Des travaux antérieurs effectués dans notre laboratoire ont démontré une corrélation entre les niveaux circulants de peroxyde d'hydrogène (H[indice inférieur 2]O[indice inférieur 2]) et d'hormone gonadotrophine chorionique humaine (hCG) chez des femmes souffrant de prééclampsie (Kharfi et al, . 2005). Le peroxyde d'hydrogène étant surtout connu pour ses effets cytotoxiques, nous avons déterminé quels étaient ses effets sur des cellules placentaires in vitro, en rapport aux conditions retrouvées in vivo chez des femmes prééclamptiques. Pour ce faire, nous avons développé un modèle d'étude de cellules trophoblastiques in vitro, avec lequel nous avons pu évaluer les effets du peroxyde d'hydrogène sur la fonction placentaire via la production d'hCG, sur la production de TNF-[alpha] comme marqueur inflammatoire et sur l'oxyde nitrique (NO) comme facteur vasodilatateur. Nos résultats nous ont permis de constater divers effets du peroxyde d'hydrogène sur les cytotrophoblastes in vitro, effets qui peuvent être corrélés avec les niveaux constatés chez la femme prééclamptique. Premièrement, nous avons pu observer une augmentation de la production d'hCG sous l'effet de faibles concentrations de peroxyde d'hydrogène (<50 [micro]M), alors que des concentrations plus élevées diminuent l'activité placentaire en diminuant la sécrétion d'hCG, démontrant son action cytotoxique à plus forte dose. Ceci a également été constaté par une augmentation de l'apoptose. Le peroxyde d'hydrogène a aussi démontré ses effets sur la production par les cytotrophoblastes de facteurs pro-inflammatoires, tel le TNF-[alpha], ainsi que de son récepteur, le TNFR2, qui sont tous deux augmentés après traitement. Le peroxyde d'hydrogène semble aussi avoir une influence sur la production de médiateurs vasodilatateurs tel l'oxyde nitrique, dont la diminution est observée in vitro avec des concentrations croissantes d'H[indice inférieur 2]O[indice inférieur 2]. Le stress oxydatif dans la prééclampsie, représenté dans notre modèle in vitro par le peroxyde d'hydrogène, semble donc être central dans la physiopathologie de cette maladie, puisqu'on peut lui attribuer en partie la variation d'importants facteurs. Toutefois, les moyens de la prise en charge de cette condition pathologique doivent être soigneusement déterminés, puisque le peroxyde d'hydrogène paraît avoir tout de même un rôle physiologique important à jouer, d'où l'intérêt de parler d'équilibre oxydatif.

Acid Sphingomyelinase (ASMase) activity has been proposed to mediate LPS signaling in various cell types. This study shows that in macrophages, ASMase is a negative regulator of LPS-induced TNFα secretion. ASMasedeficient (asm-/-) mice and isolated peritoneal macrophages produce several fold more TNFα than their wild-type (asm+/+) counterparts when stimulated with LPS. The mechanism for these differences however is not transcriptional but post-translational. The TNFα converting enzyme (TACE) catalyzes the maturation of the 26kD precursor (proTNFα) to the active 17kD form (sTNFα). In mouse peritoneal macrophages, the activity of TACE rather than the rate of TNFα mRNA synthesis was the rate-limiting factor regulating TNFα production. Substantial portion of the translated proTNFα was not processed to sTNFα; instead it was rapidly internalized and degraded in the lysosomes. TACE activity was 2 to 3 fold higher in asm-/- macrophages as compared to asm+/+ macrophages and was suppressed when cells were treated with exogenous ceramide and SMase. In asm-/- but not in asm+/+macrophages, indirect immunofluorescence experiments revealed distinct TNFα-positive structures in close vicinity of the plasma membrane. Asm-/- cells also had higher number of EEA1-positive early endosomes. Co-localization experiments that involved inhibitors of TACE and/or lysosomal proteolysis suggest that in asm-/-cells a significant portion of proTNFα is sequestered within the early endosomes, and instead of undergoing lysosomal proteolysis it is recycled to the plasma membrane and processed to sTNFα.

