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

INTRODUÇÃO: A exposição a níveis elevados de pressão sonora é a segunda causa mais comum de perda auditiva sensorioneural adquirida. Está relacionada à morte celular por apoptose, necrose e/ou necrose programada (necroptose) devido ao dano mecânico e/ou metabólico, ocasionando a degeneração de estruturas cocleares como células ciliadas, sobretudo externas, células de suporte e de fibras aferentes do nervo coclear. Estudos têm demostrado um aumento na citocina inflamatória TNFa após a exposição ao ruído, bem como a melhoria auditiva relacionada ao uso de etanercepte, que é um bloqueador destacitocina. Neste contexto, este estudo teve por objetivo avaliar o efeito do silenciamento gênico do Tnfa na audição de ratos Wistar, expostos ao ruído branco, e identificar o perfil de expressão gênica na via metabólica desse gene. METODOLOGIA: Foram incluídos ratos Wistar do sexo masculino, jovens com limiar auditivo no Peate clique de 50 dBSPL. Os animais foram submetidos à introdução do siRNA Tnfa em uma orelha, e siRNA scramble na orelha contralateral por via trans-timpânica com posterior exposição à 120 dBSPL de ruído branco por 3h. Foi realizado Peate clique e remoção das cócleas para obtenção do cDNA e avaliação da expressão gênica da via metabólica do Tnfa por meio de qRT-PCR. Um grupo de animais, não submetidos ao silenciamento, foi exposto ao ruído para análise dessa via metabólica. Para o cálculo da expressão gênica relativa (Rq) utilizamos o método do deltaCT comparativo e o teste t-Student pareado para avaliar os parâmetros no Peate clique. RESULTADOS: A taxa de silenciamento observada foi de 74,1%. A média do limiar eletrofisiológico nas orelhas silenciadas foi estaticamente melhor que a orelha scramble (p < 0,001) com valores médios de 49,5 +- 10.5. A amplitude média da onda I em 80 dBSPL e das ondas II e IV em 90 dBSPL foi estatisticamente maior na orelha siRNA Tnfa, sem alteração na latência nas intensidades audíveis,para ambos os lados. A exposição ao ruído promoveu aumento da expressão do gene Tnfa e de seu receptor Tnfrsf1A, 24h após à exposição, associado ao aumento da expressão de genes relacionados à apoptose e diminuição de genes relacionados à sobrevida celular. CONCLUSÃO: O ruído promoveu aumento de expressão do gene Tnfa e de genes envolvidos na apoptose, associado à perda auditiva em modelo experimental. A administração trans-timpânica do siRNA Tnfa promoveu preservação do limiar eletrofisiológico e da amplitude da onda I, II e IV no Peate clique, após exposição ao ruído intenso, sugerindo que a inibição deste pode ser uma estratégia de preservação auditiva promissora. O silenciamento do Tnfa inibiu a disfunção coclear após estímulo acústico, sugerindo que esta proteína é um dos principais agentes envolvidos na perda auditiva induzida pelo ruído, devendo ser considerada como alvo terapêutico na estratégia de preservação auditiva / INTRODUCTION: Exposure to high levels of sound pressure is the second most common cause of acquired sensorineural hearing loss. It is related to cell death through apoptosis, necrosis and/or programmed necrosis (necroptosis) due to mechanical and/or metabolic damage, causing the degeneration of cochlear structures, such as cilliary cells, mainly external, as well as support cells and afferent fibers of the cochlear nerve. Studies have demonstrated an increase in the inflammatory cytokine TNFalfa after exposure to noise, as well as auditory improvement related to the use of etanercept, a cytokine blocker. In this context, this study aimed to evaluate the effect of Tnfa gene silencing on the hearing of Wistar rats exposed to white noise and to identify the expression profile in the metabolic pathway of this gene. METHODOLOGY: Young male Wistar rats with 50 dBSPL threshold in auditory brainstem responses click (ABR) were included in this study and submitted to the introduction of the TnfasiRNA in one ear and the scramble siRNA in the contralateral ear by trans-tympanic route, with subsequent exposure to 120 dB SPL of white noise for 3h. ABR was measured and the cochleae were dissected and used for the extraction of total RNA to obtain cDNA and conduct evaluations of the Tnfa metabolic pathway gene expression by qRT-PCR. A group of animals, not submitted to silencing, was exposed to noise to analyze this metabolic pathway. The relative gene expression (Rq) was calculated by the comparative deltaCT method and the paired Student t-test were applied to evaluate the parameters of the ABR click. RESULTS: The silencing rate was 74.1%. The mean electrophysiological threshold in the silenced ears was statistically higher than the scrambled ear (p < 0.001) with mean values of 49.5 +- 10.5. The mean amplitudes of wave I at 80 dBSPL and waves II and IV at 90dBSPL were statistically higher in the TnfasiRNA ear, with no change in latency at audible intensities for both sides. Noise exposure promoted increased expression of the Tnfa gene and its receptor, Tnfrsf1A, 24h after exposure, associated with increased expression of genes related to apoptosis and decreased expression of genes related to cell survival. CONCLUSIONS: Noise exposure promoted increased in Tnfa gene expression as well as in genes involved in apoptosis, associated with hearing loss in an experimental model. The trans-tympanic administration of TnfasiRNA promotes the preservation of the electrophysiological threshold and the amplitude of wave I, II and IV in the ABR click, after exposure to intense noise, suggesting that this inhibition may be a promising auditory preservation strategy. The Tnfa knockdown inhibited cochlear dysfunction after acoustic injury, suggesting that this protein plays an important role in noise induced hearing loss and should be considered a therapeutic target in auditory preservation strategies

