Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-12Bitstream added on 2014-06-13T20:21:27Z : No. of bitstreams: 1 takahagi_en_dr_botib.pdf: 614550 bytes, checksum: a7740b5af41df8726b1c4a6469ca54e5 (MD5) / Receptores Toll-like RECONHECER componentes distintos da FUNGOS para iniciar a resposta imune inata. Examinamos se Paracoccidioides brasiliensis OU SEUS gp43 antígenos imunodominantes pode modular IN VITRO E TLR2 TLR4 expressão e produção TRIGGER citocinas por monócitos humanos. Monócitos de indivíduos saudáveis ​​foram incubadas com gp43 LYPOPOLYSACCHARIDE, (LPS) ou mortas pelo calor FORMAS leveduriforme do P. brasiliensis em uma proporção de 50 monócitos por célula fúngica (Pb18) a 37 º C por 4h e 18h. A EXPRESSÃO DE TLR2 E TLR4 SOBRE SUPERFÍCIE monócitos, e TNF-alfa, IL-10 e IL-12p40 PRODUÇÃO foram determinados por citometria de fluxo e ELISA respectivamente. Os resultados mostraram que a estimulação de monócitos COM OU LPS Pb18 PROMOVIDO UP-regulação da expressão TLR2 E TLR4 SOBRE SUPERFÍCIE EM RELAÇÃO monócitos às células não-estimulados EM AMBAS AS 4H e 18h de cultura, e aumentaram os níveis de TNF-alfa, IL- 10 e IL-12p40 PRINCIPALMENTE EM 18H DA CULTURA. POR OUTRO LADO, E EXPRESSÃO TLR4 BAIXA ALTA TLR2 são evocados por gp43 AT 4H DA CULTURA, associadas a altos níveis de TNF-alfa. No entanto, após 18H UMA MUDANÇA DE ALTA E BAIXA TLR2 TLR4 EXPRESSÕES foi seguido por elevados níveis de IL-10 e IL-12p40. Estes resultados sugerem que a gp43 pode induzir um desequilíbrio entre RESPOSTAS pró e anti-inflamatório em FUNGOS-monócitos interações, um efeito modulatório na CAMINHO TLR. AS TNF-alfa pode estar envolvida na patogênese da paracoccidioidomicose, O EFEITO regulatórios induzidos por gp43, VIA upregulation de TLR2 EXPRESSÃO E IL-10 produção, pode ser importante para proteger contra a lesão de tecido que é descrito neste micose. / Toll-like receptors recognize distinct components of fungi to initiate the innate immune íesponse. We examined whether Paracoccidioides brasiliensis ar its immunodominant antigen gp43 can modulate in vitro TLR2 and TLR4 expression and trigger cytokine production by human monocytes. Monocytes from healthy individuaIs were incubated with gp43, IypopoIysaccharide (LPS) or heat-killed yeast forms of P. brasiliensis in a ratio of 50 monocytes per funga! cell (pb 18) at 37°C for 4h and I8h. The expression of TLR2 and TLR4 on monocyte surface, and TNF-alpha, IL-Io and IL-I2p4o production were determined by flow cytometry and ELISA respectiveIy. The results showed that monocyte stimuIation with LPS ar Pb18 promoted up-reguIation ofthe TLR2 and TLR4 expression on monocyte surface in reIation to the non-stimuIated cells at both 4h and I8h ofculture, and induced higher IeveIs ofTNF-aIpha, IL-Io and IL-12p4o mainly at I8h of culture. On the other hand, high TLR4 and low TLR2 expression were elicited by gp43 at 4h of cuIture, associated with higher Ievels of TNF-aIpha. However, after 18h a change to high TLR2 and !ow TLR4 expressions was followed by e1evated levels of IL-Io and IL-12p4o. These results suggest that gp43 might induce an imbalance between pro- and anti-inflammatory responses in fungal-monocyte interactions by a modulatory effect on TLR pathway. As TNF-alpha may be invoIved in the pathogenesis of paracoccidioidomycosis, the regulatory effect induced by gp43, via upregulation of TLR2 expression and IL-Io production, can be important to protect against tissue injury which is described in this mycosis.

