The doctoral dissertacion is focused on the role of inflammation in the pathogenesis of preterm labor. In the first part, we describe the current view on pathophysiology of preterm labor. In the second part, we evaluated the relationship of specific markers of inflammation and endothelial dysfunction to preterm birth and fetal inflammatory response. The most important findings of our study was that we found decreased levels of MMP-2 and decreased levels of sRAGE in women with preterm labor in comparison with the control group of pregnant women. Similarly, we found decreased levels of MMP-2 in women with subsequent diagnosed fetal inflammatory response. sRAGE is currently ranked among patttern recognition receptors. In the case of sRAGE we followed the results of our pilot project, it can be assumed that the its low level are connected with tissue damage. We confirmed that it can play an important role in the pathogenesis of preterm labor. We assume abnormal regulatory mechanisms of the production of MMP-2. In both cases, however, further studies are required to elucidate the functional significance of our results.

A lesão de isquemia e reperfusão pulmonar (LIRI) é a principal causa de disfunção precoce do enxerto após o transplante pulmonar. Diferentes substâncias têm sido utilizadas na tentativa de proteger o pulmão na fase inicial pós-transplante e melhorar o desempenho do enxerto com pouco resultado tanto a curto quanto em longo prazo. O peptídeo liberador gastrina (GRP) está envolvido na indução de respostas imunes inatas adaptativas por induzir a quimiotaxia de mastócitos, migração de macrófagos e proliferação de células T e fibroblastos. Este agente foi capaz de proteger o dano tecidual em outros modelos experimentais de inflamação, incluindo sepsis pós-isquemia e perfuração de ceco, artrite e colite induzidos por drogas. O efeito protetor foi atribuído ao bloqueio na liberação de citocinas pró-inflamatórias, como da interleucina -1 beta (IL-1G) e TNF- alfa (TNF-H), e inibição de migração de mononucleares. Nesta tese o efeito protetor do antagonista do receptor de peptídeo liberador de gastrina (GRPR), o RC-3095 (RC) endovenosamente administrado antes e depois da reperfusão foi estudado em modelo animal de LIRI. Vinte ratos Wistar foram randomizados em quatro grupos: (SHAM), Isquemia-reperfusão (IR), administração de RC Pré-IR (RC-Pré) e RC Pós-IR (RC-Pós). Após a reperfusão, os animais foram observados por 120 minutos. Foi realizada a análise histológica, com verificação de escala de lesão pulmonar, e imunohistoquímica para Caspase clivada - 9 e oxido nítrico sintetase endotelial (eNOS). Expressão de IL-1G e TNF-H foram quantificadas por Enzime-Linked Immunosorbent Assay (ELISA). Uma redução significativa na pressão arterial média foi observada nos grupos IR e RC - Pré quando comparado com seus valores antes da reperfusão (p < 0.001). No final do experimento o grupo RC-Pós apresentou uma redução significativa na pressão arterial de oxigênio em relação ao início (p=0.005). Observou-se maior expressão de Caspase - 9 clivada no grupo RC-Pós (p>0.013), quando comparado aos demais grupos. Não houve diferença significativa na captação da eNOS entre os grupos (p=0.206). Os grupos RC - Pré e Pós mostraram discreta redução, não atingindo significância estatística, da IL-1G (p=0.159) e TNF- H (p= 0.260), quando comparado com o grupo IR. Não houve diferença estatística entre as médias dos escores histológicos entre os diferentes grupos. e SHAM. Notavelmente, quando administrado após reperfusão parece potencializar dano celular estimulando a apoptose. Concluindo, a utilização do antagonista do peptídeo liberador de gastrina, RC-3095, não produziu efeito protetor significativo na resposta inflamatória em pulmões de ratos. Estudos adicionais devem ser implementados para confirmar estes achados. / Lung ischemia reperfusion injury (LIRI) is the main cause of early graft dysfunction and death after lung transplantation. Several methods and substances have been used as an attempt to protect the lung during the early phase after transplantation and improve graft performance at short and long term with limited results. The selective gastrin-releasing peptide receptor (GRPR) antagonist RC-3095 (RC) was shown to protect from damage in various models of inflammation, including sepsis due to cecal ischemia and perforation, drug-induced arthritis and colitis, probably due to the inhibition in the release of proinflammatory cytokines, such as interleukin-1-beta and TNFalpha, as well as interference with mononuclear migration. In this thesis, the protective effect of the selective gastrin-releasing peptide receptor (GRPR) antagonist RC-3095, administrated intravenous before and after reperfusion was studied in an animal model of LIRI. A total of 20 Wistar rats were randomized in four groups: (SHAM), Ischemia-reperfusion (IR), RC Pre-IR (RCPre) and the RC Post-IR (RC-Post) .The IR groups were submitted to a model of ischemia/reperfusion by clamping of the left pulmonary hilum for 45 minutes followed by reperfusion for 120 minutes. We recorded the hemodynamic parameters, blood gas analysis and histology. We analyzed Cleaved Caspase- 9 and endothelial nitric oxide synthase (eNOS) by immunohistochemistry. Expression of IL-1G and TNF-H was evaluated by Enzyme-Linked Immunosorbent Assay (ELISA). We observed that the final mean arterial pressure significantly decreased in IR and RC Pre-IR compared to their values before reperfusion (p < 0.001).The RC-Post group showed significant decrease of partial pressure of arterial oxygen at the end of the observation compared to baseline (p=0.005). Caspase-9 activity was significantly higher in the RC-Post- IR as compared to the other groups (p < 0.013). No significant differences were observed of eNOS activity among the groups. The groups RC Pre-IR e RC Post-IR did not show a significant decrease of IL-1G (p=0.159) and TNF-H (p= 0.260), as compared to IR. The histological score showed no significant differences among the groups. Notably, its use after reperfusion seems to potentiate cell damage, stimulating apoptosis In conclusion, RC3095, a selective GRPR antagonist failed to demonstrate a significant protective effect in our LIRI model in male Wistar rats. Further studies to address this question are warranted.

