名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年3月25日 原真由氏の博士論文として提出された

Epidemiological studies report that infarct size is decreased and stroke outcomes are improved in young females when compared to males. However, mechanistic insight is lacking. We posit that sex-specific differences in glial cell functions occurring immediately after ischemic stroke are a source of dichotomous outcomes. In this study we assessed astrocyte Ca2+ dynamics, aquaporin 4 (AQP4) polarity, Sloop expression pattern, as well as, microglia morphology and phagocytic marker CD11b in male and female mice following 60 min of middle cerebral artery (MCA) occlusion. We reveal sex differences in the frequency of intracellular astrocyte Ca2+ elevations (F-(1,F-86) = 8.19, P = 0.005) and microglia volume (F-(1,F-40) = 12.47, P = 0.009) immediately following MCA occlusion in acute brain slices. Measured in fixed tissue, AQP4 polarity was disrupted (F-(5,F-86) = 3.30, P = 0.009) and the area of non-S100 beta immunoreactivity increased in ipsilateral brain regions after 60 min of MCA occlusion (F-(5,F-86) = 4.72, P = 0.007). However, astrocyte changes were robust in male mice when compared to females. Additional sex differences were discovered regarding microglia phagocytic receptor CD11b. In sham mice, constitutively high CD11b immunofluorescence was observed in females when compared to males (P = 0.03). When compared to sham, only male mice exhibited an increase in CD11b immunoreactivity after MCA occlusion (P = 0.006). We posit that a sex difference in the presence of constitutive CD11b has a role in determining male and female microglia phagocytic responses to ischemia. Taken together, these findings are critical to understanding potential sex differences in glial physiology as well as stroke pathobiology which are foundational for the development of future sex-specific stroke therapies. (C) 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Bordetella pertussis is an important human pathogen that causes an infection disease called whooping cough. This gram-negative bacterium produces an adenylate cyclase toxin (CyaA) that recognizes an integrin receptor CD11b/CD18 present on the surface of myeloid phagocytes and delivers an adenylate cyclase (AC) domain into the cell cytosol. This thesis deals with the endocytic machinery of CyaA and its potential use as a specific marker for endocytosis of the CD11b/CD18 receptor molecule. Detoxified mutant of CyaA, CyaA-AC- , that has the capacity to promote calcium influx as well the potassium efflux, was shown to trigger activation of the integrin receptor CD11b/CD18 followed with endocytic uptake by clathrin-dependent pathway. On the other side, the inactive mutant CyaA-KP-AC- that is unable to provoke integrin activation was endocytosed by clathrin-independent pathway. These results suggest that the various endocytic pathways of the CD11b/CD18 are determined by different conformational states of the receptor molecule.

A cavidade peritoneal de camundongos abriga uma variedade de células do sistema imune. Inicialmente, devido as limitações metodológicas, acreditava-se que, aproximadamente, 90% das células peritoneais era representada por macrófagos. Em seguida, graças aos extensos estudos com células peritoneais, observou-se que, além dos macrófagos, o peritônio abrigava muitos linfócitos B, principalmente do subtipo B-1. Utilizando metodologias contemporâneas de FACS - citometria de fluxo, este trabalho mostra que, aproximadamente, 30% das células peritoneais são macrófagos, 55% são linfócitos B-1, dos quais 40% pertencem ao subtipo B-1a e 15% ao subtipo B-1b. Os 15%-20% restantes representam outros subtipos celulares, como linfócitos T, linfócitos B-2, linfócitos NK, eosinófilos, além da presença de outras populações de células que não foram possíveis de ser identificadas com os marcadores de superfície utilizados neste trabalho. Em contraste com a literatura, nossos estudos mostraram que os macrófagos da cavidade peritoneal de camundongos representam uma população heterogênea. Por meio da co-expressão de CD11b e F4/80, foi possível descrever dois subtipos funcionais de macrófagos, denominados aqui de PM-1 (CD11bhigh, F4/80high) e