Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-02-12T10:02:46Z No. of bitstreams: 1 andreajunqueiracortes.pdf: 11746820 bytes, checksum: cd7019a0ace25082404e47c8d3645bc8 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-02-26T12:06:29Z (GMT) No. of bitstreams: 1 andreajunqueiracortes.pdf: 11746820 bytes, checksum: cd7019a0ace25082404e47c8d3645bc8 (MD5) / Made available in DSpace on 2016-02-26T12:06:29Z (GMT). No. of bitstreams: 1 andreajunqueiracortes.pdf: 11746820 bytes, checksum: cd7019a0ace25082404e47c8d3645bc8 (MD5) Previous issue date: 2014-07-14 / A matriz derivada do esmalte (EMD) é um complexo proteico de origem ectodérmica, isolado de germes dentários em desenvolvimento. Originalmente utilizada na regeneração de tecidos do periodonto, vem se mostrando um excelente recurso na regeneração de outros tecidos mesenquimais como derme, tecido ósseo e tendão. No presente estudo, o potencial cicatrizante da matriz foi analisado histopatologicamente em feridas cutâneas cirúrgicas realizadas em ratos Wistar, eutanaziados nos dias 01, 03, 07, 14 e 21. Durante o experimento, as feridas foram acompanhadas macroscopicamente e, a seu término, foram removidas e submetidas ao processamento histológico. À avaliação microscópica, foi observado que as lesões tratadas com a EMD evoluíram para o remodelamento dérmico de modo mais eficaz em tempo e qualidade em relação ao grupo controle não tratado, a partir do 3º dia pós-operatório; o amadurecimento e organização do colágeno foi bastante evidente a partir do 7º dia e, destaca-se que nos dias 14 e 21 o plano muscular, na profundidade da pele, exibiu excelente regeneração, sendo que, também neste período, o amadurecimento vascular se mostrou mais significativa nas amostras tratadas. Além da análise histomorfométrica, foi realizada a avaliação da expressão de iNOS, TGF- β2 e TNF-α por imunoistoquímica. A expressão de iNOS foi significativamente mais expressiva nas amostras tratadas, aumentando progressivamente a partir do 7º dia pós-operatório. Os resultados sugerem que a EMD, aplicada em feridas cutâneas cirúrgicas, potencializa a neoformação e o remodelamento de colágeno, amadurecimento vascular e regeneração muscular possivelmente via modulação do óxido nítrico. / The enamel matrix derivative (EMD) is a protein complex of ectodermal origin, isolated from the developing tooth germs. Originally used in the regeneration of periodontal tissues, has proved to be an excellent resource in the regeneration of other mesenchymal tissues such as dermis, bone and tendon. In the present study, the healing potential of the matrix was analyzed histologically in surgical wounds made in Wistar rats, euthanized on days 01, 03, 07, 14 and 21. During the experiment, the wounds were followed macroscopically, and at its end, were removed and subjected to histological processing. For microscopic evaluation, it was observed that the lesions treated with EMD progressed to dermal remodeling more effectively in time and quality in relation to the untreated control group, from the 3th postoperative day ; the maturation and organization of the collagen was quite evident from the 7th day , and it is noteworthy that on 14 and 21 muscle -up, the skin depth, showed excellent regeneration, and also in this period, vascular maturation showed more significantly in the treated samples. In histomorphometric analysis, evaluation of iNOS, TGF- β2, and TNF-α expression was performed by immunohistochemistry. The expression of iNOS was significantly more expressive in the treated samples, increasing progressively from the 7th postoperative day. The results suggest that EMD applied on surgical wounds enhances the new formation and remodeling of collagen, vascular maturation and muscle regeneration possibly via modulation of nitric oxide.