Este estudo teve como objetivo avaliar, in vivo, por um período de 24h, a resposta tecidual inflamatória subcutânea após a utilização de cimentos endodônticos. Realizouse a seleção de 75 ratos, machos, da linhagem Wistar, de aproximadamente 90 dias (de vida) e com cerca de 350g (de peso), que foram aleatoriamente distribuídos em 5 grupos (n=20), como se segue: G1 - ActiV GPTM, G2 - Biosealer, G3 - RealSeal®, G4 - RoekoSeal® e G5 - AH Plus®. A resposta inflamatória foi observada pelo teste edemogênico, nos tempos 6, 12 e 24 horas. Como protocolo de aplicação da metodologia, os animais depois de anestesiados, receberam uma injeção do corante vital Azul de Evans na veia lateral caudal. Em seguida, foram tricotomizados em seu dorso e, doses iguais dos materiais estudados foram inseridas subcutaneamente. Ao final de cada período de tempo, realizou-se a remoção da pele dorsal em fragmentos de 15 mm de diâmetro. Os fragmentos foram imersos em solução de formamida P.A., armazenados em estufa a 37ºC, por 48 horas. Nesse momento, os índices de absorbância do corante extraído pelo solvente foram quantificados por meio de espectrofotometria, os resultados convertidos em microgramas (?g) e a concentração de cada substância no infiltrado inflamatório obtida. Os dados obtidos foram submetidos a ANOVA e teste de Tukey (p<0,05). E, de acordo com os resultados obtidos, observou-se que houve diferenças estatisticamente significativas para os fatores em estudo material (p<0,0001) e tempo (p<0,0001), entretanto não houve interação material*tempo (p=0,128). Sendo assim, nas primeiras 6h, a ordem decrescente de irritabilidade dos materiais analisados foi: G3 ? G5 > G1 > G2 > G4. Entretanto, os materiais desempenharam-se de forma semelhante, tanto no tempo de 12h quanto no de 24h, como se segue: G1 ? G3 ? G5 > G2 ? G4. Desta forma, pôde-se concluir que: 1 - o cimento RoekoSeal demonstrou o menor grau de irritabilidade no tempo experimental de 6 horas; 2 - Os cimentos RealSeal e AH Plus apresentaram resposta inflamatória mais intensa no período experimental de 6 horas e 3 - o índice de irritabilidade nas primeiras 6 horas é significativamente superior quando comparado ao período de 24h, independente do material testado. / The aim of this study was evaluate in vivo the earliest 24 hours tissue inflammatory response to five endodontic sealers. In order to this, 75 male rats Wistar with 90 days of age and approximately 350g of weight were divided in 5 experimental groups (n=20): G1 - ActiV GPTM, G2 - Biosealer, G3 - RealSeal®, G4 - RoekoSeal® e G5 - AH Plus®. The inflammatory response was evaluated using the edemogenic test in the experimental periods of 06, 12 and 24 hours. Anesthetized mice received an Evan´s blue dye injection in the lateral caudal vein and were tricotomized in the dorsum where equal amounts of each sealer were subcutaneously inoculated. After experimental period, the mice were sacrified, the dorsal skin was excised and pieces with 15mm of diameter around each inoculated sealer were removed. Each tissue piece was reduced to fragments, immersed in formamide solution and stored at 37ºC during 48 hours. At this moment, the absorbance index of the dye extracted by the solvent was quantified using light absorption spectrometry, the results were converted to micrograms (?g) and their concentration in the inflammatory infiltrated was obtained. The data was submitted to statistical analysis using the ANOVA followed by the Tukey test (p<0.05) and the results showed statistical significant difference for analyzed factors, the material and the time (p<0.001); without interaction between both (p=0.128). Thus, in decrescent order inflammatory response, the groups were scaled: G3 ? G5 > G1 > G2 > G4 for 6 hours, and G1 ? G3 ? G5 > G2 ? G4 for 12 hours and 24 hours. Our findings indicated that: 1 - The RoekoSeal presented the minor inflammatory response value in the 6 hours experimental period; 2 - The RealSeal and AH Plus sealers showed intense inflammatory response in the experimental period of 6 hours; 3 - The inflammatory values at 6 hours were significantly higher compared with results in the period of 24 hours independently of the tested material.