IL-17 promotes inflammation through the recruitment of monocytes and induction of various chemokines and inflammatory cytokines. Monocytes respond to IL-17 through the heteromeric IL-17 receptor (IL-17R) composed of subunits IL-17RA and IL-17RC. Together, monocytes and IL-17 amplify inflammation. Controlling the cellular response to IL-17 is crucial to prevent hyperactivation of inflammatory responses, which could lead to chronic inflammatory diseases. The cellular response to increased IL-17 levels may be limited by controlling the receptor levels. Before we understand how monocytes respond to IL-17 during infection, we must first characterize the expression of IL-17R in these cells in response to LPS, a well-characterized pro-inflammatory signal. The aim of this study is to understand the mechanisms which regulate IL-17R levels in human monocytes. IL-17R mRNA and protein levels were measured in response to LPS by RT-PCR and Western blot analysis in primary human monocytes, peripheral blood mononuclear cells (PBMC), and the human monocytic cell line, THP-1. LPS enhanced IL-17RA and RC transcript levels in monocytes and PBMC. In contrast, IL-17RA protein levels decreased with LPS treatment in these cells. Investigation into mechanisms regulating IL-17RA protein levels lead to the observation that IL-17RA undergoes receptor degradation in response to LPS. This work identifies for the first time that 1) LPS enhances transcript levels of IL-17R and 2) after LPS treatment, IL-17RA protein levels are reduced via an endosome-dependent degradation pathway. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-06-21 11:53:28.706

Orientador: Maria Teresinha Serrão Peraçoli / Banca: Maria de Lourder Ribeiro de Souza da Cunha / Banca: Vanessa Lara / Banca: Iracilda Z. Carlos / Banca: Sueli Calvi / Resumo: Receptores Toll-like RECONHECER componentes distintos da FUNGOS para iniciar a resposta imune inata. Examinamos se Paracoccidioides brasiliensis OU SEUS gp43 antígenos imunodominantes pode modular IN VITRO E TLR2 TLR4 expressão e produção TRIGGER citocinas por monócitos humanos. Monócitos de indivíduos saudáveis ​​foram incubadas com gp43 LYPOPOLYSACCHARIDE, (LPS) ou mortas pelo calor FORMAS leveduriforme do P. brasiliensis em uma proporção de 50 monócitos por célula fúngica (Pb18) a 37 º C por 4h e 18h. A EXPRESSÃO DE TLR2 E TLR4 SOBRE SUPERFÍCIE monócitos, e TNF-alfa, IL-10 e IL-12p40 PRODUÇÃO foram determinados por citometria de fluxo e ELISA respectivamente. Os resultados mostraram que a estimulação de monócitos COM OU LPS Pb18 PROMOVIDO UP-regulação da expressão TLR2 E TLR4 SOBRE SUPERFÍCIE EM RELAÇÃO monócitos às células não-estimulados EM AMBAS AS 4H e 18h de cultura, e aumentaram os níveis de TNF-alfa, IL- 10 e IL-12p40 PRINCIPALMENTE EM 18H DA CULTURA. POR OUTRO LADO, E EXPRESSÃO TLR4 BAIXA ALTA TLR2 são evocados por gp43 AT 4H DA CULTURA, associadas a altos níveis de TNF-alfa. No entanto, após 18H UMA MUDANÇA DE ALTA E BAIXA TLR2 TLR4 EXPRESSÕES foi seguido por elevados níveis de IL-10 e IL-12p40. Estes resultados sugerem que a gp43 pode induzir um desequilíbrio entre RESPOSTAS pró e anti-inflamatório em FUNGOS-monócitos interações, um efeito modulatório na CAMINHO TLR. AS TNF-alfa pode estar envolvida na patogênese da paracoccidioidomicose, O EFEITO regulatórios induzidos por gp43, VIA upregulation de TLR2 EXPRESSÃO E IL-10 produção, pode ser importante para proteger contra a lesão de tecido que é descrito neste micose. / Abstract: Toll-like receptors recognize distinct components of fungi to initiate the innate immune íesponse. We examined whether Paracoccidioides brasiliensis ar its immunodominant antigen gp43 can modulate in vitro TLR2 and TLR4 expression and trigger cytokine production by human monocytes. Monocytes from healthy individuaIs were incubated with gp43, IypopoIysaccharide (LPS) or heat-killed yeast forms of P. brasiliensis in a ratio of 50 monocytes per funga! cell (pb 18) at 37°C for 4h and I8h. The expression of TLR2 and TLR4 on monocyte surface, and TNF-alpha, IL-Io and IL-I2p4o production were determined by flow cytometry and ELISA respectiveIy. The results showed that monocyte stimuIation with LPS ar Pb18 promoted up-reguIation ofthe TLR2 and TLR4 expression on monocyte surface in reIation to the non-stimuIated cells at both 4h and I8h ofculture, and induced higher IeveIs ofTNF-aIpha, IL-Io and IL-12p4o mainly at I8h of culture. On the other hand, high TLR4 and low TLR2 expression were elicited by gp43 at 4h of cuIture, associated with higher Ievels of TNF-aIpha. However, after 18h a change to high TLR2 and !ow TLR4 expressions was followed by e1evated levels of IL-Io and IL-12p4o. These results suggest that gp43 might induce an imbalance between pro- and anti-inflammatory responses in fungal-monocyte interactions by a modulatory effect on TLR pathway. As TNF-alpha may be invoIved in the pathogenesis of paracoccidioidomycosis, the regulatory effect induced by gp43, via upregulation of TLR2 expression and IL-Io production, can be important to protect against tissue injury which is described in this mycosis. / Doutor

