Abdominal aortic aneurysm (AAA) is a disease of the aorta characterized by pathological remodeling and progressive weakening of the vessel resulting in the increased risk of rupture and sudden death. In a mouse model of the disease induced by chronic Angiotensin II (AngII) infusion, progression of AAAs is associated with reduced differentiation of smooth muscle cells (SMCs) at the site of lesion development. In the mouse model, the effectiveness of cyclooxygenase-2 (COX-2) inhibition for attenuating AAA progression is associated with maintenance of a differentiated SMC phenotype. However, the safety of COX-2 inhibitors is currently in question due to the increased risk of adverse cardiovascular events. Thus, it is crucial to identify mediators downstream of COX-2 that may provide new targets for treatment of this disease. Recent studies in humans and mouse models have suggested that the microsomal prostaglandin E synthase (mPGES-1) enzyme, which acts downstream of COX-2, may also be involved in the pathogenesis of the disease. We hypothesized that increased prostaglandin E2 (PGE2) synthesis resulting from the induction of both COX-2 and mPGES-1 may result in reduced differentiation of SMCs, and that disruption of this pathway would preserve the differentiated phenotype. To test this hypothesis, human aortic smooth muscle cells (hASMCs) were utilized to examine the effects of a variety of agents involved in AAA development and the COX-2 pathway. My findings suggest that one of the effects of exposing hASMCs to AngII involves a specific induction of mPGES-1 expression. Furthermore, although different COX-2-derived products may have opposing effects, mPGES-1-derived PGE2 may be the primary prostanoid synthesized by SMCs which functions to attenuate differentiation. Therefore, mPGES-1 inhibition may provide inhibition of PGE2 that is more specific than COX-2 inhibitor treatment and may serve as a therapeutic target for attenuating AAA progression by maintaining a differentiated SMC phenotype.

The blood-brain barrier, formed by brain microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. It was previously demonstrated in our laboratory that knock down of fatty acid transport proteins FATP-1 and CD36 attenuated apical to basolateral monounsaturated fatty acid transport across human brain microvessel endothelial cells (HBMEC). Arachidonic acid (AA; 5,8,11,14 - cis-eicosatetraenoic acid) is a conditionally essential, polyunsaturated fatty acid [20:4(n-6)] and a major constituent of brain lipids. We examined transport of AA across confluent monolayers of HBMEC. Control cells or HBMEC with knock down of FATP-1 or CD36 were cultured on Transwell® plates and incubated apically with [3H]AA and incorporation of [3H]AA into the basolateral medium was determined temporally. [3H]AA was rapidly incorporated into the basolateral medium with time in control cells. Surprisingly, knock down of FATP-1 or CD36 did not alter [3H]AA movement into the basolateral medium. The increased permeability mediated by AA was likely caused by a metabolite of AA produced de novo and was confirmed by an increased movement of fluorescent dextran from apical to basolateral medium. HBMECs expressed PGE2 synthase, cyclooxygenase-1 and -2, PGE2 receptors, tight junction proteins and prostaglandin transporters. The AA-mediated increase in membrane permeability was not attenuated by cyclooxygenase inhibitor drugs (NSAIDs). Incubation of the HBMEC monolayers with exogenous PGE2 resulted in attenuation of the AA-mediated permeability increases. The results indicate that AA increases the permeability of the HBMEC monolayer likely via increased production of metabolites or by-products of the lipoxygenase or epoxygenase pathways. These observations may explain the rapid influx of AA into the brain previously observed upon plasma infusion with AA.