A lesão de isquemia e reperfusão pulmonar (LIRI) é a principal causa de disfunção precoce do enxerto após o transplante pulmonar. Diferentes substâncias têm sido utilizadas na tentativa de proteger o pulmão na fase inicial pós-transplante e melhorar o desempenho do enxerto com pouco resultado tanto a curto quanto em longo prazo. O peptídeo liberador gastrina (GRP) está envolvido na indução de respostas imunes inatas adaptativas por induzir a quimiotaxia de mastócitos, migração de macrófagos e proliferação de células T e fibroblastos. Este agente foi capaz de proteger o dano tecidual em outros modelos experimentais de inflamação, incluindo sepsis pós-isquemia e perfuração de ceco, artrite e colite induzidos por drogas. O efeito protetor foi atribuído ao bloqueio na liberação de citocinas pró-inflamatórias, como da interleucina -1 beta (IL-1G) e TNF- alfa (TNF-H), e inibição de migração de mononucleares. Nesta tese o efeito protetor do antagonista do receptor de peptídeo liberador de gastrina (GRPR), o RC-3095 (RC) endovenosamente administrado antes e depois da reperfusão foi estudado em modelo animal de LIRI. Vinte ratos Wistar foram randomizados em quatro grupos: (SHAM), Isquemia-reperfusão (IR), administração de RC Pré-IR (RC-Pré) e RC Pós-IR (RC-Pós). Após a reperfusão, os animais foram observados por 120 minutos. Foi realizada a análise histológica, com verificação de escala de lesão pulmonar, e imunohistoquímica para Caspase clivada - 9 e oxido nítrico sintetase endotelial (eNOS). Expressão de IL-1G e TNF-H foram quantificadas por Enzime-Linked Immunosorbent Assay (ELISA). Uma redução significativa na pressão arterial média foi observada nos grupos IR e RC - Pré quando comparado com seus valores antes da reperfusão (p < 0.001). No final do experimento o grupo RC-Pós apresentou uma redução significativa na pressão arterial de oxigênio em relação ao início (p=0.005). Observou-se maior expressão de Caspase - 9 clivada no grupo RC-Pós (p>0.013), quando comparado aos demais grupos. Não houve diferença significativa na captação da eNOS entre os grupos (p=0.206). Os grupos RC - Pré e Pós mostraram discreta redução, não atingindo significância estatística, da IL-1G (p=0.159) e TNF- H (p= 0.260), quando comparado com o grupo IR. Não houve diferença estatística entre as médias dos escores histológicos entre os diferentes grupos. e SHAM. Notavelmente, quando administrado após reperfusão parece potencializar dano celular estimulando a apoptose. Concluindo, a utilização do antagonista do peptídeo liberador de gastrina, RC-3095, não produziu efeito protetor significativo na resposta inflamatória em pulmões de ratos. Estudos adicionais devem ser implementados para confirmar estes achados. / Lung ischemia reperfusion injury (LIRI) is the main cause of early graft dysfunction and death after lung transplantation. Several methods and substances have been used as an attempt to protect the lung during the early phase after transplantation and improve graft performance at short and long term with limited results. The selective gastrin-releasing peptide receptor (GRPR) antagonist RC-3095 (RC) was shown to protect from damage in various models of inflammation, including sepsis due to cecal ischemia and perforation, drug-induced arthritis and colitis, probably due to the inhibition in the release of proinflammatory cytokines, such as interleukin-1-beta and TNFalpha, as well as interference with mononuclear migration. In this thesis, the protective effect of the selective gastrin-releasing peptide receptor (GRPR) antagonist RC-3095, administrated intravenous before and after reperfusion was studied in an animal model of LIRI. A total of 20 Wistar rats were randomized in four groups: (SHAM), Ischemia-reperfusion (IR), RC Pre-IR (RCPre) and the RC Post-IR (RC-Post) .The IR groups were submitted to a model of ischemia/reperfusion by clamping of the left pulmonary hilum for 45 minutes followed by reperfusion for 120 minutes. We recorded the hemodynamic parameters, blood gas analysis and histology. We analyzed Cleaved Caspase- 9 and endothelial nitric oxide synthase (eNOS) by immunohistochemistry. Expression of IL-1G and TNF-H was evaluated by Enzyme-Linked Immunosorbent Assay (ELISA). We observed that the final mean arterial pressure significantly decreased in IR and RC Pre-IR compared to their values before reperfusion (p < 0.001).The RC-Post group showed significant decrease of partial pressure of arterial oxygen at the end of the observation compared to baseline (p=0.005). Caspase-9 activity was significantly higher in the RC-Post- IR as compared to the other groups (p < 0.013). No significant differences were observed of eNOS activity among the groups. The groups RC Pre-IR e RC Post-IR did not show a significant decrease of IL-1G (p=0.159) and TNF-H (p= 0.260), as compared to IR. The histological score showed no significant differences among the groups. Notably, its use after reperfusion seems to potentiate cell damage, stimulating apoptosis In conclusion, RC3095, a selective GRPR antagonist failed to demonstrate a significant protective effect in our LIRI model in male Wistar rats. Further studies to address this question are warranted.