Ankylosing spondylitis (AS) is a chronic, inflammatory rheumatic disease that has a highly variable disease course. Three biologic agents, adalimumab, etanercept, and infliximab, have been developed for the treatment of AS. We conducted three studies: 1) an exploratory analysis of a year-long longitudinal dataset to gain insight into the variability of disease activity, physical function, and well-being and to explore the relationship between these outcome measures; 2) a systematic review of the available evidence for the efficacy of biologic treatment; 3) a systematic review of potential adverse effects of this treatment. We found that repeated measures of disease activity, function and well-being fluctuate considerably between patients, with complex patterns occurring over time within patients. There was mostly high quality evidence that these biologics are efficacious against placebo. We did not find evidence of an increase in serious adverse events or serious infections from short-term randomized controlled trials.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The involvement of the pro-inflammatory cytokine, tumour necrosis factor alpha (TNF-α) and the sympathetic nervous system in the development of delayed-onset muscle soreness has not been established. I assessed the effect of etanercept, a TNF- α inhibitor, and atenolol, a β1-receptor antagonist, on DOMS induced in the quadriceps muscle. Thirteen male subjects reported to the exercise laboratory on three separate occasions, 6-15 weeks apart. In a randomised, double-blind cross-over format, I administered etanercept (25mg), atenolol (25mg) or placebo, one hour before the exercise. Subjects then completed four sets of 15 repetitions at 80% of their one repetition maximum (1RM) on a 45° inclined leg press machine. Muscle strength changes were detected by remeasuring the subject’s 1RM 24h, 48h and 72h after the exercise. Sensitivity to pressure of the quadriceps muscle was measured using a pressure algometer before and 24h, 48h and 72h after exercise. The subject’s perception of the pain was measured with the visual analogue scale and McGill Pain Questionnaire. Muscle tumour necrosis factor-alpha concentration was measured before exercise and then 2h and 24h after exercise in four subjects. Muscle strength was impaired 24h and 48h after exercise regardless of agent administered (P < 0.001). At 72h after exercise, muscle strength was significantly improved (P < 0.01) in subjects receiving etanercept and atenolol compared to those receiving placebo. The subject’s were significantly more sensitive to pressure applied to the quadriceps 24h, 48h and 72h after exercise compared to before exercise, regardless of agent administered (P < 0.001). The VAS was elevated significantly at all three time intervals, with no difference after etanercept or atenolol administration compared to that of placebo. There was no significant difference in the muscle TNF-α concentration between any of the time intervals or between subjects receiving placebo and etanercept (P=0.065). The administration of atenolol and etanercept, at the regimen used, had no effect on the soreness associated with DOMS.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that leads to destruction of articular cartilage and subchondral bone at the synovial joints. Clinically, RA is characterized by swelling, tenderness and destruction of synovial joints, which results in severe disability and premature mortality. In the RA disease state, inflammation in the synovial compartment is regulated by a complex cytokine and chemokine network, including tumor necrosis factor α (TNFα), which has been clinically demonstrated a key mediator of RA pathogenesis. TNFα can be found in elevated levels in the synovial fluid and serum of RA patients and the role of the cytokine in both the inflammation and bone destruction of RA suggests it is important in the understanding of disease progression as well as the development of therapeutic targets. Many of the biological processes that mediate RA, including bone turnover and cartilage resorption, involve signaling pathways that are mediated by adenosine and its receptors. The A2B adenosine receptor (A2BAR) is highly expressed in the synoviocytes of RA patients and the receptor has a similar expression profile in humans and mice. The goal of this thesis was to use a mouse model of RA to understand how the A2B adenosine receptor modulates TNFα and other destructive enzymes that contribute to the progression of the disease. A collagen antibody-induced arthritis (CAIA) mouse model was used to determine the effect of A2BAR ablation on systemic and joint-specific TNFα expression. Comparable arthritic conditions were observed in CAIA mice of both A2BAR knockout (KO) and wild-type (WT) genotypes and the absence of the A2BAR gene did not result in any observable differences in the gross arthritic state created in each genotype. Immunohistochemistry analysis of TNFα expression in mouse paws revealed that paw joints from CAIA A2BAR KO mice exhibited more robust TNFα staining compared to CAIA WT specimens of the same treatment duration. ELISA analysis of the serum showed that only CAIA A2BAR KO mice had greater serum production of TNFα at day 10 after induction of arthritis. TNFα and matrix metalloproteinase-9 mRNA expression were also elevated in KO CAIA knee joints in comparison to WT CAIA knee joints; however, WT CAIA mice were found to have higher levels of aggrecanase mRNA compared to KO mice. These results suggest that while the loss of A2BAR activity leads to a hyper-inflammatory state, the A2B adenosine receptor alone is not responsible for the progressive inflammation of the synovial joints associated with rheumatoid arthritis.

