Orientador: Carlos Amilcar Parada / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T09:59:35Z (GMT). No. of bitstreams: 1 Vieira_AndreSchwambach_D.pdf: 871138 bytes, checksum: fc1ecaa5a27015a5ae9ed72d9687b540 (MD5) Previous issue date: 2012 / Resumo: A hiperalgesia inflamatória resulta da sensibilização de nociceptores aferentes periféricos induzida por mediadores inflamatórios. A prostaglandina E2 (PGE2) é uma das principais moléculas sinalizadoras envolvidas na hiperalgesia, sendo capaz de agir diretamente em nociceptores, induzindo mudanças nas propriedades de transdução sensorial destas células. A Janus Quinase 2 (JAK2) é uma molécula sinalizadora intracelular geralmente associada ao mecanismo de ação de citocinas, sendo que sua atividade pode ser induzida em nociceptores após uma inflamação periférica. Entretanto, não existem evidencias do envolvimento direto da JAK2 na sensibilização de nociceptores mediada pela PGE2. Assim o objetivo deste trabalho foi de explorar o possível papel da JAK2 na sensibilização mediada pela PGE2. Em neurônios do gânglio da raiz dorsal (DRG) em cultura foi observado que a PGE2 altera o influxo de cálcio induzido pela capsaicina, e a pré-incubação das células com o inibidor seletivo da JAK2, AG490, foi capaz de bloquear este efeito. Adicionalmente, a administração intratecal de AG490 em ratos reduziu a hiperalgesia induzida pela administração subcutânea e local de PGE2 ou carragenina. A administração intratecal de AG490 também bloqueou a ativação da PKCepsilon induzida no DRG L5 ispsilateral após inflamação na pata. Em conclusão o presente trabalho demonstra que a JAK2 expressa no DRG pode possuir um papel na sensibilização de nociceptores induzida por um evento inflamatório periférico. Desta forma a inibição da JAK2 pode ser um novo alvo farmacológico para o controle da hiperalgesia inflamatória / Abstract: Inflammatory hyperalgesia results from the sensitization of peripheral afferent nociceptors by inflammatory mediators. Prostaglandin E2 (PGE2) is one of the major signaling molecules involved in hyperalgesia, being able to act directly on nociceptors, inducing sensitization. The Janus Kinase 2 (JAK2) is an intracellular signaling molecule generally associated with cytokines signaling pathway, and its activity can be increased in nociceptors after peripheral inflammation. However, there are no evidences about the role JAK2 directly plays in PGE2-induced sensitization of nociceptors. Therefore, the aim of the present study was to explore a possible role for JAK2 in PGE2 mediated sensitization. The data of this study showed in cultured dorsal root ganglion (DRG) neurons, that the administration of PGE2 alters capsaicin-induced calcium transients, and the pre-incubation of the cells with the JAK2 selective inhibitor AG490 blocks this effect. In addition, the intrathecal administration of AG490 in rats reduces the hyperalgesia induced by local administration of PGE2 or carrageenan in the hindpaw. AG490 intrathecal administration also blocks PKCepsilon activation in the ipsilateral L5 DRG induced by inflammation of peripheral tissue. In conclusion, the present study indicates that the JAK2 expressed in the DRG may have a role in the sensitization of nociceptors by a peripheral inflammatory event. Moreover, the inhibition of JAK2 may be a possible novel pharmacological target for the control of the inflammatory hyperalgesia / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

