There is a growing literature of clinical studies of bone marrow (BM) cell therapy for liver cirrhosis. At present, the optimum choice of cell type(s) and the mechanism(s) of effect remain undefined. Cells of the monocyte-macrophage lineage have key roles in the development and resolution of liver fibrosis. Therefore, I tested the therapeutic effects of these cells in the context of experimental murine liver fibrosis. The effects of unmanipulated, syngeneic macrophages, their specific BM precursors and unfractionated (whole) BM cells were examined in the iterative carbon tetrachloride model of liver fibrosis. BM-derived macrophage (BMM) delivery resulted in early chemokine upregulation with the hepatic recruitment of endogenous macrophages and neutrophils. These cells delivered matrix metalloproteinases-13 and -9 respectively, into the hepatic scar. The effector cell infiltrate was accompanied by increased levels of the anti-inflammatory cytokine IL-10. A reduction in hepatic myofibroblasts was followed by reduced fibrosis detected 4 weeks after macrophage infusion. Serum albumin levels were elevated at this time. Upregulation of the liver progenitor cell mitogen TWEAK preceded expansion of the progenitor cell compartment. BMM delivery increased hepatic expression of cytokines with reparative effects (including colony stimulating factor-1, insulin-like growth factor-1 and vascular endothelial growth factor). In contrast to the effects of differentiated macrophages, liver fibrosis was not significantly improved by the application of macrophage precursors and was exacerbated by whole BM. BMMs did not affect liver fibrosis or regeneration in the 1% DDC model of biliary disease. These effects were only detected following the intraportal delivery of BM cells. The peripheral (tail) vein administration of BMMs, either singly or repeatedly did not recapitulate the therapeutic phenotype. This was investigated by in vivo tracking of BMMs constitutively expressing green fluorescent protein (GFP). The peripheral administration route resulted in the early (1 hour) accumulation of BMMs within the pulmonary system. This was followed by delayed hepatic engraftment, which was also numerically reduced (< 30%) compared with intraportal administration. Macrophage cell therapy improves clinically relevant parameters in experimental chronic liver injury. Paracrine signalling to endogenous cells amplifies the effect. The benefits from this single, defined cell type suggest clinical potential.

Os antimoniais pentavalentes, tais como o Glucantime, são geralmente usados como fármacos de primeira escolha para o tratamento das leishmanioses, no entanto seu mecanismo de ação não é completamente esclarecido. Atua contra formas amastigotas intracelulares de Leishmania sp, comprometendo o potencial redox levando danos ao DNA do parasito. Alguns trabalhos sugerem que o Glucantime aumenta a capacidade fagocítica e a produção de TNF-alfa por fagócitos. O objetivo deste estudo foi avaliar a capacidade do Glucantime modular a atividade do macrófago, a principal célula hospedeira da Leishmania. Inicialmente, a toxicidade do Glucantime foi testada sobre macrófagos peritoneais de camundongos BALB/c, tratando as monocamadas in vitro por 48 horas. A viabilidade celular foi avaliada pelo método do MTT. A capacidade do Glucantime (0,1, 1 e 10 mg/ml) modular os macrófagos foi avaliada tratando as monocamadas de macrófagos peritoneais por 24 horas antes da infecção com Leishmania braziliensis. Após 48 horas de incubação com meio de cultura foi avaliado o índice de infecção por contagem. Antes e após a infecção foram analisados a produção de óxido nítrico (NO) pelo método de Griess, espécies reativas de oxigênio (EROS) por fluorimetria usando a sonda H2DCFDA e a produção de citocinas por ELISA. Para avaliar se o Glucantime seria capaz de modular macrófagos in vivo, camundongos suíços foram tratados por 5 dias consecutivos com 8 mg de Glucantime pela via intraperitoneal. Macrófagos peritôneais foram avaliados quanto a sua capacidade de controlar a infecção in vitro com L. braziliensis. Os resultados mostraram que nas concentrações até 10 mg/ml, o Glucantime não alterou a viabilidade dos macrófagos in vitro. O pré-tratamento dos macrófagos com Glucantime nas concentrações de 0.1mg/mL, 1mg/mL e 10mg/mL, foi capaz de reduzir o índice de infecção em 49%, 74% e 85%, respectivamente. Em macrófagos não infectados a produção de NO foi aumentada na concentração de 10mg/ml de Glucantime. O tratamento com 1 e 10 mg/ml de Glucantime foi capaz de aumentar significativamente a produção de EROs (p<0,05 e p<0.01, respectivamente) e a produção IL-12 (p<0,05), mas a IL-10 não foi alterada. Não houve alterações significativas desses parâmetros em relação ao controle após a infecção com L. braziliensis. Os macrófagos oriundos dos animais tratados com Glucantime foram capazes de reduzir o índice de infecção por L. braziliensis (p<0,05). Esses resultados sugerem que o Glucantime é capaz de ativar os macrófagos e esse efeito pode contribuir para o mecanismo de ação desse fármaco. / The pentavalent antimonial drugs, such as Glucantime, are generally used as first choice for the treatment of leishmaniasis, however its mechanism is not fully understood. It has activity against intracellular amastigotes of Leishmania sp, compromising the redox potential