Thesis advisor: Thomas C. Chiles / For ten weeks during the summer of 2005, I was a Summer Undergraduate Research Fellow in an immunology laboratory at the Mayo Clinic. My research focused on the BLyS/APRIL system and the receptor TACI on the surface of B cells. Going into my summer research, I had very little experience in immunology. Throughout the process of writing this thesis, I have sought to improve upon my knowledge of immunology, building a cohesive story that begins with basic biology and ends with the results of the summer's experiments. The first part of this thesis covers topics in general immunology, and narrow down in focus to cover the function and development of lymphocytes and B cell maturation and activation. In the second part of the thesis, the background for my research is described in more detail, and topics such as autoimmunity and cancer, lipid rafts, cell polarization, the BLyS/APRIL system for B cell survival, and TACI are covered extensively. The final portion of this thesis discusses the experimental logic, a background on materials and methods, and the results of the experiments I conducted over the summer. By reading this thesis, anyone with a background in biology should become familiar with basic subjects in immunology, advanced concepts in the study of lymphocytes, the ligands BLyS and APRIL, and the receptor TACI in B cells. / Thesis (BS) — Boston College, 2006. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program.

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No. of bitstreams: 3 license_text: 21328 bytes, checksum: 683d9883b2ad62ac3b8bafc566b2e600 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) 2015LucianaKnabbendeOliveiraBeckerDelving.pdf: 1574790 bytes, checksum: b751dea1371239a23970a2767f3789ba (MD5) / Na medicina tradicional, as plantas tem sido utilizadas para o tratamento de diversas doenças ao longo dos séculos e são consideradas uma das maiores fontes para o desenvolvimento de novas drogas. Com o objetivo de contribuir com o conhecimento cientifico no estudo e desenvolvimento de novas drogas procurou-se com este trabalho, identificaros principais constituintes fitoquímicosda Calyptranthes grandifolia, avaliar seus potenciais citotóxicos e antioxidantes e também identificar e avaliar o efeito terapêutico, através de vias específicas e já conhecidas por seu envolvimento no processo inflamatório, como o TNF- α. Para todos os experimentos, foram utilizados extratos etanólico e hexânico das folhas da C.grandifolia, coletada no entorno do município de Lajeado.Foi realizada a análise fitoquímica de esteroides/terpenoides, taninos, flavonoides, cumarinas, quinonas, alcaloides e saponinas, utilizando metodologias descritas na literatura. O potencial citotóxico in vitro foi avaliado pelo método de Alamar Blue, utilizando células não metabolizadoras CHO-K1 e a atividade antioxidante foi realizada pelo método2,2-difenil-1-picrilhidrazila (DPPH). A análise da liberação do TNF-α utilizou células RAW 264.7, tratadas com os extratos vegetais e posterior estimulação com lipopolissacarídeo (LPS). A quantificação de liberação do TNF-α foi feita pelo método de ELISA. Os resultados dos ensaios indicaram a presença de esteroides/terpenoides, taninos e flavonoides no extrato etanólico. O potencial citotóxico, não apresentou citotoxicidade considerável no extrato etanólico e manteve a viabilidade celular aumentada na maioria das concentrações, com uma diminuição não significativa da viabilidade em 200 μg/mL, de forma semelhante para o extrato hexânico a redução da viabilidade celular pode ser observada somente na concentração de 200 μg/mL. A atividade antioxidante esteve presente apenas no extrato etanólico de maneira dose-dependente, com IC50 de 21.3±1.7μg/mL.O extrato etanólico também apresentou melhores resultados em relação à inibição do TNF-α, tendo sido significativa na concentração de 200 μg/mL, frente ao controle positivo (LPS). O extrato hexânico não apresentou inibição da liberação do TNF-α na concentração de 200 μg/mL de forma significativa quando comparada ao LPS. Os resultados deste estudo mostram indícios de atividades anti-inflamatórias nos extratos testados, havendo um melhor desempenho do extrato etanólico. A presença de polifenóis e esteroides/terpenoides encontradas neste extrato podem ser a resposta para uma melhor avaliação das atividades antioxidantes e anti-inflamatórias. Estudos futuros podem indicar um potencial anti-inflamatório promissor para esta planta, lembrando ainda que processos inflamatórios fazem parte do processo de carcinogênese, havendo portanto uma porta de entrada para maiores pesquisas avaliando o potencial anticâncer da C. grandifolia. / In traditional medicine, plants have been used in the treatment of several diseases throughout centuries and are considered as one of the largest sources for the development of new drugs. However, the bioactive components and action mechanisms are not entirely known. Aiming to contribute with scientific knowledge towards the study and development of new drugs, this work sought to identify their main phytochemical components, evaluate