L'endométriose est une maladie gynécologique touchant les femmes en âge de procréer caractérisée par la présence de tissu endométrial ectopique, c'est-à-dire en dehors de la cavité utérine. Les femmes atteintes d'endométriose peuvent présenter divers symptômes parmi lesquels des douleurs pelviennes et de l'infertilité. Des dysfonctions du système immunitaire sont de plus en plus souvent suspectées comme un des éléments responsables de la pathogenèse de cette maladie ainsi que de l'infertilité associée. L'objectif général de ce projet est d'étudier l'implication dans l'infertilité associée à l'endométriose de deux molécules pro-inflammatoires aux propriétés variées et à l'expression anormalement élevée dans cette pathologie, MIF et PGE2. Nos résultats montrent que MIF favoriserait la capacitation des spermatozoïdes dans des conditions physiologiques mais pourrait également affecter leur fonction lorsqu'il se retrouve à des niveaux anormalement élevés comme lors de l'endométriose. Ceci suggère un rôle direct possible de MIF dans l'infertilité associée à l'endométriose. La stimulation des cellules ectopiques par MIF entraîne une libération accrue de PGE2, ceci via l'induction de COX-2. De plus, les MAPKs jouent un rôle dans ce phénomène. Par son effet inducteur sur la libération de PGE2, MlF pourrait favoriser l'aspect inflammatoire et le développement ectopique des lésions d'endométriose. L'effet de MlF sur la libération de PGE2 par les cellules endométriales ectopiques pourrait également expliquer les quantités élevées de cette prostaglandine dans le liquide péritonéal des femmes atteintes d'endométriose, un phénomène suspecté dans l'infertilité associée à cette maladie. En plus d'être un médiateur de l'inflammation, PGE2 est impliquée à de nombreux niveaux dans la reproduction. Finalement, nos travaux ont porté sur les enzymes de synthèse de PGE2 dans l'endomètre eutopique des femmes normales et l'endomètre eutopique et ectopique des femmes atteintes d'endométriose. Selon nos données, l'expression de certaines de ces enzymes est perturbée durant cette maladie, ce qui peut avoir des effets délétères sur la physiologie de la reproduction. L'endométriose et ses symptômes sont des phénomènes complexes ayant probablement plus qu'une seule origine. Parmi les nombreux facteurs à l'expression altérée dans l'endométriose, notre étude montre que MIF et PGE2 pourraient être de ceux jouant un rôle dans l'infertilité reliée à cette maladie.

Yu, Le. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 171-198). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Hormonal-mediated uterine quiescence involves the maintenance of a decreased inflammatory responsiveness. However, no study has investigated whether labor induction with prostanoids is associated with changes in maternal serum hormones. The objective of this study was to determine whether changes in circulating levels of progesterone, estradiol and estriol from admission to delivery are associated with successful labor induction with dinoprostone. A cohort of 81 pregnant women at term was followed from admission to birth until delivery, during the period of 2010-2011. The study was performed at the Hospital of the Federal University of Santa Maria, a tertiary care hospital. Unselected subjects were recruited and blood samples were obtained at admission and immediately before delivery. Sixteen patients had vaginal delivery after spontaneous labor, 12 required emergency cesarean after spontaneous labor and 16 underwent elective cesarean. Thirty-seven patients had labor induction with dinoprostone. Eligible patients received a vaginal insert of dinoprostone (10 mg), and patients were followed up until delivery. Progesterone (P4), estradiol (E2) and estriol (E3) plasma level and P4/E2, P4/E3 and E3/E2 ratio changes were observed from admission to immediately before birth, and the association of these measures with the resulting clinical classification outcome (route of delivery and induction responsiveness) were assessed. Progesterone plasma level decreased from