and causing damage to the DNA of the parasite. Some studies suggesting that Glucantime enhances phagocytosis and TNF-&#945; production by phagocytes. The aim of this study is to evaluate the modulation of Glucantime on macrophages, the major host cell of Leishmania. Initially, Glucantimes toxicity was tested on peritoneal macrophages from BALB/c mice, by treating the monolayers in vitro for 48 hours. Cell viability was evaluated by MTT method. The capacity of Glucantime (0,1, 1 and 10 mg/ml) of modulate macrophages was evaluated by treating the monolayers of peritoneal macrophages for 24 hours before the infection with Leishmania braziliensis. After 48 hours of incubation with culture medium the infection index was evaluated by counting. Before and after the infection were analyzed the production of nitric oxide (NO) by Griess method, reactive oxygen species (ROS) by fluorimetry using the H2DCFDA dye and cytokines by ELISA. To evaluate if Glucantime could modulate macrophages in vivo, Swiss Webster mice were treated for 5 consecutive days with 8 mg Glucantime by intraperitoneal route. Peritoneal macrophages were evaluated about its capacity of control the in vitro infection with L. braziliensis. Results showed that until the concentration of 10 mg/ml, Glucantime did not alter the macrophages viability in vitro. The pre-treatment of macrophages with Glucantime at 0.1mg/mL, 1mg/mL and 10mg/mL was able to reduce the infection index in 49%, 74% and 85%, respectively. On non-infected macrophages the NO production was increased at 10mg/ml of Glucantime. The treatment with 1 and 10 mg/ml de Glucantime was able to significantly increase the ROS production (p<0,05 and p<0.01, respectively) and IL-12 production (p<0,05), however the IL-10 production was not altered. There were no significant changes of these parameters comparing to control after the L. braziliensis infection. The macrophages from the treated mice were capable of reduce the infection index by L. braziliensis (p<0,05). These results suggest that Glucantime is capable of activate macrophages and this effect could contribute to the mechanism of action of this drug.

A disfunção do sistema imune inato e a neuroinflamação tem sido cada vez mais reconhecidas como elementos importantes na fisiopatologia do transtorno bipolar (TB). Como componentes essenciais da imunidade inata, os macrófagos tem múltiplas funções tanto na inibição como na promoção da proliferação celular e na reparação tecidual, sendo a diversidade e a plasticidade características marcantes deste tipo celular. A polarização M1 clássica e a polarização alternativa M2 de macrófagos representam dois extremos de um estado dinâmico na mudança da ativação dos mesmos. Os macrófagos do tipo M1 sintetizam citocinas próinflamatórias que inibem a proliferação de células circundantes e danificam tecidos, enquanto os macrófagos do fenótipo M2 liberam citocinas antiinflamatórias que podem promover reparo tecidual. Um desequilíbrio da polarização M1-M2 dos macrófagos é frequentemente associado a várias doenças ou condições inflamatórias. O objetivo desta tese foi, além de revisar a importância da inflamação sistêmica na modulação da resposta inflamatória da microglia/macrófagos e consequentemente seu potencial envolvimento na fisiopatologia do TB, avaliar o perfil de polarização M1/M2 em cultura de macrófagos de sujeitos com TB comparados a indivíduos saudáveis. Monócitos foram isolados a partir de sangue periférico de dez sujeitos com TB e dez indivíduos saudáveis e diferenciados em macrófagos através da adição de fator estimulante de colônia de macrófagos (MCSF) ao meio de cultura. Para induzir a polarização M1 ou M2, as culturas foram incubadas com IFN-y e LPS ou IL-4 respectivamente. Após a incubação, recolheram-se os sobrenadantes e mediram-se as citocinas (IL-1β, IL-6, IL-10 e TNF-α) por ensaio multiplex. A secreção das citocinas IL-1β, TNF-α e IL-6 características do protótipo M1 e citocinas IL-10 do protótipo M2 foram semelhantes entre os pacientes e os controles. Utilizou-se a razão TNF-α / IL-10 do fenótipo M1 para refletir o estado inflamatório dos participantes. Não foi observada diferença entre os grupos (p=0,627). Duas hipóteses diferentes poderiam explicar esses resultados: todos os pacientes incluídos neste estudo representam um estágio inicial da doença como evidenciado pela pontuação FAST total inferior a 11. De acordo com o modelo de estadiamento em TB, as alterações biológicas (incluindo a inflamação) parecem estar relacionadas com os episódios de humor e progressão da doença. Juntamente com estudos anteriores, os nossos dados sugerem que os pacientes nos estágios iniciais ainda preservam a função do sistema imunológico sem apresentar um desequilíbrio a favor do perfil de macrófagos M1 como tem sido observado em pacientes no estágio tardio, destacando a relevância da intervenção precoce no TB. Ainda, estes pacientes estavam em tratamento com estabilizadores de humor e é plausível especular que esses fármacos exerçam efeitos sobre a polarização de macrófagos. Estudos futuros em pacientes drug-free são essenciais para avaliar esta questão. Em conclusão, nossos achados sugerem que os pacientes TB não apresentam desequilíbrio na polarização dos macrófagos em favor do fenótipo pró-inflamatório M1. O fato de todos estes pacientes estarem em estágios iniciais da