Prion diseases are fatal infectious neurodegenerative disorders hypothesised to be caused by misfolding of the prion protein. Following prion infection, the infectious agent is sequestered to and replicates upon follicular dendritic cells (FDC) within lymphoid follicles prior to neuroinvasion. The mechanism of transport of the prion infectious agent from the site of infection to FDC is unknown. One of the postulated routes of transport is the specific migration of antigen presenting cells (APC) to FDC. APC specifically capture antigenic material and transport and present that material to effector cells and FDC in order to generate an appropriate acquired immune response. FDC reside within the B-cell follicle of secondary lymphoid organs. FDC organise and maintain the B-cell follicular structure by secretion of the chemokine CXCL13 which stimulates chemotactic movement of cells which express the CXCR5 receptor, e.g. B cells. Dendritic cells are specialised APC that are commonly characterised by their expression of CD11c. Transport of the prion infectious agent from the site of infection to FDC was observed to be blocked or severely delayed following depletion of CD11c+ cells. To determine whether CD11c+ cells acquire prions and subsequently deliver them to the FDC, the chemokine receptor CXCR5 was depleted from CD11c+ cells using a conditional transgenic mouse model. These mice were characterised for normal lymphoid organogenesis and monitored for their responses to oral infection with either prions or intestinal helminths. Data in this thesis show that the CD11c-mediated depletion of CXCR5 resulted in a delay in peripheral prion pathogenesis after oral exposure and significantly reduced disease susceptibility. These data suggest that efficient prion transport to FDC requires delivery by APC and is potentially mediated by CXCR5 chemotaxis. Following oral exposure to the intestinal helminth (Trichuris muris) CD11c-mediated depletion of CXCR5 prevented the establishment of a protective TH2 response. As a consequence the mice mounted a TH1-dominated response and were unable to clear the infection. These data also confirm that the effective generation of TH2 responses to oral helminth infection also requires APC localisation to B-cell follicles via CXCR5.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative diseases that affect several species, such as scrapie in sheep or goats and CJD in humans. In several species, neurological disease is preceded by TSE agent accumulation in lymphoid tissues prior to neuroinvasion. While oral transmission is considered the most common route for scrapie, transmission can also occur through lesions to the skin or mucosa, for example in the mouth or gastrointestinal tract due to rough feed, or birth associated skin damage. Scrapie has also been experimentally transmitted through skin scarification in mice. Following scrapie infection via skin scarification, PrPSc accumulates in the draining lymph node (LN) before spreading to other organs in the lymphoreticular system. It is not yet known by what means the scrapie agent is transported from the skin to the draining LN. Dendritic cells (DCs) in the skin have been found to transport viruses, such as HIV or Dengue, from the skin, thereby raising the question whether DCs or Langerhans cells (LCs), located within the epidermis, play a role in the uptake and transport of the TSE agent from the skin to the draining LN. CD11c is a cell surface marker traditionally used to identify or isolate DCs from other cell types. Mice and rats are naturally resistant to Diphtheria toxin (DTX). A transgenic mouse line was created where the Diphtheria toxin receptor (DTR) was expressed on CD11c+ cells. The presence of this receptor on CD11c+ cells allowed for the temporary conditional depletion of CD11c+ cells following a single injection of DTX. The cells repopulate the tissues within a time frame specific to the tissues the cells are located in. These mice were used to determine whether the absence of CD11c+ cells at the time of scrapie infection via the skin had an effect on the early accumulation of PrPSc within the lymphoid tissues and on disease progression. Immunohistochemical analysis demonstrated that early PrPSc accumulation in the draining LNs was delayed following depletion of CD11c+ cells, indicating that their potential role in the transport of the scrapie agent from the skin. Scrapie incubation period was not affected by the absence of the CD11c+ cells at the time of infection. Recent findings show that CD11c is not exclusive to DCs and is also expressed on macrophage populations. Following DTX-mediated depletion, DCs repopulate the tissues much faster than CD11c+ macrophages. Scrapie infection was carried out in the skin in DTX treated mice after DCs had repopulated the tissues but before macrophage numbers had returned, to determine whether macrophages rather than DCs played a role in the early accumulation of PrPSc in the draining LNs. No differences in PrPSc accumulation were observed in mice depleted of macrophages compared to controls and there was no effect on disease incubation period. Another transgenic mouse line was used, where DTX expression on langerin+ cells (LCs and langerin+ DCs in the dermis), allowed for their temporary depletion through DTX treatment. Following langerin+ cell depletion, increased PrPSc accumulation was observed in the draining LNs 7 weeks post infection, but did not affect the incubation period of disease. These results indicate that the absence of LCs somehow accelerated PrPSc accumulation, and that LCs might play a preventative role in early stages after infection. Histopathological analysis was used to complement microarray studies aimed to determine what immune responses were associated with scarification and DTXmediated depletion of cells within the skin and whether these responses might be linked to disease transmission. DCs and LCs in the skin appear to play different roles in the early stages following scrapie infection via the skin, but the lack of effect on incubation period does not rule out the involvement of other cell types or cell-free mechanisms of scrapie agent spread from the skin.