\\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

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 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

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

Early administration of broad-spectrum antibiotics is the first goal in sepsis treatment. Besides from bacteriostatic/bactericidal effects, some antibiotics may also modify the host´s response to infection. The novel antibiotic tigecycline may exert such properties; however, this property has not been evaluated in large-animal trials. We compared tigecycline with doxycycline and placebo in relation to anti-inflammatory, circulatory and organ dysfunction effects in a sterile pig model of sepsis. Doxycycline, but not tigecycline, reduced the inflammatory response as manifested by tumor necrosis factor alpha levels in plasma. Tigecycline, however, had a stabilizing effect on the circulation not exerted by doxycycline or placebo. To achieve rapid restoration of the circulating blood volume - another major goal in sepsis treatment - fluid bolus administration of is some-times practiced. In addition to crystalloids, albumin-containing solutions are suggested. Yet, some animal-experimental data suggests that rapid bolus administration of albumin reduces albumin’s plasma-expanding effect. We compared a rapid intravenous bolus of radiolabeled albumin with a slow infusion in a sterile pig model of sepsis. Rapid bolus of administration did not reduce plasma levels of albumin following administration and did not increase the amount of albumin that left the circulation. Inadequate oxygen delivery (DO2) by the circulation to the tissues may cause increased plasma lactate, which is the most striking effect of sepsis on the metabolism. However, experimental data and clinical trials refute this link, instead, suggesting other mechanisms, including impaired oxygen extraction, mitochondrial dysfunction and accelerated aerobic glycolysis. We investigated the impact of DO2, oxygen consumption (VO2), hemodynamic parameters and inflammatory response on plasma lactate and organ dysfunction in two experimental sepsis models. In the most severe cases of shock, with DO2, there was an increase in plasma lactate, but without a decrease in VO2, invalidating the assumption that the increase in lactate is due to anaerobic metabolism. To identify critical steps in the sepsis-induced increase in lactate, we inhibited the major energy-producing step in the electron transport chain (ETC). The combination of sepsis and ETC inhibition led to a cellular energy crisis. This finding suggests that early sepsis induces a partial mitochondrial dysfunction.

In Canada alone, there were an estimated 800,000 visually impaired people in 2007, costing the federal government an annual amount of $15.8 billion in services, treatments and lost revenue. These costs are estimated to double by the year 2032, as the population ages. The leading causes of visual impairment and blindness is retinal degeneration, characterized by the progressive death of retinal cells. The research presented in this PhD thesis aimed to prevent retinal degeneration by over-expressing the X-linked Inhibitor of Apoptosis (XIAP) in retinal cells using plasmid and adeno-associated viral vectors. The work is divided into four sequential chapters targeted at developing an anti-apoptotic gene therapy strategy to prevent retinal cell death. The first chapter examines XIAP gene therapy in the treatment of Leber’s Hereditary Optic Neuropathy (LHON). In vitro studies using the 661W cone-photoreceptor cell line showed that XIAP over-expression significantly lowers cell death when 661W cells are exposed to a number of apoptotic stimuli. In a mouse model of Leber’s Hereditary Optic Neuropathy (LHON), XIAP expression in retinal ganglion cells (RGCs) protected the ultrastructure of the RGC axons within the optic nerve, in addition to providing evidence of functional protection. The second and third chapters further examine the potential for XIAP gene therapy in the treatment of retinal disease by developing an in vivo model of retinal detachment in cats, followed by evaluating the efficacy of XIAP gene therapy intervention. When XIAP was over-expressed in the photoreceptor cells, there was significant structural protection and trends in preservation of function in this model of degeneration. Finally, the fourth chapter explores an alternate method to viral gene therapy by evaluating the efficacy and toxicity of chitosan microparticles as a protein delivery system to the retina. Results show that chitosan microparticles are mucosal-adhesive and are non-toxic at low concentrations in vitro in 661W cells and in vivo in rats. This thesis work provides strong evidence that XIAP gene therapy is an effective method for preventing retinal degeneration, and works as a broad spectrum gene therapy strategy that can be applied to different forms of retinal degeneration.