Introdução: O exercício físico pode reverter o prejuízo funcional causado pela insuficiência cardíaca (IC). No entanto, os mecanismos implicados na melhora funcional e o efeito do exercício em outros biomarcadores de gravidade, incluindo microRNAs e marcadores de inflamação, são apenas parcialmente compreendidos.Objetivos: Avaliar o efeito do exercício nos níveis séricos da adiponectina, interleucina-6 (IL-6), fator de necrose tumoral alfa (TNF-alfa), galectina-3, microRNAs miR-423-5p, -221 e -155 em pacientes com IC. Analisar a associação entre estes biomarcadores e a melhora da capacidade funcional após 12 semanas de exercício em pacientes com IC. Métodos: Foram incluídos pacientes com IC, FEVE <= 40%, terapia clínica otimizada e randomizados em três grupos: exercício intervalado, exercício contínuo ou controle. Foi realizado teste de esforço cardiopulmonar (TECP) e dosados os níveis séricos de adiponectina, IL-6, TNF-alfa, galectina-3, microRNAs miR-423-5p, -221 e -155 antes e após a intervenção, com duração de 12 semanas. Resultados: Quarenta pacientes, 49±7 anos, 53% homens, FEVE 30±6%, 25% com cardiopatia isquêmica foram incluídos na análise (intervalado-12, continuo-14, controle-14). O exercício, especialmente intervalado, aumentou o tempo de tolerância ao esforço no TECP em relação ao grupo controle (intervalado - 13 ± 3 min vs contínuo - 12 ± 3 min vs controle - 11±2 min, p = 0,034), mas não teve efeito no VO2 pico. Ambas modalidades de exercício, intervalado e contínuo, tiveram efeito neutro em todos os biomarcadores séricos dosados, incluindo os microRNAs. Os parâmetros basais associados com mudança na capacidade funcional foram o tempo de tolerância ao esforço no TECP e o nível sérico de IL-6. Na análise multivariada, somente o nível sérico de IL-6 (após conversão logarítmica) foi significativamente associado com mudança no VO2 pico com o exercício [Coeficiente beta =-0,35 ± 0,11, p = 0,005]. Conclusões: Doze semanas de exercício aeróbico, tanto intervalado como contínuo, tiveram efeito neutro em biomarcadores de inflamação e fibrose e nos níveis circulantes dos microRNAs miR-423-5p, -221 e -155 em acientes com IC. Além disso, níveis séricos elevados de IL-6 foram independente associados a ausência de resposta ao treinamento físico / Background: Exercise training can revert the functional impairment caused by heart failure (HF). Nevertheless, the mechanisms underlying the improvement in functional capacity and the effect of the exercise on other biomarkers of severity, including microRNAs and inflammatory biomarkers, are only partially understood. Aims: To evaluate the effect of exercise on serum levels of adiponectin, interleucina-6 (IL-6), tumor necrosis fator-alpha (TNF-alpha), galectina-3, microRNAs miR-423-5p, -221 and -155 in patients with HF. To assess the association between these biomarkers and improvement in functional capacity after 12 weeks of exercise in patients with HF. Methods: We included patients with HF, LVEF <= 40%, under optimized clinical therapy, and randomized into three groups: interval exercise, continuous exercise and control. We performed cardiopulmonary exercise testing (CPET) and determined the serum levels of adiponectin, IL-6, TNF-alpha, galectina-3, microRNAs miR-423-5p, -221 and -155 before and after the intervention, which lasted 12 weeks. Results: Forty patients, 49±7 years old, 53% men, LVEF 30 ± 6%, 25% with ischemic cardiomyopathy were included in the analysis (interval-12, continuous-14, control-14). The exercise, particularly the interval training, increased the CPET exercise time, when compared with the control group (interval - 13 ± 3 min vs continuous - 12 ± 3 min vs control - 11±2 min, p = 0.034), but had no effect on peak VO2. Both modalities of exercise, interval and continuous, had neutral effect on all analyzed serum biomarkers, including the microRNAs. Baseline parameters associated with change in functional capacity with exercise were CPET exercise time and IL-6 serum level. In multivariate analysis, only IL-6 serum level (log-transformed) was significantly associated with modification in peak VO2 with exercise [? coefficient =-0.35 ± 0.11, p = 0.005]. Conclusions: Twelve weeks of aerobic exercise, both interval and continuous, had neutral effect on the serum biomarkers of inflammation and fibrosis and the circulant microRNAs miR-423-5p, -221 e -155 in patients with HF. Besides, increased IL-6 serum levels at baseline were independently associated with lack of response to exercise training

Kar Wing To. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 200-223). / Abstracts in English and Chinese. / Acknowledgements --- p.i / List of Abbreviations --- p.ii / Abstract --- p.v / 撮要 --- p.viii / List of Tables --- p.x / List of Figures --- p.xi / Contents --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- The General Characteristics of Glial Cells --- p.1 / Chapter 1.1.1 --- Astrocytes --- p.1 / Chapter 1.1.2 --- Oligodendrocytes --- p.5 / Chapter 1.1.3 --- Microglial --- p.6 / Chapter 1.2 --- Brain Injury and Astrocyte Proliferation --- p.6 / Chapter 1.3 --- Reactive Astrogliosis and Glial Scar Formation --- p.9 / Chapter 1.4 --- Astrocytes and Immune Response --- p.10 / Chapter 1.5 --- Cytokines --- p.10 / Chapter 1.5.1 --- Cytokines and the Central Nervous System (CNS) --- p.12 / Chapter 1.5.2 --- Cytokines and brain injury --- p.13 / Chapter 1.5.3 --- Cytokines-activated astrocytes in brain injury --- p.13 / Chapter 1.5.4 --- Tumour Necrosis Factor-a --- p.14 / Chapter 1.5.4.1 --- Types of TNF-α receptor and their sturctures --- p.16 / Chapter 1.5.4.2 --- Binding to TNF-α --- p.17 / Chapter 1.5.4.3 --- Different Roles of the TNF-a Receptor Subtypes --- p.17 / Chapter 1.5.4.4 --- Role of TNF-α and Brain Injury --- p.19 / Chapter 1.5.4.5 --- TNF-α Stimulates Proliferation of Astrocytes and C6 Glioma Cells --- p.23 / Chapter 1.5.5 --- Interleukin-1 (IL-1) --- p.26 / Chapter 1.5.5.1 --- Interleukin-1 and Brain Injury --- p.27 / Chapter 1.5.6 --- Interleukin-6 (IL-6) --- p.28 / Chapter 1.5.6.1 --- Interleukin-6 and brain injury --- p.29 / Chapter 1.5.7 --- γ-Interferon (γ-IFN) --- p.30 / Chapter 1.5.7.1 --- γ-Interferon and Brain Injury --- p.30 / Chapter 1.6 --- Ion Channels and Astrocytes --- p.31 / Chapter 1.6.1 --- Roles of Sodium Channels in Astrocytes --- p.33 / Chapter 1.6.2 --- Role of Potassium