PM-2 (F4/8010W, CD11bint). As células PM-2 encontram-se em menor abundância (-10% do total de macrófagos) no peritônio não estimulado e, diferentemente de células PM-1, espraiam-se de forma alongada (bipolar) quando colocadas em cultura. Curiosamente, PM-2 são os únicos macrófagos peritoneais que expressam MHC-II e, ainda, 30% dessa população expressa CD11c, o marcador típico de células dendríticas. Já as células PM-1 possuem morfologia arredondada, estão em abundância no peritônio e produzem altas doses de NO após estímulo com LPS. Ambas as células são capazes de fagocitar bactérias in vivo, no entanto, as células PM-2 parecem mais eficientes, sendo capazes de internalizar quantidades maiores de bactérias. Após infecção in vivo, o número absoluto e a porcentagem de PM-2 aumenta muito no peritônio, chegando a representar metade dos fagócitos da cavidade peritoneal. Os macrófagos PM-1 e PM-2 parecem não representar monócitos já que não compartilham do fenótipo de monócitos presentes no sangue periférico. Deve-se ressaltar que, além das células PM-1 e PM-2, outras subpopulações do peritônio também expressam CD11b e F4/80. Com base na literatura atual, acredita-se que todas as células B-1 do peritônio expressam CD11b, uma molécula que é co-expressa com CD18 para formar o receptor de complemento e a molécula de adesão Mac-1. Entretanto, os estudos, apresentados neste trabalho, mostram que metade de cada subtipo de B-1 (B-1a e B-1b) não expressa CD11b. As células B-1 CD11b+ são maiores, mais granulosas e expressam quantidades maiores de IgM de superfície. As células CD11b+ possuem uma curiosa tendência de se juntar e formar duplas (doublets) fortemente ligadas entre si que estão presentes em abundância no peritônio de camundongos adultos. Além de ligarem entre si, as células B-1 podem formar duplas com macrófagos PM-1 e PM-2. Durante a ontogenia, as células CD11b- surgem primeiro no peritônio e são as progenitoras das células B-1 CD11b+. Após estímulo inflamatório (LPS í. v.) os linfócitos B-1 CD11b+ migram do peritônio para o baço, onde proliferam e transformam-se em células secretoras de anticorpos (plasmócitos). No peritônio, as células B-1 não são capazes de se diferenciar em plasmócitos (células CD138high). Os resultados apresentados aqui mostraram que, diferentemente dos macrófagos PM-2, os PM-1 são os responsáveis por inibir a formação de células secretoras de anticorpos derivadas de B-1. Em suma, nossos dados sugerem que, mediante estímulo inflamatório, as células B-1 maduras (CD11b+) migram do peritônio para o baço, afastandose de macrófagos PM-1. Já no baço, as células B-1 encontram um micro-ambiente favorável para se proliferarem e diferenciarem-se em plasmócitos, secretando a maioria dos anticorpos naturais vistos logo nas primeiras horas pós-estímulo. / Mouse peritoneal cavity (PerC) represents the source for a variety of cellular subsets of the immune system. In early studies, marred by Iimited methodological tools, it was thought that macrophages comprise roughly 90% of total PerC cells. Shortly thereafter, it was recognized that, beside macrophages, the mouse peritoneal cavity shelters large amount of B-lymphocytes, specially the B-1 subset. In essence, by applying contemporary technology, studies presented here show that, roughly 30% of PerC cells comprise macrophages, 55% comprise B-1 cells, which 40% represents the B-1 a subset and roughly 15% are B-1b cells. The remaining 15%-20% of PerC cells comprise a variety of known immune cells, including B-2, T, NK and eosinophils. In addition, there were some other cellular subsets that could not be identified in these studies, probably due to limited cell surface molecules analyzed. Surprisingly, the data presented here show that peritoneal macrophages represent a highly heterogeneous population. Based on the co-expression of both CD11b and F4/80, we have identified two functionally distinct subsets of peritoneal macrophages, named here as PM-1 (CD11bhigh, F480high) and PM-2 (CD11bint, F4/80low). PM-2 are less abundant (-10% within total macrophages) in the PerC of unstimulated mouse, however, unlike PM-1, these cells are able to spread in spindle (bipolar) morphology upon sorting and