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Epilepsy is one of the most common neurologic disorders. It has been suggested that seizures may be facilitaded by inflammation. PGE2 is one of the most important inflammatory mediators, and facilitates pentylenetetrazol (PTZ)-induced seizures by stimulating EP1 and EP3 receptors. However, up to the present moment, no study has investigated whether EP1 and EP3 receptors blocking attenuate seizures induced by convulsants other than PTZ. It is also unknown whether Na+,K+-ATPase activity alterations are involved in such an effect. Therefore, in the current study we investigated whether EP1 and EP3 ligands (agonists and antagonists) modulate PTZ- and kainic acid (KA)-induced seizures, and whether alterations in Na+,K+-ATPase activity mediate such a protective effect, in mice. EP1 and EP3 antagonists (ONO-8713 and ONO-AE3-240, respectively, 10 Og/kg, s.c.) attenuated PTZ (60 mg/kg, i.p.)- and KA (20 mg/kg, i.p.)-induced seizures. The respective agonists (ONO-DI-004 and ONO-AE-248, 10 Og/kg, s.c.) facilitated seizures in both acute models, and at noneffective doses, prevented the protective effects of the antagonists. Animals injected with PTZ presented decreased Na+,K+-ATPase activity in the cerebral cortex and hippocampus. On the other hand, animals injected with KA presented increased Na+,K+-ATPase activity in the same cerebral structures at the end of the experiment. These divergent findings suggest that alterations in Na+,K+-ATPase activity in both acute models depends on the convulsant agent used and make difficult to establish a relationship between Na+,K+-ATPase activity and seizure development. Moreover, EP1 and EP3 antagonists administration abolished Na+,K+- ATPase activity alterations induced by PTZ and KA, in such a way that these alterations seem to be related more to the presence of ictal phenomenon itself than to the seizure induction mechanisms. Notwithstanding, the currrent results clearly show that EP1 and EP3 receptors might constitute novel targets for anticonvulsants development, since EP1 and EP3 decreased seizures, regardless of the convulsant agent used. / A epilepsia é uma das disfunções neurológicas mais comuns. Tem sido sugerido que as crises epilépticas podem ser facilitadas pela ocorrência de inflamação. A PGE2 é um dos mediadores inflamatórios mais importantes que, agindo por meio dos receptores EP1 e EP3, facilita as convulsões induzidas por pentilenotetrazol (PTZ). Contudo, até a presente data, nenhum estudo investigou, de maneira sistêmica, se a ativação ou bloqueio de receptores EP1 e EP3 facilitam as convulsões induzidas por outros agentes; tampouco se alterações na atividade da Na+,K+-ATPase estão envolvidas nesse efeito. Assim, no presente estudo, investigamos se ligantes (agonistas e antagonistas) de receptores EP1 e EP3 modificam as crises induzidas por PTZ e ácido caínico (KA), e se tais efeitos estão associados a alterações na atividade da enzima Na+,K+-ATPase, em camundongos. Os antagonistas EP1 e EP3 (ONO-8713 e ONO-AE3-240, respectivamente, 10 Og/Kg, s.c.) atenuaram as convulsões induzidas por PTZ (60 mg/Kg, i.p.) e KA (20 mg/Kg). Os seus respectivos agonistas (ONO-DI-004 e ONO-AE-248 de 10 Og/Kg, s.c.) facilitaram as convulsões em ambos modelos agudos de crises epilépticas e, em doses não efetivas para gerar crises, preveniram os efeitos dos antagonistas. Os animais submetidos à administração de PTZ apresentaram, ao final do experimento, a atividade Na+,K+-ATPásica diminuída no córtex cerebral e hipocampo. Por outro lado, animais tratados com KA apresentaram um aumento na atividade Na+,K+-ATPásica nestas mesmas estruturas, que se correlacionou positivamente com a vigência de status epilepticus no momento do sacrifício. Os achados divergentes no que diz respeito à alteração da atividade da Na+,K+-ATPase nos dois modelos de crises agudas sugere que tais alterações estejam relacionadas ao tipo de agente convulsivante utilizado, e dificultam estabelecer, de forma inequívoca, uma relação entre atividade desta ATPase e sensibilidade à crises agudas. Ademais, a administração de antagonistas EP1 e EP3 aboliu as alterações da atividade da Na+,K+-ATPase induzidas tanto por PTZ como por KA, de tal forma que estas parecem estar mais associadas com o fenômeno ictal em si, do que com os mecanismos de indução da crise. Contudo, os resultados mostram de forma clara que os receptores EP1 e EP3 podem se constituir possíveis novos alvos para o desenvolvimento de drogas antiepilépticas, pois antagonistas EP1 e EP3 diminuíram as crises, independente do agente convulsivante utilizado.