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-03-27T14:12:27Z No. of bitstreams: 2 Tese - Lilhian Alves de Araújo - 2017.pdf: 5322101 bytes, checksum: 57793fec5ba7f799bab9153daa5b7952 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-03-27T14:41:27Z (GMT) No. of bitstreams: 2 Tese - Lilhian Alves de Araújo - 2017.pdf: 5322101 bytes, checksum: 57793fec5ba7f799bab9153daa5b7952 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-27T14:41:27Z (GMT). No. of bitstreams: 2 Tese - Lilhian Alves de Araújo - 2017.pdf: 5322101 bytes, checksum: 57793fec5ba7f799bab9153daa5b7952 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-06 / Sepsis is an infectious disease characterized by severe systemic inflammatory response. Rupture of the complex equilibrium between inflammatory mediators in the acute phase of the disease leads to exacerbated production of proinflammatory cytokines, with consequent hypotension, increased capillary permeability, organ lesions and death. Recent research using the Hev b 13 protein derived from natural Hevea brasiliensis (rubber tree) latex has demonstrated important anti-inflammatory and immunomodulating effects. The aim of this study was to investigate the effects of Hev b 13 on the systemic and tissue inflammatory response of septic rats. To that end, cecal ligation and puncture (CLP) was performed on male Wistar rats and after six hours, the animals were randomized into groups and subcutaneously treated with doses of 0.5; 2.0 and 3.0 mg/Kg of Hev b 13. Next, the animals were subdivided into three different times (1, 6 and 24 hours after treatment) for blood sample collection and euthanasia with removal of the lungs and liver. Leukocytes, tumor necrosis factor- alpha (TNF-α) plasma and tissue levels, interleukin (IL)-6, IL-10, and IL-4 and histological slides were analyzed. The results demonstrated that treatment with the Hev b 13 protein prompted a significant decline in total and differential leukocytes, as well as the production of TNF-α and IL-6, associated with an increase in IL-10 and IL-4 in plasma and lung tissue. Moreover, it restricted the morphopathological changes found in the lungs, including neutrophil infiltration, swelling and alveolar thickening. In the liver, it increased IL-10 production and inhibited TNF-α and IL-6, in addition to reducing hemorrhage, sinusoidal inflammatory infiltrates and hydropic degeneration in histological assessment. We conclude that Hev b 13 displays anti-inflammatory and immunomodulating activity capable of attenuating lung and liver lesions in rats during acute sepsis, with potential for clinical applications. / Sepse é uma doença infecciosa caracterizada por uma resposta inflamatória sistêmica grave. A ruptura do complexo equilíbrio entre os mediadores inflamatórios na fase aguda da doença leva a uma produção exacerbada de citocinas pró-inflamatórias, com conseguinte hipotensão, aumento da permeabilidade capilar, lesões de órgãos e morte. Pesquisas recentes utilizando a Hev b 13, proteína derivada do látex natural da Hevea brasiliensis (seringueira), tem demonstrado importantes efeitos anti-inflamatórios e imunomoduladores. O objetivo deste estudo foi investigar os efeitos da Hev b 13 na resposta inflamatória sistêmica e tecidual de ratos com sepse. Para isso, foi realizado ligadura e perfuração do ceco (LPC) em ratos machos Wistar e, após seis horas, os animais foram randomizados em grupos e tratados com as doses 0,5; 2,0 e 3,0 mg/Kg de Hev b 13 subcutâneo. Posteriormente, subdividiu-se os animais em três tempos diferentes (1, 6 e 24 horas após os tratamentos) para coleta de amostras sanguíneas e eutanásia com remoção dos pulmões e fígado. Foram analisados os leucócitos, concentrações plasmáticas e teciduais de fator de necrose tumoral-α (TNF-α), interleucina (IL) 6, IL- 10, IL-4 e lâminas histológicas. Os resultados demonstraram que no tratamento com a proteína Hev b 13 houve redução significativa dos leucócitos totais e diferenciais bem como na produção de TNF-α e IL-6, associado ao aumento de IL-10 e IL-4 no plasma e tecido pulmonar. Além disso, restringiu as alterações morfopatológicas encontradas nos pulmões, incluindo infiltrado de neutrófilos, edema e espessamento alveolar. No fígado, aumentou a produção de IL-10 e inibiu TNF-α e IL-6, além de reduzir hemorragia, infiltrado inflamatório sinusoidal e degeneração hidrópica na avaliação histológica. Concluímos que a Hev b 13 possui atividade anti-inflamatória e imunomoduladora capaz de atenuar lesões nos pulmões e fígado de ratos durante a sepse aguda, com potencialidades para aplicabilidade clínica.

