik ungeeignet. Demzufolge ergaben sich die folgenden Ziele: 1. Eine alternative Methode zur Gewebsfixierung zu etablieren, die in der Routinediagnostik praktikabel einsetzbar ist, die einen guten Strukturerhalt, eine geeignete Anfärbbarkeit und ideale Auswertbarkeit gewährleistet sowie umfangreiche immunhistochemische Untersuchungen ermöglicht; 2. Die immunhistochemische Phänotypisierung von Lymphozyten- und Makrophagen-(sub-)populationen an fixierten equinen Gewebeproben zu entwickeln; 3. Eine semiquantitative Bestimmung der endometrialen Entzündungszellen ohne und mit einer NE Endometritis durchzuführen. Tiere, Material & Methoden: Gewebeproben (Lymphknoten als Referenzgewebe und endometriales Gewebe) wurden von 5 Stuten im Rahmen der Sektion entnommen, in Formalin (F), HOPE® (H) oder IHC Zinc Fixative (Z) fixiert und zusätzlich als natives Gefriermaterial aufgearbeitet. Es erfolgte eine vergleichende Beurteilung der Gewebemorphologie, des Färbeverhaltens und der Artefakte am equinen Endometrium in HE gefärbten Gefrierschnitten bzw. im fixierten, Paraffin-eingebetteten Material (F, H und Z). Equine Lymphknoten dienten als Referenzgewebe für die immunhistochemische Etablierung der Lymphozytenmarker (anti-CD3, -CD4, -CD8) an Gefrierschnitten und am fixierten, Paraffin-eingebetteten Material (F, H und Z). F- und Z-fixiertes Referenzgewebe (Leber, Dünndarm, Darmlymphknoten) von 4 Stuten wurde für die Etablierung der Makrophagenmarker (anti-CD172a, -CD14, -CD206) verwendet. Die Immunreaktionen der Primärantikörper wurden verglichen. Als Resultat aus den vergleichenden Analysen wurde die Z-Fixierung als effektivste alternative Fixierungsmethode ausgewählt. Im abschließenden Teil der Untersuchungen wurden 28 Z-fixierte Endometriumbioptate hinsichtlich der Entzündungszellen semiquantitativ ausgewertet. 4 Stuten zeigten keine Entzündungsreaktion und dienten als Kontrolle. 24 Stuten wiesen eine oberflächliche NE Endometritis auf. Die Anzahl CD3+, CD4+, CD8+, CD20+, CD172a+, CD14+ und CD206+ Zellen sowie die Anzahl von Plasmazellen mittels Methylgrün-Pyronin-Färbung wurde an Serienschnitten in 5 zufällig ausgewählten Gesichtsfeldern (400x) jeweils für das Stratum compactum, Stratum spongiosum und das luminale sowie glanduläre Epithel erhoben. Ergebnisse: 1. Die Z-Fixierung zeigt mit der F-Fixierung vergleichbare, exzellente Eigenschaften hinsichtlich des Strukturerhalts, der Anfärbbarkeit und Auswertbarkeit. Sie übersteigt insgesamt die Eigenschaften der H-Fixierung: die Z-Fixierung erweist sich einfacher in der Anwendung und zeigt eine geringere Artefaktbildung. 2. Die immunhistochemische Charakterisierung von T- und B-Lymphozyten, Helfer-T-Lymphozyten sowie zytotoxischen T-Lymphozyten und Makrophagenpopulationen sowie der Nachweis von Plasmazellen mittels Methylgrün-Pyronin-Färbung an Z-fixierten equinen Gewebeproben ist möglich. 3. Im entzündlich veränderten Endometrium sind mehr CD3+, CD4+, CD8+, CD20+, CD172a+, CD206+ Zellen sowie Plasmazellen nachweisbar als in unveränderten Gewebeproben bezogen auf das Stratum compactum und Stratum spongiosum. Die durchschnittliche Anzahl CD3+ Zellen ist im Stratum compactum NE Endometritiden knapp 3x höher als im unveränderten Endometrium. Wenige CD20+ B-Zellen und CD172a+ Makrophagen sowie eine unterschiedliche Anzahl an Plasmazellen sind innerhalb des Stratum compactum entzündlich veränderter Endometrien nachweisbar. Es existieren große individuelle Unterschiede in der Anzahl CD4+ und CD8+ T-Zellen in Endometrien ohne sowie mit einer Entzündung. Schlussfolgerungen: Eine Integration der Z-Fixierung in die Routinediagnostik mit zusätzlichen Anwendungsmöglichkeiten ist problemlos möglich. Die immunhistochemische Entzündungszelldifferenzierung im Endometrium der Stute