Resumo: O objetivo do presente estudo foi o de avaliar os efeitos do controle de placa supragengival, em pacientes com doença periodontal crônica generalizada, por meio dos parâmetros clínicos microbiológicos e imunológicos. Foram selecionados 30 pacientes, com idade entre 32 e 59 anos, apresentando 4 bolsas entre 3 e 5mm e 4 bolsas entre 6 e 10mm e sem envolvimento sistêmico. Após o exame inicial, os pacientes foram submetidos a raspagem supragengival e receberam instruções de higiene oral, sendo acompanhados semanalmente por um período de 30 dias. Os parâmetros clínicos avaliados foram profundidade de sondagem, recessão gengival, índice de placa, índice gengival e sangramento à sondagem. Amostras de fluido crevicular das bolsas foram coletadas para análise da concentração dos mediadores de inflamação interleucina-1b e prostaglandina E2 e identificação pela reação de polimerase em cadeia dos microrganismos Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Prevotella intermedia e Prevotella nigrescens. Os resultados demonstraram a ocorrência de melhoras evidenciadas pelos parâmetros clínicos avaliados. Observou-se ainda uma redução significante no número de Porphyromonas gingivalis, Bacteroides forsythus, Prevotella intermedia e Prevotella nigrescens. O microrganismo A. actinomycetemcomitans não apresentou diferença significante. Para o exame imunológico não foram encontradas diferenças nos níveis de interleucina-1b, sendo constatado ainda um aumento nas concentrações de prostaglandina E2 nas bolsas de 6 a 10mm que apresentavam sangramento no final do tratamento. Concluiu-se que decisão da necessidade de tratamento periodontal em sítios profundos, após a realização do controle de placa supragengival, baseada somente na ausência de sangramento à sondagem pode subestimar... (Resumo completo, clicar acesso eletrônico abaixo). / Abstract: The aim of the present study was to evaluate the effect of supragingival plaque control, in patients with generalized chronic periodontal disease using clinical, microbiological and immunological parameters. 30 patients were selected, aged 32 to 59 years, presenting 4 periodontal pockets between 3 and 5mm and 4 pockets between 6 and 10mm. After initial exam patients received supragingival scaling and oral hygiene instructions, which were reinforced once a week for 30 days. Clinical parameters were probing depth, gingival resection, plaque index, gingival index and bleeding upon probing. Samples of crevicular fluid were taken for analyses of interleukin-1b and prostaglandin E2 and for identification by polymerase chain reaction of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Prevotella intermedia and Prevotella nigrescens. The results demonstrated an improvement of the clinical parameters. The microbiological findings indicate reduction of Porphyromonas gingivalis, Bacteroides forsythus, Prevotella intermedia, and Prevotella nigrescens. A. actinomycetemcomitans did not show significant difference. In regard to the immunological exam, no differences were observed in interleukin-1b concentration. Prostaglandin E2 concentrations were elevated in pockets between 6 and 10mm with persistent bleeding upon probing after treatment. In conclusion the decision for treatment needs after supragingival plaque control only based on the absence of bleeding upon probing might underestimate the periodontal disease activity. / Orientador: Rosemary Adriana Chiérici Marcantonio / Coorientador: Iracilda Zepponi Carlos / Banca: Carlos Rossa Júnior / Banca: Márcio Fernando de Moraes Grisi / Banca: Luciene Cristina de Figueiredo / Banca: Magda Feres / Doutor

Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2018-05-15T15:58:32Z No. of bitstreams: 1 DISS_2015_Deise Cristine Schroder.pdf: 1269604 bytes, checksum: b4aaf38792fb2d963d422ec5bfeabadf (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2018-05-24T16:46:13Z (GMT) No. of bitstreams: 1 DISS_2015_Deise Cristine Schroder.pdf: 1269604 bytes, checksum: b4aaf38792fb2d963d422ec5bfeabadf (MD5) / Made available in DSpace on 2018-05-24T16:46:13Z (GMT). No. of bitstreams: 1 DISS_2015_Deise Cristine Schroder.pdf: 1269604 bytes, checksum: b4aaf38792fb2d963d422ec5bfeabadf (MD5) Previous issue date: 2015-03-13 / CAPES / Objetivou-se avaliar a eficácia do firocoxib em impedir a quebra da barreira hematoaquosa em gatos saudáveis e naqueles com sorologia positiva para toxoplasmose. Avaliaram-se trinta e dois gatos divididos em quatro grupos (n=8). Os grupos saudável controle (SC) e toxoplasmose controle (TC) foram compostos respectivamente por gatos saudáveis e com sorologia positiva para toxoplasmose, enquanto os grupos saudável firocoxib (SF) e toxoplasmose firocoxib (TF) foram compostos respectivamente por gatos saudáveis e com sorologia positiva para toxoplasmose que receberam tratamento prévio com firocoxib (5 mg/kg), por via oral, 24 e uma hora antes da indução da uveíte experimental, através da paracentese da câmara anterior. Após indução anestésica colheu-se 0,2 mL de humor aquoso