A ação de fosfolipases A2 (FLA2s): miotoxinas (MTs) ?II e III, isoladas de Bothrops asper (VBa) e CB2, de Crotalus durissus terrificus (VCdt) e os venenos brutos, sobre a expressão de ciclooxigenases (COXs) e síntese de prostaglandina (PG) E2 e PGD2 foi avaliada. As MTs e VBa mas não CB2 e nem VCdt induziram a expressão de COX-2 por leucócitos. Em estudos in vitro ocorreram a liberação de PGs e expressão de COX-2, após a incubação de macrófagos (M?s) com FLA22s e, de neutrófilos (N?s) e M?s, com VBa. A CB2 induziu somente a liberação de PGs. A inibição de FLA2 citosólica (cFLA2), diminuiu os níveis de PG induzidos pelas MTs, mas não pela CB2, e não afetou a expressão de COX-2 induzida pelas MTs. O envolvimento do NF-?kB na expressão de COX-2 foi mostrado com inibidores. Em conclusão, MTs, CB2 e VBa estimulam a síntese de PGs in vivo e in vitro e MTs e VBa, mas não CB2, induzem a expressão de COX-2. O VCdt não afeta estes parâmetros. O efeito das MTs sobre a expressão de COX-2 e PGs é mediado pelo NF-kB e pela cFLA2, respectivamente. Os efeitos de CB2 na produção de PGs são independentes de cFLA2s e COX-2. O fato da MT-II ser destituída de atividade enzimática sugere que a atividade catalítica per se, não seja relevante para os efeitos observados. / Action of the phospholipase A2 (PLA22): myotoxins (MTs) -II and -III, from Bothrops asper (BaV) and CB2, from Crotalus durissus terrificus (CdtV) and these venoms on cyclooxygenases (COXs) and synthesis of prostaglandins (PGs) E2 and D2 were studied in vivo and in vitro. Intraperitoneal injection of sPLA22s and BaV but not CdtV released PGD2 and PGE2. MTs and BaV but neither CB2 nor CdtV induced expression of COX-2 by leukocytes. Release of PGs and expression of COX-2 occurred in vitro after incubation of macrophages (M?s) with PLA2 and neutrophils (N?) and M?s with BaV. CB2 induced only PGs release. Inhibition of cytosolic PLA2 (cPLA2), reduced PG levels caused by MTs, but not by CB2 while did not affect MTs-induced COX-2 expression. Involvement of NF-kB in COX-2 was showed using with inhibitors. In conclusion MTs, CB2 and BaV stimulate the synthesis of PGs in vivo and in vitro and MTs and BaV, but not CB2, induce COX-2 expression. VCdt does not affect these parameters. Effect of MTs on COX-2 is mediated by NF-kB, and on PGs by cPLA2. Effects of CB2 on PGs are independent of cPLA2 and OX-2. Since MT-II lacks catalytic activity the PLA2 activity per se is not relevant for activation of this cascade.

by Lo Ming Yip. / "July 1998." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 208-247). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstract in Chinese.

O glioblastoma multiforme (GBM) é um dos gliomas mais comuns, classificado como um glioma de grau IV (Organização Mundial da Saúde - OMS) e notoriamente difícil de ser tratado. O tratamento recomendado consiste na ressecção cirúrgica seguida de radio e quimioterapia, e a sobrevida média dos pacientes é de apenas 12 meses após o diagnóstico. Portanto, novas terapias que focam a redução do volume do tumor e o aumento da morte das células tumorais são urgentemente necessárias. Estudos pré-clínicos sugerem que a inibição de COX-1 e 2 com Anti-inflamatórios não esteroidais (AINEs), como o Ibuprofeno (IBP), inibiram significantemente a proliferação e a migração celular em diferentes tumores. Assim, o presente estudo teve como objetivo avaliar in vitro o efeito da inibição de COX-1 e COX-2 através do tratamento com IBP, e também com inibidores específicos como SC-560 e NS-398 respectivamente, na migração e na proliferação da linhagem celular de glioma humano U251-MG. O presente estudo demonstrou através de diversos ensaios que a inibição de COX-1 e COX-2 através do IBP, do SC-560 e do NS-398 inibiu significativamente a migração e a proliferação celular da linhagem celular U251-MG. Dessa maneira, concluímos que a PGE2 está envolvida na migração e proliferação celular das células desta linhagem celular. / Glioblastoma Multiforme (GBM) is the most common glioma, classified as grade IV (World Health Organization WHO) and notoriously difficult to treat. The recommended treatment consists of surgery followed by radiotherapy and chemotherapy, and the median survival is ten to twelve months. Preclinical studies suggest that inhibiton of cyclooxygenase-2 by treatment with non-steroidal anti-inflammatory drugs, such as ibuprofen, significantly blocked the proliferation of different tumors including gliomas. Thus this study aimed to evaluate the migration of glioma cell line U251-MG after inhibition of cyclooxygenases 1 and 2 by treatment with ibuprofen (IBP), and specifics inhibitors such as SC-560 for COX-1 and NS-398 for COX-2. The present study demonstrated by various assays where inhibition of COX-1 and COX-2 by IBP, SC-560 and NS-398, significantly inhibited migration and cell proliferation of U251-MG cell line. Thus, we conclude that PGE2 is involved in this cell line migration and cell proliferation.