Terminally differentiated B cells are responsible for antibody generation, a key component of adaptive immunity. IgG antibodies play an important role in defence against infection but can be pathogenic in some autoimmune diseases and in solid organ transplantation. In addition to antibody generation, there is increasing interest in the antibody-independent functions of B cells, including their ability to regulate immune responses via the production of IL10. In this thesis I firstly explored the therapeutic potential of belimumab, an anti-BLyS antibody, in an experimental medicine study in kidney transplant recipients. The rationale for this study was based on published studies showing that B cells activate alloreactive T cells and secrete human leukocyte antigen (HLA) and non-HLA antibodies that negatively affect graft function and survival, but may also play a protective role by regulating alloimmune responses promoting transplant tolerance. B-Lymphocyte Stimulator (BLyS) is a cytokine that promotes B cell activation and survival. We performed the first randomized controlled trial using belimumab as early maintenance immunosuppression in kidney transplantation. In belimumab-treated subjects, we demonstrate a reduction in naïve and activated memory B cells, plasmablasts, IgG transcripts in peripheral blood and new antibody formation as well as evidence of reduced CD4 T cell activation and of a skewing of the residual B cell compartment towards an IL10-producing regulatory phenotype. This experimental medicine study highlights the potential of belimumab as a novel therapeutic agent in transplantation. In the second part of my project I performed a preclinical study investigating the potential efficacy of bromodomain inhibitors in reducing antibody-mediated immune cell activation. Immune complexed antigen can activate mononuclear phagocytes (MNP), comprising macrophages and dendritic cells (DCs), via ligation of Fc gamma receptors (FcγR), that bind the Fc region of IgG. FcγR-dependent MNP activation results in profound changes in gene expression that mediate antibody effector function in these cells. The resulting inflammatory response can be pathological in the setting of autoimmune diseases, such as systemic lupus erythematosus and in antibody-mediated rejection in transplantation. BET proteins are a family of histone modification 'readers' that bind acetylated lysine residues within histones and function as a scaffold for the assembly of complexes that regulate gene transcription. Bromodomain inhibitors (I-BET) selectively inhibit the transcription of a subset of inflammatory genes in macrophages following toll-like receptor stimulation. Since MNPs make a key contribution to antibody-mediated pathology, we sought to determine the extent to which I-BET inhibits macrophage and DC activation by IgG. We show that I-BET delays phagolysosome maturation associated with build-up of immune complex (IC) whilst selectively inhibiting IC induced cytokine production. I-BET changed MNP morphology, resulting in a less adherent phenotype, prompting an assessment of its impact on DC migration. In vitro, in a three-dimensional collagen matrix, IgG-IC induced augmentation of DC chemotaxis to chemokine (C-C motif) ligand 19 (CCL19) was abrogated by the addition of I-BET. In vivo, two photon imaging showed that systemic I-BET treatment reduced IC-induced dermal DC mobilisation. Tissue DCs and transferred DC also had reduced migration to draining lymph nodes following I-BET treatment. These observations provide mechanistic insight into the potential therapeutic benefit of I-BET in the setting of antibody-associated inflammation.