Background & Aims: Oxidative stress and apoptosis are proposed mechanisms of cellular injury in studies of xenobiotic hepatotoxicity. The aim of this work is to find early signal markers of drug-induced injury of the liver by focusing on select antioxidant/oxidant and apoptotic genes. As well, to address the relationship between conventional liver dysfunction markers and the measured mRNA and protein expressions in the D-galactosamine/lipopolysaccharide and tert-butylhydroperoxide hepatotoxicity models. Furthermore, potential hepatoprotective capabilities of antioxidant polyphenols quercetin and curcumin were evaluated in relation to its modulation of the oxidative stress and apoptotic parameters in the given xenobiotic hepatotoxicity models. Methods: Biochemical markers testing the hepatic function included aminotransferases (ALT, AST) and bilirubin. Measurements of TBARS and conjugated dienes were used to assess lipoperoxidation. Plasma levels of catalase and reduced glutathione were used as indicators of the oxidative status of the cell. Real time PCR was used to analyse the mRNA expressions of the inducible nitric oxide synthase (NOS-2), heme oxygenase-1 (HO-1), superoxide dismutase (SOD-1), glutathione peroxidase (Gpx-1), caspase 3 (Casp3), BH3 interacting domain death agonist (Bid) and Bcl-2...

Introduction: Durant la pathogenèse d’ostéoarthrose (OA), les cytokines pro-inflammatoires IL-1β (Interleukin-1 beta) et TNF-α (Tumor necrosis factor alpha) stimulent la dégradation des agrécanes par l’aggrécanase-1 ou ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motif). Ces cytokines peuvent stimuler plusieurs voies de signalisation conduisant ainsi à l’augmentation de l’expression des ADAMTS dans les chondrocytes humains. Les TIMPs (tissue inhibitor of metalloproteinases) présentent des inhibiteurs endogènes de l’ADAMTS. Nous avons démontré que la Rapamycine (un immunosuppresseur et un inhibiteur du mamalian target of Rapamycin (mTOR)) peut avoir des effets bénéfiques dans cette pathologie. Notre étude examine l’effet de la Rapamycine sur l’expression de l’ADAMTS-4 induit par les cytokines, son implication dans certaines voies de signalisation, et son effet sur l’expression du TIMP-3. Méthodes: Des chondrocytes normaux sont traités avec la Rapamycine seule ou stimulés aussi avec l’IL-1β et le TNF-α. Les effets de la Rapamycine sur l’expression de l’ADAMTS-4 et du TIMP-3 ont été étudiés par l’analyse RT-PCR et l’activité enzymatique a été étudiée par la technique d’ELISA. Les effets de la Rapamycine sur certaines voies de signalisation ont été étudiés par le Western blot. Résultats: Nous avons trouvé que la Rapamycine inhibe l’expression de l’ARNm de l’ADAMTS-4 induit par les cytokines pro-inflammatoires dans les chondrocytes humains. L’activité enzymatique de l’ADAMTS-4 induit par l’IL-1β a été légèrement diminuée par la Rapamycine. En plus, cette dernière a montré de différents effets sur plusieurs voies de signalisation stimulées par l’IL-1β et le TNF-α telles que les voies des MAPKs (Mitogen activated protein kinase), de l’AKT, et de la p70 S6 kinase. La Rapamycine a inhibé partiellement l’activation de la phosphorylation de l’ERK1/2 MAPK (extracellular signal-regulated protein kinase MAPK) en présence du TNF-α seulement. En outre, la Rapamycine a inhibé la phosphorylation des protéines p38 MAPK, JNK (c-Jun N-terminal kinase), et AKT activée par l’IL-1β seulement. En plus, la phosphorylation de la protéine p70 S6K stimulée par l’IL-1β et le TNF-α a été inhibée par la Rapamycine. D’autre part, nous avons démontré que le niveau du TIMP-3 a été augmenté en présence de la Rapamycine. Conclusion: Ces résultats suggèrent que la Rapamycine peut bloquer l’action de l’ADAMTS-4 via l’inhibition de l’activation des MAPKs, de l’AKT, et de la p70 S6K. La Rapamycine pourrait ainsi être considérée pour la prévention de la perte du cartilage chez les patients ostéoarthritiques. / Introduction: During the pathogenesis of osteoarthritis, the pro-inflammatory cytokines IL-1β (Interleukin-1 beta) and TNF-α (Tumor necrosis factor alpha) stimulate the degradation of aggrecans by aggrecanase-1 or ADAMTS-4 (A disintegrin and metalloproteinase with thrombospondin motif). These cytokines may stimulate several signaling pathways leading to an increased expression of ADAMTS-4 in human chondrocytes. The TIMPs (tissue inhibitor of metalloproteinases) are endogens inhibitors of ADAMTS-4. It has been shown that Rapamycin (an immunosuppressor and an inhibitor of the mammalian target of rapamycin or mTOR) may have beneficial effects in this pathology. Our study investigates the impact of Rapamycin on cytokine-induced expression of ADAMTS-4, his implication in certain signaling pathways, and his effects on the expression of TIMP-3. Methods: Human chondrocytes were pretreated with different doses of Rapamycin either alone or stimulated with IL-β and TNF-α. The effect on ADAMTS-4 expression was examined by RT-PCR analysis and enzyme activity by ELISA. Impact of Rapamycin on the activation of different signaling pathways was measured by Western blot analysis. Results: We have shown that Rapamycin down-regulated pro-inflammatory cytokines-induced ADAMTS-4 mRNA expression in human chondrocytes. The IL-1β-induced-enzymatic activity of ADAMTS-4 was slightly inhibited by Rapamycin. Furthermore, Rapamycin present different effects on pro-inflammatory cytokines-stimulated activation of certain signaling pathways such as MAPKs (Mitogen activated protein kinases), AKT, and P70 S6 kinase. Moreover, Rapamycin partially inhibits TNF-α-induced activation of phosphorylation of ERK1/2 MAPK (extracellular signal-regulated protein kinase MAPK). Thus, Rapamycin inhibit IL-1β-induced activation of phosphorylation of the proteins p38 MAPK, JNK (c-Jun N-terminal kinase), and AKT. Also, Rapamycin inhibit IL-1β and TNF-α-activation of phosphorylation of the protein p70 S6 kinase. In other way, we have shown that the level of TIMP-3 has been increased in the presence of Rapamycin. Conclusion: These results suggest that Rapamycin down-regulates the expression of ADAMTS-4 by inhibiting the cytokine activation of MAPKs, AKT, and p70 S6 kinase. Thus Rapamycin could be considered as potential therapeutic agent for prevention of cartilage loss in patient with osteoarthritis.