admission to delivery in patients who underwent successful labor induction with dinoprostone [vaginal and cesarean delivery after induced labor: 23% (P<0.001) and 18% (P<0.025) decrease, respectively], but not in those whose induction failed (6.4% decrease, P>0.05). Estriol and estradiol levels did not differ between groups. Successful dinoprostone-induced labor was associated with maternal progesterone level decrease along time. While a causal relationship between progesterone decrease and effective dinoprostone-induced labor can not be established, it is tempting to propose that it may contribute for progesterone withdrawal and favor labor induction in humans. / A quiescência uterina mediada por hormônios envolve a manutenção de uma responsividade inflamatória reduzida. Contudo, nenhum estudo investigou se a indução do parto com prostanóides está associada com alterações em hormônios séricos maternos. Os objetivos deste estudo foram determinar se as alterações nos níveis circulantes de progesterona, estradiol e estriol desde a admissão até o parto estão associados à indução bem sucedida do parto com dinoprostona. Uma coorte de 81 mulheres grávidas a termo foi acompanhada desde a admissão até o parto, durante o período de 2010-2011. O estudo foi realizado no Hospital da Universidade Federal de Santa Maria, um hospital de cuidados terciários. Indivíduos não selecionados foram recrutados e amostras de sangue foram obtidas na admissão e imediatamente antes do nascimento. Dezesseis pacientes tiveram parto vaginal após trabalho de parto espontâneo, 12 necessitaram a realização de cesariana de emergência após trabalho de parto espontâneo e 16 foram submetidas à cesárea eletiva. Trinta e sete pacientes tiveram indução de trabalho de parto com dinoprostona. As pacientes elegíveis receberam um pessário de inserção vaginal de dinoprostona (10 mg), e foram acompanhadas até o parto. Os níveis plasmáticos de progesterona (P4), estradiol (E2) e estriol (E3) e as relações P4/E2, P4/E3 e E3/E2 foram observadas da admissão até imediatamente antes do nascimento, e a associação destas medidas com a classificação clínica resultante foi avaliada (via de parto e resposta à indução). Os níveis plasmáticos de progesterona diminuíram desde a admissão até o nascimento em pacientes que responderam à indução com dinoprostona [parto vaginal e cesáreo após trabalho de parto induzido: redução de 23% (P<0.001) e 18% (P<0.025), respectivamente], mas não nos quais a indução falhou (redução de 6.4%, P>0.05). Os níveis de estriol e estradiol, e as relações P4/E2, P4/E3 e E3/E2 não foram diferentes entre os grupos. O sucesso da resposta à indução de parto com dinoprostona esteve associado com a redução no nível de progesterona materna ao longo do tempo. Enquanto uma relação causal entre a redução na progesterona e o trabalho de parto efetivo induzido pela dinoprostona não pode ser estabelecida, é tentador propor que possa contribuir para a retirada da progesterona e favorecer a indução do parto em humanos.

環氧合酶-2(COX-2)是在花生四烯酸(AA)轉化為前列腺素H₂(PGH₂)的過程中最重要的限速酶，PGH2再進一步被合成為各種前列腺素，包括前列腺素E₂(PGE₂), 因此，COX-2在前列腺素的合成中起著舉足輕重的作用。COX-2在受到例如感染和炎症等刺激的情況下被誘導，迅速大量地產生。越來越多的證據證明瞭COX-2在許多細胞反應和病理生理過程中起重要作用, 其中, 對COX-2在炎症中的作用研究最深入。 / 囊性纖維化病(CF)是一種由於編碼囊性纖維化跨膜轉導調節器(CFTR)基因的突變所引起的常染色體隱性遺傳疾病。CFTR是在上皮細胞中廣泛表達的環磷酸腺苷(cAMP)依賴的陰離子通道。愈來愈多的證據顯示, CF的呼吸道上皮處於過量炎症因子和前列腺素的微環境中, 最終導致了在CF肺部病變中觀察到的超炎症反應. 但其中的機制仍未闡明. 本研究觀察到, 相對於野生型人類支氣管上皮細胞系(16HBE14o-), CF的人類支氣管上皮細胞系(CFBE41o-)中NFκB的活化, COX-2的表達和PGE₂的產量增加. 此外, CFTR基因敲除小鼠顯示出升高的NFκB活性和COX-2表達水準, 提示CFTR基因的缺失介導了超炎症反應的信號. 我們還驗證了一條PKA和CREB參與介導的PGE₂產生的正回饋通路. 更重要的是, 在CFBE41o-細胞中過表達CFTR顯著地抑制了COX-2的表達. 用LPS或者PGE₂處理16HBE14o-細胞導致了野生型CFTR表達的顯著升高. 這些實驗結果提示了CFTR可能參與對COX-2/PGE₂的負調節. 因此, CFTR負調節PGE₂介導的炎症反應. 這個調節機制的缺陷可能導致在CF炎症反應的組織中觀察到的過量的NFκB活化和過量PGE₂產生. / 我們證實了睾丸中也存在這條CFTR負調節COX-2/PGE₂的通路. 