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-02-07T18:54:08Z No. of bitstreams: 1 Micely D`el-Rei Hermida... Transporte de Leishmania do sitio.pdf: 12642864 bytes, checksum: 1f1b3fd2c8676340857d3b59f352212d (MD5) / Made available in DSpace on 2014-02-07T18:54:08Z (GMT). No. of bitstreams: 1 Micely D`el-Rei Hermida... Transporte de Leishmania do sitio.pdf: 12642864 bytes, checksum: 1f1b3fd2c8676340857d3b59f352212d (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil / A infecção por Leishmania modula a função de integrinas em fagócitos inflamatórios, afetando a migração celular e disseminação do parasito. O conhecimento sobre as populações de fagócitos capazes de transportar Leishmania ou fragmentos de parasitos mortos, e os mecanismos de disseminação da Leishmania do sítio de infecção para os diferentes tecidos ainda está incompleto. Nesse trabalho, nós adaptamos um modelo de migração de células in vivo para estudar o efeito da infecção parasitária na capacidade das diferentes populações de fagócitos mononucleares de migrar do sítio inflamatório para o linfonodo drenante e para estudar a cinética de disseminação da Leishmania através do sistema linfático. Inicialmente, nós usamos um modelo de peritonite crônica induzida por tioglicolato para identificar populações de fagócitos inflamatórios susceptíveis a infecção por Leishmania e suas possíveis alterações na migração após a infecção. Células peritoneais estimuladas por tioglicolato coletadas de animais Ly5.1+, não infectadas ou infectadas por Leishmania, foram injetadas na cavidade peritoneal de animais Ly5.1- e as diferentes populações de fagócitos foram rastreadas no linfonodo drenante. Células migrantes corresponderam no linfonodo 1% dos leucócitos injetados. Em animais injetados com células peritoneais cultivadas somente com meio, 28-90% das células migrantes eram células dendríticas mielóides e 30-74% eram macrófagos. No grupo de animais injetados com células do exsudato peritoneal co-cultivadas com Leishmania, 9-65% das células migrantes eram células dendríticas mielóides e 20-69% eram macrófagos. Somente a migração da célula dendríticas foi consistentemente diminuída após a co-incubação Leishmania. Em seguida, nós determinamos a cinética de disseminação do parasito do sítio inflamatório para o linfonodo drenante e sistemicamente. Promastigotas de L. amazonensis foram injetadas na cavidade peritoneal de camundongos e depois de 15min e 30min; 1h, 2h, 4h, 6h, 12h e 24h amostras de fagócitos peritoneais, linfonodo, baço e pulmão foram cultivados para isolamento de Leishmania. Culturas de fagócitos peritoneais e células do linfonodo ficaram positivas após 15min a 6h da infecção. Parasitos foram detectados em culturas de células esplênicas de 15min a 1h após a infecção. Cultura de células do pulmão foram positivas 1h após a infecção ou, menos consistentemente, 15min e 30min após a inoculação. Parasitos na forma promastigota foram identificados no linfonodo drenante. Nossos dados mostraram que: (1) Uma variedade de fagócitos são capazes de migrar do sítio inflamatório para o linfonodo drenante. (2) Populações de células dendríticas infectadas por Leishmania parecem estar retidas no sítio inflamatório. (3) Os mecanismos de disseminação da Leishmania da cavidade peritoneal para o linfonodo ocorre em menos de 15 minutos após a injeção. (4) O trânsito para a corrente sanguínea ocorre nas primeiras horas após a infecção. Nossos dados também sugerem que mesmo antes do tempo requerido para alterações nas populações de células inflamatórias ocorra no sítio inflamatório, parasitos de Leishmania e seus antígenos podem chegar ao linfonodo drenante. / Leishmania infection modulates integrin function in inflammatory phagocytes, affecting cell migration and parasite dissemination. The knowledge on the phagocyte populations capable of transporting Leishmania or fragments of dead parasites, and the mechanisms of Leishmania dissemination from the infection site to the different tissues is still incomplete. In this work, we adapted a model of cell migration in vivo to study the effect of parasite infection upon the ability of different mononuclear phagocyte populations to migrate from the inflammatory site to the draining lymph node and to study the kinetics of Leishmania dissemination through the lymphatic system. First, we used a model of chronic peritonitis induced by