Made available in DSpace on 2014-06-11T19:31:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-12Bitstream added on 2014-06-13T19:01:46Z : No. of bitstreams: 1 graciani_apb_dr_botfm.pdf: 427562 bytes, checksum: b481f2e6f558c5bae2f08ca356c1ed60 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Paracoccidioides brasiliensis (Pb), agente etiológico da paracoccidioidomicose, é um fungo dimórfico que sobrevive no interior de monócitos/macrófagos humanos não ativados. Estudos anteriores em nosso laboratório têm demonstrado que os monócitos humanos não ativados são incapazes de realizar atividade fungicida, e esse processo está associado com a capacidade do fungo induzir a produção de prostaglandinas (PGs), uma vez que, essas células são capazes de realizar atividade fungicida significativa após o tratamento com indometacina (INDO), um inibidor da produção de ciclooxigenase. No entanto, o processo de pré-ativação com IFN-γ, resulta em um parcial efeito compensatório sobre os efeitos inibidores das PGs, principalmente quando essas células são desafiadas com a cepa de baixa virulência do fungo. Assim, a proposta deste presente estudo foi avaliar se a ativação de monócitos humanos com outras citocinas como TNF-α e GM-CSF resulta em um efeito similar ao observado com IFN-γ. Uma outra questão a ser respondida é se esse processo poderia estar associado com alterações nos níveis de H2O2 e NO, que são moléculas efetoras envolvidas na atividade fungicida contra o P. brasiliensis, bem como nos níveis das citocinas TNF-α, IL-10 e IL-6. Culturas de monócitos do sangue periférico, obtidos de 20 indivíduos normais foram tratadas somente com INDO ou ativados com IFN-γ, TNF-α ou GM-CSF na presença ou ausência de INDO por 18h, e posteriormente desafiados com cepas de alta (Pb18) ou baixa (Pb265) virulência do P. brasiliensis por 4h. Após esse período, as culturas foram avaliadas quanto à atividade fungicida, produção de H2O2 e NO e expressão de mRNA para enzima óxido nítrico sintase (iNOS) por RT-PCR em tempo real. As concentrações de TNF-α, IL-6 e IL-10 nos sobrenadantes das coculturas foram avaliadas por ELISA. Nossos resultados... / Paracoccidioides brasiliensis (Pb), the etiological agent of paracoccidioidomycosis, is a dimorphic fungus that survives within nonactivated human monocytes/macrophages. Previous studies have demonstrated that the lack of fungicidal activity by nonactivated human monocytes is associated to fungus capacity to inducing prostaglandins release, since a significative fungicidal activity was detected after monocytes treatment with indomethacin (INDO), a cyclooxigenase inhibitor. However, cells activation with IFN-γ seems to partially compensating this inhibitory effect, mainly when cells were challenged with low virulent strain of the fungus. Here, we extended our studies, addressing whether monocytes activation with other cytokines such as TNF-α and GM-CSF results in a similar effect to that observed with IFN-γ. Moreover, we asked if this process could be associated with alterations on H2O2 and NO levels, the molecules involved in Pb killing, as well as in the levels of the cytokines TNF-α, IL-10 and IL-6. Peripheral blood monocytes obtained from 18 healthy donors were treated only with INDO or activated with IFN-γ, TNF-α or GM-CSF in presence or absence of INDO for 18h, and further challenged with high (Pb18) or low (Pb265) virulent strain of Pb for 4h. After, cultures were evaluated for fungicidal activity, H2O2 and NO release and expression of inducible nitric oxide synthase (iNOS) mRNA by real-time RT-PCR. The concentrations of TNF-α, IL-6 and IL-10 on supernatants of cocultures were evaluated by ELISA. Our results provided evidence that human monocytes challenged with both strains of P. brasiliensis release prostaglandins that via induction of IL-10 and IL-6 inhibits TNF-α production. This process results in defective cell activation with consequent release of low H2O2 levels and lack of fungicidal activity by cells. However the inhibitory effect of PGs may be... (Complete abstract click electronic access below)

No description available.