A destruição crônica do aparelho de inserção periodontal devido a uma intensa resposta inflamatória a bactérias conduz a uma condição clínica conhecida como doença periodontal. A inflamação periodontal geralmente provoca úlceras superficiais no sulco gengival, onde os capilares sanguíneos estão expostos a bactérias. Patógenos periodontais, assim como seus produtos podem ser translocados e liberados do sulco gengival para a corrente sanguínea e, portanto, ter efeitos periféricos. A bolsa periodontal é também importante reservatório de mediadores inflamatórios que podem atingir a corrente sanguínea. Diversos estudos avaliam a relação entre a doença periodontal e as doenças cardiovasculares. Sabe-se que muitas doenças cardiovasculares têm início em desordens vasculares, principalmente na camada endotelial dos vasos sanguíneos de grande condutância. Assim, o foco deste estudo foi investigar a reatividade vascular de aortas de animais com periodontite. Tanto a contração para a norepinefrina quanto o relaxamento para a acetilcolina estão diminuídos em animais com periodontite, mas os mecanismos para estes efeitos são diferentes. A diminuição da resposta contrátil à norepinefrina parece estar associada a um aumento da produção de óxido nítrico através da isoforma induzível da óxido nítrico sintase e a diminuição da resposta relaxante à acetilcolina parece estar associada a produção de prostanóides principalmente derivados da isoforma do tipo II da enzima ciclooxigenase. / The chronic destruction of the periodontal attachment apparatus due to an intense inflammatory response to bacteria leads to a condition known as periodontal disease. The periodontal inflammation usually causes superficial ulcers in the gingival sulcus, where blood capillaries are exposed to bacteria. Periodontal pathogens, as well as their products can be translocated and released into the gingival sulcus, subsequently into the bloodstream and thus have peripheral effects. The periodontal pocket is also an important reservoir of inflammatory mediators that can reach the bloodstream. Several studies have evaluated the relationship between periodontal disease and cardiovascular disease. It is known that many cardiac diseases begin as vascular disorders, especially in the endothelial layer of blood vessels of large conductance. Thus, the focus of this study was to investigate the vascular reactivity of aortas from animals with periodontitis. Both norepinephrine-mediated contraction and acetylcholine-mediated relaxation are decreased in animals with periodontitis, but the mechanisms are different for these responsess. The contraction appears to be associated with increased production of nitric oxide by inducible isoform of nitric oxide synthase and relaxation with prostanoids primarily derived type II isoform of the enzyme cyclooxygenase.