basal. Decorrido uma hora, realizou-se nova paracentese para colheita de 0,2 mL de humor aquoso inflamado. As amostras de humor aquoso foram acondicionadas a -80°C para posterior mensuração dos níveis de prostaglandina E2 (PGE2) e proteínas totais. No humor aquoso dos grupos TC e TF, realizou-se ainda, titulação para anticorpos IgG anti-Toxoplasma gondii. Mediante análise das amostras, observou-se aumento significativo dos níveis de PGE2 e proteína total do humor aquoso basal para o inflamado (p<0,05). No humor aquoso basal, os níveis de PGE2 no grupo TC, assim como os níveis de proteína total nos grupos SF e TF foram significativamente superiores aos demais (p<0,05). Os grupos TC e TF não apresentaram títulos IgG anti-Toxoplasma gondii no humor aquoso basal. No humor aquoso inflamado, os níveis de PGE2 e proteína total, bem como os títulos IgG anti-Toxoplasma gondii, não diferiram significativamente entre os grupos (p>0,05). Admite-se que gatos com títulos de anticorpos IgG anti-Toxoplasma gondii possuem níveis de PGE2 no humor aquoso basal superiores aos apresentados por indivíduos saudáveis. Entretanto, a concentração de PGE2 no humor aquoso destes indivíduos não é suficiente para ensejar a quebra da barreira hematoaquosa e causar uveíte anterior. O firocoxib não é capaz de evitar a quebra da barreira hematoaquosa em gatos saudáveis e naqueles soropositivos para toxoplasmose. / The objective was to evaluate the efficacy of firocoxib in preventing the blood-aqueous barrier breakdown in healthy cats and those with positive serology for toxoplasmosis. Thirty-two cats divided into four groups (n=8/each). The healthy control (HC) and toxoplasmosis control (TC) groups, were composed respectively of healthy cats and cats with positive serology for toxoplasmosis, while healthy firocoxib (HF) and toxoplasmosis firocoxib (TF) groups, were composed respectively of healthy cats and cats with positive serology for toxoplasmosis who had received previous treatment with orally firocoxib (5 mg/kg), twenty-four and one hour before the experimental induction of uveitis. Under anesthesia 0.2 mL of baseline aqueous humor was collected via aqueocentesis. One hour later, the same procedure was repeated and inflamed aqueous samples were collected. Aqueous samples were conditioned at -80°C for subsequent measurement of the levels of prostaglandin E2 (PGE2) and total proteins. In the aqueous samples of TC and TF groups, anti-Toxoplasma gondii IgG specific antibodies were titrated. PGE2 and total protein levels increased significantly in inflamed aqueous humor in comparison to baseline aqueous samples (p<0.05). At baseline aqueous humor, levels of PGE2 in TC group and total protein in HF and TF groups were increased significantly, in comparison with the other groups (p<0.05). Anti-Toxoplasma gondii IgG specific antibodies were found only in inflamed aqueous humor, and aqueous titers did not change significantly between TC and TF (p=0.1051). Although we have observed that aqueous humor PGE2 levels were significantly higher in seropositive cats in baseline aqueous humor, such increase was not able to break the blood-aqueous barrier and cause anterior uveitis. Firocoxib did not prevent intraocular inflammation after aqueocentesis, in healthy and toxoplasmosis-seropositive cats.

Resultados obtidos pelo nosso grupo demonstraram, in vitro, que a PGE2 é capaz de modular a sobrevivência de linfócitos TCD4+ protegendo essas células da morte. Dentro do modelo de EAE, nossa hipótese é que a PGE2 liberada pelas APCs, durante a fase de indução, module a sobrevivência de linfócitos autorreativos específicos induzindo a doença. Realizamos o tratamento de camundongos submetidos à EAE com indometacina durante 5 dias e notamos que houve redução da EAE associada à redução de linfócitos produtores de IFN-&gamma;, IL-17 e GM-CSF, e macrófagos infiltrantes e microglias ativadas, no SNC. O tratamento alterou a freqüência de células em proliferação e a frequência de células produtoras de IFN-&gamma; e IL-17 na periferia e a concentração dessas citocinas. Esses resultados sugerem que a indometacina reduz o desenvolvimento da EAE e sua resposta antígeno-específica demonstrando a sua importância na modulação das respostas de linfócitos T na autoimunidade. / Results obtained by our group demonstrated in vitro that PGE2 is able to modulate CD4+ T cells survival protecting these cells from death. Within the EAE model, we hypothesized that PGE2 released by APCs during the induction phase, modulate survival of autoreactive specific lymphocytes by induction the disease. We carried out the treatment of EAE in mice subjected to indomethacin for 5 days and noticed that there is reduction of EAE associated with decreased IFN-&gamma;, IL-17 and GM-CSF producing T cells, and infiltrating macrophages and activated microglia in the CNS. The results suggest that indomethacin reduces EAE and its antigen-specif response demonstrating their importance in the modulation of T lymphocyte responses in autoimmunity.

