Submitted by Amanda Silva (amanda.osilva2@ufpe.br) on 2015-04-08T13:58:16Z No. of bitstreams: 2 TESE_completa_em_PDF.pdf: 1415623 bytes, checksum: 2b87aa820a2d8db28f2ec936e6a6d764 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-04-08T13:58:17Z (GMT). No. of bitstreams: 2 TESE_completa_em_PDF.pdf: 1415623 bytes, checksum: 2b87aa820a2d8db28f2ec936e6a6d764 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012-02-27 / FACEPE / Entre os fármacos que são utilizados na terapêutica do lúpus eritematoso sistêmico (LES) e artrite reumatoide (AR) destacam-se os anti-maláricos como a cloroquina (CQ) e hidroxicloroquina (HCQ) e os glicocorticoides que exercem graves efeitos colaterais. Entre os efeitos biológicos da CQ e da HCQ que são descritos na literatura destaca-se à inibição de um número restrito de citocinas pró-inflamatórias como, por exemplo, IFNg, TNFa e IL-6. No entanto, ainda não está relatado o efeito desses compostos diante das citocinas pró-inflamatórias da via Th17, como IL-17 e IL-22. Também são estudadas as citocinas que inibem a via Th17 como, por exemplo, a IL-27 considerada como antiinflamatória, em alguns estudos. Porém existem poucos trabalhos da relação desta citocina com os níveis séricos em pacientes portadores de doenças autoimunes. A necessidade de novos fármacos anti-inflamatórios com menos efeitos adversos direcionam estudiosos à descoberta de novos compostos. As tiazolidinas-2,4-dionas são substâncias promissoras devido aos efeitos anti-inflamatórios via PPARg. Foram sintetizados dez novos compostos derivados da tiazolidina-2,4-diona para posterior avaliação anti-inflamatória in vitro, em células esplênicas, e in vivo, através do protocolo experimental de air-pouch. Partindo-se da tiazolidina-2,4-diona, os derivados sintetizados foram obtidos por reações de N-alquilação, na posição 3 da tiazolidina-2,4- diona, e por reações de Michael, na posição 5 do núcleo. As estruturas químicas dos compostos sintetizados foram devidamente comprovadas por RMN 1H, IV e EM. Os resultados da avaliação anti-inflamatória in vitro demonstraram que os compostos 3-(2- bromo-benzil)-5-(3-bromo-6-metóxi-benzilideno)-tiazoldina-2,4-diona (LPSF/GQ- 113B), 3-(2-bromo-benzil)-5-(4-metóxi-benzilideno)-tiazolidina-2,4-diona (LPSF/GQ- 92) e 3-(3-flúor-benzil)-5-(4-metil-sulfonil-benzilideno)-tiazoldina-2,4-diona (LPSF/GQ- 57) apresentaram atividade de inibição da síntese das citocinas pró-inflamatórias IFN-g e IL-17 em células esplênicas provenientes de camundongos BALB/c. Na avaliação da atividade anti-inflamatória in vivo, no modelo de air-pouch induzido por carragenina, os mesmos compostos apresentaram percentuais de inibição da migração celular superior a 50%, na dose de 3 mg/kg. Os compostos mencionados possuem ação inibitória da resposta inflamatória induzida por carragenina revelando a potencial ação antiinflamatória dos derivados das tiazolidinas-2,4-dionas 3,5-dissubstituídos. Em paralelo, o efeito da CQ e da HCQ foi avaliado em sobrenadantes de cultura de células esplênicas, em PBMCs de voluntários sadios e, em pacientes com LES ou AR. Os resultados indicaram que houve inibição da síntese de IL-17, em esplenócitos, assim como de IL-17 e IL-22 em PBMCs de voluntários sadios e de pacientes com LES ou AR. Também foi avaliada a correlação dos níveis de IL-27 em soro de pacientes portadores de LES com a atividade da doença (SLEDAI) e nefrite. Os resultados demonstraram que há níveis reduzidos de IL-27 nos pacientes com LES quando comparados ao grupo controle. No entanto, os resultados da correlação com SLEDAI e nefrite não foram significativos, quando comparados ao controle.