A sepse é caracterizada por uma produção excessiva de mediadores inflamatórios, acompanhada de taquicardia e hipotensão. Experimentalmente, a administração de endotoxina (Lipopolissacarídeo, LPS) em doses relativamente elevadas induz choque endotoxêmico, sendo um bom modelo de estudo da sepse. Diversos grupos têm demonstrado ações antiinflamatórias e antitumorais do citral, um composto do óleo essencial de Cymbopogon citratus. Nosso laboratório demonstrou ação antipirética do citral em modelo de febre induzida por LPS, acompanhada de redução nos níveis de citocinas plasmáticas e de prostaglandina E2 (PGE2) no plasma e área pré óptica do hipotálamo (POA), importante região termorregulatória. A hipótese testada neste trabalho foi a de que o citral atenua a hipotensão provocada pela endotoxina, além de amenizar as alterações termoregulatórias. Todos os procedimentos foram executados de acordo com os princípios éticos de experimentação animal, aprovados pelo comitê de ética local (CEUA 2015.1 1214 58-2). Foi realizado o implante de cânulas na artéria e veia femoral para registro da pressão arterial e administração de LPS (1,5 mg/kg) ou salina apirogênica 0,9% além do implante de datalogger na cavidade peritoneal de ratos Wistar, para registro da temperatura corporal. No dia do registro, 30 minutos antes da administração de LPS ou salina, os animais receberam citral (100 mg/kg) ou tween 80 a 1% (veículo) por via oral. Os parâmetros cardiovasculares e temperatura corporal foram registrados por 300 minutos após os respectivos tratamentos. Os valores de pressão arterial média (PAM) e frequência cardíaca (FC) foram coletados a cada 10 minutos após o tratamento e a temperatura corporal foi registrada pelo datalogger em intervalos de 5 minutos. Em outro protocolo foi realizado apenas o implante de cânula na veia femoral dos animais de todos os grupos para administração de LPS ou salina, coleta de sangue para dosagem de interleucina 6, PGE2, nitrito e nitrato e corticosterona e coleta do encéfalo para dosagem de PGE2 e PGD2. As diferenças estatísticas entre os grupos foram analisadas pelo teste ANOVA two-way seguido por pós teste de Newman-Keuls, com o nível de significância adotado de p < 0,05. A administração de LPS provocou queda na PAM eaumento na FC. Tais respostas não foram afetadas pela administração prévia de citral. O LPS também induziu febre e aumento nas concentrações plasmáticas de interleucina - 6 (IL-6), óxido nítrico (NO), PGE2 e corticosterona. Esses parâmetros não foram alterados pela pré- administração de Citral. No entanto, o citral provocou redução na produção de PGD2 naPOA, sem alterar a de PGE2 nesta região. Podemos concluir que o citral não previne as alterações nos parâmetros cardiovasculares no modelo de endotoxemia em ratos, porém reduz a produção de um mediador termorregulatório e inflamatório do sistema nervoso central (a PGD2), sem alterar a produção de outros mediadores inflamatórios a nível periférico (no plasma). Portanto, em um modelo mais agressivo de inflamação sistênica o citral não se mostrou suficiente para proteger o organismo das ações deletérias do LPS. / Sepsis is characterized by the overproduction of inflammatory mediators, accompanied by tachycardia and hypotension. Experimentally, administration of endotoxin (Lipopolysaccharide, LPS) in relatively high doses induces endotoxemic shock, a widely used model of sepsis in rats. Several groups have demonstrated anti-inflammatory and antitumor roles of citral, an essential oil compound of Cymbopogon citratus. Emilio-Silva et al. (2017) have shown an antipyretic role of citral in a model of LPS-induced fever, accompanied by a reduction of cytokines and prostaglandin E2 plasma levels and in the preoptic area of hypothalamus (POA), the hierarchically most important thermoregulatory region. We hypothesized that citral attenuates the LPS-induced hypotension, besides mitigating the thermoregulatory adjustments in rats. All procedures were performed in agreement with ethical guidelines for animal experimentation aproved by the local ethical committee (CEUA 2015.1 1214 58-2). Femoral artery and vein were implanted with cannulas for blood pressure recording and LPS (1.5 mg/kg) or 0.9% apyrogenic saline injection. In a second surgical procedure a datalogger was implanted into the peritoneal cavity for measurements of body temperature. All surgical procedures were performed under Ketamin/xilazin (100/10 mg/kg) anesthesia. One day after arterial catheterization, 30 minutes prior to LPS or saline administration, the animals received either citral (100 mg / kg) or 1% tween 80 (vehicle) oralstarly. The cardiovascular parameters and body temperature were recorded for 300 minutes after the respective treatments. Mean blood pressure (MAP) and heart rate (HR) were collected every 10 minutes after treatments and body temperature was recorded by the datalogger at 5 minute intervals. Blood samples were obtained in another set of rats for interleukin-6, PGE2, nitrite and nitrate and corticosterone analyses. The brain was removed for PGE2 and PGD2 analyses. Statistical differences between groups were analyzed by the two-way or one-way ANOVA test followed by Newman-Keuls post-test, with significance level adopted at p <0.05. As expected, LPS administration caused a decrease in MAP and an increase in HR, and these responses were not affected by citral. LPS also induced fever and increased plasma levels of interleukin - 6 (IL - 6), nitric oxide (NO), prostaglandin E 2 andcorticosterone. These parameters were also not altered by citral. On the other hand, citral caused a reduction in prostaglandin D2 concentration in the POA, but failed to alter PGE2 levels in this region. Our data are consistent with the notion that citral does not affect changes in cardiovascular and thermoregulatory parameters. Consistently, citral also caused no changes in both LPS-induced peripheral inflammatory mediators (in plasma) and in the POA, except PGD2. Therefore, in our model which mimetic a fairly critical situation, citral may not be sufficient to protect the organism from the deleterious actions of LPS. Financial support: FAPESP / CAPES.