INTRODUÇÃO: A doença pneumocócica invasiva (DPI) tem alta mortalidade sendo o pulmão órgão de intenso acometimento. Na DPI caracterizou-se localmente importante processo inflamatório agudo com expressivo aumento de macrófagos, polimorfonucleares e fenômenos exsudativos como edema e hemorragia intra-alveolar. Concretizou-se uma resposta inflamatória proeminente com redução dos fenômenos de apoptose que se traduziu por aumento significativo de citocinas pró-inflamatórias, exceto IL-6 e IL-8, aumento de Toll-2, ativação do complemento, aumento de expressão de ICAM- 1 e CD 14 que em conjunto favorecem o estabelecimento dos fenômenos inflamatórios. A diminuição significativa das células NK e das células de Langherhans, IL-6 e IL-8 reflete comprometimento da imunidade inata. Tal comprometimento poderia ser responsável pela diminuição dos linfócitos TCD4+ e TCD8+ com consequente baixa produção de IFN. Em resumo, as lesões teciduais graves na DPI seriam decorrentes do comprometimento parcial da imunidade inata, em especial das células NK e das células de Langherhans, do prejuízo da imunidade adaptativa e da redução da apoptose como possível estratégia defensiva do pneumococo / INTRODUCTION: Invasive pneumococcal disease (IPD) is a condition with high mortality rates, the lungs being intensely attacked. The in situ immune response was determined, in blocks recovered from 22 necropsies of adults who died from IPD in the lungs, by quantitative immune cell phenotype (CD57-NK, CD1a, CD68, antigen S-100, TCD4, TCD8, CD20), Complement-C3, ICAM-1, CD14, Caspase-3 and cytokine (interferon , TNF, TGF, interleukin - IL-1, IL-2, IL-4, IL-6, IL-8, IL- 10), Toll-2 and SP-A (surfactant). A locally important acute inflammation process was characterized in IPD, with significant rise in macrophages, neutrophils and exsudative phenomena such as edema and intra-alveolar hemorrhage. Compared with the lungs from age-matched controls, results from patients with IPD showed significant depletion of NK, CD1a,CD4+, CD8+, CD20+ cells, interferon , IL-4, IL- 6 , IL-8, TGF and Caspase-3 (apoptosis). On the other hand, S-100, Toll-2, IL-1, IL-2R, IL-10, ICAM-1, CD14 and SP-A were more frequently seen in the alveoli of patients with IPD than in controls. A pronounced inflammatory response was detected, with decrease in apoptosis phenomena that translated into significant increase of pro-inflammatory cytokines, except for IL-6 and IL-8, increase in Toll-2, complement activation, increased ICAM-1 and CD-14 expression, which altogether favored installation of the inflammatory processes. A significant decrease in NK and Langherhans cells, IL-6 and IL-8 reflect the harm to the innate immune system. This could respond for the decrease in TCD4+ and TCD8+ lymphocytes, with a consequent low IFNy output. Briefly, the severe tissue lesions in IPD could be a consequence of the partial damage to the innate immunity, particularly of NK and Langherhans cells, of adaptive immune dysfunction, and of apoptosis reduction possibly as a defense strategy of the pneumococcus

Tous les êtres vivants présentent un arsenal de défenses contre les pathogènes, et la réponse inflammatoire constitue le processus initial de cette défense, qui s’achève par la réparation des tissus lésés. Paradoxalement, un processus inflammatoire prolongé est également associé à de nombreuses pathologies comme l’athérosclérose, l’asthme, les maladies auto-immunes mais aussi certains cancers. Le recrutement excessif de leucocytes au site de l’inflammation est un processus commun à ces pathologies. Dès lors, la compréhension et la maîtrise du phénomène complexe et finement orchestré de la migration sélective des populations leucocytaires, appelée chimiotactisme, sont des enjeux majeurs de la recherche médicale contemporaine. <p>Les récepteurs aux peptides formylés bactériens et mitochondriaux (FPRs) forment la première famille de récepteurs chimiotactiques identifiée. Elle comprend trois membres, FPR1, 2 et 3, présentant un haut niveau de similitude et partageant certains de leurs multiples ligands. Le troisième membre de ce groupe, FPR3, reste actuellement le moins bien connu. Récemment, un agoniste de FPR3, affin et spécifique, a été identifié dans le laboratoire. Il s’agit du peptide F2L, qui correspond aux 21 premiers acides aminés de la protéine intracellulaire HEBP1.