<p>Prostaglandin (PG) E₂ is an eicosanoid derived from the polyunsaturated twenty carbon fatty acid arachidonic acid (AA). PGE₂ has physiological as well as pathophysiological functions and is known to be a key mediator of inflammatory responses. Formation of PGE₂ is dependent upon the activities of three specific enzymes involved in the AA cascade; phospholipase A₂ (PLA₂), cyclooxygenase (COX) and PGE synthase (PGEs). Although the research within this field has been intense for decades, the regulatory mechanisms concerning the PGE₂ synthesising enzymes are not completely established.</p><p>PGE₂ was investigated in human monocytes with or without lipopolysaccharide (LPS) pre-treatment followed by stimulation with calcium ionophore, opsonised zymosan or phorbol myristate acetate (PMA). Cytosolic PLA₂a (cPLA₂a) was shown to be pivotal for the mobilization of AA and subsequent formation of PGE₂. Although COX-1 was constitutively expressed, monocytes required expression of COX-2 protein in order to convert the mobilized AA into PGH₂. The conversion of PGH₂ to the final product PGE₂ was to a large extent due to the action of microsomal PGEs-1 (mPGEs-1). In addition, experiments with inhibitors of extracellular signal regulated kinase and p38 activation, indicated that phosphorylation of cPLA₂α was markedly advantageous for the formation of PGE₂.</p><p>Ellagic acid, a natural polyphenolic compound found in fruits and nuts, was shown to inhibit stimuli induced release of PGE₂ in human monocytes. The effect of ellagic acid was not due to a direct effect on the activities of the enzymes but rather to inhibition of the LPS-induced protein expression of COX-2, mPGEs-1 and cPLA₂a.</p>

Resumo: Paracoccidioides brasiliensis (Pb), agente etiológico da paracoccidioidomicose, é um fungo dimórfico que sobrevive no interior de monócitos/macrófagos humanos não ativados. Estudos anteriores em nosso laboratório têm demonstrado que os monócitos humanos não ativados são incapazes de realizar atividade fungicida, e esse processo está associado com a capacidade do fungo induzir a produção de prostaglandinas (PGs), uma vez que, essas células são capazes de realizar atividade fungicida significativa após o tratamento com indometacina (INDO), um inibidor da produção de ciclooxigenase. No entanto, o processo de pré-ativação com IFN-γ, resulta em um parcial efeito compensatório sobre os efeitos inibidores das PGs, principalmente quando essas células são desafiadas com a cepa de baixa virulência do fungo. Assim, a proposta deste presente estudo foi avaliar se a ativação de monócitos humanos com outras citocinas como TNF-α e GM-CSF resulta em um efeito similar ao observado com IFN-γ. Uma outra questão a ser respondida é se esse processo poderia estar associado com alterações nos níveis de H2O2 e NO, que são moléculas efetoras envolvidas na atividade fungicida contra o P. brasiliensis, bem como nos níveis das citocinas TNF-α, IL-10 e IL-6. Culturas de monócitos do sangue periférico, obtidos de 20 indivíduos normais foram tratadas somente com INDO ou ativados com IFN-γ, TNF-α ou GM-CSF na presença ou ausência de INDO por 18h, e posteriormente desafiados com cepas de alta (Pb18) ou baixa (Pb265) virulência do P. brasiliensis por 4h. Após esse período, as culturas foram avaliadas quanto à atividade fungicida, produção de H2O2 e NO e expressão de mRNA para enzima óxido nítrico sintase (iNOS) por RT-PCR em tempo real. As concentrações de TNF-α, IL-6 e IL-10 nos sobrenadantes das coculturas foram avaliadas por ELISA. Nossos resultados... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Paracoccidioides brasiliensis (Pb), the etiological agent of paracoccidioidomycosis, is a dimorphic fungus that survives within nonactivated human monocytes/macrophages. Previous studies have demonstrated that the lack of fungicidal activity by nonactivated human monocytes is associated to fungus capacity to inducing prostaglandins release, since a significative fungicidal activity was detected after monocytes treatment with indomethacin (INDO), a cyclooxigenase inhibitor. However, cells activation with IFN-γ seems to partially compensating this inhibitory effect, mainly when cells were challenged with low virulent strain of the fungus. Here, we extended our studies, addressing whether monocytes activation with other cytokines such as TNF-α and GM-CSF results in a similar effect to that observed with IFN-γ. Moreover, we asked if this process could be associated with alterations on H2O2 and NO levels, the molecules involved in Pb killing, as well as in the levels of the cytokines TNF-α, IL-10 and IL-6. Peripheral blood monocytes obtained from 18 healthy donors were treated only with INDO or activated with IFN-γ, TNF-α or GM-CSF in presence or absence of INDO for 18h, and further challenged with high (Pb18) or low (Pb265) virulent strain of Pb for 4h. After, cultures were evaluated for fungicidal activity, H2O2 and NO release and expression of inducible nitric oxide synthase (iNOS) mRNA by real-time RT-PCR. The concentrations of TNF-α, IL-6 and IL-10 on supernatants of cocultures were evaluated by ELISA. Our results provided evidence that human monocytes challenged with both strains of P. brasiliensis release prostaglandins that via induction of IL-10 and IL-6 inhibits TNF-α production. This process results in defective cell activation with consequent release of low H2O2 levels and lack of fungicidal activity by cells. However the inhibitory effect of PGs may be... (Complete abstract click electronic access below) / Orientador: Ângela Maria Victoriano de Campos Soares / Coorientador: Luciene Alarcão Dias Melicio / Banca: Maria Terezinha Serrão Peraçoli / Banca: João pessoa Araújo Junior / Banca: Heloisa Blotta / Banca: Gil Bernard / Doutor

Prostaglandin (PG) E2 is an eicosanoid derived from the polyunsaturated twenty carbon fatty acid arachidonic acid (AA). PGE2 has physiological as well as pathophysiological functions and is known to be a key mediator of inflammatory responses. Formation of PGE2 is dependent upon the activities of three specific enzymes involved in the AA cascade; phospholipase A2 (PLA2), cyclooxygenase (COX) and PGE synthase (PGEs). Although the research within this field has been intense for decades, the regulatory mechanisms concerning the PGE2 synthesising enzymes are not completely established. PGE2 was investigated in human monocytes with or without lipopolysaccharide (LPS) pre-treatment followed by stimulation with calcium ionophore, opsonised zymosan or phorbol myristate acetate (PMA). Cytosolic PLA2a (cPLA2a) was shown to be pivotal for the mobilization of AA and subsequent formation of PGE2. Although COX-1 was constitutively expressed, monocytes required expression of COX-2 protein in order to convert the mobilized AA into PGH2. The conversion of PGH2 to the final product PGE2 was to a large extent due to the action of microsomal PGEs-1 (mPGEs-1). In addition, experiments with inhibitors of extracellular signal regulated kinase and p38 activation, indicated that phosphorylation of cPLA2α was markedly advantageous for the formation of PGE2. Ellagic acid, a natural polyphenolic compound found in fruits and nuts, was shown to inhibit stimuli induced release of PGE2 in human monocytes. The effect of ellagic acid was not due to a direct effect on the activities of the enzymes but rather to inhibition of the LPS-induced protein expression of COX-2, mPGEs-1 and cPLA2a.