doença reforça os efeitos protetores da intervenção precoce no TB na prevenção de alterações do sistema imune e, consequentemente, na progressão da doença. / Innate immune system dysfunction and neuroinflammation have been recognized as important elements in the pathophysiology of bipolar disorder (BD). As essential players of innate immunity, macrophages have multiple roles in inhibition and promotion of cell proliferation and tissue repair. The classical M1 polarization and the M2 alternative polarization of macrophages represent two extremes of a dynamic state in their change of activation. M1 macrophages synthesize proinflammatory cytokines that inhibit the proliferation of surrounding cells and damage tissues, whereas macrophages of the M2 phenotype release antiinflammatory cytokines that may promote tissue repair. An imbalance of the M1-M2 polarization of macrophages is often associated with various diseases or inflammatory conditions. The aim of this thesis was to review the importance of systemic inflammation in modulating the inflammatory response of microglia/ macrophages and consequently their potential involvement in the pathophysiology of BD, and also evaluate the M1/M2 polarization profile in macrophages of patients with BD compared to healthy individuals. Blood monocytes were obtained from ten BD patients and ten healthy controls. These cells were activated/polarized into the M1 (IFNγ + LPS) or M2(IL-4) phenotype. Supernatants were collected and the cytokines (IL-1β, IL-6, IL-10 and TNF-α) were measured by multiplex assay. Secretion of the IL- 1β, TNF-α, IL-6 and IL-10 were similar between patients and controls. The TNF-α/IL- 10 ratio of the M1 phenotype was used to reflect the inflammatory state of the participants. There was no difference between groups (p = 0.627). Two hypotheses could explain these results: all patients included in this study represent an early stage of disease as evidenced by the FAST score below 11. According to the BD staging model, biological changes (including inflammation) appear to be related to mood episodes and disease progression. Together with previous studies, our data suggest that patients in early stages of BD still preserve immune system function without presenting an imbalance in favor of M1 macrophages as has been observed in latestage patients, highlighting the relevance of early intervention. Moreover, these patients were under treatment with mood stabilizers and it is plausible to speculate that these drugs have effects on macrophage polarization. Future studies in drug-free patients are essential to assess this issue. In conclusion, our findings suggest that BD patients do not present imbalance in macrophage polarization in favor of the M1 proinflammatory phenotype. The fact that all these patients are in the early stages of the disease reinforces the protective effects of early intervention in BD to prevent changes in the immune system and, consequently, prevent the progression of the disease.

We aimed to characterize well-know PIDs, novel genetic defects and immunopathological mechanisms in Brazilian patients with susceptibility to mycobacterial diseases. The patients developed different mycobacterial diseases and M. tuberculosis was the most frequent species. Molecular and genetic analysis revealed mutations in different genes: RAG1 (P1), CD40LG (P2, P3, P4), NEMO (P5), NCF1 (P6), TLR2 (P7) IL-12Rb2 (P8), IL-12Rb1 (P9), TLR10 (P10), DKC1(P11), SOCS-1(P12) and IRAK2 (P13). Finally, MDMs from patients phagocytose normally but were unable to appropriately control intracellular M. tuberculosis growth in comparison to MDMs from healthy subjects. We concluded that the Brazilian patients have heterogeneous mutations previously associated with susceptibility to mycobacterial diseases and novel genetic variations were identified suggesting novel PIDs. In addition, the inability of MDMs to control the intracellular growth of M. tuberculosis indicates this contributes to patients´ susceptibility to mycobacterial infections. / Objetivamos identificar novos defeitos genéticos e mecanismos imunopatológicos em pacientes brasileiros com suscetibilidade a infecções por micobactérias. Os pacientes foram investigados se portadores de imunodeficiencias previamente caracterizadas tais como SCID, deficiência de CD40L, MSMD, defeitos na sinalização via TLRs e CGD. A análise genética foi realizada por sequenciamento Sanger e \'\'whole exome sequencing\'\' para identificar possíveis novas imunodeficiências primárias. Além disso a função dos macrófagos dos pacientes foi avaliada. Infecções por diferentes espécies de micobactérias foram apresentadas pelos pacientes, sendo M. tuberculosis a espécie mais frequentemente identificada. Mutações em diferentes genes foram encontradas: RAG1 (P1), CD40LG (P2, P3, P4), NEMO (P5), NCF1 (P6), TLR2 (P7), IL-12Rb2 (P8), IL-12Rb1 (P9), IRAK2 (P10), SOCS-1 (P11) e TLR10 (P12). MDMs dos pacientes fagocitaram normalmente M. tuberculosis, porém reduzida capacidade em inibir o crescimento da M. tuberculosis foi observada. Concluímos que os pacientes estudados possuem defeitos moleculares heterogêneos e que os MDMs desses indivíduos apresentam falhas no controle do crescimento da M. tuberculosis. Nossos dados sugerem que esses são fatores subjacentes à susceptibilidade a infecções por micobactérias nesses indivíduos.