Le glaucome est la principale cause de cécité irréversible dans le monde. Chez les patients atteints de cette pathologie, la perte de la vue résulte de la mort sélective des cellules ganglionnaires (CGR) de la rétine ainsi que de la dégénérescence axonale. La pression intraoculaire élevée est considérée le facteur de risque majeur pour le développement de cette maladie. Les thérapies actuelles emploient des traitements pharmacologiques et/ou chirurgicaux pour diminuer la pression oculaire. Néanmoins, la perte du champ visuel continue à progresser, impliquant des mécanismes indépendants de la pression intraoculaire dans la progression de la maladie. Il a été récemment démontré que des facteurs neuroinflammatoires pourraient être impliqués dans le développement du glaucome. Cette réponse est caractérisée par une régulation positive des cytokines pro-inflammatoires, en particulier du facteur de nécrose tumorale alpha (TNFα). Cependant, le mécanisme par lequel le processus neuroinflammatoire agit sur la mort neuronale reste à clarifier. L’hypothèse principale de ce doctorat propose que les facteurs pro-inflammatoires comme le TNFα et la phosphodiestérase 4 (PDE4) interagissent avec les mécanismes moléculaires de la mort neuronale, favorisant ainsi la survie et la protection des CGRs au cours du glaucome. Dans la première partie de ma thèse, J’ai utilisé un modèle in vivo de glaucome chez des rats Brown Norway pour montrer que l’expression du TNFα est augmentée après l'induction de l'hypertension oculaire. L'hypothèse spécifique de cette étude suggère que les niveaux élevés de TNFα provoquent la mort des CGRs en favorisant l'insertion de récepteurs AMPA perméables au calcium (CP-AMPAR) à la membrane cytoplasmique. Pour tester cette hypothèse, j’ai utilisé un inhibiteur sélectif de la forme soluble du TNFα, le XPro1595. L'administration de cet agent pharmacologique a induit une protection significative des somas et des axones des neurones rétiniens. L'évaluation de la perméabilité au cobalt a montré que le TNFα soluble est impliqué dans l'insertion de CP-AMPAR à la membrane des CGRs lors du glaucome. L’exposition des neurones à une pression oculaire élevée est à l’origine de la hausse de la densité membranaire des CP-AMPARs, grâce à une diminution de l’expression de la sous-unité GluA2. La présence de GluA2 au sein du récepteur ne permet pas l’entrée du calcium à l’intérieur de la cellule. L'administration intraoculaire d’antagonistes spécifiques des CP-AMPARs promeut la protection des somas et des axones des CGRs. Ces résultats montrent que les CP-AMPARs jouent un rôle important dans la pathologie du glaucome. Dans la deuxième partie de ma thèse, j’ai caractérisé l'effet neuroprotecteur d’un inhibiteur de la PDE4, l’ibudilast, dans notre modèle de glaucome. L'hypothèse spécifique s’oriente vers une atténuation de la réponse neuroinflammatoire et de la gliose par l’administration d’ibudilast, favorisant ainsi la protection neuronale. Les résultats montrent que dans les rétines glaucomateuses, l’ibudilast diminue la gliose et l'expression de plusieurs facteurs tels que le TNFα, l'interleukine-1β (IL-1β), l’interleukine-6 (IL-6) et le facteur inhibiteur de la migration des macrophages (MIF). Chez les rats glaucomateux, nous avons observé une expression notable de PDE4A dans les cellules de Müller, qui est en corrélation avec l'accumulation de l’AMP cyclique (AMPc) dans ces cellules après un traitement d’ibudilast. Finalement, nous avons démontré que la protection des CGRs via l’administration d’ibudilast est un mécanisme dépendent de l’AMPc et de la protéine kinase A (PKA). En conclusion, les résultats présentés dans cette thèse identifient deux mécanismes différents impliqués dans la perte des CGRs au cours du glaucome. Ces mécanismes pourraient fournir des perspectives potentielles pour le développement de nouvelles stratégies de traitement du glaucome. / Glaucoma is the leading cause of irreversible blindness worldwide. Loss of vision in glaucoma results from the selective death of retinal ganglion cells (RGCs) and axonal degeneration. Elevated intraocular pressure (IOP) is the major risk factor for developing glaucoma, and current therapies have focused on pharmacological or surgical strategies to lower IOP. However, visual field loss continues to progress in spite of effective pressure control, indicating that mechanisms other than elevated IOP contribute to disease progression. Recent data demonstrate a neuroinflammatory component in glaucoma, characterized by upregulation of proinflammatory cytokines, most notably tumor necrosis factor α (TNFα). However, the mechanism by which the neuroinflammatory response acts on RGC death needs to be clarified. The main hypothesis of this thesis is that targeting pro-inflammatory factors including TNFα and phosphodiesterase-type 4 (PDE4), interferes with molecular mechanisms that contribute to RGC death and this will thus successfully promote neuronal protection. In the first part of my thesis, I used an in vivo glaucoma model in Brown Norway rats to show that TNFα is upregulated early after induction of ocular hypertension. The specific hypothesis of this study is that high levels of TNFα promote RGC death by mediating the membrane insertion of Ca2+-permeable AMPA receptors (CP-AMPARs). I blocked TNFα function with XPro1595, a selective inhibitor of soluble TNFα. Administration of XPro1595 effectively protected RGC soma and axons. The cobalt permeability assay was used to show that soluble TNFα triggers the membrane insertion of CP-AMPAR in RGCs of glaucomatous retinas. This CP-AMPAR activation is caused by the downregulation of GluA2 which occurs when neurons are exposed to elevated IOP. Finally, intraocular administration of specific CP-AMPAR antagonists promoted RGC soma and axon protection. Taken together, these results show that CP-AMPARs play an important role in in the pathology of glaucoma. In the second