thioglycollate to identify inflammatory phagocytes populations susceptible to Leishmania infection and the potential changes in their migratory patterns after infection. Uninfected and Leishmania-infected, thioglycollate-elicited peritoneal exudate cells from Ly5.1+ mice were injected into the peritoneal cavity of Ly5.1- mice, and the different phagocyte populations were tracked to the draining lymph node. Migrating cells corresponded 1% of the injected leukocytes. In the animals injected with peritoneal cells cultivated with medium alone, 28-90% of the migrating cells were myeloid dendritic cells and 30-74% were macrophages. In the group of animals injected with peritoneal exudate cells co-cultivated with Leishmania, 9-65% of the migrating cells were myeloid dendritic cells and 20-69% were macrophages. Only dendritic cell migration was consistently decreased after co-incubation with Leishmania. Afterwards, we determined the kinetic of parasite dissemination from the inflammatory site to the draining lymph node and systemically. L. amazonensis promastigotes were injected into BALB/c mice peritoneal cavity and after 15min and 30min; 1h, 2h, 4h, 6h, 12h and 24h samples of peritoneal phagocytes, lymph nodes, spleen and lungs were cultivated for Leishmania isolation. Peritoneal phagocytes and lymph nodes were positive after 15min to 6h of infection. Parasites were detected in splenic cells cultures 15min to 1h after infection. Lungs cultures were positive after 1h of infection or, less consistently, 15min and 30min after inoculation. Labeled parasites were identified in the draining lymph nodes. Our data show that: (1) A variety of phagocytes are able to migrate from the inflammatory site to the draining lymph node. (2) Populations of Leishmania-infected dendritic cells appear to be retained in the inflammatory site. (3) The mechanisms of Leishmania dissemination from peritoneal cavity to the lymph node occur in less than 15 minutes after injection. (4) The transit to bloodstream occurs in the first hour of infection. Our data also suggest that even before the time required for deep changes in inflammatory cell population takes place in the inflammatory site, Leishmania parasites and their antigens can reach the draining lymph node.

Localisé entre l'Epithélium Pigmentaire Rétinien (EPR) et les segments externes des photorécepteurs, l'espace sous-rétinien est une zone immunosuppressive ; régulée par des signaux comme la thrombospondine-1 (TSP-1) ou Fas Ligand (FasL), qui empêchent l'accumulation des phagocytes mononucléés (PMs), en particulier des monocytes inflammatoires. La Dégénérescence Maculaire Liée à l'Age (DMLA) est associée à une rupture de l'immunosuppression de cet espace, et s'accompagne d'une accumulation de PMs ; causant la mort des photorécepteurs, la dédifférenciation de l'EPR et une néovascularisation pathologique. Des études d'associations génétiques ont établi un lien entre la DMLA et un haplotype qui affecte le locus 10q26, qui contient trois gènes : PLEKHA1, ARMS2 et HTRA1. L'haplotype est associé à une augmentation de la transcription de HTRA1 dans les lymphocytes ou les cellules de l'EPR. HTRA1 code pour une sérine protéase qui a une multitude de substrats ; mais le mécanisme par lequel elle pourrait être impliquée dans la pathogenèse de la DMLA reste inconnu. TSP-1 est une glycoprotéine exprimée par l'EPR, les macrophages résidents et inflammatoires. Le domaine C-terminal de TSP-1 contient deux séquences VVM qui peuvent chacune interagir avec un récepteur CD47. Dans cette étude, nous montrons que HTRA1 clive TSP-1 et inhibe l'élimination des PMs régulée par l'interaction entre TSP-1 et CD47 à l'état physiologique, in vitro et in vivo. L'activation pharmacologique de CD47 nous a permis d'annuler les effets pro-inflammatoires de HTRA1 et pourrait représenter un espoir thérapeutique pour le contrôle de la progression de la DMLA chez les patients porteurs de l'haplotype à risque. / Localized between the Retinal Pigment Epithelium (RPE) and the photoreceptors