A Mycobacterium abscessus subspécie abscessus é um pesadelo quando envolvida em infecção pulmonar que são incuráveis, a despeito do uso de antimicrobianos com atividade in vitro, caso o tratamento não inclua a ressecção cirúrgica da área afetada. É a micobactéria patogência de crescimento rápido mais frequentemente isolada de culturas de sítios pulmonares. Há um número reduzido de opções terapêuticas para o tratamento dessas infecções, e é ainda mais reduzido o número de antimicrobianos que atingem concentrações terapêuticas no compartimento intracelular, em particular no fagossomo. O número limitado de antimicrobianos disponíveis para tratamento apontam a necessidade de determinação do perfil de susceptibilidade frente a antimicrobianos isolados e em combinação, nos compartimentos intra e extracelular. Os objetivos deste estudo foram avaliar: a sensibilidade de M. abscessus estruturadas em biofilmes e presentes no interior dos macrófagos; a ocorrência de sinergismo quando da associação entre fármacos, inibidores de betalactamase e o anti-inflamatório. As combinações entre os antimicrobianos foram apenas indiferente quanto ao FIC e a atividade dos fármacos em biofilme e em macrófagos é bacteriostático. / Mycobacterium abscessus subspecies abscessus is a nightmare when involved in lung infection that is incurable, despite the use of antibiotics with in vitro activity, if the treatment does not include surgical resection of the affected area. It is a MCR - rapidly growing mycobacteria pathogenic most frequently isolated from cultures of lung sites. There are a small number of therapeutic options for the treatment of such infections is further reduced and the number of drugs that reach therapeutic concentrations in the intracellular compartment, particularly in the phagosome. The limited number of antimicrobials available for treatment indicate the need for determining the susceptibility profile against antimicrobials alone and in combination, in the intra and extracellular compartments. The objectives of this study were sensitivity of MCR structured biofilms and present in macrophages, the occurrence of synergism when the association between drugs, beta-lactamase inhibitors and anti-inflammatory. Combinations of antimicrobials were just indifferent and the activity of drugs on biofilms and macrophages was bacteriostatic.

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No. of bitstreams: 2 Dissertação - Camila Imai Morato - 2013.pdf: 4002125 bytes, checksum: 75c6fdada46a41f441e1dc6455c95483 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The American Tegumentary Leishmaniasis (ATL) is an infectious disease caused by parasitic protozoa of the genus Leishmania, whose most prevalent species in Brazil is L. (Viannia) braziliensis. The human macrophage infection with L. (V.) braziliensis has been poorly investigated and is not known whether leukotrienes, lipid mediators produced by macrophages, may modulate this infection. The objective of this study was to evaluate whether leukotrienes modulate infection of human macrophages with L. (V.) braziliensis IMG3, a field clinical isolate. Human monocyte-derived macrophages were infected with promastigote forms of MHOM/BR/2003/IMG (IMG3) at a ratio of ~ 10:1 (parasite:cell). Cultures were incubated for 4 h and then washed to remove excess extracellular parasites, and incubated for additional 24, 48 or 72 h. To inhibit leukotriene synthesis, the cultures were treated with MK0591 and to antagonize the LTB4 receptor, CP-105, 696. LTB4 was also added to the cultures. The measurement of LTB4 was performed on culture supernatants using enzyme immunoassay. To evaluate the involvement of reactive oxygen intermediates (ROI) and nitric oxide (NO) their respective inhibitors were used, apocynin and aminoguanidine. To assess the production of ROIs it was used nitroblue tetrazolium salt (NBT). The IMG3 infected human macrophages, being selected periods of 4 h and 48 h incubation to assess phagocytosis and microbicidal activity, respectively. Treatments with MK0591 or CP105, 696 significantly increased the infection index after 4 h or 48 h incubation (p <005). The results show that the presence of MK0591 is need during whole incubation time (48 h) but not the LTB4 antagonist, whose effect is maintained after incubation for 4 h. The addition of LTB4 to the cultures significantly decreased macrophage infection, both during the first 4 h and after 48 h of incubation (p <0.05). The parasites induced LTB4 production in macrophage cultures during the first 30 min, but this production decreased after 4 h (p <0.05). The ROI and NO inhibitors significantly increased the infection index, before (ROI and NO) or after treatment with LTB4 (ROI). Preliminary results showed that production of superoxide anion is induced by parasites and this is further increased by LTB4. In conclusion, the results suggest that human macrophages produce