O Histoplasma capsulatum é um fungo dimórfico, patogênico e responsável por graves lesões pulmonares. A infecção é adquirida pela inalação de conídios e posterior conversão para leveduras nos alvéolos e bronquíolos, onde são fagocitadas por macrófagos alveolares residentes e leucócitos que migram para o local da infecção. Recentemente, demonstramos que animais infectados com H. capsulatum e tratados com inibidor da síntese de prostaglandinas apresentaram diminuição de carga fúngica nos pulmões e baço, aumento da produção de nitrito e da fagocitose de leveduras por macrófagos alveolares, e maior sobrevivência, quando comparados com os animais somente infectados. Porém, neste estudo não foram determinados quais subtipos de prostaglandinas participam na patogênese da histoplasmose. Vários grupos de pesquisa têm demonstrado que PGD2 e PGE2 podem ter ações biológicas distintas quanto à remoção de microrganismos no hospedeiro. Desta maneira, é fundamental o entendimento do papel da PGD2 e da PGE2 nos mecanismos efetores dos macrófagos na defesa do hospedeiro, especialmente na histoplasmose. Portanto, o objetivo deste estudo foi investigar a participação da PGD2 e PGE2 na infecção experimental por H. capsulatum. Assim, demonstramos que a PGD2 aumentou a fagocitose e mecanismos microbicidas de macrófagos alveolares infectados in vitro com H. capsulatum. Observamos ainda que a 15dPGJ2, metabólito da PGD2, aumentou somente a fagocitose, e PGE2 inibiu os mecanismos efetores do macrófago. Mostramos ainda o aumento de BLT1 em macrófagos alveolares após adição de PGD2, e a possível ligação desta ao BLT1, e de LTB4 em DP2. Além disso, caracterizamos micropartículas de PLGA contendo PGD2 (MS-PGD2), e investigamos seus efeitos. O tamanho, carga elétrica e morfologia das micropartículas foram adequados para um tratamento intranasal e para fagocitose por macrófagos alveolares. As MS-PGD2 foram fagocitadas e capazes de ativar NF-B, e consequentemente, influenciar na produção de nitrito, IL-1, TNF-, IL-6 e TGF-. Com base nestes dados, avaliamos os efeitos do tratamento da MS-PGD2 ou da MS-PGE2 em animais infectados com H. capsulatum. Estas foram administradas via intranasal em animais infectados e tratados ou não com celecoxibe. Verificamos a diminuição da carga fúngica nos pulmões e baço, diminuição do infiltrador celular no espaço broncoalveolar e de citocinas inflamatórias no pulmão após tratamento com MS-PGD2. Contrariamente, após tratamento da MS-PGE2 observamos maior carga fúngica nos pulmões e baço, e aumento da inflamação no tecido e maior produção de IL-10. Além disso, demonstramos que no 21° dia após infecção, referente ao 7° dia após o término do tratamento com MS-PGD2, a carga fúngica manteve-se reduzida nos pulmões, comprovando assim a eficácia deste tratamento. Posteriormente, utilizando inibidores específicos, HQL-79 e CAY10526, mostramos respectivamente o papel protetor da PGD2 e o deletério da PGE2 na histoplasmose. Em conjunto, nossos dados contribuíram para o entendimento das funções antagônicas da PGD2 e PGE2 nesta micose. / Histoplasma capsulatum is a pathogenic dimorphic fungus and responsible for severe pulmonary lesions. Infection is acquired by inhalation of conidia and posterior conversion to yeasts in the alveoli and bronchioles, in which they are phagocyted by resident alveolar macrophages and leukocytes that migrate to the local infection. Recently, we demonstrate that mice infected by H. capsulatum and treated with inhibitor of prostaglandins synthesis presented a decrease in fungal burden in lungs and spleen, increase in nitrite production and uptake of yeasts by alveolar macrophages, and more survival, when compared with animals only infected. However, in this study, it was not determined what subtypes of prostaglandins participate in pathogenesis of histoplasmosis. Many research groups have demonstrated that PGD2 and PGE2 can have different biological effects regarding to microorganisms elimination in the host. Thus, it is primordial the understanding about the role of PGD2 and PGE2 on effector mechanisms of macrophages in host defense, especially in histoplasmosis. Therefore, the aim of this study was to investigate the role of PGD2 and PGE2 on experimental infection by H. capsulatum. So, we verify that PGD2 increased the uptake and microbicidal mechanisms of alveolar macrophages infected in vitro by H. capsulatum. 