INTRODUÇÃO: A cardiopatia chagásica crônica (CCC) apresenta características específicas, tais como: disfunção autonômica e atividade inflamatória exacerbada. Esta fisiopatologia sugere que alguns parâmetros metabólicos podem estar alterados em pacientes chagásicos. O objetivo deste estudo foi avaliar os parâmetros metabólicos e inflamatórios nas diversas formas evolutivas de doença de Chagas e sua correlação com medidas de avaliação do Sistema Nervoso Autônomo (SNA). MÉTODOS: Foram avaliados 60 indivíduos divididos em 4 grupos (n=15): Grupo controle (GC), Grupo FI - forma indeterminada, Grupo ECG- cardiopatia chagásica com alteração eletrocardiográfica sem disfunção ventricular e Grupo IC - cardiopatia chagásica com disfunção ventricular e insuficiência cardíaca. Todos os grupos foram pareados de acordo com sexo, idade e índice de massa corporal. Os pacientes realizaram dosagens sanguíneas de insulina, leptina, adiponectina, interleucina-6 (IL- 6) e fator de necrose tumoral-alfa (TNF-alfa) pelo método de ELISA. O SNA foi avaliado através da variabilidade da frequência cardíaca no holter 24 horas e no teste de inclinação postural. Os valores de RMSSD, pNN50 e do componente alta frequência (AF) foram utilizados como estimativa da atividade parassimpática. Os valores do componente de baixa frequência (BF) estimaram a atividade simpática. A análise estatística foi feita utilizando-se a ANOVA ou teste de Kruskal-Wallis para a comparação entre os grupos, o coeficiente de Spearman para a análise das correlações e a regressão linear múltipla para a análise multivariada. RESULTADOS: A leptina e insulina não apresentaram diferenças significativas entre os grupos [Leptina: GC=3,42 (7,43); FI=3,03 (6,53); ECG=5,56 (6,2); IC=2,86 (2,67) ng/ml; p=0,626. Insulina: GC=3,41 (1,98); FI=4,31 (2,85); ECG=4,30 (3,06); IC=4,58 (2,88) ng/ml; p=0,901] A adiponectina apresentou níveis maiores nos grupos ECG e IC [GC=4766,5 (5529,5); FI= 4003,5 (2482,5); ECG= 8376,5 (8388,5); IC= 8798 (4188) ng/ml; p < 0,001]. IL-6 e TNF-alfa foram maiores no Grupo IC [IL-6: GC=1,85 (6,41); FI=1,58 (1,91); ECG=1,0 (1,57); IC= 31,44 (72,19) pg/ml; p=0,001. TNF-?: GC=22,57 (88,2); FI=19,31 (33,16); ECG=12,45 (3,07); IC=75,15 (278,57) pg/ml; p=0,04]. A insulina, leptina e TNF-alfa não apresentaram correlações significativas com medidas de avaliação do SNA. A adiponectina apresentou correlação positiva com o componente AF (r= 0,336; p= 0,009) e correlação negativa com o componente BF (r= -0,336; p= 0,009). A interleucina-6 apresentou correlação positiva com o componente AF (r= 0,419; p=0,004) e correlação negativa com o componente BF (r= -0,393; p= 0,007). Porém, na análise multivariada apenas a adiponectina apresentou correlação significativa com medidas de função do SNA. CONCLUSÃO: A adiponectina foi maior nos grupos ECG e IC. A IL-6 e o TNF-alfa foram maiores no grupo IC. O aumento dos níveis de adiponectina esteve associado a diminuição da atividade simpática e predomínio da atividade parassimpática. / BACKGROUND: Chagas disease (CD) has specific characteristics such as autonomic dysfunction and increased inflammatory activity. This pathophysiology suggests that metabolic parameters can be altered in patients with CD. The aim of this study was to evaluate the metabolic and inflammatory parameters in different forms of CD and their correlation with Autonomic Nervous System (ANS) measures. METHODS: We evaluated 60 subjects divided into 4 groups (n=15): control group (CG), group IF (indeterminate form); group ECG (ECG abnormalities and normal left ventricular function in echocardiogram) and HF group (heart failure with left ventricular dysfunction). All groups were matched for age, sex and body mass index. The patients underwent insulin, adiponectin, leptin, interleukin-6 (IL-6) and tumor necrosis factor-alfa (TNF-alfa) measurements by ELISA. The Autonomic Nervous System was assessed by heart rate variability in 24-hour Holter and tilt test. RMSSD, pNN50 and High Frequency (HF) component values were used to estimate parasympathetic activity and low frequency (LF) components were used to estimate sympathetic activity. Statistical analyses were performed using ANOVA or Kruskal- Wallis tests to compare groups. Spearman coefficient was used for correlation analysis and linear regression for multivariate analysis. RESULTS: No significant differences were observed in leptin and insulin levels between groups. [Leptin: CG=3.42 (7.43); IF=3.03 (6.53); ECG=5.56 (6.2); HF=2.86 (2.67) ng/ml; p=0.626. Insulin: CG=3.41 (1.98); IF=4.31 (2.85); ECG=4.30 (3.06); HF=4.58 (2.88) ng/ml; p=0.901]. Adiponectin was higher in ECG and HF groups. [CG=4766.5 .