由於隱睾處於比陰囊溫度高的腹腔中, 在隱睾中, 我們觀察到了高溫導致的CFTR下調,伴隨著COX-2的上調以及緊密連接蛋白(ZO-1, occludin)的下調. 這種CFTR和COX-2的負相關在小鼠睾丸高熱動物模型以及CFTR基因敲除小鼠模型中也被證實. 為了模擬隱睾的病理狀況, 我們提高原代睾丸支援細胞的培養溫度至37°C. 與在32°C培養條件下的對照細胞相比, 37C培養的支持細胞中CFTR表達顯著下調, 而COX-2表達顯著上調. 用CFTR的抑制劑CFTRinh-172處理支持細胞48小時後, COX-2的表達也上升了. 抑制或者敲除支持細胞中的CFTR都引起了ZO-1和occludin表達水準的下降, 從而損傷了支持細胞間的緊密連接. NFκB或者PGE₂的抑制劑都能逆轉ZO-1和occludin表達水準的下降. PGE₂同樣導致了支援細胞間緊密連接的損傷. 以上結果提示CFTR對緊密連接的調節作用是通過NFκB/COX-2/PGE₂通路實現的. 本研究闡明了在支持細胞中, CFTR通過負調節NFκB/COX-2/PGE₂通路調節緊密連接, 從而參與了隱睾導致的生精障礙的病理過程. / 總之, 本研究論證了CFTR/COX-2/PGE₂通路在CF呼吸道的超炎症反應以及隱睾導致的生精障礙兩個病理過程中的作用, 說明了CFTR在呼吸系統和男性生殖系統中維持細胞因子穩態的重要作用. CF肺中CFTR的缺失或者隱睾病中CFTR表達水準的下降可能導致了呼吸道中過剩炎症反應和生精障礙. / Cyclooxygenase-2 (COX-2) is a pivotal rate-limiting enzyme responsible for the production of prostaglandins by converting arachidonic acid (AA) to prostaglandin H₂ (PGH₂), which is further metabolized to various prostaglandins, including PGE₂. COX-2 is inducible and increases dramatically upon stimulation, such as infection and inflammation. Accumulating evidences have demonstrated the important role of COX-2 in many cellular responses and pathophysiological processes, especially inflammation. / Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a cAMP-dependent anion channel expressed in many epithelia. Accumulating evidence suggests that CF airway epithelia are overwhelmed by excessive inflammatory cytokines and prostaglandins (PGs), which eventually lead to the over-inflammatory condition observed in CF lung disease. However, the exact underlying mechanism remains elusive. In this study, we observed increased COX-2 expression and over-production of prostaglandin E₂ (PGE₂) in human CF bronchial epithelial cell line (CFBE41o-) with elevated NFκB activity compared to a wild-type bronchial epithelial cell line (16HBE14o-). Moreover, we demonstrated that CFTR knockout mice had inherently higher levels of COX-2 and NFκB activity, supporting the notion that lack of CFTR results in hyper-inflammatory signaling. In addition, we identified a positive feedback loop for production of PGE₂ involving PKA and transcription factor, CREB. More importantly, overexpression of wild-type CFTR significantly suppressed COX-2 expression in CFBE41o- cells, and wild-type CFTR protein expression was significantly increased when 16HBE14o- cells were challenged with LPS as well as PGE₂, indicating possible involvement of CFTR in the negative regulation of COX-2/PGE₂. These results suggest that CFTR is a negative regulator of PGE₂-mediated inflammatory response, defect of which may result in excessive activation of NFκB, leading to over production of PGE2 as seen in inflammatory CF tissues. / This negative regulation of COX-2/PGE₂ pathway by CFTR was also identified in the testis in the present study. Downregulation of CFTR accompanied by upregulation of COX-2/PGE₂ and downregulation of tight junction proteins, including ZO-1 and occludin, were observed in a cryptorchidism mouse model with elevated testis in the abdomen, at which the temperature is several degrees higher than that in the scrotum. The inverse correlation of CFTR and COX-2 was further confirmed in a mouse testis hyperthermia model and in CF mice. Culturing primary Sertoli cells at a temperature of 37°C, which mimics the pathological condition of cryptorchidism, led to a significant decrease in CFTR and increase in COX-2 expression compared to the physiological condition of 32°C. Increase of COX-2 expression was also detected 48 hours after administrating CFTRinh-172 to the cells. Inhibition or knockdown of CFTR led to decreased ZO-1 and occludin expression and impaired tight junction in Sertoli cells, which could be mimicked by PGE₂, but reversed by NFκB and COX-2 inhibitors, suggesting that