leukotrienes following infection with L. (V.) braziliensis, and the main of them is LTB4. The LTB4 inhibits phagocytosis and enhances the microbicidal activity of macrophages, contributing to control of the infection. Results suggest that L. (V.) braziliensis induces LTB4, NO and ROI production and, in turn, LTB4 increases the microbicidal activity of macrophages via increase of ROI. Understanding the involvement of leukotrienes in the control of human macrophage infection with L. (V.) braziliensis can lead to the development of novel therapeutic targets for ATL. / A Leishmaniose Tegumentar Americana (LTA) é uma doença infecto-parasitária causada por protozoários do gênero Leishmania, cuja espécie mais prevalente no Brasil é L. (Viannia) braziliensis.A infecção de macrófagos humanos com L. (V.) braziliensis tem sido pouco estudada e não é conhecido se os leucotrienos, mediadores lipídicos produzidos por macrófagos, podem modular a infecção destas células por este parasito. O objetivo deste trabalho foi avaliar se os leucotrienos modulam a infecção de macrófagos humanos pelo isolado L. (V.) braziliensis IMG3. Macrófagos humanos foram derivados de monócitos do sangue periférico e infectados com formas promastigotas do isolado MHOM/BR/2003/IMG (IMG3) na proporção de ~10:1 (parasitos:célula). As culturas foram incubadas por 4 h, sendo em seguida lavadas para retirar o excesso de parasitos extracelulares, e incubadas por adicionais 24, 48 ou 72 h. Para inibir a síntese dos leucotrienos, as culturas foram tratadas com MK0591 e para antagonizar o receptor de LTB4, CP-105,696. O LTB4 também foi adicionado às culturas. A dosagem de LTB4 foi realizada nos sobrenadantes das culturas, usando ensaio imunoenzimático. Para avaliar a participação de espécies intermediárias do oxigênio (ROI) e do óxido nítrico (NO), foram utilizados os seus respectivos inibidores, apocinina e aminoguanidina. Para avaliar a produção de ROIs foi utilizada a técnica do azul de tetrazólio (NBT). O isolado IMG3 infectou os macrófagos humanos, sendo selecionados os períodos de 4 h e 48 h de incubação para avaliar a fagocitose e a atividade microbicida, respectivamente. Os tratamentos com MK0591 ou CP105,696 aumentaram significantemente o índice de infecção, após 4 h ou 48 h de incubação (p < 005). Os resultados mostraram que há necessidade da presença do composto MK0591 durante todo o período de incubação (48 h), mas não a do antagonista do LTB4, cujo efeito é mantido após incubação por 4 h. A adição de LTB4 às culturas diminuiu significantemente a infecção dos macrófagos, tanto durante as primeiras 4 h quanto após 48 h de cultura (p < 0,05). Os parasitos induziram a produção de LTB4 nas culturas de macrófagos nos primeiros 30 min, caindo esta produção após 4 h (p < 0,05). Além disso, o uso de inibidores de ROI e NO aumentaram significantemente a infecção de macrófagos antes (ROI e NO) ou após a ativação com LTB4 (ROI). Os resultados preliminares mostram a produção de ânion superóxido, induzida pelos parasitos e uma tendência no aumento pelo LTB4. Em conclusão, os resultados sugerem que os macrófagos humanos produzem leucotrienos após a infecção por L. (V.) braziliensis, sendo o LTB4 o principal leucotrieno produzido. O LTB4 inibe a fagocitose e aumenta a atividade microbicida dos macrófagos, contribuindo para diminuir a infecção. Os resultados sugerem que há produção de ROI e NO após infecção com L. (V.) braziliensis e que o LTB4 aumenta a atividade microbicida dos macrófagos via aumento da produção de ROI. Entender a participação dos leucotrienos no controle da infecção de macrófagos humanos por L. (V.) braziliensis pode levar à descoberta de novos alvos terapêuticos para a LTA.

Indivíduos diabéticos apresentam incidência elevada de doenças infecciosas. Isto pode estar relacionado às alterações na capacidade da resposta destes indivíduos aos agentes agressores. Em animais diabéticos, algumas destas alterações já foram descritas, assim como sua reversão pela administração de insulina. Este hormônio regula o metabolismo celular, modulando a atividade de proteínas e mediadores inflamatórios envolvidos neste processo. Sabemos que o lipopolissacarídeo (LPS) estimula, em macrófagos alveolares (MA) de animais não-diabéticos, a liberação do fator de necrose tumoral (TNF)-&#945; e do óxido nítrico (NO). O pré-tratamento deste MA com insulina inibiu todos estes efeitos. Assim, neste projeto, avaliamos o papel da insulina em MA e macrófagos peritoneais (MP) de camundongos, tornados diabéticos pela indução com aloxana (60 mg/kg, i.v.). Uma suspensão contendo 1x106 células foi estimulada com LPS (100 ng/mL) na presença ou não de insulina (1mU/mL). Realizamos a evolução temporal (0,5; 1; 3; 6; 24 horas) para a dosagem de NO, TNF-&#945; e interleucina (IL)-10. Nos tempos de maior produção destas citocinas (0,5 e 3 horas), também quantificamos IL-6, interferon (IFN)-&#947; e IL-4. Nossos resultados mostram que a