(5529.5); IF= 4003.5 (2482.5); ECG= 8376.5 (8388.5); HF= 8798 (4188) ng/ml; p < 0.001)]. IL-6 and TNF-alfa were higher in HF group. [IL-6: CG=1.85 (6.41); IF=1.58 (1.91); ECG=1.0 (1.57); HF= 31.44 (72.19) pg/ml; p=0.001. TNF-alfa: CG=22.57 (88.2); IF=19.31 (33.16); ECG=12.45 (3.07); HF=75.15 (278.57) pg/ml; p=0.04]. Insulin, leptin and TNF-alfa did not correlate with autonomic dysfunction. Adiponectin correlated positively with HF component (r=0.336; p= 0.009) and inversely with LF component (r= -0.336; p=0.009). IL-6 correlated positively with HF component (r= 0.419; p=0.004) and inversely with LF component (r= -0.393; p= 0.007). However, in multivariate analysis only adiponectin correlated significantly with ANS measures. CONCLUSION: Adiponectin levels were higher in ECG and HF groups. IL-6 and TNF-alfa were higher in HF group. Higher levels of adiponectin were associated with reduced sympathetic activity and predominance of parasympathetic activity

<p>Reumatoidni artritis (RA) je hronično inflamatorno oboljenje zglobova koji nastaje usled poremećaja u regulaciji imunskih mehanizama. TNF-alfa jedan je od ključnih medijatora inflamacije u RA, a koji preko složenih mehanizama podstiče aktivnost osteoklasta koji dovodi do poremećaja u procesu ko&scaron;tanog remodelovanja u pravcu povećane ko&scaron;tane resorpcije koji se klinički može pratiti određivanjem nivoa markera ko&scaron;tane resorpcije i ko&scaron;tanog formiranja u urinu i serumu. Primenom TNF inhibitora započeo je novi koncept lečenja RA. Cilj rada: Utvrditi razliku mineralne ko&scaron;tane gustine (BMDg/cm2) i vrednosti ko&scaron;tanih biohemijskih markera-prokolagen tip 1N-terminalni propeptid (P1NP) i beta-crosslapsa pre uvođenja terapije, i nakon godinu dana sprovedene terapije TNF inhibitorima. Metode: Studija je sprovedena u Specijalnoj bolnici za reumatske bolesti Novi Sad jednim delom kao retrospektivno, a drugim delom prospektivno istraživanje, koje je obuhvatilo 50 bolesnica sa dijagnozom reumatoidnog artritisa kod kojih je postojala indikacija za uvođenje lekova iz grupe TNF inhibitora. Da bi u&scaron;le u studiju bolesnice su morale da ispune određene uključne/isključne kriterijume koji su bili vezani za dužinu trajanja RA i menopauze, način lečenja RA, stepen o&scaron;tećenja zglobova i prisutnost drugih oboljenja sa reperkusijom na ko&scaron;tano tkivo. Pored reumatolo&scaron;kog i fizikalnog pregleda određivani su faktori rizika za osteoporozu i prelome. Na početku i na kraju godinu dana po uvođenju terapije TNF inhibitora rađena je osteodenzitometrija na aparatu tipa &bdquo;Lunar&ldquo; merena na lumbalnoj kičmi i kuku kao i određivanje biohemijskih markera u serumu prokolagen tip 1 N-terminalni propeptid (P1NP) i betacrosslapsa ECLIA metodom. Rezultati: Prosečna starost bolesnica bila je 51,5 godina koje su u 84%, bolovale od RA do 5 godina kod kojih je u najvećem procentu dužina trajanja menopauze bila do dve godine, a u svojoj terapiji pored metotreksata su imale uključen TNF inhibitor, Etanercept 34%, Adalimubam 46%, Golimubam 9% i 2% Infliksimab.Pre uvođenja biolo&scaron;ke terapije najveći broj bolesnica 80% imalo je osteopeniju, 14% normalan nalaz, dok je osteoporoza zabeležena kod 6% bolesnica. Na kraju jednogodi&scaron;nje primene TNF inhibitora 18% bolesnica je imalo normalan osteodenzitometrijski nalaz, 78 % osteopeniji, a 4% osteoporozu. Ova promena je statistički značajna ( p=0,000). Nakon jednogodi&scaron;nje primene TNF inhibitora nije do&scaron;lo do smanjenja