kann an fixierten equinen Endometriumbioptaten durchgeführt werden und bietet darüber hinaus die Möglichkeit, diese Methode an anderen equinen Organen anzuwenden. Die Ergebnisse der semiquantitiven Auswertung deuten auf das mögliche Vorliegen unterschiedlicher Formen der nicht-eitrigen Entzündung hin. Das etablierte Verfahren gibt möglicherweise nähere Hinweise auf immunologische Konstellationen, die das Auftreten einer nicht-eitrigen Endometritis begünstigen.:INHALTSVERZEICHNIS I ABKÜRZUNGSVERZEICHNIS II 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 2 2.1 Das Endometrium der Stute 2 2.1.1 Endometritiden 4 2.1.1.1 Eitrige Endometritis 4 2.1.1.2 Nicht-eitrige Endometritis 5 2.1.1.3 Sonderformen 6 2.1.2 Resistant und susceptible mares 8 2.1.3 Entzündungszelltypisierung im Endometrium der Stute 10 2.1.4 Diagnostische Bedeutung des Endometriumbioptates 13 2.2 Immunologie 14 2.2.1 T-Lymphozyten 14 2.2.2 B-Lymphozyten 17 2.2.3 Makrophagen 18 2.2.4 Immunhistochemische Charakterisierung von equinen Entzündungszellen 21 2.3 Fixierungsmethoden 24 2.4 Fazit aus der Literatur bezüglich der Fragestellung dieser Arbeit 26 3 PUBLIKATIONEN 28 3.1 Publikation 1 inkl. Stellungnahme zum Eigenanteil 28 3.2 Publikation 2 inkl. Stellungnahme zum Eigenanteil 39 4 DISKUSSION 53 4.1 Etablierung alternativer Methoden zur Gewebefixierung 54 4.2 Immunhistochemische Detektion von Immunzell- (sub-)populationen 59 4.3 Erkenntnisse im Hinblick auf die Pathogenese nicht-eitriger Endometritiden 60 5 ZUSAMMENFASSUNG 63 6 SUMMARY 65 7 LITERATURVERZEICHNIS 67 8 DANKSAGUNG 81 / Clinically unapparent inflammatory alterations of the equine endometrium can only be diagnosed by the histopathological examination of a biopsy and are the main cause of subfertility in mares. The non-suppurative endometritis is characterised by infiltration of the endometrium with lymphocytes, plasma cells and/or macrophages. So far, the cause and pathogenesis of non-suppurative endometritis are unclear. The subclassification of the immune cell populations involved will likely provide important information on the etiology and pathogenesis of this disease. Available antibodies are often established exclusively for immunohistochemistry on cryostat sections of native frozen equine tissue. However, this method is impractical for the routine diagnostic work-up. The aim of the present study was: 1. To reveal the method most suitable for a routine diagnostic work-up in combination with proper tissue preservation, staining results as well as histological and immunohistochemical analysis; 2. To establish an immunohistochemical method for the detection of lymphocytes and macrophages including their subpopulations in fixed equine tissue samples; 3. To characterize the immune cell populations in fixed endometria of mares without and with non-suppurative endometritis. Material & methods: Equine endometrial tissue and intestinal lymph node were obtained from five mares during post mortem examination and were either fixed in formalin, HOPE® or IHC Zinc Fixative. Aditionally snap frozen tissue samples were processed for cryostat sectioning. HE stained cryostat sections and fixed paraffin embedded tissue samples (formalin, HOPE®, IHC Zinc Fixative) of the equine endometrium were analysed regarding tissue preservation, staining results and artefacts. To establish lymphocyte markers (anti-CD3, anti-CD4 and anti-CD8) immunohistochemically on cryostat sections and fixed paraffin embedded tissue samples (formalin, HOPE®, IHC Zinc Fixative) equine lymph node was used as reference. Formalin fixed and zinc fixed tissue samples with macrophage populations (liver, small intestine, intestinal lymph node) from four mares were used for the immunohistochemical establishment of the