15dPGJ2, a PGD2 metabolite, increased only the phagocytosis, and PGE2 inhibited the effector mechanisms of macrophages. Among these results, we showed an increase of BLT1 expression on alveolar macrophages after addition of PGD2, and a possible binding of this mediator to BLT1, and of LTB4 to DP2. Later, as tool of therapeutic investigation, we used PGD2 encapsulation in biodegradable polymer, PLGA, in order to preserve its stability. Size, zeta potential and morphology were adequate for a possible intranasal treatment and uptake by alveolar macrophages. MS-PGD2 were phagocyted and able to activate NF-B, and consequently, to modulate nitrite, IL-1, TNF-, IL-6 and TGF- production. In this context, we purpose a treatment of the infection with MS-PGD2, in comparison to treatment with PGE2. MS-PGD2 were administrated via intranasal in infected mice, treated or not with celecoxib. We verify a decrease of fungal burden in lungs and spleen, less cellular infiltrate and decrease of some inflammatory cytokines. In contrast, after treatment of MS-PGE2, we observed greater fungal burden in the lungs and spleen, and an increase of the tissue inflammation and production of IL-10. Furthermore, we show that on day 21 after infection, referring to the 7th day after the treatment with MS-PGD2, fungal burden remained reduced in the lungs, thus proving the effectiveness of the treatment. Subsequently, using specific inhibitors, HQL-79 and CAY10526, respectively show the protective role of PGD2 and in deleterious to PGE2 in histoplasmosis. Together, our data contribute to the understanding of the antagonistic functions of PGD2 and PGE2 in this mycosis.

Les sensibilisants de contact sont des molécules réactives électrophiles qui ont la capacité de modifier des protéines de la peau pour former un antigène. Au delà de ce mécanisme d'hapténisation, le signal de danger induit par les sensibilisants conduisant à l'activation des cellules dendritiques (DC) est un élément déterminant dans l'induction de cellules T spécifiques de l'haptène. Dans le contexte du 7ième amendement à la directive cosmétique européenne, la mise en place d'une batterie de tests in vitro permettant de prédire le potentiel sensibilisant de molécules est indispensable pour l'industrie cosmétique. Tandis que la plupart des études in vitro étudient les signaux de danger induits par les sensibilisants dans des modèles homéostasiques, nous nous sommes intéressés à l'effet des sensibilisants sur la mise en place d'une réponse inflammatoire. Lorsque la lignée U937 est différenciée avec du PMA et stimulée avec du LPS, les facteurs de transcription NF-κB et Nrf2 sont activés et l'acide arachidonique (AA) est métabolisé au travers de la cascade cPLA2 / COX-2. L'ensemble de ces voies activées conduit à la production par les U937 d'un grand nombre de médiateurs inflammatoires (IL-1β, TNF-α, IL-6, IL-10, IL-8, PGE2, PGD2, TxB2). Dans ce modèle, nous avons analysé l'effet de 6 sensibilisants de potentiels variés (DNCB, PPD, HQ, PG, CIN, EUG) et montré que de façon inattendue, tous les sensibilisants étudiés diminuent significativement et de façon spécifique la production de tous les prostanoïdes et en particulier de PGE2 induite par PMA/LPS. Nous avons de plus démontré que selon les sensibilisants, les cibles de cette inhibition au sein de la cascade métabolique de l'AA diffèrent, même si elles se focalisent la plupart du temps (sauf pour le DNCB) sur l'enzyme COX-2 (inhibition de son expression et/ou de son activité). Pour le DNCB, le mécanisme d'inhibition semble plutôt impliquer sa capacité à réagir fortement avec les groupements résidus thiols, ce qui se traduit en particulier par la déplétion du GSH intracellulaire et engendrerait l'inhibition des synthases dépendantes du GSH pour leurs activités. En parallèle de cette étude mécanistique, nous avons appréhendé la problématique du point de vue statistique et vérifié sur un set plus important et diversifié de molécules (160 molécules) que le paramètre « inhibition de PGE2 » pouvait être un bon test de prédiction de l'HSRC. L'étude statistique a permis de déterminer le modèle prédictif du test PGE2 et de mettre en évidence de bonnes performances (78%) par rapport aux prédictions du LLNA. Au-delà, une certaine complémentarité du test PGE2 avec d'autres tests in vitro (MUSST, Nrf2-HTS) a pu être mise en évidence. En conclusion, au travers de cette étude, nous avons pu mettre en évidence de nouvelles propriétés biochimiques des sensibilisants. Même si la signification biologique de la