<p><p>Dans le cadre de ce travail de thèse, nous nous sommes attelé à la caractérisation approfondie du récepteur FPR3 et son ligand peptidique F2L. <p>Dans un premier temps, et à l’aide d’anticorps validés dans le cadre de ce travail, nous avons montré que le peptide F2L induit le chimiotactisme d’un ensemble de populations leucocytaires qui expriment FPR3, dont les sous-populations de macrophages des poumons, du colon et de la peau, les éosinophiles et les cellules dendritiques plasmacytoïdes. Cette distribution suggère, pour FPR3, une fonction dans la réponse inflammatoire. <p>Nous avons pu montrer ensuite que F2L peut être généré par la protéolyse de son précurseur, HEBP1, sous l’action de la cathepsine D des macrophages. La cathepsine D est une aspartique protéase lysosomiale impliquée dans l’homéostasie cellulaire, les processus apoptotiques et inflammatoires physiologiques et pathologiques, et dans le développement tumoral. Il s’agit désormais d’identifier dans quel compartiment et sous quelles conditions F2L est produit et sécrété. <p>Enfin, parallèlement à ces travaux, nous avons démontré que la cathepsine G, une sérine protéase contenue dans les granules azurophiles des neutrophiles, active également le récepteur FPR3. Des résultats préliminaires suggèrent un mode d’activation alternatif du récepteur, impliquant la protéolyse d’un troisième partenaire et la génération d’un agoniste actuellement non identifié. <p><p>Le couple FPR3-F2L semble dès lors impliqué dans l’induction ou la résolution de la réponse inflammatoire en recrutant les éosinophiles, monocytes, macrophages et cellules dendritiques au site de la lésion. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

It is a challenge to assess the toxicity of environmental air particulate matter (PM) because PM composition is complex and variable, due to source contribution and atmospheric transformation. The goal of this study is to establish an in vitro model that can fingerprint the cytotoxic effects of airborne PM and their associated toxicity mechanisms. For this purpose, the cytotoxic effects of different reference and environmental particles on A549 human lung epithelial cells were characterized using multiple endpoint assays (cytokine release, LDH release, BrdU incorporation, cellular ATP and resazurin reduction) and proteomic analyses (2D-GE and MALDI-TOF-TOF-MS/MS). The results of this study demonstrated that proteomic analyses can distinguish the influences of different (carbon black and titanium dioxide) and similar (cristobalite and α-quartz) particles on various pathways in A549 cells (e.g., cell death and cell proliferation); and the cytotoxicity assays were capable of differentiating the phenotypic outcomes of the particles, which were complementary and supportive to pathway analyses. The ability of in vitro toxicoproteomics to differentiate the toxicity of environmental particles was tested on Ottawa urban dust (EHC-93) and its water-insoluble and soluble fractions. Findings from both cytotoxicity assays and proteomic analyses consistently indicated that the insoluble materials explained most of the toxic effects of the total PM. Interestingly, the toxic potency of EHC-93 total was not equal to the sum of its insoluble and soluble fractions, implying inter-component interactions between insoluble and soluble materials that may be reflected through synergistic or antagonistic in vitro responses. The insoluble and soluble fractions uniquely altered the expression patterns of the proteins involved in pathways such as cell death, cell proliferation and inflammation. For example, the insoluble and soluble fractions oppositely altered the expression of the proteins (e.g., TREM1, PDIA3, PKM and ENO1) involved in an inflammatory response pathway in A549 cells, and the insoluble fraction was more potent than the soluble fraction in increasing secretion of pro-inflammatory cytokines MCP-1 and IL-8 from A549 cells. In essence, in vitro toxicoproteomics is a valuable tool in relating the physicochemical characteristics of ambient air particles to their biological reactivity through understanding their mechanisms of toxicity.