Channels in Astrocytes --- p.33 / Chapter 1.6.3 --- Importance of Calcium Ion Channels in Astrocytes --- p.34 / Chapter 1.6.3.1 --- Function of Cellular and Nuclear Calcium --- p.34 / Chapter 1.6.3.2 --- Nuclear Calcium in Cell Proliferation --- p.36 / Chapter 1.6.3.3 --- Nuclear Calcium in Gene Transcription --- p.36 / Chapter 1.6.3.4 --- Nuclear Calcium in Apoptosis --- p.38 / Chapter 1.6.3.5 --- Spatial and Temporal Changes of Calcium-Calcium Oscillation --- p.39 / Chapter 1.6.3.6 --- Calcium Signalling in Glial Cells --- p.39 / Chapter 1.6.3.7 --- Calcium Channels in Astrocytes --- p.41 / Chapter 1.6.3.8 --- Relationship Between [Ca2+]i and Brain Injury --- p.43 / Chapter 1.6.3.9 --- TNF-α and Astrocyte [Ca2+]i --- p.45 / Chapter 1.6.3.10 --- Calcium-Sensing Receptor (CaSR) --- p.46 / Chapter 1.7 --- Protein Kinase C (PKC) Pathways --- p.49 / Chapter 1.7.1 --- PKC and Brain Injury --- p.50 / Chapter 1.7.2 --- Role of Protein Kinase C Activity in TNF-α Gene Expression in Astrocytes --- p.51 / Chapter 1.7.3 --- PKC and Calcium in Astrocytes --- p.52 / Chapter 1.8 --- Intermediate Early Gene (IEGs) --- p.54 / Chapter 1.8.1 --- IEGs Expression and Brain Injury --- p.54 / Chapter 1.8.2 --- IEGs Expression and Calcium --- p.55 / Chapter 1.9 --- The Rat C6 Clioma Cells --- p.56 / Chapter 1.10 --- The Aim of This Project --- p.58 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.61 / Chapter 2.1.1 --- Sources of the Chemicals --- p.61 / Chapter 2.1.2 --- Materials Preparation --- p.65 / Chapter 2.1.2.1 --- Rat C6 Glioma Cell Line --- p.65 / Chapter 2.1.2.2 --- C6 Glioma Cell Culture --- p.65 / Chapter 2.1.2.2.1 --- Complete Dulbecco's Modified Eagle Medium (CDMEM) --- p.65 / Chapter 2.1.2.2.2 --- Serum-free Dulbecco's Modified Eagle Medium --- p.66 / Chapter 2.1.2.3 --- Phosphate Buffered Saline (PBS) --- p.66 / Chapter 2.1.2.4 --- Recombinant Cytokines --- p.67 / Chapter 2.1.2.5 --- Antibodies --- p.67 / Chapter 2.1.2.5.1 --- Anti-TNF-Receptor 1 (TNF-R1) Antibody --- p.67 / Chapter 2.1.2.5.2 --- Anti-TNF-Receptor 2 (TNF-R2) Antibody --- p.67 / Chapter 2.1.2.6 --- Chemicals for Signal Transduction Study --- p.68 / Chapter 2.1.2.6.1 --- Calcium Ionophore and Calcium Channel Blocker --- p.68 / Chapter 2.1.2.6.2 --- Calcium-Inducing Agents --- p.68 / Chapter 2.1.2.6.3 --- Modulators of Protein Kinase C (PKC) --- p.69 / Chapter 2.1.2.7 --- Reagents for Cell Proliferation --- p.69 / Chapter 2.1.2.8 --- Reagents for Calcium Level Measurement --- p.70 / Chapter 2.1.2.9 --- Reagents for RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.71 / Chapter 2.1.2.10 --- Sense and Antisense Used --- p.72 / Chapter 2.1.2.11 --- Reagents for Electrophoresis --- p.74 / Chapter 2.2 --- Methods --- p.74 / Chapter 2.2.1 --- Maintenance of the C6 Cell Line --- p.74 / Chapter 2.2.2 --- Cell Preparation for Assays --- p.75 / Chapter 2.2.3 --- Determination of Cell Proliferation --- p.76 / Chapter 2.2.3.1 --- Determination of Cell Proliferation by [3H]- Thymidine Incorporation --- p.76 / Chapter 2.2.3.2 --- Measurement of Cell Viability Using Neutral Red Assay --- p.77 / Chapter 2.2.3.3 --- Measurement of Cell Proliferation by MTT Assay --- p.77 / Chapter 2.2.3.4 --- Protein Assay --- p.78 / Chapter 2.2.3.5 --- Data Analysis --- p.79 / Chapter 2.2.3.5.1 --- The Measurement of Cell Proliferation by [3H]- Thymidine Incorporation --- p.79 / Chapter 2.2.3.5.2 --- The Measurement of Cell growth in Neutral Red and MTT Assays --- p.79 / Chapter 2.2.3.5.3 --- The Measurement of Cell Proliferationin Protein Assay --- p.79 / Chapter 2.2.4 --- Determination of Intracellular Calcium Changes --- p.80 / Chapter 2.2.4.1 --- Confocal Microscopy --- p.80 / Chapter 2.2.4.1.1 --- Procedures for Detecting Cell Activity by CLSM --- p.81 / Chapter 2.2.4.1.2 --- Precautions of CLSM --- p.82 / Chapter 2.2.5 --- Determination of Gene Expression by Reverse- Transcription Polymerase Chain Reaction (RT-PCR) --- p.83 / Chapter 2.2.5.1 --- RNA Preparation --- p.83 / Chapter 2.2.5.1.1 --- RNA Extraction --- p.83 / Chapter 2.2.5.1.2 --- Measurement of RNA Yield --- p.84 / Chapter 2.2.5.2 --- Reverse Transcription (RT) --- p.84 / Chapter 2.2.5.3 --- Polymerase Chain Reaction (PCR) --- p.85 / Chapter 2.2.5.4 --- Separation of PCR Products by Agarose Gel Electrophoresis --- p.85 / Chapter 2.2.5.5 --- Quantification of Band Density --- p.86 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- Effects of Different Drugs on C6 Cell Proliferation --- p.87 / Chapter 3.1.1 --- Effects of Cytokines on C6 Cell Proliferation --- p.87 / Chapter 3.1.1.1 --- Effect of TNF-α on C6 Proliferation --- p.88 / Chapter 3.1.1.2 --- Effects of Other Cytokines on C6 Cell Proliferation --- p.92 / Chapter 3.1.2 --- The Signalling Pathway of TNF-α induced C6 Cell Proliferation --- p.92 / Chapter 3.1.2.1 --- The Involvement of Calcium Ions in TNF-α-induced C6Cell Proliferation --- p.95 / Chapter 3.1.2.2 --- The Involvement of Protein Kinase C in TNF-α- induced C6 Cell Proliferation --- p.96 / Chapter 3.1.3 --- Effects of Anti-TNF Receptor Subtype Antibodies on C6 Cell Proliferation --- p.102 / Chapter 3.2 --- The Effect of in Tumour Necrosis Factor-α on Changesin Intracellular Calcium Concentration --- p.102 / Chapter 3.2.1 --- Release of Intracellular Calcium in TNF-α-Treated C6 Cells --- p.104 / Chapter 3.2.2 --- Effects of Calcium Ionophore and Calcium Channel Blocker on TNF-α-induced [Ca2+]i Release --- p.107 / Chapter 3.2.3 --- Effects of Other Cytokines on the Change in [Ca2+]i --- p.109 / Chapter 3.2.4 --- The Role of PKC in [Ca2+]i release in C6 Glioma Cells --- p.109 / Chapter 3.2.4.1 --- Effects of PKC Activators and Inhibitors on the Changes in [Ca2+]i --- p.114 / Chapter 3.3 --- Determination of Gene Expression by RT-PCR --- p.114 / Chapter 3.3.1 --- Studies on TNF Receptors Gene Expression --- p.117 / Chapter 3.3.1.1 --- Effect of TNF-α on TNF Receptors Expression --- p.117 / Chapter 3.3.1.2 --- Effects of Other Cytokines on the TNF Receptors Expression --- p.119 / Chapter 3.3.1.3 --- Effects of Anti-TNF Receptor Subtype Antibodies on the TNF-a-induced Receptors Expression --- p.121 / Chapter 3.3.1.4 --- Effect of Calcium Ions on TNF Receptors Expression --- p.121 / Chapter 3.3.1.4.1 --- Effect of Calcium Ionophore on TNF Receptors Expression --- p.126 / Chapter 3.3.1.4.2 --- Effect of TNF-α Combination with A23187 on the Expression of TNF Receptors --- p.128 / Chapter 3.3.1.4.3 --- Effects of Calcium Ionophore and Channel Blocker on TNF Receptors Expression --- p.130 / Chapter 3.3.1.4.4 --- Effects of Calcium-Inducing Agents on TNF Receptors Gene Expressions --- p.130 / Chapter 3.3.1.5 --- Effects of PKC Activator and Inhibitor on TNF-α- induced TNF Receptors Expressions --- p.135 / Chapter 3.3.1.6 --- Effect of PKC and Ro31-8220 on IL-l-induced TNF Receptors Expressions --- p.138 / Chapter 3.3.2 --- Expression of Calcium-sensing Receptor upon Different Drug Treatments --- p.138 / Chapter 3.3.2.1 --- Effect of TNF-α on the Calcium-sensing Receptor Expression --- p.141 / Chapter 3.3.2.2 --- Effects of Other Cytokines on CaSR Expression --- p.143 / Chapter 3.3.2.3 --- Effect of A23187 on CaSR Expression --- p.143 / Chapter 3.3.2.4 --- Effect of TNF-α and A23187 Combined Treatment on CaSR Expression --- p.146 / Chapter 3.3.2.5 --- Effects of Calcium-inducing Agents on CaSR Expression --- p.149 / Chapter 3.3.2.6 --- Effects of PKC Activator and PKC Inhibitor on CaSR Expression --- p.149 / Chapter 3.3.3 --- Expression of PKC Isoforms upon Treatment with Different Drugs --- p.153 / Chapter 3.3.3.1 --- Effect of TNF-α on the PKC Isoforms Expression --- p.155 / Chapter 3.3.3.2 --- Effect of A23187 on the PKC Isoforms Expression --- p.155 / Chapter 3.3.3.3 --- Effect of TNF-α and Calcium Ionophore Combined Treatment on PKC Isoforms Expression --- p.157 / Chapter 3.3.3.4 --- Effects of Calcium-inducing Agents on PKC Isoforms Expression --- p.159 / Chapter 3.3.4 --- Expression of the Transcription Factors c-fos and c-junin Drug Treatments --- p.161 / Chapter 3.3.4.1 --- Effect of TNF-a on c-fos and c-jun Expression --- p.163 / Chapter 3.3.4.2 --- Effect of A23187 on c-fos and c-jun Expression --- p.163 / Chapter 3.3.4.3 --- Effect of TNF-a in Combination with A23187 on c- fos and c-jun Expression --- p.165 / Chapter 3.3.4.4 --- Effects of Calcium-inducing Agents on c-fos and c- jun Expression --- p.167 / Chapter 3.3.5 --- Effects of Different Drugs on Endogenous TNF-α Expression --- p.167 / Chapter 3.3.5.1 --- Effect of TNF-α on Endogenous TNF-α Expression --- p.169 / Chapter 3.3.5.2 --- Effect of A23187 on Endogenous TNF-α Expression --- p.169 / Chapter 3.3.5.3 --- Effect of TNF-α in Combination with A23187 on the Expression of Endogenous TNF-α --- p.172 / Chapter 3.3.5.4 --- Effects of Calcium-inducing Agents on Endogenous TNF-α Expression --- p.172 / Chapter 3.3.6 --- Effect of Different Drugs on LL-1 Expression --- p.175 / Chapter 3.3.6.1 --- Effect of TNF-a on IL-lα Expression --- p.177 / Chapter 3.3.6.2 --- Effect of A23187 on the IL-lα Expression --- p.177 / Chapter 3.3.6.3 --- Effect of Calcium Ionophore and TNF-α Combined Treatment on IL-1α Expression --- p.179 / Chapter 3.3.6.4 --- Effects of Calcium-inducing Agents on IL-lα Expression --- p.179 / Chapter 3.3.6.5 --- Effect of PKC Activator on the IL-1α Expression --- p.181 / Chapter CHAPTER 4 --- DISCUSSIONS AND CONCLUSIONS / Chapter 4.1 --- "Effects of Cytokines, Ca2+ and PKC and Anti-TNF-α Antibodies on C6 Glioma Cells Proliferation" --- p.184 / Chapter 4.2 --- The Role of Calcium in TNF-α-induced Signal Transduction Pathways --- p.186 / Chapter 4.3 --- Gene Expressions in C6 Cells after TNF-a Stimulation --- p.187 / Chapter 4.3.1 --- "Expression of TNF-α, TNF-Receptors and IL-1" --- p.187 / Chapter 4.3.2 --- CaSR Expression --- p.190 / Chapter 4.3.3 --- PKC Isoforms Expressions --- p.192 / Chapter 4.3.4 --- Expression of c-fos and c-jun --- p.193 / Chapter 4.4 --- Conclusion --- p.194 / REFERENCES --- p.200

Base teórica: A introdução de agentes biológicos, especialmente os bloqueadores do fator de necrose tumoral (anti-TNF), para o tratamento de doenças reumáticas aumentou o risco de desenvolver tuberculose (TB). O rastreio para infecção tuberculosa latente (ILTB) é fortemente recomendado antes de iniciar a terapia com agentes anti-TNF. Os objetivos deste estudo foram identificar a prevalência de ILTB e TB entre pacientes com doenças reumáticas em uso dos medicamentos anti-TNF. Métodos: Estudo transversal. Foram revisados os registros médicos eletrónicos de todos os doentes adultos (≥ 18 anos) em uso da terapia anti-TNF. Todos os pacientes foram submetidos ao teste tuberculínico (TT) antes de iniciar o tratamento com os medicamentos anti-TNF. Resultados: No total, 176 pacientes foram incluídos no estudo. A idade média de todos os pacientes foi de 51,9 ± 12,4 anos, 34,7% eram do sexo masculino e 90,9% eram brancos. As doenças subjacentes mais comuns foram: Artite Reumatóide (AR) em 89 pacientes (50,6%), Espondilite Anquilosante (EA) em 49 (27,8%) e Artrite Psoriática (AP) em 31 (17,6%). A prevalência de TT positivo foi de 29,5%. O contato domiciliar com TB foi significativamente associado com TT positivo (p = 0,020). Os pacientes com AR apresentaram reações TT menores do que os pacientes com EA (p = 0,022). Houve seis casos de TB (3,4%) diagnosticados durante a terapia anti-TNF. Conclusões: Demonstrou-se alta prevalência de TT positivo (29,5%) em pacientes com doenças reumáticas em uma região com alta prevalência de TB. Nossos dados corroboram a recomendação do Colégio Americano de Reumatologia (ACR) de que os pacientes que vivem em configurações de alta incidência de TB devem ser testados anualmente para ILTB. / Background: The introduction of biological agents, especially the blockers of tumor necrosis factor (anti-TNF), for the treatment of rheumatic diseases increased the risk of developing tuberculosis (TB). Screening for latent TB infection (LTBI|) is strongly recommended before starting therapy with anti-TNF agents. The objectives of this study were to identify the prevalence of LTBI and TB among patients with rheumatic diseases on anti-TNF drugs. Methods: Cross-sectional study. The electronic medical records of all adult patients (≥ 18 years