vrednosti BMD (g/cm&sup2;) merenog na lumbalnom delu kičme i kuka. Beleži se statističko značajno povećanje vrednosti T- skora (SD) merenog na lumbalnom delu kičme i vratu butne kosti. Vrednost ko&scaron;tanih biohemijskih markera P1NP i beta crosslapsa značajno su povećani nakon jednogodi&scaron;nje primene TNF inhibitora, pri čemu se beleži veće povećanje biohemijskog markera ko&scaron;tane sinteze, P1NP. Zaključak: Savremeni pristup lečenja reumatoidnog artritisa podrazumeva primenu biolo&scaron;kih lekova kao &scaron;to su TNF inhibitori koji značajno suzbijaju inflamaciju i dovode do smanjenja odnosa RANKL/OPG sistema, čime se inhibira dejstvo osteoklasta i sprečava gubitak mineralne ko&scaron;tane gustine. Primena TNF inhibitora nakon godinu dana sprečila je pad vrednosti BMD (g/cm&sup2;), povećana je vrednost T- skora (SD) i vrednosti ko&scaron;tanih biohemijskih markera, posebno markera ko&scaron;tane sinteze. Uprkos velikom broju studija vezanih za dejstvo TNF inhibitora na kost, za sada nema dovoljan broj istraživanja o njegovom uticaju na sprečavanju osteoporoze i preloma kostiju i nivou vrednosti ko&scaron;tanih biohemijskih markera posebno u dužem periodu praćenja, &scaron;to će biti verovatno predmet daljih istraživanja.</p> / <p>Rheumatoid arthritis (RA) is a chronic inflammatory joint disease resulting from compromised regulation of immune mechanisms. TNF-alpha is one of the key inflammation mediators in RA that, through complex mechanisms stimulates osteoclast activity, thereby modifying the bone remodeling process in the direction of increased bone resorption that can be clinically monitored by determining the level of bone resorption and bone formation markers in urine and serum. Use of TNF has initiated a new concept in RA treatment. Aims: To determine the differences in bone mineral density (BMD, g/cm2) and values of biochemical markers in bone procollagentype 1 N-terminal propeptide(P1NP) and betacrosslaps before and after yearlong TNF inhibitor therapy. Methods: The study was conducted at the Special Hospital for Rheumatic Diseases Novi Sad partly as retrospective and partly as prospective research, which involved 50 female patients diagnosed with rheumatoid arthritis in whom introduction of medications from the TNF inhibitor group was indicated. To be included in the study, patients had to meet certain inclusion/exclusion criteria related to RA and menopause duration, RA treatment, degree of joint impairment, and presence of comorbidities with repercussions for bone tissues. In addition to rheumatological and physical examinations, risk factors for osteoporosis and fractures were determined. At the beginning and one year after commencing TNF inhibitor therapy, osteodensitometry was performed using &ldquo;Lunar&rdquo; apparatus, taking measurements on lumbar spine and hip, and serum levels of biochemical markers procollagentype 1 Nterminal propeptide(P1NP) and beta-crosslaps were determined via ECLIA method. Results: Mean patient age was 51.5 years, 84% of whom suffered from RA for up to 5 years, and in the greatest percentage experienced menopause for two years, receiving therapy that in addition to methotrexate included a TNF inhibitor, Etanercept 34%, Adalimumab 46%, Golimumab 9%, and 2% Infliximab. Prior to commencing biological therapy, majority of patients 80% suffered from osteopenia, 14% had normal findings, and osteoporosis was recorded in 6% of patients. At the end of yearlong TNF inhibitor therapy, 18% of patients had normal osteodensitometry findings, 78% had osteopenia and 4% osteoporosis. This change was statistically significant (p = 0.000). As a result of yearlong TNF inhibitor therapy no reduction occurred in BMD (g/cm&sup2;) values in lumbar spine and hip. Statistically significantly higher T scores (SD) pertaining to lumbar spine and femur were measured. Values of biochemical markers P1NP and beta-crosslaps significantly improved after yearlong TNF inhibitor therapy, whereby a greater increase was recorded in the biochemical bone synthesis marker, P1NP. Conclusion: Advanced rheumatoid