To increase diversity of enzymes and proteins, cells mix and match exonic and intronic regions retained in mature mRNAs by alternative splicing. An estimated 94% of all multi-exon genes express one or more alternatively spliced transcripts generating proteins with similar or modified functions. Cyclooxygenase is a signaling enzyme that catalyzes the rate-limiting step in the synthesis of diverse bioactive lipids termed prostaglandins. Prostaglandins are involved in myriad physiological and pathopysiological processes including vasoregulation, stomach mucosal maintenance, parturition, pain, fever, inflammation, neoplasia and angiogenesis and are inhibited by aspirin-like drugs known as NSAIDs. In 2002 an alternatively spliced, intron-1 retaining variant of COX-1 was cloned from canine brain tissue. This new variant, termed COX-3 or COX-1b, is an enzymatically active prostaglandin synthase expressed at relatively high levels in a tissue and cell type dependant manner in all species examined. In humans and most rodent species intron-1 is 94 and 98 nucleotides long respectively. Retention of the intron in these species introduces a frameshift and is predicted to result in translation of a very small 8-16kD protein with little similarity to either 72kD COX-1 or COX-2, calling into question the role of this variant. In this dissertation, I present my results from cloning and ectopically expressing a complete and accurate COX-3 cDNA from both rat and human. I confirmed that COX-3 mRNA encodes multiple large molecular weight cyclooxygenase-like proteins in the same reading frame as COX-1. Translation of these proteins relies on several recoding mechanisms including cap-independent translation initiation, alternative start site selection, and ribosomal frameshifting. Using siRNA and Western blotting I have identified some of these proteins in tissues and cells. Two COX-3 encoded proteins are active prostaglandin synthase enzymes with activities similar to COX-1 and represent novel targets of NSAIDs. Other COX-3 proteins have unknown function, but their size and cellular location suggest potential roles as diverse as cytosolic enzymes and nuclear factors.

Prostaglandins (PG) are ubiquitous lipid mediators that play key roles in pathophysiological responses to infections. They are considered to have both pro and anti-inflammatory roles depending upon the time of inflammation, the receptors that they bind to and the tissues that they act upon. Hence given their pleiotropic effects, a perfect balance between the pro and anti-inflammatory functions of PGs are required to ensure that a controlled timely immune response is elicited to mediate protection and to avoid immunopathology. PGD2 is one such PG that was reported to increase with age in the lungs of mice and to mediate an anti-inflammatory effect thereby blunting the immune response following Severe Acute Respiratory Syndrome – Coronavirus (SARS-CoV). Increase in PGD2 with age incapacitates respiratory dendritic cells (rDC) to migrate from lungs to the draining lymph node following SARS-CoV infection due to down regulation of CCR7 (a receptor for chemokines CCL19/21). Migration of rDCs to draining lymph nodes requires high expression of CCR7 and it's binding to CCL19/21, a chemokine that mediates migration of dendritic cells along its gradient. Although increase in levels of PGD2 might prove beneficial in high inflammatory conditions, it should be noted that high levels of such a potent anti-inflammatory mediator during the initiation of an immune response could prove detrimental. In chapter II of this thesis I show that age-related increases in oxidative stress result in the upregulation of a single phospholipase (PLA2) group II D (G2D) (PLA2G2D) with anti-inflammatory roles. PLA2G2D functions by releasing Arachidonic acid (AA) from the lipid membrane, which will be further metabolized to other pro-resolving/ anti-inflammatory lipid mediators including PGD2. I show that inducing oxidative stress in young mice as well as in human peripheral blood macrophages, results in the upregulation of PLA2G2D (probably as a counter mechanism against oxidative stress). Also increase in the expression levels of this gene during the course of SARS-CoV infection results in the upregulation of PGD2, which is completely abrogated in Pla2g2d-/- mice. I also show Pla2g2d/- middle-aged mice have low levels of PGD2 and that they are capable of mounting a strong immune response and survive the otherwise lethal SARS-CoV infection. PGD2 is also a major PG in the brain and its role has been investigated in many non-infectious setting such as stroke and Alzheimer' disease. The PGD2 binding to one of its receptors DP1 has been shown to have primarily a neuro-protective role. In chapter III, I show that PGD2/DP1 signaling has beneficial effects in the brain of mice infected with a neurotropic strain of murine hepatitis virus (MHV) (rj2.2). In agreement with the neuro-protective role of PGD2, at least 60% of DP1-/- mice succumb to a sublethal dose of rj2.2. rj2.2 infection in these mice is characterized by a delay in the induction of IFN I response and lower activation status of microglia and macrophages in the brain. I also show that abrogation of DP1 signaling results in global defects in the immune system response to infection. Notably, a genome wide expression analysis using microarray, shows that a gene, Pydc3 with putative inflammasome inhibiting function is upregulated in WT mice compared to DP1-/- mice in the CD11b population of cells which primarily comprises microglia and macrophages. In line with the predicted function of Pydc3, DP1-/- mice have higher frequency and number of IL-1β+ producing microglia in the brain. Studies are underway to determine the exact role of DP1 signaling in Pydc3 expression as well as the role of this gene in inflammasome function. Overall these studies emphasize the immuno-modulatory roles of PGs in the context of a viral infection. Thus, altering the levels of these lipid mediators at appropriate times during the course of infection might prove useful as an effective therapeutic strategy to decide the fate of an infection.