old) undergoing anti-TNF treatment were reviewed. Every patient underwent TST test before starting anti-TNF treatment. Results: In total, 176 patients were included in the study. The mean age of all patients was 51.9 ± 12.4 years, 34.7% were males, and 90.9% were white. The most common underlying diseases were: RA in 89 patients (50.6%), AS in 49 (27.8%), and PA in 31 (17.6%). The prevalence of positive TST was 29.5%. Household contact with TB was significantly associated with a positive TST (p=0.020). RA patients had lower TST reactions than AS patients (p=0.022). There were six cases of TB (3.4%) diagnosed during anti-TNF therapy. Conclusions: We demonstrated a high prevalence of positive TST (29.5%) among patients with rheumatic diseases in a region with high TB prevalence. Our data corroborates the ACR’s recommendation that patients who live in high TB incidence settings should be tested annually for LTBI.

Objetivos: avaliar a associação entre dor oncológica crônica e as citocinas pró-inflamatórias interleucina-6 (IL-6), IL-8, IL-1&#946; e TNF-&#945;, e a interferência destas citocinas na relação entre dor, qualidade de vida relacionada à saúde (QVRS) e desempenho funcional (DF). Método: 220 pacientes ambulatoriais com câncer, que não haviam recebido nenhum tratamento antineoplásico nos últimos 30 dias, foram avaliados pelo Inventário Breve de Dor, Questionário de Dor McGill (MPQ), Inventário de Depressão de Beck, Escala de Desempenho Funcional de Karnofsky e a escala de QVRS, EORTC-QLQ-c30. Os níveis plasmáticos das citocinas foram dosados através do teste imunoenzimático ELISA e comparados entre pacientes com dor leve (G1), moderada a intensa (G2) e sem dor (G3) usando a ANOVA ou o teste de Kruskal-Wallis seguido por análise de múltiplas comparações. Os pacientes do G1 e G2 apresentavam apenas dor oncólogica e estavam em uso de analgésicos. Os do G3 tinham câncer, mas não apresentaram dor ou fizeram uso de analgésicos nos últimos 14 dias. 23 voluntários saudáveis (G4) foram incluídos como controle. A ANCOVA foi utilizada para avaliar o efeito das citocinas na relação dor, QVRS e DF. A análise de Árvore de Classificação e Regressão (CART) avaliou a relação entre citocinas e níveis de dor, ajustada por características clínicas, demográficas e sintomas. As correlações foram avaliadas pelos testes de Spearman e Pearson. Resultados: Os pacientes do G2 (n=49) apresentaram significativamente (p<0,05) maiores níveis de IL-6 e IL-8 que todos os demais grupos. Os níveis do TNF-&#945; e da IL-1&#946; foram maiores no G2 que no G1 (n=76) e G4, mas não diferiram significativamente do G3 (n=95). Entre pacientes com dor (n=125) foram observadas correlações significativas (p<0,05) ou com tendência a significância entre: IL-6 e a pior dor (r=0,23) e o escore total do MPQ (r=0,18); TNF-&#945; e os descritores afetivos do MPQ (r=0,33); IL-8 e escore total do MPQ (r=0,16); dimensão emocional da QVRS e IL-8 (p=-0,26) e IL-6 (r=-0,17); escalas de sintomas de dor e IL-6 (r=0,21), e de fadiga com IL-8 (r=0,14). A ANOVA mostrou que os pacientes do G2 tiveram significativamente pior DF e QVRS que os do G1, G3 e G4, na maioria das escalas. Segundo a ANCOVA apenas a IL-8 moderou o efeito da dor sobre a escala de perda de apetite; e independentemente aumentou a fadiga. A análise de CART selecionou o estádio da doença, a IL-8, a insônia moderada a intensa, a fadiga leve a intensa e a idade <=48 anos como preditoras de dor. O maior percentual de casos com dor moderada a intensa foi observado entre os com estádio IV da doença e IL-8 > 5,20 pg/ml. Conclusões: o aumento das citocinas pró-inflamatórias IL-6, IL-8, IL-1&#946; e TNF-&#945; esteve relacionado ao aumento da dor. A IL-6 e IL-8 estavam associadas à ocorrência de dor moderada a intensa. A IL-8 moderou o efeito da dor sobre a perda de apetite em pacientes com dor, não interferindo no impacto da dor sobre o desempenho funcional, a QVRS geral e os domínios físico, emocional, social e cognitivo da QVRS. A IL-8 e IL-6 estavam independentemente correlacionadas com redução da QVRS emocional e a IL-8 com piora da fadiga em pacientes com dor oncológica. Os resultados sugerem que tratamento com antagonistas/inibidores das citocinas IL-6, IL-8, IL-1&#946; e TNF-&#945; pode contribuir para o alívio da dor em pacientes com câncer / Aims: to examine the association between chronic cancer pain and the pro-inflammatory cytokines interleukin-6 (IL-6), IL-8, IL-1&#946; and TNF-&#945;, as well as the interference of these cytokines in the relationship between pain, health-related quality of life (HRQOL), and performance status (PS). Methods: 220 cancer outpatients, who didn`t receive any antineoplastic treatment in the last 30 days, were evaluated by the Brief Pain Inventory (BPI), McGill Pain Questionnaire (MPQ), Beck Depression Inventory (BDI), Karnofsky Performance Scale (KPS), and a HRQOL measurement, the EORTC-QLQ-30. Plasma cytokine levels were measured using an enzyme-linked immunosorbent assay (ELISA) and were compared among patients with mild (G1), moderate to severe (G2) and without pain (G3) using one-way analysis of variance (ANOVA) or Kruskal-Wallis followed by multiple comparison tests. Patients in G1 and G2 had only cancer pain and were using analgesics. G3 members had cancer but felt no pain and didn`t use analgesics in the last 14 days. Twenty-three healthy volunteers (G4) were included as controls. ANCOVA was used to assess the effect of cytokines on the pain, HRQOL and PS relationship. Associations between pain and cytokines, adjusted by cancer symptoms and clinical and demographic characteristics were also examined using Classification and Regression Tree (CART) analysis. Correlations were assessed by Spearman\'s and Pearson\'s tests. Results: the IL-6 and IL-8 levels in G2 (n=49) patients was significantly (p<0.05) higher than of those in all other groups. The IL-1&#946; and TNF-&#945; levels were significantly higher in G2 than in G1 (n=76) and G4, but not significantly different when compared with G3 (n=95). Among patients with pain (n=125), it was observed significant, or almost