L'intestin est la première barrière de l'organisme contre les contaminants alimentaires, dont les mycotoxines. Le déoxynivalenol (DON) est un contaminant majeur des céréales, souvent retrouvé en association avec d'autres trichothécènes B (TCTs B), le 3- et 15-acétyldéoxynivalénol (3-ADON et 15-ADON), le nivalénol (NIV) et la fusarénone X (FX). Au niveau cellulaire, le DON interagit avec l'ARN ribosomique, bloquant ainsi la synthèse protéique et activant la cascade de la voie de signalisation de MAPKinases impliquée dans des mécanismes de la réponse inflammatoire. Au niveau intestinal, cette mycotoxine pourrait donc perturber le renouvellement continu de l'épithélium, et l'homéostasie de la réponse inflammatoire. On suggère ainsi qu'elle pourrait jouer un rôle dans la pathogénie des maladies inflammatoires chroniques de l'intestin. Si les effets du DON sont relativement connus, ceux du NIV et de leurs dérivés acétylés sont moins bien documentés. De même, peu de données existent quant à la toxicité combinée de ces mycotoxines dont la co-occurrence est avérée. Sur des modèles in vitro de cellules épithéliales intestinales humaines et porcines et sur un modèle ex vivo d'explants de jéjunum de porc, nous avons comparé les toxicités individuelles de cinq TCTs B (DON, 3- et 15-ADON, NIV et FX) et analysé leur toxicité combinée en termes de synergie, additivité ou antagonisme vis-à-vis de l'intestin. Les résultats montrent qu'à des concentrations de l'ordre du micromolaire, les TCTs B inhibent la croissance des cellules épithéliales intestinales par ordre croissant de toxicité 3-ADON, DON, 15-ADON, NIV et FX. Aux faibles doses correspondant à des niveaux d'exposition rencontrés chez le consommateur français ou européen, des synergies d'un facteur 3 à 10 ont été observées. Ces travaux ont également permis de caractériser l'activité pro-inflammatoire au niveau intestinal des TCTs B, et l'analyse benchmark de données de transcriptomique a montré que l'exposition de l'intestin à des doses aussi faibles que 0.04µM de FX, 0.1µM de DON ou 0.1µM de NIV s'accompagne d'une activation significative des mécanismes de l'inflammation. Ces doses sont de l'ordre des concentrations attendues dans le chyle sur la base des valeurs toxicologiques de référence actuelles. En conclusion, ces données montrent que le renouvellement de l'épithélium intestinal et l'activité pro-inflammatoire au niveau intestinal pourraient être des marqueurs très sensibles dans le cadre de l'évaluation de la toxicité individuelle et des interactions entre TCTs B. / As for other food-born contaminants, the gastro-intestinal tract represents the first barrier against deoxynivalenol (DON). This mycotoxin frequently co-occurs with other type B trichothecenes (TCTs B) namely 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon-X (FX). At the cellular level, DON binding to ribosomal RNA results in the inhibition of protein synthesis and triggers the mitogen-activated protein kinases (MAPKs) pathway that have been linked to immune response mechanisms. Thus, intestinal epithelial cell renewing is considered a putative target in DON toxicity. Moreover, based on the ability of DON to disturb the state of homeostasis of the inflammatory response in the intestine mimicking what is found in inflammatory bowel diseases (IBD), it is proposed that this mycotoxin may play a role in such diseases. However, very few is known about the intestinal toxicity of the other co-occuring TCT B, and their combined effects eventually. By means of in vitro human and porcine intestinal epithelial cells models and an ex vivo porcine jejunal explants model, we assessed the individual toxicity of five TCT B (DON, 3- and 15-ADON, NIV and FX) toward the intestine and we analyzed their combined toxicity in terms of additivity, synergy or antagonism. The tested TCT B significantly impaired the intestinal epithelial cell growth in the micromolar range, in increasing order of potency 3-ADON, DON, 15-ADON, NIV and FX. The toxicity of low doses of TCT B was synergistic. For mycotoxin concentrations corresponding to exposure levels reported for French and European consumers, the amplitude of this synergy ranged between 3 and 10. Benchmark dose analyses of the transcriptional data also showed that the exposure of the intestine to mycotoxin concentrations as low as 0.04µM for FX, 0.1µM for DON and 0.1µM for NIV could be associated to a significant activation of the inflammatory response mechanisms. Taken together, these results suggest that epithelial cell renewing and pro-inflammatory effects at the intestinal level may be consider very sensitive biomarkers for the assessment of the individual toxicity and interactions between the co-occurring TCTs B.