diminution de PGE2 par les sensibilisants de contact demeure complexe d'interprétation, ce paramètre a permis le développement d'un test qui prédit avec de bonnes performances le caractère sensibilisant de molécules et dont la position au sein d'une batterie prédictive d'évaluation de l'allergie de contact reste à être précisée. / Contact sensitizers are defined as reactive molecules (electrophilic) which have the ability to modify skin proteins to form an antigen (hapten). In addition to the haptenation mechanism, danger signals, leading to the activation of dendritic cells, are described to be crucial for the effective induction of an hapten-specific T cell immune response. In the context of the 7th amendment to the Cosmetic Directive, the cosmetic industry is concerned by the challenge of finding non-animal approaches to assess the sensitizing potential of chemicals. While danger signals induced by sensitizers in steady-state conditions have already been analyzed, we chose to investigate the impact of sensitizers on the course of an inflammatory response. For this purpose we used the U937 cell line differentiated with PMA and activated with LPS. In these conditions, cells produce a large amount of inflammatory mediators (IL-β, TNF-α, IL-6, IL-10, IL-8, PGE2, PGD2, TxB2) through the activation of pathways leading to the activation of the transcription factors NF-κB and Nrf2 and through AA metabolism by the cPLA2/COX-2 cascade. Interestingly, we showed that 6 contact sensitizers with various potential (DNCB, PPD, HQ, PG, CIN, EUG) significally and specifically decrease the production of prostanoïds and in particular of PGE2 induced by PMA/LPS. We further demonstrated that there is no unique inhibition profile of the sensitizers even if the majority (except for DNCB) of the effects applies on COX-2 (i.e. inhibition of the expression and/or activity). For DNCB, inhibition mechanism appears to be dependant of its capacity to react with thiols residues and in particular to deplete intracellular glutathione possibly leading to the inactivation of the PG-synthases. In parallel, we assess a statistical analysis on 160 molecules that allow us to define the test parameters (a molecule is a sensitizer if the PGE2 inhibition at 24h is more than 60%) and to calculate the test performance toward LLNA (78%). Moreover we demonstrated that the PGE2 test could be complementary to other already existing in vitro tests like MUSST or Nrf2-HTS. In summary, we add here a new insight into the multiple biochemical effects described so far for sensitizers. Even if the underlying biological relevance remains unclear, the parameter “PGE2 inhibition” is good test for skin sensitization evaluation. Further studies will precise how this parameter could be implemented into an alternative testing strategy for the evaluation of skin sensitization.

Diese Arbeit verdeutlicht die Wirkung von Chondroitinsulfat auf die Synthese von Prostaglandin E2 in humanen mesenchymalen Stromazellen in Abhängigkeit ihres Sulfatierungsgrades. MSC zeichnen sich durch ihre antiinflammatorischen Eigenschaften aus und haben damit einen modulierenden Effekt auf Wundheilungsprozesse. Als Vorläuferzellen von Osteoblasten sind sie direkt an der Knochenneubildung beteiligt. Eine persistierende Entzündung hat eine kontinuierliche Freisetzung von Zytokinen, wie IL-1 zur Folge. Es konnte gezeigt werden, dass IL-1 in hMSC zu einer Freisetzung von PGE2 führt. Unter kurzzeitiger Wirkung stimuliert PGE2 die Knochenneubildung. Eine langanhaltende Präsenz leitet dagegen die Bildung des Faktors RANKL, einen die Osteoklastogenese stimulierenden Faktor, ein. Seit langem ist der positive Effekt von Chondroitinsulfat in chronischen Entzündungsprozessen, wie Rheumatoider Arthritis, bekannt. Zudem werden sie in aktuellen Studien als Beschichtungsbestandteile von Knochenimplantaten verwendet. Sie führten hier zu einer besseren Bioinduktivität und Biokonduktivität. Bisher ist dennoch der molekulare Wirkmechanismus nicht genau beschrieben. Die Schwierigkeit besteht darin, dass die molekularen Signalkaskaden für die einzelnen Kulturmoldelle Unterschiede aufweisen und kein ubiquitärer Mechanismus dargestellt wird. In hMSC führte die Stimulation mit IL-1 unter vorheriger Zugabe von Chondroitinsulfat zu einer Reduktion der PGE2 Freisetzung. Der Effekt des hochsulfatierten sCS3 war gegenüber dem nativen C4S verstärkt. Die reduzierende Wirkung von C4S setzte verzögert ein. Es ist bereits