Made available in DSpace on 2014-06-12T15:55:53Z (GMT). No. of bitstreams: 2 arquivo9638_1.pdf: 1415158 bytes, checksum: 99d72178b3b8199614795e307f81954f (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2012 / Entre os fármacos que são utilizados na terapêutica do lúpus eritematoso sistêmico (LES) e artrite reumatoide (AR) destacam-se os anti-maláricos como a cloroquina (CQ) e hidroxicloroquina (HCQ) e os glicocorticoides que exercem graves efeitos colaterais. Entre os efeitos biológicos da CQ e da HCQ que são descritos na literatura destaca-se à inibição de um número restrito de citocinas pró-inflamatórias como, por exemplo, IFNg, TNFa e IL-6. No entanto, ainda não está relatado o efeito desses compostos diante das citocinas pró-inflamatórias da via Th17, como IL-17 e IL-22. Também são estudadas as citocinas que inibem a via Th17 como, por exemplo, a IL-27 considerada como antiinflamatória, em alguns estudos. Porém existem poucos trabalhos da relação desta citocina com os níveis séricos em pacientes portadores de doenças autoimunes. A necessidade de novos fármacos anti-inflamatórios com menos efeitos adversos direcionam estudiosos à descoberta de novos compostos. As tiazolidinas-2,4-dionas são substâncias promissoras devido aos efeitos anti-inflamatórios via PPARg. Foram sintetizados dez novos compostos derivados da tiazolidina-2,4-diona para posterior avaliação anti-inflamatória in vitro, em células esplênicas, e in vivo, através do protocolo experimental de air-pouch. Partindo-se da tiazolidina-2,4-diona, os derivados sintetizados foram obtidos por reações de N-alquilação, na posição 3 da tiazolidina-2,4- diona, e por reações de Michael, na posição 5 do núcleo. As estruturas químicas dos compostos sintetizados foram devidamente comprovadas por RMN 1H, IV e EM. Os resultados da avaliação anti-inflamatória in vitro demonstraram que os compostos 3-(2- bromo-benzil)-5-(3-bromo-6-metóxi-benzilideno)-tiazoldina-2,4-diona (LPSF/GQ- 113B), 3-(2-bromo-benzil)-5-(4-metóxi-benzilideno)-tiazolidina-2,4-diona (LPSF/GQ- 92) e 3-(3-flúor-benzil)-5-(4-metil-sulfonil-benzilideno)-tiazoldina-2,4-diona (LPSF/GQ- 57) apresentaram atividade de inibição da síntese das citocinas pró-inflamatórias IFN-g e IL-17 em células esplênicas provenientes de camundongos BALB/c. Na avaliação da atividade anti-inflamatória in vivo, no modelo de air-pouch induzido por carragenina, os mesmos compostos apresentaram percentuais de inibição da migração celular superior a 50%, na dose de 3 mg/kg. Os compostos mencionados possuem ação inibitória da resposta inflamatória induzida por carragenina revelando a potencial ação antiinflamatória dos derivados das tiazolidinas-2,4-dionas 3,5-dissubstituídos. Em paralelo, o efeito da CQ e da HCQ foi avaliado em sobrenadantes de cultura de células esplênicas, em PBMCs de voluntários sadios e, em pacientes com LES ou AR. Os resultados indicaram que houve inibição da síntese de IL-17, em esplenócitos, assim como de IL-17 e IL-22 em PBMCs de voluntários sadios e de pacientes com LES ou AR. Também foi avaliada a correlação dos níveis de IL-27 em soro de pacientes portadores de LES com a atividade da doença (SLEDAI) e nefrite. Os resultados demonstraram que há níveis reduzidos de IL-27 nos pacientes com LES quando comparados ao grupo controle. No entanto, os resultados da correlação com SLEDAI e nefrite não foram significativos, quando comparados ao controle

In recent years, the subsets of mammalian CD4+ T cells and their repertoire of effector cytokines has expanded beyond the original Th1/Th2 paradigm, to include natural (n) and inducible (i) regulatory T cells (Treg), Th17, Th9, Th22 and follicular T helper (Tfh) cells. Whilst Th1, Th2 and nTreg immune responses have been described in the chicken, the existence of other Th cell subsets is yet to be determined. To investigate Th17 immune responses in the chicken, the mammalian components of these responses currently unannotated in the chicken genome, IL-23 p19 and the IL-23R, were identified and cDNAs cloned. A chicken IL-23 flexiconstruct, containing IL-23 p19 and p40 joined by a linker, was designed. Recombinant chicken IL-23 protein (rchIL-23) was expressed and purified. Bioactivity of rchIL-23 was demonstrated by increased mRNA expression of chIL- 17F and chIL-22 in rchIL-23-stimulated splenocytes. Monoclonal antibodies which identify chIL-12/chIL-23 p40 also recognised purified rchIL-23. Further, chIL-23 p19 mRNA levels were measured and detected in a wide range of tissues but was not up-regulated in stimulated splenocytes, thymocytes or bursal cells. Messenger RNA (mRNA) expression levels of Th17 cytokines (chIL-17A, chIL-17F, chIL-21, chIL-22 and chIL-23) were measured in a chicken tissue panel, in stimulated splenocytes, thymocytes and bursal cells, as well as during infections previously described as initiating typical Th1 or Th2 adaptive immune responses in the chicken. Chicken IL-17A mRNA expression levels were up-regulated in susceptible chickens during infection with Marek’s disease virus (a disease which typically drives a Th1 immune response), but were down-regulated in resistant birds. Chicken CD4+ T cells were sorted by fluorescence-activated cell sorting (FACS) and recombinant Th17-associated cytokines used to attempt to drive the cells towards a Th17 phenotype, as measured by expression of mRNA for chIL-17A and chIL-23R. The sorted chicken CD4+ cells failed to proliferate or respond to Th17 cytokine stimulation. ChIL-23R was also correctly identified and cloned as cDNA, and its mRNA expression measured in a panel of unstimulated and stimulated tissues and cells. The chIL-23R mRNA levels were detected in a wide range of tissues as well as stimulated splenocytes, thymocytes and bursal cells. Future work would seek to positively identify Th17 cells in the chicken and determine the role of Th17 immune responses against avian diseases.

Les hypothèses retraçant les mécanismes physiopathologiques de la maladie de Behçet (MB), une vascularite inflammatoire non liée aux anticorps anti-cytoplasme des polynucléaires neutrophiles (ANCA), sont multiples. Cette étude propose une compilation exhaustive des mécanismes immunopathologiques décrits dans la littérature contemporaine, et fournit un résumé détaillé des aspects cliniques de la maladie et de ses différents traitements. Cette étude inclut également une analyse statistique de 20 signatures de protéines proposées comme biomarqueurs potentiels de la MB. Vingt-deux patients avec une MB active (MBA) et 46 patients avec une MB inactive (MBI), répondant aux critères de l’International Criteria for Behçet Disease (2013), ainsi que 47 donneurs sains (DR) et 98 patients subissant une angiographie coronaire (AC) ont fourni des échantillons de sérums pour une étude de dosage multiplex. Les résultats indiquent que les protéines sériques ICAM-1, SAA, THBD, et VCAM-1 jouent un rôle essentiel dans la différenciation entre les patients MB et les DR. De même, Caldesmon, Clusterin, CRP, IL-8, SELP et SICMA3 permettent un tri entre les patients MB et AC. Les modèles de signatures des biomarqueurs proposés dans cette étude et qui séparent entre les patients atteints par la MB, les DR et / ou les AC, représentent une nouvelle piste pour le développement de tests sériques pour la MB, avec une sensibilité et une spécificité élevées. Ceci peut éventuellement compléter les outils de diagnostic clinique établis. Ces résultats apportent une contribution significative à l’interprétation actuelle de la pathogénie de la MB en tant que vascularite auto-immune non-ANCA. Cette enquête fournit un bilan à la fois qualitatif et quantitatif aux cliniciens et aux chercheurs dans ce domaine. / Hypotheses concerning the specific pathophysiological mechanisms of Behçet’s Disease (BD), a non-antineutrophil cytoplasmic antibodies (ANCA) inflammatory vasculitis, are numerous. This study offers an exhaustive review of the disease in an attempt to recap the immunopathological pathways described by extant literature, and provides a detailed summary of the clinical aspects of, and treatment options for the disease. In addition, this investigation completed a statistical analysis of 20 protein signatures that were proposed as potential biomarkers for BD. Twenty-two patients with active BD (BDA) and 46 patients with inactive BD (BDI) fulfilling the International Criteria for Behçet's Disease, 47 healthy donors (HD), and 98 coronary angiography patients (CA) provided serum samples for a multiplex assay study. Findings indicate that serum proteins ICAM-1, SAA, THBD, and VCAM-1 play a significant role in differentiating BD patients from HD. Likewise, Caldesmon, Clusterin, CRP, IL-8, SELP, and sICAM-3 segregate between BD and CA. The biomarker predictive models proposed in this study that segregate between BD, HD, and / or CA represent a significant avenue for the development of sera testing specific to BD with a high level of sensitivity and specificity. This may serve as a supplement to established clinical diagnostic tools. These results represent a noteworthy complement to the current interpretation of the pathogenesis of BD as an autoimmune non-ANCA vasculitis. This investigation provides expert clinicians and researchers with both qualitative and quantitative outcomes.