in vitro culture. Curiously, PM-2 is the only PerC macrophage that expresses surface MHC-II. In addition, one third of PM-2 population expresses CD11c, a universal marker for dendritic cells. In turn, PM-1 cells are round shaped cells, represent the majority of PerC macrophages (-90%) and secrete high amounts of NO upon LPS stimulation. In vivo phagocytosis assay revealed that both cells could internalize bacteria, however, PM-2 cells showed to be more efficient, in that it was able to phagocyte higher amounts of bacteria when compared to PM-1. After i.p. in vivo stimulation, the absolute number and percentage of PM-2 cells increase drastically in the peritoneum, reaching almost half of the total PerC macrophages. Importantly, PM-1 and PM-2 macrophages seem not to represent PerC monocytes since it does not share any of the phenotype expressed by blood monocytes. Interestingly, in addition to PM-1 and PM-2, some other cellular subsets in the PerC, such as eosinophils, are able to express CD11b and F4/80. PerC B-1 cells have long been known to express CD11b, which is co-expressed with CD18 to form the Mac-1/CR3 complement receptor and adhesion molecule. However, although all PerC B-1 cells are commonly believed to express CD11b, we show here that nearly half of the cells in each of the PerC B-1 subsets (B-1a and B-1b) do not express this surface receptor. The CD11b+ cells in each B-1 subset are larger, more granular and express higher levels of surface IgM than the CD11b- B-1 cells. Surprisingly, the CD11b+ B-1 subset has the curious tendency to initiate the formation of tightly associated doublets that are present at high frequency in adult PerC. In addition to binding to each other, B-1 cells can also form doublets with PM-1 and PM-2. During ontogeny, CD11b- B-1 cells arise earlier in the mouse PerC and are the progenitors of CD11b+ B-1 cells. Upon LPS stimulation, PerC CD11b+ B-1 cells migrate to the spleen where they proliferate and differentiate into antibody-secreting cells (plasma cells). Within the PerC, B-1 cells are not able to differentiate into plasma cells (CD138high cells). The data shown in here reveal that, unlike PM-2, PM-1 macrophages are the cellular subset responsible for inhibiting the formation of B-1-derived antibody-secreting cells. Altogether, our data suggest that, upon inflammatory stimuli, mature CD11b+ B-1 cells migrate from the PerC to the spleen, avoiding the inhibitory effect of PM-1 cells. In the spleen, B-1 cells find an appropriate environment to proliferate and terminally differentiate into antibody-secreting cells, thus, secreting the majority of immunoglobulin seen in few hours after in vivo stimulation.

A cavidade peritoneal de camundongos abriga uma variedade de células do sistema imune. Inicialmente, devido as limitações metodológicas, acreditava-se que, aproximadamente, 90% das células peritoneais era representada por macrófagos. Em seguida, graças aos extensos estudos com células peritoneais, observou-se que, além dos macrófagos, o peritônio abrigava muitos linfócitos B, principalmente do subtipo B-1. Utilizando metodologias contemporâneas de FACS - citometria de fluxo, este trabalho mostra que, aproximadamente, 30% das células peritoneais são macrófagos, 55% são linfócitos B-1, dos quais 40% pertencem ao subtipo B-1a e 15% ao subtipo B-1b. Os 15%-20% restantes representam outros subtipos celulares, como linfócitos T, linfócitos B-2, linfócitos NK, eosinófilos, além da presença de outras populações de células que não foram possíveis de ser identificadas com os marcadores de superfície utilizados neste trabalho. Em contraste com a literatura, nossos estudos mostraram que os macrófagos da cavidade peritoneal de camundongos representam uma população heterogênea. Por meio da co-expressão de CD11b e F4/80, foi possível descrever dois subtipos funcionais de macrófagos, denominados aqui de PM-1 (CD11bhigh, F4/80high) e PM-2 (F4/8010W, CD11bint). As células PM-2 encontram-se em menor abundância (-10% do total de macrófagos) no peritônio não estimulado e, diferentemente de células PM-1, espraiam-se de forma alongada (bipolar) quando colocadas em cultura. Curiosamente, PM-2 são os únicos macrófagos peritoneais que