regulation of tight junction by CFTR is mediated by NFκB /COX-2/PGE₂ pathway. This study illustrates that CFTR may be involved in regulating testicular tight junctions through its negative regulation of NFκB/COX-2/PGE₂ pathway in Sertoli cells, defect of which may result in spermatogenesis defect in cryptorchidism. / Taken together, the present study has demonstrated the role of CFTR/ NFκB /COX-2/PGE₂ pathway in two pathological processes, exaggerated inflammation in CF airway and defective spermatogenesis in cryptorchidism, indicating that CFTR is critical for maintaining cytokine homeostasis in respiratory system and male reproductive system. Defect of CFTR in CF lung and downregulation of CFTR in cryptorchidism may contribute to the excessive lung inflammation and impaired spermatogenesis respectively. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Jing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 109-121). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENT --- p.vi / LIST OF PUBLICATIONS --- p.vii / ABBREVIATIONS --- p.xii / LIST OF FIGURES AND TABLES --- p.xvi / Chapter 1 --- Chpter 1: Overview --- p.1 / Chapter 1.1 --- CFTR and Cystic Fibrosis --- p.1 / Chapter 1.1.1 --- Cystic Fibrosis --- p.1 / Chapter 1.1.2 --- Structure of CFTR --- p.2 / Chapter 1.1.3 --- Mutations of CFTR --- p.2 / Chapter 1.1.4 --- Channel and signal transduction function of CFTR --- p.3 / Chapter 1.1.5 --- Interaction of CFTR with other proteins --- p.4 / Chapter 1.1.6 --- Regulation of CFTR --- p.5 / Chapter 1.2 --- COX-2 and PGE₂ --- p.6 / Chapter 1.2.1 --- Biosynthesis of PGE₂ --- p.6 / Chapter 1.2.2 --- Pathophysiologic roles of COX-2 and PGE₂ --- p.7 / Chapter 1.2.3 --- Role of COX-2/PGE₂ in inflammation --- p.7 / Chapter 1.2.4 --- Regulation of COX-2 --- p.8 / Chapter 1.2.4.1 --- Regulation of COX-2 by NF-κB --- p.9 / Chapter 1.2.4.2 --- Regulation of COX-2 by CREB --- p.10 / Chapter 1.3 --- Link between CFTR and NF-κB --- p.11 / Chapter 1.4 --- General hypothesis and aims of study --- p.12 / Chapter 2 --- Chapter 2: CFTR negatively regulates COX-2/PGE₂ positive loop in feedback loop in inflammation --- p.13 / Chapter 2.1 --- Introduction --- p.13 / Chapter 2.1.1 --- Airway inflammation in Cystic Fibrosis --- p.13 / Chapter 2.1.2 --- Current theories on the causes of pulmonary inflammation in CF --- p.13 / Chapter 2.1.2.1 --- Theory one --- p.14 / Chapter 2.1.2.2 --- Theory two --- p.16 / Chapter 2.1.3 --- Role of airway epithelia in CF airway inflammation --- p.16 / Chapter 2.1.4 --- Link between CFTR and NF-κB in pulmonary inflammation --- p.17 / Chapter 2.1.5 --- Link between CFTR and COX-2/PGE₂ in pulmonary inflammation --- p.18 / Chapter 2.1.6 --- Hypothesis and aims of study --- p.18 / Chapter 2.2 --- Materials and methods --- p.20 / Chapter 2.2.1 --- Cell culture materials --- p.20 / Chapter 2.2.2 --- Animals --- p.20 / Chapter 2.2.3 --- Chemicals, drugs and assay kits --- p.20 / Chapter 2.2.4 --- Antibodies --- p.22 / Chapter 2.2.5 --- Cell culture. --- p.22 / Chapter 2.2.6 --- Animal models and procedures --- p.23 / Chapter 2.2.7 --- Manipulation of RNA and QRT-PCR --- p.23 / Chapter 2.2.8 --- Manipulation of protein and Western blot --- p.25 / Chapter 2.2.9 --- Histological and morphological --- p.27 / Chapter 2.2.9.1 --- Tissue section. --- p.28 / Chapter 2.2.9.2 --- Hematoxylin and eosin staining --- p.28 / Chapter 2.2.9.3 --- Immunohistochemistry --- p.28 / Chapter 2.2.10 --- PGE₂ EIA --- p.29 / Chapter 2.2.11 --- Statistical analysis --- p.30 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- Increased expression of NF-κB and COX-2 in the lung of CF mice --- p.31 / Chapter 2.3.2 --- Defect of CFTR leads to increased COX-2 expression in CF cell line --- p.31 / Chapter 2.3.3 --- Increased