The Interleukin (IL)-17 cytokine family, expressed by T helper (Th)17 cells and γδ T cells, plays pivotal roles in adaptive immune responses. They have been implicated in autoimmune and allergic diseases as well having roles in bacterial and fungal clearance. Importantly, IL-17 producing γδ T cells have been shown to be critical for the development of adaptive Th17 responses in the murine model of multiple sclerosis, experimental autoimmune encephalomyelitis. Interestingly, natural ligands of the aryl hydrocarbon receptor (AhR) are known to influence the development of Th17 cells. It has been shown previously that prolonged topical exposure of mice to the contact allergen 2-4 dinitrochlorobenzene (DNCB) or to the respiratory sensitiser trimellitic anhydride (TMA) causes the preferential development of a preferential T helper (Th)1 or Th2 response, respectively. The presence of the novel IL-17 family cytokines and their cellular source was investigated following both single and prolonged exposure to allergen. Exposure only to the contact allergen DNCB resulted in up-regulation of the expression of IL-17 by dermal γδ T cells. It was shown also that topical application of a range of contact allergens and of the respiratory allergen TMA resulted in IL-17 expression by γδ T cells in the lymph node draining the site of exposure. However, differential kinetics were observed between the two classes of allergens. Exposure to the contact allergens resulted in rapid expression of IL-17 within 6-16 h, whereas the respiratory allergen displayed considerably delayed kinetics, with maximal levels detected 48 h post exposure. Treatment with DNCB only was shown to be associated with the development of Th17 cells following prolonged exposure to chemical allergen. Thus DNCB provoked a Tc1/Th1/Th17 profile, in contrast prolonged exposure to TMA resulted in a very selective Th2 cytokine pattern. The influence of γδ T cells on polarised responses to chemical allergens was investigated also using γδ T cell knockout mice; here the adaptive Th17 response induced by DNCB was completely abrogated. These data demonstrate that the absence of IL-17 production by γδ T cells during the early innate immune response affects the subsequent adaptive Th17 response stimulated by chemical contact allergens. Finally, the importance of the affinity of the AhR for endogenous ligands during in vitro Th17 polarisation was assessed. Using three strains of mice with differential AhR affinities the contribution of ligation of these receptors in Th17 cell development was investigated. In all three strains AhR ligation was required for optimal polarisation of Th17 cells, even in strains that are reported to express a low affinity receptor. These data suggest that across a range of receptor affinities, including low affinity receptors analogous to that of humans, endogenous AhR ligands may play a major role in driving Th17 cell differentiation, regardless of receptor phenotype.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22150号 / 医博第4541号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 稲垣 暢也, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Tim-3 has been linked to several inflammatory diseases by regulation on both adaptive and innate immunities. Here, we assessed the augmented expression of Tim-3 in brain tissue of ischemia-reperfusion mice and PBMCs of ischemic stroke (IS) patients. The augmented expression of Tim-3 significantly correlated with abnormal lipid levels. In vitro studies showed that plasma from ischemic stroke patients induced Tim-3 expression in THP- 1 cells. More importantly, our results revealed a significant correlation of Tim-3 expression on CD4 + T cells with systemic IL-17 in patients with ischemic stroke. Consistently, we also found a positive correlation of Tim-3 expression on CD14 + monocytes and serum TNF-a in IS patients. Collectively, augmented expression of Tim-3 may play an important role in the pathogenesis of ischemic stroke by regulation of proinflammatory cytokines. Further studies will give us new insights on the pathogenesis of ischemic stroke and potentially provide a new target at the medical therapy.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21634号 / 医博第4440号 / 新制||医||1034(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 三森 経世, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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