outer segments, the subretinal space is an immunosuppressive zone, mediated by signals such as Thrombospondin-1 (TSP-1), Fas Ligand (FasL) that prevent the accumulation of Mononuclear Phagocytes (MPs) and in particular pathogenic inflammatory monocytes. Age related Macular Degeneration (AMD) is associated with a breakdown of this immunosuppressivity and an accumulation of MPs, which causes photoreceptor degeneration, RPE dedifferentiation and pathological neovascularization. Genome association studies showed a strong link between AMD and a relatively common haplotype of 10q26 locus that contains the PLEKHA1, ARMS2 and HTRA1 genes. The disease haplotype is associated with increased HTRA1 transcription in cell types such as lymphocytes and RPE cells. HTRA1 is a serine protease with a number of substrates, but the mechanism by which it might be involved in AMD pathogenesis is unknown. TSP-1 is a glycoprotein expressed by RPE, resident macrophages and inflammatory macrophages. The C-terminal domain of TSP-1 contains two VVM sequences that can each interact with a CD47 receptor. We show that HTRA1 induced subretinal MP accumulation is dependent on TSP-1 deactivation in an RPE/Mo co-culture model and in a laser induced inflammation model in vivo. This pathogenic effect of HTRA1 was reversible by synthetic CD47 agonists. Our study reveals a comprehensive mechanism how the risk-allele 10q26 participates in the pathogenesis of AMD and opens new therapeutic avenues to restore subretinal immunosuppressivity and inhibit the inflammation-dependent neurodegeneration.

Changes to the composition and function of the gut mononuclear phagocyte (MNP) compartment are associated with the development of intestinal inflammation. Much work has focused on the role of MNPs in gut-associated lymphoid tissue in maintaining homeostasis, however little is known regarding the roles of MNPs during colitis. We have investigated MNPs in the large intestinal lamina propria during the steady state and inflammation. One of our primary aims was to determine the contribution of MNP subsets to intestinal pathology. For our studies of inflammation, we focused mainly on the Helicobacter hepaticus infection + anti-IL-10R model, which induces inflammation of the colon and caecum (typhlocolitis). We defined the composition of the MNP compartment alongside intestinal pathology scores throughout Hh + anti-IL-10R typhlocolitis. Peak pathology, 2-3 weeks after induction of colitis, coincided with peak frequencies of CX₃CR1^int Ly6C^+ MNPs. Having observed the accumulation of CX₃CR1^int CD64^+ monocyte/macrophage MNPs in the inflamed lamina propria, we conducted comparative whole genome microarray analysis of these cells isolated from the large intestine three weeks after Hh + anti-IL-10R treatment. CX₃CR1^int CD64^+ MNPs selectively expressed a variety of pro- and anti-inflammatory genes, including a number of genes which individually can both promote and negatively regulate inflammation. IL-23 is essential for Hh + anti-IL-10R-induced intestinal pathology. We investigated the role of MNPs as a source of IL-23 which drives Hh + anti-IL-10R colitis. Unexpectedly, our results indicate that normally hyporesponsive CX₃CR1^hi macrophages may act as the initial source of IL-23, which induces development of colitis. Recruitment of Ly6C^+ MHCII^+ MNPs to the lamina propria was IL-23-dependent, and these cells also expressed IL-23, which may establish a positive feedback loop of immune cell recruitment, activation and IL-23 production. Finally, we also examined how MNPs might be recruited to the colonic lamina propria during inflammation. Our studies support the conclusion that CCR6 is not required for accumulation of monocyte-derived populations in the inflamed intestine. We cannot rule out a role for CCR2, however preliminary data from the Hh + anti-IL-10R colitis model suggest a potential role for CCR1 or its close relation CCRL2. Such pathways could represent new therapeutic targets in inflammatory bowel disease.