produção/liberação dos mediadores imunes por MA e MP estimulados por LPS, quando tratados simultaneamente com insulina, tiveram uma redução. Assim, a produção/liberação de NO foi reduzida durante 0,5; 1; 3; 6, 24 horas, em MA e em MP; a liberação de TNF-&#945; foi reduzida durante 0,5 hora, em MP; a liberação de IL-6 foi reduzida durante 3 horas, em MA, e 0,5 hora, em MP. Estes dados mostram que a insulina reduziu a liberação destes mediadores inflamatórios, tanto para os MA quanto para os MP, durante o estímulo com LPS, de animais diabéticos já nos primeiros minutos de estímulo com LPS, com um pico de redução, na maioria das vezes, em 3 horas. / Diabetic patients exhibit high incidence of infectious diseases, at least in part, by due impaired immune response against aggressive agents. Lipopolysaccharide (LPS) triggers the releasing of tumor necrosis factor (TNF)-alpha and nitric oxide (NO) by alveolar macrophages (AM) of non-diabetic animals. The pretreatment using insulin inhibited of these cytokine release. The aim in the present study was to evaluate the insulin role on releasing of cytokines by MA and peritoneal macrophages (MP) of diabetic mouse. Resident AM and MP from diabetic (alloxan 60 mg/kg, i.v.) male C57BL/6 mice (CEUA/FCF/USP-339) stimulated by LPS (100 ng/mL). Insulin (1 mU/mL) insulin treatment was simultaneously with stimulus by LPS. Cytokines were measured by ELISA, and NO by Griess reaction. We performed a time course (0,5; 1; 3; 6; 24 hours), and measured NO, TNF-alpha and interleukin (IL)-10. We also measured IL-6, interferon (IFN)-&#947;, and IL-4 in 0.5 and 3 hours. Results were evaluated by analysis of variance (ANOVA) followed by multiple comparison Tukey-Kramer or the nonparametric Kruskal-Wallis test. P value were considered when <0.05. Before the induction of diabetes by alloxan injection in day 0 animals showed: weight (26±0,3 g) and blood glucose concentration (164±2,6 mg/dL) - and 10 days after the onset of the disease, diabetic mice presented a lower weight gain (24 ± 0,4 g*) and elevated blood glucose concentration (546±9,7 mg/dL*). An increase was seen NO release levels and TNF-alpha in the time course (Figure 1); an increase of IL-6 in 3 hours (Figure 3), and a decrease of IL-4 in 3 hours (Figure 5) after LPS stimulation of MA in diabetic animals. In MP, there was an increase of NO levels, and TNF-alpha in the time course (Figure 2); in 3 hours increased IL-6 levels (Figure 4) - under the same conditions. Insulin treatment under stimulation by LPS in MA reduced the levels of NO, IL-6, and TNF-alpha compared to the group stimulated by LPS. Regarding MP, insulin treatment also decreased NO levels, TNF-alpha, and IL-6. IL-4 levels produced by MP, and IFN-&#947; levels were not be detected in MA and MP under stimulation by LPS, or in the presence of hormone. These findings suggest that insulin reduce the release of these inflammatory mediators by both resident macrophages of diabetic animals concomitantly stimulation by LPS

Sabe-se que a desnutrição modifica a resposta imune específica e inespecífica do organismo frente a agentes infecciosos comprometendo a produção e a função de células linfo-hemopoéticas, estando associada a modificações da resposta imune, resultando em maior suscetibilidade à infecções, porém os mecanismos exatos que comprometem o sistema imune em estados de desnutrição ainda estão para serem esclarecidos. A literatura relata que aproximadamente 60% das infecções que evoluem para sepse são adquiridas no ambiente hospitalar, envolvendo geralmente bactérias Gram negativas e incidindo especialmente em indivíduos com nutrição inadequada. Considerando tais aspectos e em função da complexidade da interação do estado nutricional e resposta do organismo frente a agentes patogênicos, envolvendo controles celulares e moleculares múltiplos ainda pouco conhecidos, propusemo-nos a estudar alguns aspectos da resposta inflamatória em desnutrição. Camundongos Swiss machos adultos, submetidos a desnutrição protéica-energética, após perda de aproximadamente 25% do peso corpóreo, foram inoculados com lipopolissacarideo de Escherichia coli: Hemograma, mielograma, esplenograma e dosagens sistêmicas de citocinas, foram realizadas. Células coletadas da cavidade peritonial de animais que não foram estimulados com LPS foram utilizadas para as determinações de citocinas e NO in vitro, bem como para quantificação dos receptores CD14 e TLR-4/MD-2 e do fator de transcrição NF&#954;B. Células da cavidade peritonial foram usadas, também, para realização dos testes de espraiamento, fagocitose e atividade cida com Candida albicans. Animais desnutridos apresentaram anemia, leucopenia; severa redução na celularidade da medula óssea, do baço e da cavidade peritonial. A capacidade de espraiamento, fagocitose, atividade cida e síntese de citocinas e NO foram significativamente menores nos animais dos grupo desnutrido. O número de receptores de CD14 