expression of COX-2 in CF cells is attributed to NF-κB activation --- p.33 / Chapter 2.3.4 --- A positive feedback loop from PGE₂ to COX-2 is mediated by PGE₂/cAMP/PKA/p-CREB pathway --- p.34 / Chapter 2.3.5 --- PGE₂ increase the expression of CFTR protein in 16HBE14o- but not in CFBE41o- cells --- p.35 / Chapter 2.4 --- Discussion --- p.47 / Chapter 2.5 --- Conclusion --- p.51 / Chapter 3 --- Chapter 3: Role of CFTR/COX-2/PGE₂ Pathway in the Regulation of Junctional Complex Proteins in Sertoli Cells and its Implication in Spermatogenesis Defect in Cryptorchidism --- p.53 / Chapter 3.1 --- Introduction --- p.53 / Chapter 3.1.1 --- Spermatogenesis.p53 / Chapter 3.1.1.1 --- Structure of the seminiferous tubules --- p.53 / Chapter 3.1.1.2 --- Role of Sertoli cells in spermatogenesis --- p.55 / Chapter 3.1.1.3 --- Role of junctional complexes in spermatogenesis --- p.55 / Chapter 3.1.2 --- Junctional complexes in the testis --- p.59 / Chapter 3.1.2.1 --- Tight Junction --- p.59 / Chapter 3.1.2.2 --- Anchoring Junction. --- p.60 / Chapter 3.1.2.3 --- Cross talk between TJs and AJs --- p.60 / Chapter 3.1.3 --- Cryptorchidism --- p.61 / Chapter 3.1.3.1 --- Causes and consequences of Cryptorchidism --- p.61 / Chapter 3.1.3.2 --- Elevated temperature caused by cryptorchidism greatly contributes to defective spermatogenesis --- p.62 / Chapter 3.1.3.3 --- Changes of Sertoli cells in cryptorchidim contributing to defective spermatogenesis. --- p.62 / Chapter 3.1.3.4 --- Disruption of junctional complexes in heat shock and cryptorchidism. --- p.65 / Chapter 3.1.4 --- CFTR and spermatogenesis --- p.66 / Chapter 3.1.4.1 --- Expression of CFTR in Sertoli cells in testis --- p.66 / Chapter 3.1.4.2 --- Temperature sensitive processing of CFTR protein --- p.66 / Chapter 3.1.4.3 --- CFTR and junctional complex --- p.67 / Chapter 3.1.4.4 --- CFTR and male reproduction --- p.68 / Chapter 3.1.4.5 --- Role of CFTR in spermatogenesis --- p.68 / Chapter 3.1.5 --- Prostaglandins and male fertility --- p.69 / Chapter 3.1.5.1 --- Expression of COX-2 in testis. --- p.69 / Chapter 3.1.5.2 --- Role of prostaglandins in spermatogenesis --- p.70 / Chapter 3.1.5.3 --- Regulation of junctional complexes by PGE₂ --- p.70 / Chapter 3.1.5.4 --- Prostaglandins in cryptorchidism --- p.72 / Chapter 3.1.6 --- Hypothesis and aims of study --- p.73 / Chapter 3.2 --- Materials and Methods --- p.74 / Chapter 3.2.1 --- Cell culture materials --- p.74 / Chapter 3.2.2 --- Drugs and Reagents --- p.74 / Chapter 3.2.3 --- Antibodies --- p.74 / Chapter 3.2.4 --- Animals --- p.75 / Chapter 3.2.4.1 --- Mice artificial cryptorchidism model --- p.75 / Chapter 3.2.4.2 --- Mice testes hyperthermia model --- p.75 / Chapter 3.2.5 --- Sertoli cell primary culture --- p.76 / Chapter 3.2.6 --- siRNA against CFTR and transfection --- p.76 / Chapter 3.2.7 --- Examination of assembly and destruction of assembly of inter-Sertoli TJs --- p.77 / Chapter 3.2.8 --- Manipulation of RNA and Real-Time Quantitative RT-PCR (QRT-PCR) --- p.77 / Chapter 3.2.9 --- Manipulation of protein and western blot --- p.77 / Chapter 3.2.10 --- Histological and morphological studies --- p.78 / Chapter 3.2.10.1 --- Immunofluorescence of ZO-1 Staining in Sertoli cells --- p.78 / Chapter 3.2.10.2 --- Immunofluorescent staining of ZO-1, Occludin and β-Catenin in testes --- p.78 / Chapter 3.2.11 --- PGE₂ EIA --- p.79 / Chapter 3.2.12 --- Statistical Analysis --- p.79 / Chapter 3.3 --- Results --- p.79 / Chapter 3.3.1 --- Downregulation of CFTR is associated with upregulation of COX-2 in mice cryptorchidism model, mice testes hyperthermia model, and CF mice testes --- p.79 / Chapter 3.3.2 --- Negative regulation of COX-2 by CFTR is mediated by NF-κB --- p.81 / Chapter 3.3.3 --- Decreased tight junction proteins