arthritis treatment involves the use of biological compounds such as TNF inhibitors that significantly suppress inflammation and reduce the RANKL/OPG ratio, thereby inhibiting osteoclast activity and preventing bone mineral loss. TNF inhibitor therapy after one year prevented reduction in the BMD (g/cm&sup2;) levels, while increasing the T score (SD) and bone biochemical marker values, bone synthesis marker in particular. Despite a large number of studies related to the TNF inhibitor effect on bone, there is presently not enough research on its influence on osteoporosis and bone fracture prevention and bone biochemical marker levels, especially over longer periods, which will likely be the topic of further research.</p>

Die vorliegende publikationsbasierte Dissertationsschrift erörtert auf der Basis experimenteller Untersuchungen im Tiermodell die Auswirkungen von Stress auf die Zytokinproduktion und depressionsähnliches Verhalten. Außerdem wird getestet, ob die Blockade des Zytokins Tumornekrosefaktor-α (TNF‑α) eine Möglichkeit zur antidepressiven Intervention darstellt. Einleitend werden die Zusammenhänge von Stress, Zytokinen und Depression referiert sowie das hypothetische Modell erläutert, das den publizierten Untersuchungen zugrunde liegt. Es wird hypothetisiert, dass Stress zur Erhöhung der Produktion proinflammatorischer Zytokine führt und dass die vermehrte Zytokinproduktion depressive Verhaltensweisen hervorruft. Aus dieser Annahme leitet sich die Möglichkeit ab, durch Blockade der Wirkung des proinflammatorischen Zytokins TNF‑α antidepressive Effekte zu erzielen. In den beiden Arbeiten „The impact of social isolation on immunological parameters in rats“ (Archives of Toxicology) und „Stress-induced cytokine changes in rats“ (European Cytokine Network) wurde der Einfluss von sozialer Isolation, chronischem, milden und akutem Stress auf die Zytokinproduktion untersucht. In diesen Untersuchungen führten die verschiedenen Stressarten zu einer Modulation der Produktion proinflammatorischer Zytokine. Die dritte Publikation „Antidepressant effects of TNF‑α blockade in an animal model of depression“ (Journal of Psychiatric Research) berichtet von einem Experiment, in dem untersucht wurde, ob der TNF‑α-Inhibitor Etanercept antidepressive Effekte aufweist. Tatsächlich zeigte sich unter Etanercept ein Rückgang des depressionsähnlichen Verhaltens im forced swim test (FST) analog zu Verhaltensänderungen durch das in Tierversuchen als Standard-Antidepressivum geltende Imipramin. Die Autoren schlussfolgern, dass das Zytokinsystem durch Stress moduliert wird und so in die pathophysiologische Entwicklung einer Depression involviert sein könnte. Zytokininhibitoren könnten eine neue Klasse der Antidepressiva bei Therapieresistenz werden, wenn sich die Ergebnisse dieser Tierversuche in Studien an Probanden und an Patienten replizieren lassen.:Abkürzungsverzeichnis 5 1. Einführung 6 1.1. Zytokine 6 1.1.1. Tumornekrosefaktor-α 7 1.1.2. Interleukin‑1β 9 1.1.3. Interleukin‑2 9 1.1.4. Interleukin‑4 10 1.1.5. Interleukin‑6 10 1.1.6. Interleukin‑10 10 1.1.7. Interleukin‑17 11 1.1.8. Interleukin‑22 11 1.1.9. Interferon‑γ 12 1.2. Stress 12 1.2.1. Sympathikus und Zytokine 12 1.2.2. Hypothalamus-Hypophysen-Nebennieren-Achse und Zytokine 12 1.3. Stress und Immunsystem 13 1.4. Stressarten und ihre Paradigmen im Tierversuch 14 1.4.1. Stress durch Isolation bei Ratten 14 1.4.2. Forced Swim Test: Paradigma zur Messung depressionsähnlichen Verhaltens und Modell für akuten Stress 15 1.4.3. Restraint Stress 16 1.5. Depression und Stress 16 1.6. Depression und Entzündung 17 1.7. Neue Therapieansätze gegen Depression 18 1.8. Theoretisches Modell und Fragestellung 19 1.9. Bibliografie zur Einführung 20 2. Originalarbeiten 34 „The impact of social isolation on immunological parameters in rats“ 35 „Stress-induced cytokine changes in rats“ 38 „Antidepressant effects of TNF-α blockade in an animal model of depression“ 45 3. Zusammenfassung 51 4. Anhang 55 Erklärung über die selbständige Abfassung der Arbeit 55 Lebenslauf 56 Publikationen 57 Danksagung 58