Orientador: Flávio Henrique Caetano / Banca: Hércules Menezes / Banca: Ricardo José Gomes / Resumo: Quando um tecido ou órgão é alvo de algum tipo de injúria, dá-se início ao processo de inflamação tecidual. As reações celulares e humorais que tomam parte desta reação são evocadas a fim de exterminar ou neutralizar o agente infeccioso e seus produtos, permitindo que o reparo tecidual se dê da melhor forma possível. Contudo, algumas vezes o resultado desta ação pode ser prejudicial, levando ao comprometimento ou a perda das funções do tecido ou órgão em questão, com conseqüências que podem afetar a saúde do indivíduo como um todo. Em contrapartida, a atividade física moderada tem sido apontada como um grande aliado no combate à várias doenças, potencializando a ação do sistema imune e reduzindo os possíveis danos e minimizando os efeitos nocivos das respostas inflamatórias. Desta forma, nos propomos a verificar os efeitos da atividade física moderada no processo inflamatório e de reparação tecidual, comparando 10 ratos Wistar que realizaram exercícios físicos moderados com 10 sedentários, por meio da análise numérica (microscopia de luz) e ultra-estrutural (microscopia eletrônica de transmissão) de mastócitos, macrófagos e fibroblastos, células muito importantes para tais fenômenos. O modelo de estudo escolhido foi o implante subcutâneo de esponjas de PVC, que mimetiza situações clínicas importantes como, por exemplo, implantes de marcapasso. Nossos resultados indicam que as células do grupo treinado respondem de maneira mais ágil (precoce) e efetiva à injúria, que o número de cada tipo celular foi significativamente maior neste grupo e ainda que, para quaisquer dos grupos, os tipos celulares em estudo agem estreitamente interrelacionados...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: When a tissue or organ is target of some offense, it feels beginning to the process of tissue inflammation. Cellular and humoral reactions that participate of this reaction are evoked to exterminate or to neutralize the infectious agent and your products, allowing the tissue repair to feel in the best possible way. However, the result of this action can sometimes be harmful, taking to the compromising or the loss of the functions of the tissue or organ in subject, with consequences that can affect the individual's health as a completely. On the other hand, the moderate physical activity has been pointed as a great ally in the combat to several diseases, improving the action of the immune system and reducing the possible damages and minimizing the noxious effects of the inflammatory answers. Thus, we intend to verify the effects of the moderate physical activity in the inflammatory and tissue repair process, comparing 10 Wistar rats that had carried through moderate physical exercises with 10 sedentary ones, through the numeric analysis (light microscopy) and ultrastructural (transmission electronic microscopy) of mast cells, macrophages and fibroblasts, which are very important cells for such phenomena. The model of chosen study was the subcutaneous implantation of PVC sponges, that simulates important clinical situations as, for instance, implant of pacemaker. Our results indicate that the cells of the trained group answer in a more agile (precocious) and effective way to the offense, that the number of each cellular type was significantly larger in this group and although, for any of the groups, the cellular types in study act narrowly interrelated. Therefore, we ended in our work that the moderate physical activity can contribute positive and significantly for the inflammation and tissue repair process, making possible that the homeostasis is reestablished more easily and, like this, avoiding larger damages to the individual. / Mestre

Made available in DSpace on 2014-06-11T19:22:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-04-20Bitstream added on 2014-06-13T19:49:27Z : No. of bitstreams: 1 rodrigues_rf_me_rcla.pdf: 5396854 bytes, checksum: 53dc5df0dd553ffb8f11bf6074d163cc (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Quando um tecido ou órgão é alvo de algum tipo de injúria, dá-se início ao processo de inflamação tecidual. As reações celulares e humorais que tomam parte desta reação são evocadas a fim de exterminar ou neutralizar o agente infeccioso e seus produtos, permitindo que o reparo tecidual se dê da melhor forma possível. Contudo, algumas vezes o resultado desta ação pode ser prejudicial, levando ao comprometimento ou a perda das funções do tecido ou órgão em questão, com conseqüências que podem afetar a saúde do indivíduo como um todo. Em contrapartida, a atividade física moderada tem sido apontada como um grande aliado no combate à várias doenças, potencializando a ação do sistema imune e reduzindo os possíveis danos e minimizando os efeitos nocivos das respostas inflamatórias. Desta forma, nos propomos a verificar os efeitos da atividade física moderada no processo inflamatório e de reparação tecidual, comparando 10 ratos Wistar que realizaram exercícios físicos moderados com 10 sedentários, por meio da análise numérica (microscopia de luz) e ultra-estrutural (microscopia eletrônica de transmissão) de mastócitos, macrófagos e fibroblastos, células muito importantes para tais fenômenos. O modelo de estudo escolhido foi o implante subcutâneo de