expression and increased anchoring junction proteins expression in cryptorchid testes. --- p.81 / Chapter 3.3.4 --- Elevation of culture temperature results in downregulation of CFTR and upregulation of COX-2 in primary cultured rat sertoli cells --- p.82 / Chapter 3.3.5 --- Defect of functional CFTR leads to increased COX-2 expression. --- p.83 / Chapter 3.3.6 --- CFTR regulates TJ protein expression and TJ formation through NF-κB/COX-2/PGE₂. --- p.83 / Chapter 3.4 --- Discussion --- p.100 / Chapter 3.5 --- Conclusion --- p.104 / Chapter 4 --- Chapter 4: General Discussion --- p.105 / Chapter 4.1 --- The immunosuppressive function of PGE₂ in CF lung disease and cryptorchidism-induced infertility. --- p.105 / Chapter 4.2 --- Importance of CFTR/ NF-κB /COX-2/PGE₂ pathway in inflammation-based diseases. --- p.106 / Chapter 4.3 --- Possible implications of CFTR/NF-κB /COX-2/PGE₂ pathway in cancer --- p.107 / Chapter 4.4 --- Concluding remarks --- p.108

Diss. (sammanfattning) Linköping : Linköpings universitet, 2009. / Härtill 5 uppsatser.

The mechanisms involved in human parturition, and the molecular changes that occur during the transition from pregnancy and birth are not completely elucidated. Endogenous or administered prostaglandins, including the PGE1 and PGE2, are related to contractile activity and cervical ripening, playing an important role on labor. Due to maternal or fetal causes, some pregnancies require labor induction. In many cases, prostaglandins, including Dinoprostone (PGE2) and Misoprostol (PGE1 analog), are used for labor induction. However, the response to labor induction is variable, and the reasons why this occurs are unknown. Once delivery also involves modulation of oxidative metabolism, that can be potentially affected by administered drugs, in the present study, we analyzed prostaglandin E2 receptor (EP1, EP2, EP3 and EP4) gene expression of myometrial and cervical cells, in vivo and in vitro, as well as oxidative markers in myometrial cells exposed in vitro to different concentrations of Misoprostol. In both studies, tissue biopsies were obtained from pregnant women at term. In the in vivo study, from women with spontaneous deliveries or Dinoprostone induced labors, responsive or non-responsive to labor induction, and in the in vitro study, from women with spontaneous and non-spontaneous labors, these induced with misoprostol. Gene expression was analyzed by qtRT-PCR and oxidative biomarkers by spectrophotometric and fluorimetric analysis. The results obtained from the in vivo study showed a concurrent and antagonic regulation of EP1 and EP3 mRNA expression in cervical and myometrial tissues in pregnant women at term in Dinoprostone induced labors. EP1 mRNA was upregulated in the cervical tissue of women who did not respond to Dinoprostone induction. In addition, in the myometrium, significantly higher levels of EP3 mRNA were observed in women treated with Dinoprostone, independent of their responsiveness, indicating a possible regulation of the EP3 gene at a transcriptional level. In vitro analysis revealed that myometrial cells derived from women with spontaneous labors showed greater capacity for misoprostol genomic response, since an overexpression of genes associated with muscle contraction (EP1 and EP3) was observed. In addition, Misoprostol was able to differentially modulate two important oxidative metabolism markers (protein carbonylation and lipid peroxidation). However, this effect was dependent on cells source (whether obtained from spontaneous or non-spontaneous labors) and drug concentration. The results suggest that, in term pregnant women, there is modulation of the PGE2 receptors genes in myometrial and cervical tissues in Dinoprostone or Misoprostol-induced labors, and