part of my thesis, I characterized the neuroprotective effect of ibudilast, an inhibitor of PDE4, in the Brown Norway glaucoma model. We hypothesized that ibudilast promotes neuron protection by attenuating gliosis and the neuroinflammatory response. The results show that in glaucomatous retinas, ibudilast attenuates gliosis and the expression of TNFα, interleukin-1β (IL-1β), interleukin-6 (IL-6) and macrophage migration inhibitory factor (MIF). Interestingly, elevated IOP leads to substantial expression of PDE4A in Müller cells, which correlates with the accumulation of cAMP in these cells after ibudilast treatment. Lastly, ibudilast promoted RGC soma and axons protection through the activation of the cAMP/PKA pathway. In conclusion, the findings presented in this thesis identify two different mechanisms underlying RGC loss in glaucoma. These mechanisms can potentially provide new insights to develop novel strategies for the treatment of glaucoma

Introdução: Estudos epidemiológicos têm estabelecido que a artrite reumatoide está associada com um aumento de doença cardiovascular. A avaliação de anti-TNF sobre a redução do risco de infarto agudo do miocárdio e óbito por causa cardiovascular tem demonstrado resultados promissores. A avaliação econômica para esses desfechos ainda não está estabelecida. Objetivo: Realizar uma análise de custo efetividade de anti-TNF versus Dmards para evitar um caso novo de morbidade e óbito cardiovascular na artrite reumatoide. Método: Foi realizada uma análise de custo efetividade por meio do modelo de Markov, ciclo de transição de 6 meses e horizonte temporal de 30 anos sob perspectiva do sistema público de saúde do Brasil mensurado pela razão de custo efetividade incremental. Custo em Reais no ano de 2015 e efetividade em evitar um caso novo de doença coronariana isquêmica aguda e óbito cardiovascular. Resultados: O custo médio em 30 anos de Dmards e anti-TNF foi de R$ 14.291.105,28 e R$ 96.151.873,86, respectivamente. A efetividade incremental para doença arterial coronariana foi de 2,69 e consequente razão incremental de custo efetividade de R$ 30.527.502,27, enquanto para óbito cardiovascular a efetividade incremental foi de 1,33 casos evitados e uma razão incremental de custo efetividade de R$ 61.634.231,69. A análise univariada identificou que o parâmetro de maior impacto na razão incremental de custo efetividade de ambos os desfechos foi o medicamento anti-TNF. A análise de sensibilidade estabeleceu que, para atingir o valor de disposição a pagar por semestre para evitar um infarto agudo do miocárdio, o custo médio do anti-TNF deveria ser de R$ 1.337,47 ou diferença de incidência entre as estratégias de 0,032. Ainda, para evitar um óbito cardiovascular, o custo médio deveria ser de R$ 954,22 ou diferença de incidência de 0,071. Todas as análises realizadas estabeleceram uma relação desfavorável da estratégia do tratamento medicamentoso com anti-TNF. Conclusão: Os achados da análise de custo efetividade entre pacientes com artrite reumatoide para desfecho cardiovascular quando comparada a estratégia de tratamento medicamentoso de anti-TNF em relação à estratégia dominante com Dmards após os 6 primeiros meses de exposição aos medicamentos apontaram para uma relação desfavorável, ultrapassando o valor de disposição a pagar recomendado pelo Ministério da Saúde do Brasil no ano de 2015. Descritores: artrite reumatoide; doenças cardiovasculares; infarto do miocárdio; óbito; avaliação de custo-efetividade; anti fator de necrose tumoral; drogas anti-reumáticas modificadoras de doença. / Introduction: Epidemiological studies have established that rheumatoid arthritis is associated with an increase in cardiovascular disease. The evaluation of anti-TNF on the reduction of the risk of acute myocardial infarction and death due to cardiovascular causes has shown promising results. The economic evaluation for these outcomes is not yet established. Objective: To perform a cost-effectiveness analysis of anti-TNF versus Dmards to avoid a new case of cardiovascular morbidity and death in rheumatoid arthritis. Method: A cost effectiveness analysis was performed using the Markov model, a 6-month transition cycle and a 30-year time horizon under the perspective of the Brazilian public health system measured by the incremental cost-effectiveness ratio. Cost in Reais in the year 2015 and effectiveness in avoiding a new case of acute ischemic coronary disease and cardiovascular death. Results: The average cost in 30 years of Dmards and anti-TNF was R$ 14,291,105.28 and R$ 96,151,873.86, respectively. The incremental effectiveness for coronary artery disease was 2.69 and a consequent incremental cost-effectiveness ratio of R$ 30,527,502.27, while for cardiovascular death, incremental effectiveness was 1.33 cases avoided and an incremental cost-effectiveness ratio of R$ 61,634,231.69. The univariate analysis identified that the parameter of greatest impact in the incremental cost-effectiveness ratio of both outcomes was the anti-TNF drug. The sensitivity analysis established that the average cost of anti-TNF should be R$ 1,337.47 or incidence difference between strategies of 0.032 in order to reach the amount of willingness to pay per semester to avoid an acute myocardial infarction. Also, to avoid a cardiovascular death, the average cost should be R$ 954.22 or incidence difference of 0.071. All the analyzes carried out established an unfavorable relationship of the drug treatment strategy with anti-TNF. Conclusions: The findings of the cost-effectiveness economic analysis among individuals with rheumatoid arthritis for cardiovascular outcome when compared to the strategy of anti-TNF drug treatment compared to the dominant strategy with Dmards after the first 6 months of drug exposure pointed to an unfavorable relationship, surpassing the amount of willingness to pay recommended by the Ministry of Health of Brazil in the year 2015.