Orientador: José Antonio Dell´Aqua Junior / Resumo: O objetivo do presente estudo foi avaliar o fluxo sanguíneo luteal e os efeitos colaterais em jumentas após administração de dois agentes luteolíticos. Cinco dias após a ovulação, oito fêmeas asininas foram randomizadas em crossover design, em dois grupos experimentais. No grupo 1 (GI) foi utilizado Dinoprost Trometamina, e no grupo 2 (G2) Cloprostenol sódico. Foram realizados exames ultrassonográficos modo B e modo Doppler 15 minutos antes (-15) da administração dos análogos da PGF2α, e nos tempos 0, 15, 30, 45 e 60 minutos e 2, 3, 4, 5, 6, 7, 8, 12 e 24 horas pós-aplicação. Já os efeitos colaterais foram observados nos tempos 0, 15, 30, 45 e 60 minutos pós-administração dos agentes luteolíticos. O fluxo sanguíneo e a área do corpo lúteo reduziram gradativamente durante as primeiras 24 horas em ambos os grupos. Em relação a observação dos efeitos colaterai, a aplicação de Dinoprost Trometamina provocou um maior grau de sudorese, enquanto que, após aplicação de Cloprostenol Sódico, maior desconforto abdominal e diarreia foram observados como efeitos colaterais. Conclui-se que ambos os tratamentos (dinoprost e cloprostenol) são eficientes na promoção da luteólise, entretanto os efeitos colaterais diferem em resposta à administração de cada ativo. Novos estudos se fazem necessários, com redução das doses luteolíticas efetivas visando minimizar os efeitos colaterais observados. / Abstract: The aim of the study was to evaluate the blood flow and side effect in jennies after treatment with two luteolytic drugs. Five days after ovulation, eight jennies included were randomized in crossover design into two experimental groups. In group 1 (GI) Dinoprost Tromethamine was used, and in group 2 (G2) Cloprostenol sodium. Mode B and Doppler ultrasound examinations were performed 15 minutes before (-15) administration of PGF2α, and at times 0, 15, 30, 45 and 60 minutes and 2, 3, 4, 5, 6, 7, 8, 12 and 24 hours after application. Side effects were observed at 0, 15, 30, 45 and 60 minutes after luteolytic agents administration. Blood flow and corpus luteum area decreased gradually during the first 24 hours in both groups. Regarding the observation of side effects, G1 presented major score of sweating, while in G2, greater abdominal discomfort and diarrhea was evidenced as a major side effect. It is concluded that both treatments (dinoprost and cloprostenol) are efficient in promoting luteolysis, however side effects differ between each active. Further studies are needed to determine whether low doses may be effective in inducing luteolysis to minimize side effects. Keywords: jennies, corpus luteum, Doppler ultrasonography, prostaglandins. / Mestre

No description available.

Bibliography: leaves 161-204 / xv, 204 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, Dept. of Medicine and Pathology, 1985