e TLR-4/MD-2 e do fator de transrição NF&#954;B, também foram significativamente menores nos animais desnutridos. Estes achados sugerem que. animais desnutridos apresentam resposta deficiente frente ao LPS. A menor expressão de receptores CD14 e TLR-4/MD-2 pode ser responsável, em parte pela imunodepressão observada. Os dados nos levam a inferir que o estado nutricional interfere no estado de ativação de macrófagos e na capacidade de resposta dos animais. / Malnutrition modifies the specific and non-specific immune response of the organism to infectious agents, hampering the production and function of Iympho-hemopoietic cells leading to a higher susceptibility of the orgonism to infections. However, the exact mechanisms by which the immune system is undermined has not yet been fully elucidated. Approximately 60% of infections that evolve to bacteremia are nosocomial, and usually involve Gram negative bacteria in individuais that have inadequate nutrition. Taking this into consideration, and in view of the complexity of the interaction between nutritional state and the organism\' s response to infection, which involves poorly known multiple cellular and molecular controls, we proposed to study a few aspects of the inflammatory response in protein malnutrition. Male, outbred Swiss mice were sumbitted to protein malnutrition and after the loss of about 25% of total body weight they were inoculad whith lipopolissacharide of Escherichia coli: Hemogram, mielogram, splenogram and the determination of systemic production of cytokines were used to evaluate. Cells from the peritoneal cavity of animais who were not inoculated were collected for determination of cytokines and NO production in vitro and to evaluate the expression of NF&#954;B and CD14 and TLR-4/MD-2 receptors. Cells from the peritoneal cavity were used too for the spreading, phagocytosis and killing tests with Candida albicans. Malnourished animais presented anemia, leucopenia a severe reduction on bone marrow, spleen and peritoneal cavity cellularity. The spreading, phagocytosis, killing and the production of cytokines and NO was significantly lower in malnourished animais. The number of CD14 and TLR-4/MD-2 receptors and NF&#954;B was found to be significantly lower in malnourished animais. These findings suggest that malnourished mice present a deficient response to LPS. The smaller expression of CD14 and TLR-4/MD-2 receptors may be partly responsible for the immunodeficiency observed. These data lead us to infer that the nutritional state interferes in the activation of macrophages and in the immune response capacity.

Abstract Glutaredoxins (Grx) are small thiol disulphide oxidoreductases with a conserved active site sequence -CXXC/S- and a glutathione (GSH) binding site. They catalyze the reduction of protein disulphides, preferring protein-GSH mixed disulphides as substrates. The accumulation of protein-GSH mixed disulphides has been observed during oxidative stress, where they may serve both a regulatory and an antioxidant function by protecting the enzymes from irreversible oxidation. Once oxidative stress has been removed the GSH-protein mixed disulphides are reduced by GSH or, more efficiently, by Grx. The present study showed for the first time that Grx1 and Grx2 can be detected in healthy human lung. Highly specific expression of Grx1 was observed in alveolar macrophages, but it could also be detected from sputum supernatant. Grx1 levels in alveolar macrophages were lower in selected inflammatory diseases than in control lung samples. Grx1 was also mainly negative in the fibrotic areas in usual interstitial pneumonia, an aggressive fibrotic lung disease. Overall, the present study suggests that Grx1 is a potential redox modulatory protein regulating the intracellular as well as extracellular homeostasis of glutathionylated proteins and GSH not only in healthy lung, but also in inflammatory and fibrotic lung diseases. In order to study the mechanism of action of glutaredoxins in vitro, a new real-time fluorescence-based method for measuring the deglutathionylation activity of glutaredoxins using a glutathionylated peptide as a substrate was developed. The first reaction intermediate in the deglutathionylation reaction was shown to be exclusively Grx-GSH mixed disulphide and this specificity was solely dependent on the unusual γ-linkage present in glutathione. The study also demonstrated the role of conserved residues in the proximity of proposed GSH binding site to the GSH binding specificity of E. coli Grx1. Opening the binding groove and removing charged residues enabled Grx to form more readily mixed disulfides with other molecules besides GSH. Different members of the PDI family showed considerably lower activity levels compared to glutaredoxins and, in contrast to the glutaredoxin-GSH mixed disulphide, the only intermediate in the PDI catalysed reaction was PDI-peptide mixed disulphide.