A Coagulação Intravascular Disseminada (CID) é uma síndrome caracterizada pela ativação sistêmica da coagulação sanguínea, levando à trombose microvascular difusa e podendo comprometer a função de múltiplos órgãos. O acelerado consumo de plaquetas e fatores de coagulação pode, no entanto, dar origem a um estado de hipocoagulabilidade, o que confere à CID uma característica paradoxal na qual o excesso de coagulação pode causar uma diátese hemorrágica. Doenças que levam à Síndrome de Resposta Inflamatória Sistêmica (SIRS) estão entre os principais gatilhos da CID. A pancreatite é uma dessas doenças. O maior desafio para o médico veterinário é diagnosticar a CID na fase precoce, silenciosa e de hipercoagulabilidade, visto que os testes laboratoriais de rotina, como contagem de plaquetas, tempo de protrombina (TP) e tempo de tromboplastina parcial ativada (TTPA), detectam apenas o estado de hipocoaguabilidade, que se estabelece na fase mais avançada da síndrome. Nesse contexto ganham importância os analisadores tromboelastográficos, equipamentos que avaliam a coagulação em sangue total e que, ao menos em tese, podem informar a velocidade de formação do coágulo, a força máxima que ele atinge e os padrões de sua dissolução. Este estudo é o primeiro realizado em cães com o aparelho ReoRox G2 (MediRox), uma da marcas disponíveis no mercado. Limites de referência para as variáveis do aparelho foram definidos a partir da análise do sangue de 49 animais clinicamente saudáveis para três tipos de reação: acelerada com fator tecidual (TF), acelerada com TF e um antagonista de agregação plaquetária (abciximab) e apenas com sangue recalcificado. Em seguida, foram comparados a esse intervalo de referência os valores obtidos pela análise tromboelastográfica do sangue de seis pacientes com pancreatite recém-diagnosticada. Nos três tipos de reação pelo menos 50% dos pacientes do Grupo Pancreatite apresentaram alterações sugestivas de hipercoagulabilidade. A variável MAXELAST (força máxima do coágulo) foi a que esteve alterada com mais frequência entre os animais doentes. Não houve alteração nos marcadores de velocidade de fibrinólise. Estudos prospectivos que associem outras variáveis de trombose, protocolos de tratamento e prognóstico de pacientes com doenças subjacentes que predisponham à CID são necessários para que se possa afirmar que o traçado obtido pela tromboelastografia realmente representa um estado de hipercoagulabilidade in vivo em pacientes com pancreatite. / Disseminated Intravascular Coagulation (DIC) is a syndrome characterized by systemic activation of blood clotting, leading to diffuse microvascular thrombosis and may compromise multiple organ function. The accelerated consumption of platelets and coagulation factors may, however, originate a state of hypocoagulability, which gives the DIC a paradoxical characteristic in which excess coagulation can lead to a hemorrhagic diathesis. Diseases which cause Systemic Inflammatory Response Syndrome (SIRS) are among the major triggers of DIC, including pancreatitis. The greatest challenge for veterinarians is to diagnose DIC in the early, silent and hypercoagulable phase, since routine laboratory tests, such as platelet count, prothrombin time (PT) and activated partial thromboplastin time (APTT), detect only the state of hypocoagulability, which occurs in the most advanced stage of the syndrome. In this context, thromboelastography analyzers stand out. They are equipment which evaluate coagulation in whole blood and, at least in theory, inform the speed of clot formation, its maximum force and how it dissolves. This is the first study performed in dogs with the ReoRox G2 (MediRox), one of the brands available in the market. Limits of reference were defined from blood analysis of 49 healthy animals for three reaction types: accelerated with tissue factor (TF), accelerated with TF and a platelet aggregation antagonist (abciximab) and with only recalcified blood. Next, values obtained by blood thromboelastographic analysis of six patients with newly diagnosed pancreatitis were compared to this reference range. In all three types of reactions, at least 50% of patients in the Pancreatitis Group presented alterations suggestive of hypercoagulability. The variable MAXELAST (maximum clot strength) was the one that was most frequently altered among ill animals. There was no change in fibrinolysis rate markers. Prospective studies associating other thrombosis variables, treatment protocols, and prognosis of patients with underlying diseases predisposing to DIC are necessary to confirm that the pathway obtained by thromboelastography actually represents a state of hypercoaguability in vivo in patients with pancreatitis.