macrophage markers (anti-CD172a, anti-CD14, anti-CD206). The immunoreactivity of the primary antibodies was compared. The results of the comparative analysis revealed the most suitable alternative fixation method (zinc fixation). Finally, 28 zinc fixed endometrial biopsies were evaluated semiquantitatively with regard to the numbers of CD3+, CD4+, CD8+, CD20+ lymphocytes, CD172+, CD14+, CD206+ macrophages as well as plasma cells. Four mares had no evidence of endometritis and represent the control group. The remaining 24 mares showed a mild superficial non-suppurative endometritis. The comparative analysis was performed in serial sections within five randomly selected high power fields (400x). Positive cells were counted separately within the luminal and glandular epithelium, the stratum compactum and the stratum spongiosum. Results: 1. The zinc fixation provides excellent staining results, tissue preservation and allows histological and immunohistochemical analysis. It has even some advantages compared to the HOPE® fixation regarding the routine diagnostic work-up. The zinc fixation produces less artefacts. 2. The zinc fixation allows the immunohistochemical detection of all applied T- and B-lymphocyte-, helper- and cytotoxic-T-cell- and macrophage-markers and the histochemical detection of plasma cells (methyl green-pyronin stain) within equine fixed tissue. 3. Endometria with non-suppurative endometritis have higher numbers of CD3+, CD4+, CD8+, CD20+, CD172a+, CD206+ as well as plasma cells within the stratum compactum and stratum spongiosum than endometria without inflammation. The average cell count of CD3+ lymphocytes within the stratum compactum was approximately 3 x higher within inflamed endometria than this value obtained from endometria without endometritis. Few CD20+ B cells and CD172+ macrophages as well as variable numbers of plasma cells could be detected within the stratum compactum of mares with non-suppurative endometritis. Endometria with and without inflammation showed marked differences in the numbers of CD4+ and CD8+ T cells. Conclusion: The zinc fixation can be easily incoorporated into the routine diagnostic work-up and offers additional application possibilities. Immunohistochemical phenotyping of immune cells in the equine endometrium can be performed on fixed endometrial biopsies of the mare. Furthermore, a widespread applicability is possible, e. g. on other equine organs. These findings suggest the existence of different forms of non-suppurative endometritis. The established method will likely assist to reveal possible etiologies and predisposing conditions for non-suppurative endometritis.:INHALTSVERZEICHNIS I ABKÜRZUNGSVERZEICHNIS II 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 2 2.1 Das Endometrium der Stute 2 2.1.1 Endometritiden 4 2.1.1.1 Eitrige Endometritis 4 2.1.1.2 Nicht-eitrige Endometritis 5 2.1.1.3 Sonderformen 6 2.1.2 Resistant und susceptible mares 8 2.1.3 Entzündungszelltypisierung im Endometrium der Stute 10 2.1.4 Diagnostische Bedeutung des Endometriumbioptates 13 2.2 Immunologie 14 2.2.1 T-Lymphozyten 14 2.2.2 B-Lymphozyten 17 2.2.3 Makrophagen 18 2.2.4 Immunhistochemische Charakterisierung von equinen Entzündungszellen 21 2.3 Fixierungsmethoden 24 2.4 Fazit aus der Literatur bezüglich der Fragestellung dieser Arbeit 26 3 PUBLIKATIONEN 28 3.1 Publikation 1 inkl. Stellungnahme zum Eigenanteil 28 3.2 Publikation 2 inkl. Stellungnahme zum Eigenanteil 39 4 DISKUSSION 53 4.1 Etablierung alternativer Methoden zur Gewebefixierung 54 4.2 Immunhistochemische Detektion von Immunzell- (sub-)populationen 59 4.3 Erkenntnisse im Hinblick auf die Pathogenese nicht-eitriger Endometritiden 60 5 ZUSAMMENFASSUNG 63 6 SUMMARY 65 7 LITERATURVERZEICHNIS 67 8 DANKSAGUNG 81