La synthèse des protéines de choc thermique (HSPs) est un moyen de défense développé par la cellule pour faire face aux diverses agressions auxquelles elle peut être soumise. En tant que chaperons, les HSPs participent aux mouvements intracellulaires des protéines, préviennent l'agrégation des protéines altérées, éliminent les protéines anormales et contribuent à la conformation correcte des peptides nouvellement synthétisées. Mon équipe d’accueil s’intéresse aux rôles des HSPs dans des processus cellulaires tels que l’apoptose et la différenciation cellulaire. Le but de mon travail de thèse consiste à étudier le rôle des protéines de choc thermique HSP90 et HSP70 au cours de la différenciation des monocytes en macrophages. J’ai dans un premier temps étudié l’implication de HSP90 dans la différenciation macrophagique. c-IAP1 est un membre de la famille des protéines inhibitrices de l’apoptose impliqué dans la régulation de l’apoptose, dans le cycle cellulaire et dans la signalisation cellulaire. Nous avons précédemment montré que c-IAP1 migre du noyau vers le cytoplasme au cours de la différenciation cellulaire. Nous démontrons dans ce travail que c-IAP1 est une protéine cliente de la protéine de choc thermique HSP90β. Dans trois différents modèles de différenciation, ces protéines interagissent et migrent ensemble du noyau vers le cytoplasme au cours de la différenciation cellulaire. L’inhibition de HSP90 ou la déplétion spécifique de l’isoforme β par des siRNA conduisent à sa dégradation par le protéasome. La fonction de chaperon moléculaire de HSP90 envers c-IAP1 est spécifique de l’isoforme β car la déplétion de l’isoforme α n’a pas d’effets sur c-IAP1. De plus l’inhibition de HSP90 ou la déplétion de HSP90β bloquent la différenciation cellulaire tout comme la déplétion de c-IAP1 par siRNA. La deuxième partie de montre travail a consisté à étudier le rôle de HSP70 dans la différenciation macrophagique. Nous montrons que cette protéine est fortement induite après stimulation des cellules par le facteur de croissance M-CSF et que son inhibition bloque la différenciation des monocytes en macrophage. HSP70 interagit avec la protéine Spi-1/Pu.1, facteur de transcription clé de la différenciation macrophagique. L’expression de Spi-1/Pu.1 augmente également au cours de la différenciation macrophagique et ce de manière similaire à celle de HSP70. Ceci suggère l’implication des facteurs de transcription responsables de l’induction des HSPs, les Heat Shock Factor (HSF). L’étude du promoteur de Spi-1/Pu.1 a révélé la présence d’une séquence ressemblant fortement aux éléments de réponse classiques sur lesquels se fixe HSF1. HSF1 est capable de se fixer sur le promoteur de Spi-1/Pu.1 et l’inhibition de HSF1 bloque l’expression de Spi-1/Pu.1. HSF1 participe donc au contrôle de l’expression de Spi-1/Pu.1 lors de la différenciation macrophagique. HSP90 et HSP70 sont donc essentielles à la différenciation macrophagique. Comprendre les mécanismes cellulaires impliqués dans les voies de différenciation se révèle extrêmement important puisque des altérations des mécanismes de l’hématopoïèse sont retrouvées dans plusieurs types de leucémies (leucémies aiguës myéloblastiques et leucémies myélo-monocytaires chroniques). Connaître le rôle des HSPs dans la différenciation cellulaire permettrait donc de développer de nouvelles stratégies thérapeutiques pour le traitement de ces pathologies. / Heat shock proteins (HSPs) are molecular chaperones whose expression is increased after many different stresses. They have a protective function helping the cell to cope with lethal conditions. These proteins play an essential role as molecular chaperones by assisting the correct folding of nascent and stress-accumulated misfolded proteins and by preventing their aggregation. My team is interested in understanding the roles of HSPs in two physiological related processes: apoptosis and cell differentiation. The aim of my work is to study the functions of HSP90 and HSP70 in macrophagic differentiation. I first studied the role of HSP90 in macrophagic differentiation. We previously reported that cellular inhibitor of apoptosis protein-1 (c-IAP1), migrated from the nucleus to the surface of the Golgi apparatus in cells undergoing differentiation. c-IAP1 is a member of the inhibitor of apoptosis protein (IAP) family which has demonstrated functions in cell death, cell signaling and mitosis. Here, we show that c-IAP1 is a client protein of the stress protein HSP90β. In three distinct cellular models, the two proteins interact and migrate from the nucleus to the cytoplasm during the differentiation process through a leptomycin B-sensitive pathway. Inhibition of HSP90 proteins by small chemical molecules and specific depletion of HSP90isoform by siRNA both leads to c-IAP1 degradation by the proteasomal machinery. This chaperone function of HSP90 towards c-IAP1 is specific of its β isoform as specific depletion of HSP90α isoform does not affect c-IAP1 content. Chemical inhibition of HSP90 or siRNA-mediated depletion of HSP90both inhibit cell differentiation, which can be reproduced by siRNA-mediated depletion of c-IAP1. Altogether, these results suggest that HSP90prevents auto-ubiquitination and degradation of its client protein c-IAP1, whose depletion would be sufficient to inhibit cell differentiation. The second part of my work consisted in the study of the role of HSP70 in macrophagic differentiation. We used two models of differentiation: human peripheral monocytes exposed to M-CSF and THP1 cells induced to differentiate by TPA. We found that in both models, HSP70 expression was induced during differentiation. Interestingly, the expression of Spi-1/Pu.1, a transcription factor essential for monocytes to differentiate, was similarly induced. Upon differentiation, both proteins co-localized in the nucleus and associated. Inhibition or down regulation of HSP70 induced Spi-1/Pu.1 degradation and blocks the differentiation process, indicating that the necessity of Spi-1/Pu.1 to be chaperoned by HSP70 during differentiation. Since Spi-1/Pu.1 promoter has a HSE-like, we studied whether transcription factors responsible for HSPs induction, HSF, could be involved. We show that although HSF2 do not seem involved, HSF1 binds to Spi-1/Pu.1 promoter and its inhibition blocks Spi-1/Pu.1 expression and monocytes differentiation. So HSP90 and HSP70 are essentials for macrophagic differentiation. Understanding monocyte differentiation regulation is important since defects of the differentiation process can lead to the development of leukemias (acute myeloid leukemia, chronic myelomonocytic leukemia). A better understanding of the roles of HSPs may provide new therapeutic strategies for the treatment of these pathologies.