significant, correlations between: IL-6 with worst pain (r=0.23) and with the total score of MPQ (r=0.18); TNF-&#945; with MPQ affective domain (r=0.33); IL-8 with total score of MPQ (r=0.16); emotional HRQOL domain and IL-8 (p=-0.26) and IL-6 (r=-0.17) and; between HRQOL pain scale and IL-6 (r=0.21), and fatigue scale and IL-8 (r=0.14). ANOVA showed that PS and HRQOL were significantly worse in G2 than in G1, G3 and G4 in most scales. According to ANCOVA, there was an interaction between pain and IL-8 that increased loss of appetite. IL-8 independently increased fatigue. CART analysis selected disease stage, IL-8, moderate to severe insomnia, mild to severe fatigue and age <=48 years as markers for pain. The highest percentage of patients with moderate to severe pain was observed among those with disease stage IV and plasma level of IL-8 > 5.20 pg/ml. Conclusions: increase of pro-inflammatory cytokines IL-6, IL-8, IL-1&#946; and TNF-&#945; was related to increase in pain. IL-6 and IL-8 were related to moderate to severe pain occurrence. IL-8 was a moderator to the pain effect on loss of appetite in patients with pain but has not interfered neither on pain effect over performance status, nor on general HRQOL nor its physical, emotional, social and cognitive domains. IL-8 and IL-6 were found to be independently correlated with the decrease of the emotional domain scores of HRQOL and the IL-8 with increased fatigue on patients with cancer pain. Results suggest that treatment with IL-6, IL-8, IL-1&#946; and TNF-&#945; cytokine inhibitors/antagonists may provide pain relief in cancer patients

INTRODUÇÃO: A exposição a níveis elevados de pressão sonora é a segunda causa mais comum de perda auditiva sensorioneural adquirida. Está relacionada à morte celular por apoptose, necrose e/ou necrose programada (necroptose) devido ao dano mecânico e/ou metabólico, ocasionando a degeneração de estruturas cocleares como células ciliadas, sobretudo externas, células de suporte e de fibras aferentes do nervo coclear. Estudos têm demostrado um aumento na citocina inflamatória TNFa após a exposição ao ruído, bem como a melhoria auditiva relacionada ao uso de etanercepte, que é um bloqueador destacitocina. Neste contexto, este estudo teve por objetivo avaliar o efeito do silenciamento gênico do Tnfa na audição de ratos Wistar, expostos ao ruído branco, e identificar o perfil de expressão gênica na via metabólica desse gene. METODOLOGIA: Foram incluídos ratos Wistar do sexo masculino, jovens com limiar auditivo no Peate clique de 50 dBSPL. Os animais foram submetidos à introdução do siRNA Tnfa em uma orelha, e siRNA scramble na orelha contralateral por via trans-timpânica com posterior exposição à 120 dBSPL de ruído branco por 3h. Foi realizado Peate clique e remoção das cócleas para obtenção do cDNA e avaliação da expressão gênica da via metabólica do Tnfa por meio de qRT-PCR. Um grupo de animais, não submetidos ao silenciamento, foi exposto ao ruído para análise dessa via metabólica. Para o cálculo da expressão gênica relativa (Rq) utilizamos o método do deltaCT comparativo e o teste t-Student pareado para avaliar os parâmetros no Peate clique. RESULTADOS: A taxa de silenciamento observada foi de 74,1%. A média do limiar eletrofisiológico nas orelhas silenciadas foi estaticamente melhor que a orelha scramble (p < 0,001) com valores médios de 49,5 +- 10.5. A amplitude média da onda I em 80 dBSPL e das ondas II e IV em 90 dBSPL foi estatisticamente maior na orelha siRNA Tnfa, sem alteração na latência nas intensidades audíveis,para ambos os lados. A exposição ao ruído promoveu aumento da expressão do gene Tnfa e de seu receptor Tnfrsf1A, 24h após à exposição, associado ao aumento da expressão de genes relacionados à apoptose e diminuição de genes relacionados à sobrevida celular. CONCLUSÃO: O ruído promoveu aumento de expressão do gene Tnfa e de genes envolvidos na apoptose, associado à perda auditiva em modelo experimental. A administração trans-timpânica do siRNA Tnfa promoveu preservação do limiar eletrofisiológico e da amplitude da onda I, II e IV no Peate clique, após exposição ao ruído intenso, sugerindo que a inibição deste pode ser uma estratégia de preservação auditiva promissora. O silenciamento do Tnfa inibiu a disfunção coclear após estímulo acústico, sugerindo que esta proteína é um dos principais agentes envolvidos na perda auditiva induzida pelo ruído, devendo ser considerada como alvo terapêutico na estratégia de preservação auditiva / INTRODUCTION: Exposure to high levels of sound pressure is the second most common cause of acquired sensorineural hearing loss. It is related to cell death through apoptosis, necrosis and/or programmed necrosis (necroptosis) due to mechanical and/or metabolic damage, causing the degeneration of cochlear structures, such as cilliary cells, mainly external, as well as support cells and afferent fibers of the cochlear nerve. Studies have demonstrated an increase in the inflammatory cytokine TNFalfa after exposure to noise, as well as auditory improvement related to the use of etanercept, a cytokine blocker. In this context, this study aimed to evaluate the effect of Tnfa gene silencing on the hearing of Wistar rats exposed to white noise and to identify the expression profile in the metabolic pathway of this gene. METHODOLOGY: Young male Wistar rats with 50 dBSPL threshold in auditory brainstem responses click (ABR) were included in this study and submitted to the introduction of the TnfasiRNA in one ear and the scramble siRNA in the contralateral ear by trans-tympanic route, with subsequent exposure to 120 dB SPL of white noise for 3h. ABR was measured and the cochleae were dissected and used for the extraction of total RNA to obtain cDNA and conduct evaluations of the Tnfa metabolic pathway gene expression by qRT-PCR. A group of animals, not submitted to silencing, was exposed to noise to analyze this metabolic pathway. The relative gene expression (Rq) was calculated by the comparative deltaCT method and the paired Student t-test were applied to evaluate the parameters of the ABR click. RESULTS: The silencing rate was 74.1%. The mean electrophysiological threshold in the silenced ears was statistically higher than the scrambled ear (p < 0.001) with mean values of 49.5 +- 10.5. The mean amplitudes of wave I at 80 dBSPL and waves II and IV at 90dBSPL were statistically higher in the TnfasiRNA ear, with no change in latency at audible intensities for both sides. Noise exposure promoted increased expression of the Tnfa gene and its receptor, Tnfrsf1A, 24h after exposure, associated with increased expression of genes related to apoptosis and decreased expression of genes related to cell survival. CONCLUSIONS: Noise exposure promoted increased in Tnfa gene expression as well as in genes involved in apoptosis, associated with hearing loss in an experimental model. The trans-tympanic administration of TnfasiRNA promotes the preservation of the electrophysiological threshold and the amplitude of wave I, II and IV in the ABR click, after exposure to intense noise, suggesting that this inhibition may be a promising auditory preservation strategy. The Tnfa knockdown inhibited cochlear dysfunction after acoustic injury, suggesting that this protein plays an important role in noise induced hearing loss and should be considered a therapeutic target in auditory preservation strategies

Introdução: O exercício físico pode reverter o prejuízo funcional causado pela insuficiência cardíaca (IC). No entanto, os mecanismos implicados na melhora funcional e o efeito do exercício em outros biomarcadores de gravidade, incluindo microRNAs e marcadores de inflamação, são apenas parcialmente compreendidos.Objetivos: Avaliar o efeito do exercício nos níveis séricos da adiponectina, interleucina-6 (IL-6), fator de necrose tumoral alfa (TNF-alfa), galectina-3, microRNAs miR-423-5p, -221 e -155 em pacientes com IC. Analisar a associação entre estes biomarcadores e a melhora da capacidade funcional após 12 semanas de exercício em pacientes com IC. Métodos: Foram incluídos pacientes com IC, FEVE <= 40%, terapia clínica otimizada e randomizados em três grupos: exercício intervalado, exercício contínuo ou controle. Foi realizado teste de esforço cardiopulmonar (TECP) e dosados os níveis séricos de adiponectina, IL-6, TNF-alfa, galectina-3, microRNAs miR-423-5p, -221 e -155 antes e após a intervenção, com duração de 12 semanas. Resultados: Quarenta pacientes, 49±7 anos, 53% homens, FEVE 30±6%, 25% com cardiopatia isquêmica foram incluídos na análise (intervalado-12, continuo-14, controle-14). O exercício, especialmente intervalado, aumentou o tempo de tolerância ao esforço no TECP em relação ao grupo controle (intervalado - 13 ± 3 min vs contínuo - 12 ± 3 min vs controle - 11±2 min, p = 0,034), mas não teve efeito no VO2 pico. Ambas modalidades de exercício, intervalado e contínuo, tiveram efeito neutro em todos os biomarcadores séricos dosados, incluindo os microRNAs. Os parâmetros basais associados com mudança na capacidade funcional foram o tempo de tolerância ao esforço no TECP e o nível sérico de IL-6. Na análise multivariada, somente o nível sérico de IL-6 (após conversão logarítmica) foi significativamente associado com mudança no VO2 pico com o exercício [Coeficiente beta =-0,35 ± 0,11, p = 0,005]. Conclusões: Doze semanas de exercício aeróbico, tanto intervalado como contínuo, tiveram efeito neutro em biomarcadores de inflamação e fibrose e nos níveis circulantes dos microRNAs miR-423-5p, -221 e -155 em acientes com IC. Além disso, níveis séricos elevados de IL-6 foram independente associados a ausência de resposta ao treinamento físico / Background: Exercise training can revert the functional impairment caused by heart failure (HF). Nevertheless, the mechanisms underlying the improvement in functional capacity and the effect of the exercise on other biomarkers of severity, including microRNAs and inflammatory biomarkers, are only partially understood. Aims: To evaluate the effect of exercise on serum levels of adiponectin, interleucina-6 (IL-6), tumor necrosis fator-alpha (TNF-alpha), galectina-3, microRNAs miR-423-5p, -221 and -155 in patients with HF. To assess the association between these biomarkers and improvement in functional capacity after 12 weeks of exercise in patients with HF. Methods: We included patients with HF, LVEF <= 40%, under optimized clinical therapy, and randomized into three groups: interval exercise, continuous exercise and control. We performed cardiopulmonary exercise testing (CPET) and determined the serum levels of adiponectin, IL-6, TNF-alpha, galectina-3, microRNAs miR-423-5p, -221 and -155 before and after the intervention, which lasted 12 weeks. Results: Forty patients, 49±7 years old, 53% men, LVEF 30 ± 6%, 25% with ischemic cardiomyopathy were included in the analysis (interval-12, continuous-14, control-14). The exercise, particularly the interval training, increased the CPET exercise time, when compared with the control group (interval - 13 ± 3 min vs continuous - 12 ± 3 min vs control - 11±2 min, p = 0.034), but had no effect on peak VO2. Both modalities of exercise, interval and continuous, had neutral effect on all analyzed serum biomarkers, including the microRNAs. Baseline parameters associated with change in functional capacity with exercise were CPET exercise time and IL-6 serum level. In multivariate analysis, only IL-6 serum level (log-transformed) was significantly associated with modification in peak VO2 with exercise [? coefficient =-0.35 ± 0.11, p = 0.005]. Conclusions: Twelve weeks of aerobic exercise, both interval and continuous, had neutral effect on the serum biomarkers of inflammation and fibrosis and the circulant microRNAs miR-423-5p, -221 e -155 in patients with HF. Besides, increased IL-6 serum levels at baseline were independently associated with lack of response to exercise training

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