that the EPs have an important role in the success of spontaneous delivery and in the pharmacological response to PGE1 analog administration. Addicionally, oxidative metabolism also seems to play an important role in the parturition process, requiring further studies to define its real function in this process. / Os mecanismos pelo qual a parturição humana é iniciada espontaneamente e as mudanças moleculares que ocorrem durante a transição entre a gestação e o parto não são completamente elucidados. Um dos principais fatores envolvidos são as prostaglandinas, endógenas ou administradas, entre elas a PGE1 e PGE2, que estão relacionadas à atividade contrátil e ao amadurecimento cervical. Em casos de necessidade de antecipação do nascimento, as prostaglandinas, entre elas a Dinoprostona (PGE2) e o Misoprostol (análogo da PGE1), são utilizadas para indução do parto. Entretanto, a resposta à indução do parto é variável e os motivos pelos quais isto ocorre é desconhecido. Uma vez que o parto também envolve modulação do metabolismo oxidativo, que pode ser potencialmente afetado por ação de fármacos, no presente estudo analisamos a expressão dos genes dos receptores de prostaglandina E2 (EP1, EP2, EP3 e EP4) em células miometriais e cervicais, in vivo e in vitro, bem como marcadores oxidativos em células miometriais expostas in vitro a diferentes concentrações de Misoprostol. Para a realização dos dois estudos, foram obtidas biópsias teciduais em parturientes a termo; no estudo in vivo, em mulheres com parto espontâneo, responsivas e não responsivas à indução do parto com Dinoprostona e no estudo in vitro, em mulheres com partos espontâneos e não espontâneos, estes induzidos com Misoprostol. A expressão gênica foi analisada através de qtRT-PCR e no estudo in vitro, além das análises da modulação gênica, foram conduzidas análises complementares de biomarcadores oxidativos, através de ensaios espectrofotométricos e fluorimétricos. Nos resultados obtidos a partir do estudo in vivo, observou-se regulação concomitante e antagônica da expressão do mRNA de EP1 e EP3 nos tecidos cervical e miometrial em gestantes a termo com partos induzidos com Dinoprostona. O mRNA do EP1 foi superexpresso no tecido cervical de mulheres que não responderam à indução com Dinoprostona. Além disso, no miométrio, níveis significativamente mais elevados de mRNA do EP3 foram observados em mulheres tratadas com Dinoprostona, independente da sua capacidade de resposta, indicando uma possível regulação da expressão do gene EP3 a nível transcricional. A análise in vitro evidenciou que as células miometriais oriundas de mulheres com parto espontâneo apresentaram maior capacidade de resposta genômica ao Misoprostol, uma vez que uma superexpressão dos genes relacionados com a contração muscular (EP1 e EP3) foi observada. Adicionalmente, o Misoprostol foi capaz de modular diferencialmente dois importantes marcadores do metabolismo oxidativo (lipoperoxidação e carbonilação de proteínas). Porém, este efeito foi dependente da origem das células (se obtida de partos espontâneos ou não espontâneos) e da concentração do fármaco. Os resultados obtidos sugerem que, em gestantes a termo, existe modulação dos genes dos receptores de PGE2 no miométrio e colo em partos induzidos com Dinopostona ou Misoprostrol, e que os EPs possuem um papel relevante no sucesso do parto espontâneo e na resposta farmacológica aos análogos a prostaglandina E1. Adicionalmente, o metabolismo oxidativo também parece desempenhar um papel importante no processo da parturição, necessitando de estudos complementares para esclarecimento da sua real função neste processo.

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

Introdução: 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