\\OBJETIVOS: Avaliar a imunogenicidade e a segurança a curto prazo da vacina H1N1 pandêmica em pacientes com artrite reumatóide (AR) e espondiloartrites [ESa - artrite psoriática (AP) e espondilite anquilosante (EA)] recebendo classes distintas de terapia anti-TNF, assim como comparação com pacientes que receberam drogas modificadoras de doenças reumáticas (DMARDs) e controles saudáveis. MÉTODOS: Cento e vinte pacientes (AR, n=41; EA, n=57 e artrite psoriática - AP, n=22) em uso de agentes anti-TNF (monoclonal, n=94 e receptor solúvel, n=26) foram comparados com 116 pacientes com artrite inflamatórias em uso de DMARDs e 117 controles saudáveis. Soroproteção (SP), soroconversão (SC), médias geométricas dos títulos (MGTs), fator de aumento (FI) das MGT e eventos adversos foram avaliados 21 dias após a vacinação. RESULTADOS: Após a imunização, as taxas de SC (58,2% vs 74,3%, p=0,017) foram significativamente menores nos pacientes com espondiloartrites que receberam a terapia anti-TNF, enquanto nenhuma diferença foi observada em pacientes com AR que recebem esta terapia, em comparação com controles saudáveis (p=0,067). Pacientes com espondiloartrites que receberam anticorpos monoclonais (infliximabe/adalimumabe) tiveram uma taxa de SC significativamente menor em comparação com controles saudáveis (51,6% vs. 74,3%, p=0,002) ou para aqueles em uso de DMARDs (51,6% vs. 74,7%, p=0,005), por sua vez não houve diferença para pacientes em uso de etanercepte (86,7% vs. 74,3%, p=0,091). Uma análise dos pacientes com espondiloartrites que apresentaram SC e os que não apresentaram SC revelou que o primeiro grupo teve maior média de idade (p=0,003), maior frequência de anti-TNF (p=0,031) e anticorpos monoclonais (p=0,001), e uma menor frequência de metotrexate (p=0,028). Na regressão logística multivariada, apenas a idade avançada (p=0,015) e tratamento anticorpos monoclonais (p=0,023) permaneceram fatores importantes para a não SC em pacientes com espondiloartrites. CONCLUSÕES: Este estudo mostrou um padrão distinto da resposta imune à vacina contra a gripe pandêmica em pacientes com artrite inflamatória que receberam agentes anti-TNF, com uma imunogenicidade reduzida apenas em pacientes com espondiloartrites usando anticorpos monoclonais / OBJECTIVES: To evaluate the immunogenicity of the anti-influenza A H1N1/2009 vaccine in rheumatoid arthritis (RA) and spondyloarthritis patients receiving distinct classes of anti-TNF agents compared with patients receiving DMARDs and healthy controls. METHODS: One hundred and twenty patients (RA, n=41; ankylosing spondylitis - AS, n=57 and psoriatic arthritis - PsA, n=22) under anti-TNF agents (monoclonal, n=94 and soluble receptor, n=26) were compared to 116 inflammatory arthritis patients under DMARDs and 117 healthy controls. Seroprotection (SP), seroconversion (SC), geometric mean titre (GMT), factor increase (FI) in GMT and adverse events were evaluated 21 days after vaccination. RESULTS: After immunisation, SC rates (58.2% vs. 74.3%, p=0.017) were significantly lower in spondyloarthritis patients receiving anti-TNF therapy, whereas no difference was observed in RA patients receiving this therapy compared to healthy controls (p=0.067). Spondyloarthritis patients receiving monoclonal antibodies (infliximab/adalimumab) had a significantly lower seroconversion rate compared to healthy controls (51.6% vs. 74.3%, p=0.002) or to those under DMARDs (51.6% vs. 74.7%, p=0.005), whereas no difference was observed for patients under etanercept (86.7% vs. 74.3%, p=0.091). Further analysis of non-seroconverting and seronconverting spondyloarthritis patients revealed that the former group had a higher mean age (p=0.003), a higher frequency of anti-TNF (p=0.031) and monoclonal antibodies (p=0.001), and a lower frequency of methotrexate (p=0.028). In multivariate logistic regression, only older age (p=0.015) and monoclonal antibodies treatment (p=0.023) remained significant factors for nonseroconversion in spondyloarthritis patients. CONCLUSIONS: This study revealed a distinct disease pattern of immune response to the pandemic influenza vaccine in inflammatory arthritis patients receiving anti-TNF agents, illustrated by a reduced immunogenicity solely in spondyloarthritis patients using monoclonal antibodies

OBJETIVOS: Avaliar as alterações clínicas da superfície ocular, citologia de impressão (CI) e sintomas de olho seco (OS) dos pacientes com Espondilite Anquilosante (EA) e Artrite Reumatoide (AR). Classificar a intensidade de OS. Avaliar prospectivamente os parâmetros clínicos, laboratoriais e da superfície ocular dos pacientes com EA e AR submetidos a tratamento com drogas anti-TNF. MÉTODOS: Estudo prospectivo envolvendo inicialmente (pré-tratamento) 36 pacientes com EA e 20 pacientes com AR comparados com grupo controle de 39 voluntários saudáveis para o grupo de EA e 24 voluntários saudáveis para o grupo de AR. Do total inicial, 14 pacientes consecutivos com EA e 20 pacientes consecutivos com AR foram submetidos a terapia anti-TNF. Foram realizados os seguintes exames: teste de Schirmer I, tempo de rompimento do filme lacrimal, tingimento com corantes vitais e questionário dos sintomas de OS (Ocular Surface Disease Index-OSDI), citologia de impressão (CI) conjuntival, avaliação laboratorial inflamatória: velocidade de hemossedimentação e proteína C reativa (VHS e PCR) e atividade da doença pelas medidas de Bath Ankylosing Spondylitis Activity Index e Bath Ankylosing Spondylitis Functional Index (BASDAI e BASFI respectivamente) na EA e Disease Activity Score 28 (DAS 28) para AR. Além disso, avaliou-se a qualidade de vida pelo Health Assessment Questionnaire (HAQ) para ambas as doenças. As avaliações foram realizadas pré-tratamento e repetidas aos 3 meses (3M) e 12 meses (12M) após o início da terapia. RESULTADOS: Na avaliação pré-tratamento com drogas anti-TNF, os pacientes com EA apresentaram OS de intensidade leve a moderada (80,5% versus 43,6%, p=0,01) e maior escore de alteração da CI (55% versus 12,8%, p=0,007) associados a provas de atividade inflamatórias elevadas (p < 0,001) quando comparados com controles saudáveis. A avaliação longitudinal do tratamento com terapia anti-TNF demonstrou melhora da produção aquosa lacrimal (pré-tratamento: 13,7 ? 