<p>Uvod: Reumatoidni artritis (RA) je bolest koja dovodi do ireverzibilnog o&scaron;tećenja zglobova usled čega je neophodno pri postavljanju dijagnoze započeti lečenje. TNF-alfa inhibitori predstavljaju revolucionarno otkriće u lečenju RA, pri čemu su najče&scaron;će kori&scaron;ćeni Etanercept i Adalimumab. Oni nisu efikasni kod svih pacijenata kod kojih se primene, a mehanizmi gubitka odgovora nisu jasni. Cilj rada je odrediti uticaj Etanercepta i Adalimumaba na aktivnost bolesti (merenjem DAS28 SE i DAS28 CRP skora) i funkcionalni status pacijenata (merenjem HAQ-DI upitnika), broj bolnih i otečnih zglobova pre i tokom godinu dana primene ovih lekova, kao i utvrditi povezanost koncentracije Etanercepta i Adalimumaba u krvi sa vrednostima DAS28 SE u momentu odreĎivanja koncentracije leka. Praćena je i učestalost neželjenih efekata kod pacijenata lečenih sa ova dva leka. Ispitan je i uticaj primene Metotreksata na nivoe lekova u krvi, kao i doza Metotreksata pre i 6 meseci nakon uvoĎenja Etanercepta ili Adalimumaba. Metode: Studija je sprovedena u Specijalnoj bolnici za reumatske bolesti i Klinici za nefrologiju i kliničku imunologiju, Kliničkog centra Vojvodine u Novom Sadu i obuhvatila je 88 pacijenata kod kojih je postavljena dijagnoza RA, od kojih je 49 bilo lečeno Etanerceptom, a 39 Adalimumabom. Analizirana je medicinska dokumentacija, a nakon početka primene TNF-alfa inhibitora svim ispitanicima je u toku godinu dana svaka tri meseca raĎena kontrola koja je podrazumevala anamnezu i fizički pregled, analizu biohemijskih nalaza krvi, merena je aktivnost bolesti merenjem indeksa aktivnosti bolesti DAS28 SE i DAS28 CRP i raĎena procena funkcionalnog statusa tako &scaron;to je pacijent popunjavao HAQ-DI upitnik. Rezultati: Aktivnost RA merena DAS28 SE i DAS28 CRP indeksima, funkcionalni status meren HAQ-DI upitnikom, broj bolnih i otečenih zglobova i vrednosti reaktanata akutne faze značajno su veći pre početka terapije Etanerceptom i Adalimumabom i smanjuje se tokom prvih 6 meseci lečenja ovim lekovima i potom se taj efekat terapije održava do kraja perioda praćenja. Nema statistički značajne razlike u poreĎenju Etanercepta i Adalimumaba u odnosu na učestalost neželjenih dejstava. Doza Metotreksata je statistički značajno manja 6 meseci nakon upotrebe biolo&scaron;kog leka Etanercept i Adalimumab. Pacijenti lečeni Metotreksatom uz Adalimumab imali su statistički značajno veće nivoe leka, nego oni koji ga nisu koristili. Zaključak: TNF-alfa inhibitori ne dovode do zaustavljanja bolesti kod svih pacijenata kod kojih se primene. Mehanizam gubitka odgovora na terapiju TNF-alfa inhibitorima nije jasan. Kako bi se donela najbolja odluka za pacijenta, neophodno je odrediti nivo leka u krvi, kao i nivo antitela na lek prilikom svake promene stanja pacijenta. Za sada nema dovoljno studija koje ukazuju da li postoji veza izmeĎu ekspresije TNF-alfa gena i nivoa TNF-alfa u krvi, te da li bi se merenjem TNF-alfa u krvi mogla korigovati terapija i doza TNF-alfa inhibitora &scaron;to će verovatno biti predmet budućih istraživanja.</p> / <p>Rheumatoid Arthritis (RA) is a disease that leads to irreversible joint damage, which makes necessary to start treatment when the diagnosis is set. TNF-alpha inhibitors represent a revolutionary discovery in the treatment of RA, and the most commonly used are Etanercept and Adalimumab. They are not effective in all patients, and the mechanisms of loss of response are not clear. The aim of this study is to determine the effect of Etanercept and Adalimumab on disease activity (by measuring DAS28 SE and DAS28 CRP score) and the functional status of patients (by measuring the HAQ-DI questionnaire), the number of painful and swollen joints before and during the first year of administration of these drugs. Also, it was determined a correlation between the concentration of Etanercept and Adalimumab in blood and the values of DAS28 SE at the moment of drug concentration measurement. The incidence of adverse effects in patients treated with these two drugs was also observed. It was examined the effect of Methotrexate on drug levels in the blood as well as the dose of Methotrexate before and 6 months after the introduction of Etanercept or Adalimumab. Methods: The study was conducted at the Special Hospital for Rheumatic Diseases and the Clinic of Nephrology and Clinical Immunology, Clinical Center of Vojvodina in Novi Sad. It included 88 patients with RA, 49 were treated with Etanercept and 39 with Adalimumab. Medical documentation was analyzed, and during the first year of TNF-alpha inhibitor administration, every three months were done anamnesis and physical examination, analysis of blood biochemical findings, measurements of the disease activity with DAS28 SE and DAS28 CRP score and a functional status assessment with the HAQ-DI questionnaire. Results: Disease activity measured by DAS28 SE and DAS28 CRP scores, functional status measured with HAQ-DI questionnaire, number of painful and swollen joints and acute phase reactant values are significantly higher before Etanercept and Adalimumab therapy and decreased during the first 6 months of treatment with these drugs and then this effect of therapy is maintained until the end of the monitoring period. There is no statistically significant difference in the comparison of Etanercept and Adalimumab with respect to the frequency of adverse events. The dose of Methotrexate was statistically significantly lower for 6 months after the use of Etanercept and Adalimumab. Patients treated with Methotrexate and Adalimumab had statistically significantly higher drug levels than those who did not use it. Conclusion: TNF-alpha inhibitors are not effective in all patients who used them. The mechanism of loss of response to TNF-alpha inhibitors is not clear. In order to make the best decision for the patient, it is necessary to determine the drug level in the blood as well as the level of antibodies to the drug in each change in the patient&#39;s condition. For now, there are not enough studies to indicate whether there is a link between expression of the TNF-alpha gene and the level of TNF-alpha in the blood, and whether the measurement of the TNF-alpha in blood could be used for therapy correction and change of dose of TNF-alpha inhibitor, which is likely to be the subject of the future research.</p>