Résumé : Durant les dernières années, l’encapsulation d’îlots pancréatiques endocriniens a reçu une grande attention parce qu’elle pourrait constituer une solution pour diminuer les taux d'échecs des transplantations. Dans le contexte de la perte de la matrice extracellulaire (MEC) native des îlots lors de leur isolation et le rejet de greffes par le système immunitaire du receveur, cette thèse vise à améliorer la compréhension des interactions entre la MEC et les cellules des îlots pancréatiques endocriniens ainsi qu’à étudier les effets de stress apoptotiques associés à des éléments du système immunitaire sur la survie et les fonctions des îlots. Ces études pourraient permettre de raffiner notre compréhension des mécanismes associés au rejet des greffes d'îlots de Langerhans. Dans cette thèse, le premier chapitre constitue une revue de la littérature permettant de mettre en lumière les rôles réciproques de la MEC dans l'action des cellules immunitaires et l'influence de ces rôles sur le diabète de type 1 (DT1) et sur la transplantation d'îlots. Ce premier chapitre a été publié dans la revue Pathologie Biologie. Le premier travail expérimental comprend la culture de cellules d'insulinomes de rat (INS-1) sur des surfaces composées de carboxyméthyl dextrane (CMD) recouvertes de fibronectine, RGD ou YIGSR, un peptide synthétique de la laminine. Dans cette étude, l'effet bénéfique d’éléments de la MEC sur ces cellules productrices d'insuline a été démontré. Les cellules INS-1 ont davantage proliféré sur ces surfaces et sécrétaient plus d’insuline que les cellules INS-1 cultivées sur les surfaces contrôle de CMD, CMD+RGE et dans les plaques à multi-puits de polystyrène vendues pour la culture tissulaire (TCPS). Cette première étude a été publiée dans Acta Biomaterialia. La deuxième étude expérimentale avait pour objectif d’étudier l’effet protecteur de gels de fibrine pour enrober des îlots pancréatiques endocriniens isolés de jeunes porcs et exposés à deux concentrations de peroxyde d'hydrogène (H[indice inférieur 2]O[indice inférieur 2]). L’enrobage dans la fibrine a permis de réduire l'apoptose chez les cellules des îlots et d’améliorer la sécrétion d'insuline par ceux-ci lorsque les résultats étaient comparés à ceux des îlots non-enrobés. Ce travail a été publié dans la revue Islets. Dans la troisième étude expérimentale, des îlots porcins étaient enrobés dans des gels de fibrine et d'alginate et exposés à des monocytes humains pour comparer l’effet de l’enrobage par ces deux matériaux sur la survie et les fonctions des îlots. Les monocytes sécrétaient des concentrations importantes de cytokines TNFα, IL-6, IL-1β en réponse à la fibrine seule et aux îlots. Les cellules des îlots enrobés dans les gels de fibrine et d'alginate étaient moins apoptotiques et sécrétaient plus d'insuline que leurs contrôles respectifs non-enrobés. Cette étude a été acceptée dans la revue Pathologie Biologie. // Abstract : In recent years, the encapsulation of endocrine pancreatic islets has received enhanced attention as it might constitute a solution for islet transplantation failure. In the context of the loss of the native islet extracellular matrix (ECM) and graft rejection by the recipient’s immune system, this thesis aims to improve the understanding of ECM-islet cell interactions and immune system-related implications in islet survival and function in the context of type 1 diabetes mellitus (T1DM) and islet graft rejection. In the first chapter, a literature review introduces the reciprocal roles of the ECM in immune cell action and the influence of these interactions on T1DM and islet transplantation. The most important ECM components are discussed followed by an overview of immune cells and their possible implication in diabetes. Immune cell integrins and cytokines and their communication with and influence on ECM are highlighted, concluding in a brief discussion of the significance of these interactions for islet transplantation and encapsulation. This review has been accepted for publication by Pathologie Biologie. The first experimental work comprises the culture of rat insulinoma cells (INS-1) on welldefined low-fouling carboxymethyl-dextran (CMD) surfaces covalently grafted with fibronectin, RGD and YIGSR, a synthetic laminin peptide, resulting in higher cell proliferation and insulin secretion of INS-1 cells when compared to the controls CMD, CMD+RGE and tissue culture polystyrene (TCPS) plates. With this work, the beneficial effect of ECM cues on insulin-producing cells was proven. This study has been published in Acta Biomaterialia. The second experimental work aimed to study the effect of fibrin gels when used to embed endocrine pancreatic islets isolated from young pigs and exposed to hydrogen peroxide (H[subscript 2]O[subscript 2]). Fibrin-embedded islets showed less apoptosis and higher relative insulin secretion than islets on TCPS, verifying the protective effect of fibrin towards islets. This study has been published in Islets. In the third experimental study, porcine islets were encapsulated in fibrin and alginate gels and exposed to human monocytes to compare the two materials and to further investigate the immune protective properties of fibrin and alginate. Monocytes secreted high concentrations of TNFα, IL-6, and IL-1β in response to fibrin, but at the same time islets in both fibrin and alginate gels were less apoptotic and secreted more insulin then their TCPS controls. This study has been submitted to Pathologie Biologie.