11,3 mm, aos 3M: 18,3 +- 11,1 mm e aos 12M: 19,3 +- 9,0 mm, p=0,04) assim como da CI conjuntival (pré-tratamento: 78,6% alterada, aos 3M: 57,1%, e aos 12M: 35,7%, p=0,03). Houve, paralelamente, melhora dos parâmetros inflamatórios e da atividade da doença (p < 0,05). No grupo de pacientes com AR, no momento pré-tratamento com drogas anti-TNF, foram observados maior frequência (75% versus 4%, p < 0,001) e intensidade leve de OS (65% versus 4%, p < 0,001) associados a sintomas moderados (escore OSDI 24,0 +- 17,6 versus 7,5 +- 14,3, p=0,001) quando comparados com controles saudáveis. Esses pacientes também apresentaram maior frequência de disfunção das glândulas de meibômio (55,0% versus 8,3%, p=0,001), maior escore de alteração da CI conjuntival (1,0 +- 0,6 versus 0.0 +- 0,2, p=0,001) e menor densidade de células caliciformes (431,3 +- 209,5 células/mm2 versus 804,8 +- 383,2 células/mm2, p < 0,001) quando comparados com o grupo controle. A análise prospectiva dos pacientes com AR tratados com drogas anti-TNF mostrou um aumento dos valores do teste de Schirmer (prétratamento: 11,8 +- 6,7 mm, aos 3M: 21,0 +- 10,4 mm, e aos 12M: 23,0 +- 9,7mm, p < 0,001), melhora da CI conjuntival (pré-tratamento: 1,0 +- 0,6, aos 3M: 0,8 +- 0,6, e aos 12M: 0,5 +- 0,5, p=0,005) e da densidade de células caliciformes (pré-tratamento: 429 +- 211,7 células/mm2, aos 3M: 908 +- 291,4 células/mm2, e aos 12M: 1265,4 +- 430,6 células/mm2, p=0,001). Os marcadores de atividade inflamatória sistêmicos (VHS e PCR) também melhoraram ao longo do tratamento (p=0,005 e p=0,006, respectivamente). CONCLUSÃO: Os pacientes com EA e AR avaliados nesse estudo apresentaram prevalência elevada de OS de intensidade leve a moderada associada à alteração da citologia conjuntival. A recuperação precoce e manutenção de longo prazo na produção aquosa da lágrima e na CI conjuntival, em especial, das células caliciformes, nos pacientes submetidos a terapia anti-TNF, pode refletir a melhora da condição inflamatória. Esse resultado histológico pode ter influência como biomarcador da inflamação na superfície ocular / OBJECTIVES: Evaluate ocular surface parameters, impression cytology (IC) and dry eye (DE) symptoms of patients with Ankylosing Spondylitis (AS) and Rheumatoid Arthritis (RA). Classify DE severity grade. Analyse prospectively clinical and laboratory, as well as ocular surface parameters of AS and RA patients, submitted to anti-TNF therapy. METHODS: This prospective study initially (baseline) enrolled 36 AS patients and 20 RA patients who were compared to a control group of 39 and 24 healthy volunteers for the AS group and RA group, respectively. From the initial group, 14 consecutive AS and 20 consecutive RA patients received anti-TNF therapy. They underwent the following exams: Schirmer I test, tear break-up time, vital dyes staining of the ocular surface, a questionnaire for dry eye symptoms- Ocular Surface Disease Index (OSDI), and conjunctival IC. Laboratory tests for inflammatory activity were assessed by erythrocyte sedimentation rate and C- reactive protein (ESR and CRP). The Bath Ankylosing Spondylitis Activity Index and Bath Ankylosing Spondylitis Functional Index (BASDAI and BASFI, respectively) in AS and Disease Activity Score 28 (DAS 28) in RA analyzed disease activity parameters. Besides, the Health Assessment Questionnaire evaluated the quality of life in both group of diseases. These measurements were taken at baseline (BL) and repeated at 3 months and 12 months (3M and 12M, respectively) after the beginning of anti-TNF therapy. RESULTS: At the baseline moment, AS patients presented mild to moderate DE (80.5% vs 43.6%, p=0.01) and a higher score of altered IC (55% vs 12.8%, p=0.007) associated with the systemic inflammatory activity (ESR and CRP, p < 0.001) when compared to healthy volunteers. The longitudinal evaluation of anti- TNF treatment showed an improvement of aqueous tear production (BL: 13.7 +- 11.3 mm, 3M: 18.3 +- 11.1 mm and 12M: 19.3 +- 9,0 mm, p=0.04). The IC also improved (BL: 78.6% altered IC, 3M: 57.1% and 12M: 35.7%, p=0.03). There was a parallel amelioration of systemic inflammatory markers and disease activity (p < 0.05). Concerning the RA group of patients, at the baseline moment, there was a higher frequency of DE (75% vs 4%, p < 0.001) as well as mild DE severity grade (65% vs 4%, p < 0,001) associated with moderate symptoms of DE (OSDI score: 24.0 +- 17.6 vs 7.5 +- 14.3, p=0.001) when compared to healthy volunteers. This group of patients also presented higher frequency of meibomian gland dysfunction (55% vs 8.3%, p=0.001), a worse score of IC (1.0 +- 0.6 vs 0.0 ? 0.2, p=0.001) and lower goblet cells count (431.3 +- 209.5 cells/mm2 vs 804.8 +- 383.2 cells/mm2, p< 0.001) when compared to the control group. The prospective analysis of RA patients treated with anti-TNF drugs demonstrated an increase of Schirmer\'s test (BL: 11.8 +- 6.7, 3M: 21.0 +- 10.4, 12M: 23.0 +- 9.7, p < 0.001) and an improvement of cytological grade (BL: 1.0 +- 0.6, 3M: 0.8 +- 0.6, 12M: 0.5 +- 0.5, p=0.005) and goblet cells density (BL: 429,0 +- 211.7 cells/mm2, 3M: 908,0 +- 291.4 cells/mm2, 12M: 1265.4 +- 430.6 cells/mm2, p=0.001). The systemic inflammatory markers (ESR and CRP) also improved throughout the treatment period (p=0.005 and p=0.006, respectively). CONCLUSION: Patients with AS and RA enrolled in this study presented a higher prevalence of mild to moderate DE associated with altered IC. The prompt and maintained aqueous tear and conjunctival cytology recovery, especially the goblet cells, in patients submitted to anti-TNF therapy seem to represent the improvement of inflammatory condition. This histological outcome may have an influence as a biomarker of ocular surface inflammation