expressam MHC-II e, ainda, 30% dessa população expressa CD11c, o marcador típico de células dendríticas. Já as células PM-1 possuem morfologia arredondada, estão em abundância no peritônio e produzem altas doses de NO após estímulo com LPS. Ambas as células são capazes de fagocitar bactérias in vivo, no entanto, as células PM-2 parecem mais eficientes, sendo capazes de internalizar quantidades maiores de bactérias. Após infecção in vivo, o número absoluto e a porcentagem de PM-2 aumenta muito no peritônio, chegando a representar metade dos fagócitos da cavidade peritoneal. Os macrófagos PM-1 e PM-2 parecem não representar monócitos já que não compartilham do fenótipo de monócitos presentes no sangue periférico. Deve-se ressaltar que, além das células PM-1 e PM-2, outras subpopulações do peritônio também expressam CD11b e F4/80. Com base na literatura atual, acredita-se que todas as células B-1 do peritônio expressam CD11b, uma molécula que é co-expressa com CD18 para formar o receptor de complemento e a molécula de adesão Mac-1. Entretanto, os estudos, apresentados neste trabalho, mostram que metade de cada subtipo de B-1 (B-1a e B-1b) não expressa CD11b. As células B-1 CD11b+ são maiores, mais granulosas e expressam quantidades maiores de IgM de superfície. As células CD11b+ possuem uma curiosa tendência de se juntar e formar duplas (doublets) fortemente ligadas entre si que estão presentes em abundância no peritônio de camundongos adultos. Além de ligarem entre si, as células B-1 podem formar duplas com macrófagos PM-1 e PM-2. Durante a ontogenia, as células CD11b- surgem primeiro no peritônio e são as progenitoras das células B-1 CD11b+. Após estímulo inflamatório (LPS í. v.) os linfócitos B-1 CD11b+ migram do peritônio para o baço, onde proliferam e transformam-se em células secretoras de anticorpos (plasmócitos). No peritônio, as células B-1 não são capazes de se diferenciar em plasmócitos (células CD138high). Os resultados apresentados aqui mostraram que, diferentemente dos macrófagos PM-2, os PM-1 são os responsáveis por inibir a formação de células secretoras de anticorpos derivadas de B-1. Em suma, nossos dados sugerem que, mediante estímulo inflamatório, as células B-1 maduras (CD11b+) migram do peritônio para o baço, afastandose de macrófagos PM-1. Já no baço, as células B-1 encontram um micro-ambiente favorável para se proliferarem e diferenciarem-se em plasmócitos, secretando a maioria dos anticorpos naturais vistos logo nas primeiras horas pós-estímulo. / Mouse peritoneal cavity (PerC) represents the source for a variety of cellular subsets of the immune system. In early studies, marred by Iimited methodological tools, it was thought that macrophages comprise roughly 90% of total PerC cells. Shortly thereafter, it was recognized that, beside macrophages, the mouse peritoneal cavity shelters large amount of B-lymphocytes, specially the B-1 subset. In essence, by applying contemporary technology, studies presented here show that, roughly 30% of PerC cells comprise macrophages, 55% comprise B-1 cells, which 40% represents the B-1 a subset and roughly 15% are B-1b cells. The remaining 15%-20% of PerC cells comprise a variety of known immune cells, including B-2, T, NK and eosinophils. In addition, there were some other cellular subsets that could not be identified in these studies, probably due to limited cell surface molecules analyzed. Surprisingly, the data presented here show that peritoneal macrophages represent a highly heterogeneous population. Based on the co-expression of both CD11b and F4/80, we have identified two functionally distinct subsets of peritoneal macrophages, named here as PM-1 (CD11bhigh, F480high) and PM-2 (CD11bint, F4/80low). PM-2 are less abundant (-10% within total macrophages) in the PerC of unstimulated mouse, however, unlike PM-1, these cells are able to spread in spindle (bipolar) morphology upon sorting and in vitro culture. Curiously, PM-2 is the only PerC macrophage that expresses surface MHC-II. In addition, one third of PM-2 population expresses CD11c, a universal marker for dendritic cells. In turn, PM-1 cells are round shaped cells, represent the majority of PerC macrophages (-90%) and secrete high