Les adénovirus humains (HAdVs) provoquent généralement une infection bénigne chez l'hôte sain. En revanche, chez les patients immunodéprimés et immunocompétents, ils peuvent causer des infections sévères à létales. Les vecteurs HAdVs sont couramment utilisés dans les domaines de la thérapie génique et de la vaccination. L'immunité pré-existante de l’hôte protège généralement contre les infections de type sauvage mais peut entraîner des effets indésirables lors du relargage des virus tel que l’induction de processus inflammatoire locale ou général. Après l'infection, l'inflammation va principalement entraîner le recrutement de cellules dendritiques (DCs), de monocytes et de neutrophiles. Les DCs ont la capacité unique de présenter l'antigène et d’activer les cellules T, qui, par la suite, aideront les cellules B à produire des anticorps. En plus de leurs activités phagocytaires, des peptides antimicrobiens dérivés des neutrophiles (AMPs) jouent un rôle central dans l'immunité innée. Les AMPs peuvent neutraliser les microbes infectieux et / ou activer différents types de cellules immunitaires. Dans ce contexte, nous avons étudié l'interaction ex vivo entre le facteur hôte (anticorps anti-HADV et AMPs) contre les HAdVs dans les DCs ainsi que le rôle des DCs activés indirectement (indir-DCs) lors de la réponse immunitaire. Nous avons caractérisé le profil des cytokines et des chimiokines sécrétées par les DCs stimulées par différents HAdVs, AMPs et les combinaisons qui en découlent. Enfin, nous avons constaté que l’opsonisation d’HAdV5 par les IgG accroît la capacité de capture antigénique des MoDCs (cellules dendritiques dérivées de monocytes) et induit la mort cellulaire par pyroptose. Je me suis donc concentrée sur les caractéristiques et la fonction des indir-DCs dans l’immunité contre les HAdVs. Afin de mieux comprendre les propriétés et les phénotypes des indir-DCs et DCs activés directement (dir-DCs), nous avons caractérisé leurs profils de maturation, les facteurs influençant cette maturation, et leur capacité fonctionnelle de recruter les leucocytes. Ainsi nous avons pu mettre en évidence que les dir-DCs empêchent le recrutement leucocytaire tandis que les indir-DCs favorisent la migration des monocytes. L’ensemble de ces données contribue à comprendre comment l'immunité pré-existante contre les HAdVs peut impacter l’efficacité des traitements contre les maladies HAdVs ainsi que la conception de vaccins. / Human adenoviruses (HAdV) generally cause mild infection in healthy host, but in immunocompromised and immunocompetent patients, they cause severe on lethal infections. HAdV vector are also commonly used for gene transfer and vaccination. We know that pre-existing immunity can protect from wild type infection and cause adverse effects during vector delivery including local and system inflammation. Following HAdV infection on vector use, inflammation leads to the recruitment of dendritic cells (DCs), monocyte and neutrophils. DCs have the unique ability to present antigens and activate T cells, that subsequently aid B cells to produce antibodies. In addition to their phagocytic activities, neutrophil-derived antimicrobial peptides (AMPs) play a central role in innate immunity. AMPs can kill invading microbes and/or activate various cell types. Here I studied the ex vivo interaction between host factor (anti-HAdV antibodies and AMPs) to HAdV in DCs and the role of indirect-activated DC (indir-DCs) in immune response. I characterized the profile of cytokines and chemokines of DC stimulated with different HAdV, AMPs and their combination. We recently found that IgG-oposonization of HAdV5 increase the update by MoDCs (monoctyes-derived dendritic cells) and induced pyroptotic cell death. I therefore focused on the characteristic and function of indir-DCs in anti-HAdV immunity. To better understand the properties and phenotypes of indir-DCs and direct-activated DCs (dir-DCs), we characterize their maturation profile, the factors influencing their maturation, and the functional ability to recruit leukocyte. We found that dir-DC prevent leucocyte recruitment while indir-DC increases monocyte migration. These data contribute to understand how the pre-existing immunity to HAdV impacts treatment for HAdV diseases and vaccine design.