Introdução: Apesar dos recentes avanços no entendimento da fisiopatologia e no tratamento da espondilite anquilosante (EA), pouco se sabe acerca da influência das medicações anti-fator de necrose tumoral (anti-TNF) sobre as novas vias inflamatórias descritas na patogênese das espondiloartrites. Objetivo: Dessa forma, o objetivo desse estudo é investigar e descrever a influência a longo prazo das medicações anti-TNF sobre o eixo da IL-23/IL-17 em pacientes com EA e sua possível correlação com o tratamento, parâmetros clínicos, laboratoriais e radiológicos. Métodos: Oitenta e seis pacientes com EA sem exposição prévia a medicações anti-TNF foram recrutados. Desses, 47 possuíam Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) >= 4 (grupo EA-ativo) e haviam sido encaminhados para iniciar tratamento anti-TNF e 39 possuíam BASDAI < 4 (grupo EA-controle) em uso de anti-inflamatório não hormonal (AINH) e/ou drogas antirreumáticas modificadoras do curso de doença tradicionais. O grupo EA-ativo foi avaliado clinicamente e laboratorialmente no tempo basal e após 12 e 24 meses de uso das medicações anti-TNF e foi comparado com o grupo EA-controle e com 47 controles saudáveis (CS) pareados por idade e sexo. O escore de uso de AINH foi calculado no tempo basal, 12 meses e 24 meses. Os níveis plasmáticos das interleucinas (IL) IL-17A, IL-22, IL-23 e PGE2 e a dosagem sérica da velocidade de hemossedimentação (VHS) e proteína C-reativa (PCR) foram realizados no grupo EA-ativo no tempo basal, 12 meses e 24 meses e somente no tempo basal nos grupos EA-controle e CS. No grupo EA-ativo, a progressão radiológica foi medida pelo modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) no tempo basal e após 24 meses de tratamento. Resultados: No tempo basal, o grupo EA-ativo apresentou maiores níveis plasmáticos de IL-23 e PGE2 quando comparado ao grupo EA-controle (p < 0,001 e p=0,008) e ao grupo controle saudável (p < 0,001 e p=0,02). Após 24 meses de uso de anti-TNF, os níveis plasmáticos de IL-23 e PGE2 ainda se mantiveram elevados quando comparado ao grupo CS (p < 0,001 e p=0.03) apesar da melhora de todos os parâmetros clínicos e laboratoriais (VHS/PCR) (p < 0,001). A subanálise de 27 pacientes do grupo EA-ativo que obtiveram boa resposta ao uso de anti-TNF (atingiram ASDAS-PCR< 2,1 em 24 meses, com queda >= 1,1 em relação ao ASDAS-PCR basal) revelou que, ainda assim, os níveis plasmáticos de IL-23 eram superiores aos encontrados nos CS (p < 0,001) e superiores ao grupo EA-controle com atividade de doença similar (ASDAS-PCR < 2,1; p=0,01). No grupo EA-ativo foi encontrada uma correlação positiva entre os níveis plasmáticos de IL-23 e IL-17A no tempo basal, 12 meses e 24 meses do estudo (p <= 0,001). Conclusão: Os dados apresentados sugerem que o eixo da IL-23/IL-17 não é influenciado pelas medicações anti-TNF apesar da melhora dos parâmetros clínicos e marcadores de atividade inflamatória estudados / Background: Advances in pathophysiology and treatment of ankylosing spondylitis (AS) was recently demonstrated. However, the effect of anti-tumor necrosis fator (TNF) in the newly described inflammatory pathways involved in this disease remains to be determined. Objective: The aim of our study was, therefore, investigate long-term influence of anti-TNF drugs in IL-23/IL-17 axis of AS patients and their possible correlation with treatment, clinical, laboratory and radiographic parameters. Methods: Eighty six AS anti-TNF naïve patients, 47 referred for anti-TNF therapy (active-AS group; Bath Ankylosing Spondylitis Activity Index (BASDAI) >= 4) and 39 with BASDAI < 4 (control-AS group) were included. The active group was evaluated clinically and laboratorially at baseline, 12-months and 24-months after TNF blockade and compared at baseline to control-AS group and to 47 healthy age- and gender-matched controls. Plasma levels of interleukin (IL)17A, IL-22, IL-23 and PGE2 and serum levels of erythrocyte sedimentation rate (ESR) and c-reactive protein (CRP) were measured at three study times in active-AS and at baseline in control-AS and healthy-controls. Non-steroidal anti-inflammatory drugs (NSAIDs) intake were recorded at baseline, 12 months and 24 months. Radiographic severity and progression was assessed by modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) at baseline and 24 months after therapy in active-AS patients. Results: At baseline, active-AS group presented higher IL-23 and PGE2 levels compared to control-AS group (p < 0.001 and p=0.008) and to healthy controls (p < 0.001 and p=0.02). After 24-months of TNF blockade, IL-23 and PGE2 remained elevated with higher levels compared with the healthy-control group (p < 0.001 and p=0.03) in spite of significant improvements in all clinical/inflammatory parameters (p < 0.001). Further analysis of 27 anti-TNF-treated patients who achieved a good response (ASDAS-CRP < 2.1, with a drop >= 1.1) at 24-months revealed that IL-23 plasma levels remained higher than healthy controls (p < 0.001) and higher than control-AS group with similar disease activity (ASDAS-CRP < 2.1, p=0.01). In active-AS group (n=47), there was a correlation between IL-23 and IL-17A at baseline, 12-months and 24-months after anti-TNF therapy (p <= 0.001). Conclusion: This study provides novel data demonstrating that the IL-23/IL-17 axis is not influenced by TNF blockade drugs in AS patients despite clinical and inflammation improvements and NSAID intake

INTRODUÇÃO: Os hepatócitos apresentam papel potencial em iniciar e amplificar a resposta inflamatória aguda no fígado, através da liberação de citocinas pró-inflamatórias, em papel complementar ao exercido pelas células de Kupffer e endoteliais. A invasão bacteriana da célula hepática é um estímulo para que o hepatócito produza citocinas como o TNF-alfa, capaz de induzir sua própria morte por apoptose. O TNF-alfa pode ser tanto um agente citotóxico (induzindo a morte celular), quanto um agente protetor (através da ativação de NF-kB). A morte por apoptose do hepatócito libertaria as bactérias que seriam destruídas pelo sistema imunológico hepático ativado. Salmonella Typhimurium (ST) e Listeria monocytogenes (LM) são patógenos responsáveis por importantes doenças de origem alimentar. O hepatócito é o maior local de replicação bacteriana no fígado. As conseqüências da invasão bacteriana dos hepatócitos e sua repercussão na produção de TNF-alfa e na morte celular necessitam ser melhor xxix avaliadas. MÉTODOS: Nesse estudo procuramos investigar o comportamento dos hepatócitos invadidos por ST e LM sorogrupos 4a, 4b e 1/2a, analisando os seguintes parâmetros: a) morfologia = por microscopia óptica (MO) (hematoxilina e eosina) e por microscopia eletrônica (ME); b) dosagem do TNF-alfa liberado pelos hepatócitos invadidos = o TNF-alfa liberado foi detectado por técnica ELISA no sobrenadante das culturas; c) pesquisa da morte celular por apoptose = avaliada através das técnicas TUNEL e anexina (citometria de fluxo). Para todos os parâmetros foi realizada análise comparativa estatística entre os quatro tipos de