their cytotoxic and antioxidant potentials, as well as identifying and evaluating the therapeutic effect of Calyptranthesgrandifolia, through specific and known paths for their involvement in the inflammatory and carcinogenic process, such as TNF- α. Ethanolic and hexanic extracts from C. grandifolialeaves, gathered within the Lajeado municipality, were used in all experiments. The phytochemical analysis of steroids/terpenoids, tannins, flavonoids, coumarins, quinones, alkaloids and saponins were characterized using methodologies described in literature. The in vitro cytotoxic potential was evaluated through the Alamar Blue method using non drug-metabolizing CHOK1 cells and the antioxidant activity was performed through the DPPH method. The analysis of TNF-α release used RAW 264.7 cells, treated with vegetal extracts and posterior stimulation with LPS. The quantification of TNF-α release was quantified using ELISA method. The experiment results indicated the presence of steroids/terpenoids, tannins and flavonoids in the ethanolic extract. The cytotoxic potential did not present considerable cytotoxicity in the ethanolic extract and maintained increased cellular viability in most concentrations, only presenting a slight reduction in viability at 200 μg/mL. Similarly, cellular viability reduction could only be observed in the hexanic extract at the 200 μg/mL concentration. The antioxidant activity was only present in the ethanolic extract at a dose-dependant manner, with an IC50 value of 21.3±1.7μg/mL. The ethanolic extract also presented better results concerning the inhibition of TNF-α, given that it was significant at the 200 μg/mL concentration in comparison with the positive control (LPS). The hexanic extract also presented higher TNF-α inhibition at the 200 μg/mL concentration, however, there was no significant difference in comparison with LPS. The results of this study demonstrate that there are evidences of antiinflammatory activity in the tested extracts, with better performance in the ethanolic extract. The presence of polyphenols and steroids/terpenoids found in this extract may be the answer for its better evaluation concerning antioxidant and anti-inflammatory activities. Future studies may indicate a promising anti-inflammatory potential for this plant, also reminding that inflammatory processes are part of the carcinogenesis process, therefore, this is an entry path for larger researches evaluating the anticarcinogenic potential in C. grandifolia extracts.

Sepsis is defined as potentially fatal systemic inflammation, caused by an infection. It is the leading cause of ICU mortality and the 10th leading cause of death in the United States. Several models exist to mimic this disorder, and have demonstrated differential mortality rates between the models as well as the individual animals. Previous studies have shown that elevated levels of plasma mitochondrial DNA (mtDNA) correlated with mortality in septic patients, and cell-free mitochondrial DNA can elicit toll-like receptor mediated immune responses similar to LPS-mediated septicemia. However, the role of mtDNA in the pathophysiology sepsis is still unknown. The focus of this study was to create sepsis in a mouse model using the murine Cecal Ligation and Puncture (CLP) model, and measure plasma mtDNA levels. After CLP was performed on experimental mice, blood plasma was collected 24 hours later. Elevated amounts of circulating mtDNA were detectable in the plasma using real time PCR and cytochrome B2 as a marker of mitochondria. These data were correlated with plasma IL-6 levels, which were used to predict mortality within 5 days of CLP to stratify mice into two populations of those predicted to live or die following the procedure. We also aimed to investigate the effect of mtDNA and mitochondrial debris on naïve mouse macrophages in an in vitro study of the regulation of inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1β). In order to observe the effects of mtDNA on murine macrophages, mitochondria was purified from mouse liver and used to stimulate these cells alongside positive control, LPS. Stimulation with mtDNA and mitochondrial debris resulted in increased levels of TNF-α mRNA in lysed cells as well as their surrounding media as compared to control cells, as well as increased transcript half life as measured over four hours post stimulation with transcription inhibitor actinomycin D. The increases in mRNA half-life elicited by mtDNA were comparable to those observed after LPS addition. Stimulation also caused increased binding of TNF-α mRNA to the RNA binding protein, AUF1, as measured by immunoprecipitation of RNA-protein complexes and assayed for TNF-α binding by PCR. These results demonstrate that mitochondrial damage-associated molecular patterns regulate TNF-α mRNA expression at the post-transcriptional level through AUF1, an mRNA destabilizing factor. This is a novel mechanism that likely contributes to sepsis pathophysiology, and demonstrates the involvement of the mitochondrial fission and fusion balance and its regulation in the sepsis innate immune response.