bekannt, dass die negative Ladung der CS zu einer Bindung von Zytokinen führt. Dadurch wird eventuell die Konzentration der Zytokine, wie IL-1 im Bereich der Zellrezeptoren erniedrigt und führt zu einer verringerten Stimulation der Zelle. Denkbar ist auch die Beeinflussung der intrazellulären Signaltransduktionskaskade durch die Bindung der CS an einen speziellen, bisher unbekannten, Membranrezeptor. Die entscheidenden Enzyme der PGE2 Synthese sind die Cyclooxygenase-2 (Cox-2) und die mikrosomale Prostaglandin E Synthase 1 (mPGES1). Die mRNA beider Enzyme war unabhängig vom Sulfatierungsgrad der CS reduziert. Dieser Effekt konnte auf Protein-ebene nicht belegt werden. Die produzierte Proteinmenge an mPGES1 wird durch IL-1 induziert, bleibt aber auch durch Zugabe von CS unverändert. Somit kann von einer erhöhten Translationseffizient und mRNA Stabilität der mPGES1 RNA ausgegangen werden. MAPK Kinasen sind entscheidende Schnittstellen bei der Regulation der mRNA Stabilität als auch der Aktivität von Transkriptionsfaktoren. In dieser Studie konnte die MAPK p38 als entscheidendes Enzym bei der Wirkung von CS auf die PGE2 Synthese ermittelt werden. Dabei führten sowohl das natürliche C4S als auch das hochsulfatierte sCS3 zu einer verringerten Aktivierung. Der Transkriptionsfaktor NfkB ist einer von mehreren, die an den Promotorbereichen der beiden induzierbaren PGE2 Enzyme, Cox-2 und mPGES1, binden. Es ist anzunehmen, dass die hier aufgezeigte verringerte Aktivität von NfkB als auch die verhinderte Translokation in den Zellkern eine reduzierte Transkription der jeweiligen mRNA bedingten. Abhängig vom untersuchten Modell und den verwendeten Kulturbedingungen können diese Prozesse moduliert sein. Die Erkenntnisse dieser experimentellen Arbeit liefern einen weiteren wichtigen Baustein zum Verständnis der molekularbiologischen Abläufe während entzündlicher Prozesse. Die Verwendung von Chondroitinsulfat, insbesondere hochsulfatiertes CS, in Kombination mit hMSC kann gezielt zu einer Verringerung der Entzündungsreaktion während der Implantateinheilung führen. Die durch PGE2 hervorgerufenen Symptome, wie erhöhte Gefäßpermeabilität, Schwellung und verstärktes Schmerzempfinden begründen diese positiven Effekte.

Este estudo compara quatro diferentes intensidades com o volume total similar no exercício supino. Avaliou-se a dor muscular de início tardio (DMIT), atividade de creatina quinase (CK), as concentrações sangüíneas de interleucina (IL)-1<font face=\"symbol\">b, IL-6, fator de necrose tumoral-<font face=\"symbol\">a (TNF-<font face=\"symbol\">a), prostaglandina E2 (PGE2) e o perfil hormonal. A amostra foi composta de soldados do exército brasileiro, divididos em cinco grupos: 50%-1RM, 75%-1RM, 90%-1RM, 110%-1RM e o controle. A DMIT e a atividade plasmática de CK aumentaram significativamente (P<0,05) após a sessão de exercício. A concentração de PGE2 também teve aumento significativo (P<0,05) após a sessão (P<0,05). A concentração plasmática de cortisol após 1h do término do exercício aumentou apenas no grupo 75%-1RM (p < 0,05). Esses resultados sugerem que a intensidade no exercício supino não afeta a magnitude da DMIT, marcadores de lesão muscular, inflamação e na resposta hormonal geral, desde que haja a equalização do volume total, com exceção da concentração plasmática do cortisol, grupo 75%-1RM. / This study compared four different intensities with similar total volume of a bench press exercise for muscle soreness, creatine kinase (CK) activity, interleukin (IL)-1<font face=\"symbol\">b, IL-6, tumor necrosis factor-<font face=\"symbol\">a (TNF-<font face=\"symbol\">a), prostaglandin E2 (PGE2) and hormonal concentrations in the blood. Brazilian Army male soldiers were placed into five groups: 50%-1RM, 75%-1RM, 90%-1RM, and 110%-1RM, and control that did not perform the exercise. Muscle soreness and plasma CK activity increased significantly (p<0.05) after exercise. Serum PGE2 concentration also increased significantly (p<0.05) after exercise. After one hour post exercise cortisol increased in 75%-1RM group, with this response also exceeding the other intensities (p<0.05). These results suggest that the intensity of bench press exercise does not affect the magnitude of muscle soreness and blood markers of muscle damage, inflammation and largely similar hormonal responses, which may be attributed to the equalization of total volume, exception made for the 75%-1RM group for serum cortisol concentration.