bactéria. RESULTADOS: As monocamadas de hepatócitos agredidas por Listeria monocytogenes e Salmonella Typhimurium apresentam ruptura em sua distribuição, e sinais de desorganização citoplasmática e nuclear. Para as bactérias ST, LM 4a, LM 4b e LM 1/2a obtivemos os seguintes valores em seqüência: a) taxa de liberação de TNF-alfa (pg/mL): 146,9±18,38; 94,71±13,89; 94,52±15,66 e 58,16±15,49; b) capacidade de produção de TNF-alfa (pg/mL): -67,20±71,56; -46,49±54,10; -106,3±61,0 e 58,16±15,49; c) taxa de apoptose avaliada por TUNEL em unidade de área (UA): 23,86±1,614; 15,92±0,9343; 21,14±1,421 e 23,93±1,263; d) capacidades de produção de apoptose por TUNEL em UA: -50,67±12,42; 10,81±7,186; - 17,22±10,93 e -40,27±9,712; e) taxas de apoptose por anexina em UA: 12,51±2,052; 23,10±3,481; 26,61±3,414 e 18,57±2,497; f) capacidades de produção de apoptose por anexina em UA: -63,31±15,79; -126,4±26,78; - 142,0±26,26 e -97,75±19,21. CONCLUSÕES: a) ocorre liberação de TNFxxx alfa pelos hepatócitos invadidos, sendo que a Salmonella Typhimurium foi responsável pela maior taxa de liberação de TNF-alfa, e Listeria monocytogenes 4b pela maior capacidade de produção de TNF-alfa; b) ocorre morte por apoptose dos hepatócitos invadidos por bactérias, avaliada através da técnica TUNEL, sendo que Salmonella Typhimurium e Listeria monocytogenes 1/2a foram responsáveis pelas maiores taxas e capacidades de produção de apoptose; c) ocorre morte dos hepatócitos invadidos por apoptose, avaliada através da técnica da anexina, sendo que Listeria monocytogenes 4b foi responsável pelas maiores taxas e capacidades de produção de apoptose; d) os hepatócitos cultivados invadidos pelas bactérias Salmonella Typhimurium e Listeria monocytogenes apresentam alterações morfológicas, com ruptura da distribuição da monocamada, e sinais de desorganização citoplasmática e nuclear / INTRODUCTION: Hepatocytes can play an important role in the initiation or amplification of the hepatic acute inflammatory response, through the release of proinflammatory cytokines. The bacterial invasion of hepatocyte is a stimulus for production of TNF-alpha by these cells, and this phenomenon induces its own death by apoptosis. TNF-alpha is as a cytotoxic agent (inducing cellular death), as a protector agent (through NF-kB activation). The hepatocyte death by apoptosis may release intracellular bacteria that would be destroyed by hepatic immunological system. Salmonella Typhimurium (ST) and Listeria monocytogenes (LM) are important foodborne pathogens. The hepatocyte is the major site of bacterial replication in the liver. The consequences of hepatocytes bacterial invasion must be better evaluated. METHODS: In the present work we show the behavior of hepatocytes invaded by ST and LM serotypes 4a, 4b and 1/2a, through: a) morphology = by optic microscopy (OM) (hematoxylin-eosin staining) and electronic microscopy (EM); b) quantification of TNF-alpha released by hepatocytes = TNF-alpha released was determined by ELISA in culture supernatants; c) evaluation of apoptotic cell death by TUNEL and annexin techniques (flow cytometry). For all parameters were made a statistical comparative analysis among the four types of bacteria. RESULTS: The hepatocytes monolayers invaded by LM and ST presented ruptures in your organization, and signs of nuclear and cytoplasmic disorder. For the bacteria ST, LM 4a, LM 4b and LM 1/2a we obtained the following values respectively: a) rate of TNF-alpha released (pg/mL): 146,9±18,38; 94,71±13,89; 94,52±15,66 and 58,16±15,49; b) capacities of TNF-alpha production (pg/mL): -67,20±71,56; -46,49±54,10; -106,3±61,0 and 58,16±15,49; c) rate of apoptosis by TUNEL in unit of area (UA): 23,86±1,614; 15,92±0,9343; 21,14±1,421 and 23,93±1,263; d) capacities of apoptosis production by TUNEL in UA: -50,67±12,42; 10,81±7,186; - 17,22±10,93 and -40,27±9,712; e) rate of apoptosis by annexin in UA: 12,51±2,052; 23,10±3,481; 26,61±3,414 and 18,57±2,497; f) capacities of apoptosis production by annexin in UA: -63,31±15,79; -126,4±26,78; - 142,0±26,26 and -97,75±19,21. CONCLUSIONS: a) ST was responsible for the major rate of TNF-alpha released and LM 4b was responsible for the major capacity of TNF-alpha production; b) ST and LM 1/2a caused the major rates and capacities of apoptosis,production, evaluated by TUNEL technique; c) LM 4b was responsible for the major rates and capacities of apoptosis production, evaluated by annexin technique; d) the cultured hepatocytes invaded by bacteria ST and LM presented morphological alterations, with monolayer rupture, and signs of nuclear and cytoplasmic disorder

Pancreatite aguda é uma das principais causas de síndrome da resposta inflamatória sistêmica (SRIS). Complicações sistêmicas são os principais fatores responsáveis pela ocorrência de falência de múltiplos órgãos e sistemas e morte durante a primeira etapa da doença. Os níveis de mediadores inflamatórios possuem uma relação com a gravidade da pancreatite. O TNF-alfa tem sido descrito como o agente iniciador da resposta inflamatória da pancreatite aguda. Estudos prévios usando a pentoxifilina para bloquear a produção de TNF-alfa têm demonstrado efeitos benéficos quando usada em modelos experimentais de sepse ou choque. A infecção pancreática é a mais grave complicação da pancreatite aguda com índices de mortalidade que podem chegar a 80%. Os mecanismos que determinam a ocorrência da infecção pancreática não são bem esclarecidos. Objetivo: Determinar o efeito da inibição da produção de TNF-alfa na pancreatite aguda com a administração de pentoxifilina. Métodos: Foi utilizado um modelo experimental de pancreatite aguda grave através da injeção intraductal de taurocolato de sódio a 2,5%. Cento e vinte e quatro ratos Wistar machos foram divididos em três grupos: Controle (animais submetidos ao procedimento cirúrgico sem a administração de taurocolato de sódio), Pancreatite (animais submetidos à indução da pancreatite aguda) e Pentoxifilina (animais submetidos à indução da pancreatite aguda seguida da administração intraperitoneal de 25 mg/Kg de pentoxifilina). Foi realizada a dosagem sérica das interleucinas 6 e 10 e TNF-alfa duas horas após a indução da pancreatite. Foram analisadas a ocorrência de translocação bacteriana e a incidência de infecção pancreática, através de culturas realizadas 24 horas após a indução da pancreatite