esponjas de PVC, que mimetiza situações clínicas importantes como, por exemplo, implantes de marcapasso. Nossos resultados indicam que as células do grupo treinado respondem de maneira mais ágil (precoce) e efetiva à injúria, que o número de cada tipo celular foi significativamente maior neste grupo e ainda que, para quaisquer dos grupos, os tipos celulares em estudo agem estreitamente interrelacionados... / When a tissue or organ is target of some offense, it feels beginning to the process of tissue inflammation. Cellular and humoral reactions that participate of this reaction are evoked to exterminate or to neutralize the infectious agent and your products, allowing the tissue repair to feel in the best possible way. However, the result of this action can sometimes be harmful, taking to the compromising or the loss of the functions of the tissue or organ in subject, with consequences that can affect the individual's health as a completely. On the other hand, the moderate physical activity has been pointed as a great ally in the combat to several diseases, improving the action of the immune system and reducing the possible damages and minimizing the noxious effects of the inflammatory answers. Thus, we intend to verify the effects of the moderate physical activity in the inflammatory and tissue repair process, comparing 10 Wistar rats that had carried through moderate physical exercises with 10 sedentary ones, through the numeric analysis (light microscopy) and ultrastructural (transmission electronic microscopy) of mast cells, macrophages and fibroblasts, which are very important cells for such phenomena. The model of chosen study was the subcutaneous implantation of PVC sponges, that simulates important clinical situations as, for instance, implant of pacemaker. Our results indicate that the cells of the trained group answer in a more agile (precocious) and effective way to the offense, that the number of each cellular type was significantly larger in this group and although, for any of the groups, the cellular types in study act narrowly interrelated. Therefore, we ended in our work that the moderate physical activity can contribute positive and significantly for the inflammation and tissue repair process, making possible that the homeostasis is reestablished more easily and, like this, avoiding larger damages to the individual.

Neste trabalho avaliamos a formação de oxigênio singlete in vitro em fagócitos, (células mononucleares e neutrófilos) isolados de sangue periférico humano, e eosinófilos, de lavado bronco alveolar de camundongos balb/c, ativados por estímulo partículado: zimosan opsonizado contendo o 9,10difenilantraceno (DPA) adsorvido como sonda captadora de 1O2. Por este método, a formação do 1O2 pode ser verificada pela formação do 9,10-difenilantraceno endoperóxido (DPAO2), que é detectado por HPLC. Observamos, que os fagócitos formam 1O2 e que esta formação parece ocorrer de forma diferenciada para os dois tipos celulares (neutrófilos e células mononucleares). Visando ampliar os estudos anteriores sobre o papel da melatonina (MLT) no processo inflamatório, foi testado seu efeito em fagócitos e a relação na produção de 1O2 destas células. Observamos que MLT inibe a formação de 1O2 totalmente no caso de neutrófilos e parcialmente no caso de células mononucleares e eosinófilos. Paralelamente, foi desenvolvida a síntese de um novo captador químico de 1O2, o éster 9,10-antracenil-3-bispropionato de etila (ABPE), cuja finalidade principal é o acúmulo no interior da célula, depois de sofrer hidrólise enzimática. Esta sonda, terá facil acesso ao interior das células em sua forma ester. Este novo captador de 1O2 foi testado em células mononucleares e neutrófilos estimulados de formas diferentes: via receptor independente e dependente. Os resultados demonstraram produção equivalente de 1O2 nestes fagócitos. / In this study, we evaluated the singlet oxygen (1O2) formation in vitro from phagocytes (neutrophils and mononuclear cells) isolated from human blood cells and eosinophils isolated from bronchoalveolar lavage fluid of mice balb/c activated, by opsonized zymosan. To determine whether singlet oxygen is produced by phagocytes, zymosan particles were coated with a specific chemical trap for 1O2, 9,10-diphenylanthracene (DPA). The production of 1O2 was followed using HPLC, to measure its product, 9,10-diphenylanthracene endoperoxide (DPAO2). We also noticed that the 1O2 production occurs at different levels of for two cell types, neutrophils and mononuclear cells. In order to broaden previous studies on the role of melatonin (MLT) in inflammatory processes, its effect was tested in phagocytes was tested in relation to 1O2 formation by these cells. We observed that MLT inhibits the 1O2 formation totallymt neutrophils and partiallym mononuclear cells and eosinophils. At the some time, it was also developed the synthesis of a new probe for 1O2, the 9,10-anthracene-bis-3-ethyl-propionate (ABEP), with the purpose to accumulate inside the cells, after its enzymatic hydrolysis. This probe presents easy acess to the inferior of the cells in its ester form. This new probe for trapping 1O2 was tested in mononuclear cells and neutrophils stimulated in two ways: via independent and dependent receptor. The results showed equivalent production of 1O2 for both cell types.