aguda e a mortalidade global. A ocorrência de infecção pancreática foi considerada como positiva quando a concentração de bactérias expressa em UFC/g foi maior que 105. Resultados: Quando comparado ao grupo Pancreatite, o grupo Pentoxifilina apresentou redução significativa da lesão histológica pancreática, dos níveis de IL-6, IL-10 e TNF-alfa e da ocorrência de infecção pancreática (p < 0,05), tais alterações se associam a redução significativa da mortalidade no grupo Pentoxifilina. Conclusão: A inibição da produção de TNF-alfa através da administração de pentoxifilina foi capaz de reduzir o processo inflamatório local e sistêmico, reduzir a translocação bacteriana e infecção pancreática e melhorar a sobrevida com redução dos índices de mortalidade desta grave doença / Acute pancreatitis (AP) is considered one of the typical conditions causing systemic inflammatory response (SIRS). Systemic complications are the most important contributors to multiple organ failure and death during the first stages of severe acute pancreatitis. Levels of pro-inflammatory cytokines increase during the course of AP, and these levels appear to be correlated with the severity of pancreatic inflammation. TNF-alfa may be an initiator of inflammatory process in AP. Previous studies using pentoxifylline to block TNF-alfa production have showed beneficial effects in experimental models of sepsis and shock. The gut is a target organ of the SIRS causing gut barrier dysfunction allowing bacteria and toxin translocation. Bacterial translocation has been implicated in the development of multiple organ failure and is one of the major causes of pancreatic infection in patients with pancreatic necrosis. Aim: To determinate the effects of inhibition of TNF-? on the pancreatic and systemic inflammatory response, pancreatic infection, and mortality rate in necrotizing acute pancreatitis in rats. Methods: An experimental model of severe AP by injection of 0.5ml of 2.5% sodium taurocholate into the pancreatic duct was utilized. A hundred and twenty four male Wistar rats were divided in 3 groups: Sham (surgical procedure without AP induction), Pancreatitis (AP Induction), and Pentoxifylline (AP induction plus administration of 25 mg/kg pentoxifylline). Pancreatic inflammatory response was measured by histological studies and systemic inflammatory response was analyzed measuring the production of inflammatory cytokines (IL-6, IL-10, and TNF-alfa). Pancreatic infection was evaluated with bacterial cultures performed 24 h after the AP induction. The numbers of organisms were expressed as colony forming units (CFU) per gram. The occurrence of pancreatic infection was also analyzed and considered positive when the CFU/g was > 105. A parallel survival study was also performed. Results: Inhibition of TNF-alfa by pentoxifylline shows beneficial effects in this experimental model. The Pentoxifylline group had a statistically significant reduction of histological damage in the pancreas, inflammatory cytokines levels (IL-6, IL-10, and TNF-?), and occurrence of pancreatic infection (p < 0.05). These changes were associated with a significant reduction of mortality rate. Conclusions: Inhibition of TNF-alfa reduced local and systemic inflammatory response, reduced systemic complication as pancreatic infection, and decrease mortality rate in this model. Pentoxifylline may provide a useful therapy in the treatment of acute pancreatitis

Die Haut ist das größte Organ des Menschen und bildet die Barriere gegenüber Einwirkungen aus der Umwelt. Die Störung der Hautbarriere durch exogene und endogene Reize führt zu einer Entzündungsreaktion in der Haut. In der Folge können Hauterkrankungen wie die irritative oder Atopische Dermatitis entstehen. Der Tumor Nekrose Faktor-α (TNF-α) ist ein pleiotrop wirksames Zytokin, das eine zentrale Rolle bei entzündlichen Prozessen spielt. Ziel der vorgelegten Promotionsarbeit war zu untersuchen, ob und wie TNF-α zu Entzündungsgeschehen, ausgelöst durch exogene und endogene Faktoren, beiträgt. Die Bedeutung von TNF-α wurde in TNF-ko Mäusen in verschiedenen Hautmodellen untersucht. Für das Irritationsmodell wurden chemische und physikalische Reize verwendet. TSLP (Thymic stromal lymphopoietin) wurde durch die verschiedenen Stimuli signifikant induziert. Diese Induktion war unabhängig von der endogenen TNF-α Produktion, gezeigt durch den Einsatz von TNF- ko Mäusen . Da endogenes TNF-α für die Hautirritation keine notwendige Bedingung darstellte, wurde die Bedeutung von TNF-α bei der atopischen Dermatitis (AD) untersucht. TNF-α defiziente Mäuse zeigen verstärkt Ekzeme im Vergleich zu Wildtyp Mäusen. Die Behandlung von TNF-ko Mäusen mit einem TSLP Antikörper führte zu einer Verminderung des Ekzems. Mastzellen wurden vermehrt in läsionaler Haut gefunden und korrelierten mit dem Schweregrad des atopischen Ekzems sowie der TSLP-Expression. / The skin is the largest organ of an individuum and builds the barrier for a host against the environment. Skin barrier disruption by exogenous or endogenous stimuli can lead to skin inflammation. As a consequence, irritant or atopic eczema, frequent skin diseases, may evolve. Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine which plays a central role in inflammatory processes. The main aim of this thesis was to clarify whether and how endogenous TNF-α is contributing to skin inflammation driven by exogenous and endogenous triggers. The role of endogenous TNF-α was studied using TNF knockout (-/-) mice. In an irritation model, chemical and physical stimuli were applied on to mouse skin. Thymic stromal lymphopoietin (TSLP) was significantly induced by the used irritants. This TSLP induction was independent from endogenous TNF-α proven by using TNF-/- mice. Next the role of TNF-α in atopic dermatitis (AD) promoting an allergic skin inflammation was investigated. TNF-/- mice developed more severe AD compared to the wildtype mice and TSLP was significantly increased and correlated with the severity of the eczema. To prove the pathophysiological role of TSLP for AD progression, TNF-/- mice were pretreated with an TSLP antibody. Indeed, these mice developed less AD symptoms compared to the control mice. Mast cells (MCs) were also significantly increased in lesional skin in the AD model and moreover, correlated with AD severity, but also with TSLP expression.