La Dégénérescence Maculaire Liée à l’Age (DMLA) est la première cause de cécité légale dans les pays industrialisés chez les personnes âgées. L’atrophie géographique – l’une des formes tardives de la DMLA - est caractérisée par la perte de l’épithélium pigmentaire et la dégénérescence des photorécepteurs. Nous groupe a montré précédemment que dans l’atrophie géographique, les phagocytes mononucléés (PMs) s’accumulent dans l’espace sous-rétinien, et induisent la dégénérescence rétinienne via la production d’IL-1β. Dans un premier temps, nous montrons que la présence de PMs sous-rétiniens est associée à la perte des bâtonnets et la dégénérescence des segments de cônes dans la zone de transition de patients atrophiques. Nous montrons ensuite dans différents modèles in vivo et ex vivo que les macrophages récapitulent ces effets, et qu’IL-1β est nécessaire à la perte des segments externes des cônes induite par les PMs. Dans un deuxième temps, nos résultats montrent qu’IL-1β induit indirectement la mort des bâtonnets, en perturbant l’homéostasie rétinienne du glutamate. L’inhibition des récepteurs glutamatergiques pour prévenir l’excitotoxicité du glutamate, ou la supplémentation en cystine favorisant la restauration de la machinerie neuronale antioxydante, permettent de protéger les bâtonnets de la toxicité induite par IL-1β. L’ensemble de nos résultats démontre le rôle joué par IL-1β dans la dégénérescence des segments de cônes et la perte des bâtonnets dans l’inflammation sous-rétinienne. Cette étude permettra la mise au point de thérapies innovantes, afin de lutter contre la forme atrophique de la DMLA, pour laquelle il n’existe actuellement aucun traitement. / In geographic atrophy (GA), one of the late forms of Age-related Macular Degeneration (AMD), an extending atrophic zone forms, characterized by the loss of retinal pigment epithelium and photoreceptor degeneration. Subretinal mononuclear phagocytes (MPs) accumulate in GA, and are associated with IL-1β-dependent retinal degeneration. First, we confirmed that subretinal accumulation of MPs is associated with rod degeneration and cone segment loss in the transitional zone in GA human samples. Using ex vivo and in vivo models, we then demonstrated that MPs-derived IL-1β leads to severe cone segment degeneration. Therefore, inhibiting subretinal MP accumulation or IL-1β might protect the cone segment, and help preserve high acuity daytime vision in conditions characterized by subretinal inflammation. Second, we showed that IL-1β effect on rod degeneration is indirect, and mediated by glutamate. Our results indicate that IL-1β impairs Müller glial cells glutamate recycling, and subsequently leads to the extracellular increase in glutamate content. Inhibiting glutamate receptors to prevent excitotoxicity, or exogenous cystine supplementation to supply antioxidant metabolism, are sufficient to protect rods from IL-1β-induced neurotoxicity. Our results provide new perspectives to treat pathologies associated with subretinal inflammation such as late AMD. Our results collectively demonstrated that MP-derived IL-1β induces cone segment loss, and glutamate homeostasis disruption associated with rod degeneration. This study will help with the development of new therapeutic strategies in dealing with inflammatory retinal pathologies as geographic atrophy.