amounts of NO upon LPS stimulation. In vivo phagocytosis assay revealed that both cells could internalize bacteria, however, PM-2 cells showed to be more efficient, in that it was able to phagocyte higher amounts of bacteria when compared to PM-1. After i.p. in vivo stimulation, the absolute number and percentage of PM-2 cells increase drastically in the peritoneum, reaching almost half of the total PerC macrophages. Importantly, PM-1 and PM-2 macrophages seem not to represent PerC monocytes since it does not share any of the phenotype expressed by blood monocytes. Interestingly, in addition to PM-1 and PM-2, some other cellular subsets in the PerC, such as eosinophils, are able to express CD11b and F4/80. PerC B-1 cells have long been known to express CD11b, which is co-expressed with CD18 to form the Mac-1/CR3 complement receptor and adhesion molecule. However, although all PerC B-1 cells are commonly believed to express CD11b, we show here that nearly half of the cells in each of the PerC B-1 subsets (B-1a and B-1b) do not express this surface receptor. The CD11b+ cells in each B-1 subset are larger, more granular and express higher levels of surface IgM than the CD11b- B-1 cells. Surprisingly, the CD11b+ B-1 subset has the curious tendency to initiate the formation of tightly associated doublets that are present at high frequency in adult PerC. In addition to binding to each other, B-1 cells can also form doublets with PM-1 and PM-2. During ontogeny, CD11b- B-1 cells arise earlier in the mouse PerC and are the progenitors of CD11b+ B-1 cells. Upon LPS stimulation, PerC CD11b+ B-1 cells migrate to the spleen where they proliferate and differentiate into antibody-secreting cells (plasma cells). Within the PerC, B-1 cells are not able to differentiate into plasma cells (CD138high cells). The data shown in here reveal that, unlike PM-2, PM-1 macrophages are the cellular subset responsible for inhibiting the formation of B-1-derived antibody-secreting cells. Altogether, our data suggest that, upon inflammatory stimuli, mature CD11b+ B-1 cells migrate from the PerC to the spleen, avoiding the inhibitory effect of PM-1 cells. In the spleen, B-1 cells find an appropriate environment to proliferate and terminally differentiate into antibody-secreting cells, thus, secreting the majority of immunoglobulin seen in few hours after in vivo stimulation.

A desnutrição é uma condição nutricional que pode afetar muitos aspectos da resposta imunológica, como alterações na migração celular, na fagocitose, na resposta bactericida, mudanças na produção de radicais livres e espécies de nitrogênio e na produção de citocinas pró-inflamatórias. Logo, indivíduos desnutridos apresentam maior susceptibilidade a infecções. Visto que a glutamina é um aminoácido de extrema importância para a funcionalidade de diversas células do sistema imune e que as mesmas apresentam aumento da utilização desse aminoácido durante processos infecciosos, investigou-se, neste trabalho, quais os efeitos da glutamina sobre alguns aspectos da mobilização, migração e sinalização celular em um modelo experimental de desnutrição proteica. Para tanto, utilizou-se camundongos da linhagem BALB/c machos, os quais foram divididos em dois grupos, Controle e Desnutrido, que passaram a receber dietas isocalóricas contendo 12% (normoproteica) e 2% de caseína (hipoproteica), respectivamente, durante 5 semanas. Para as avaliações in vivo, animais de ambos os grupos receberam por via endovenosa 100µL de solução contendo 1,25µg de LPS e após 1 hora 0,75mg/Kg de L-glutamina (GLUT). Após o período de desnutrição ou de indução ao processo inflamatório, os animais foram eutanasiados e as amostras biológicas coletas. Foram avaliados nos animais estimulados in vivo hemograma, mielograma, as citocinas IL-10 e TNF-α circulantes e a expressão de CD11b/CD18 nos granulócitos do sangue periférico. Foi avaliado, in vitro, a capacidade migratória, a expressão de CD11b/CD18 de polimorfonucleados da medula óssea e do sangue periférico, bem como a síntese de citocinas IL-1α, IL-6, IL-10, IL-12 e TNF-α e a expressão de NF-κB e IκBα em células cultivadas em meio com 0; 0,6; 2 e 10 mM de GLUT. Os animais desnutridos apresentaram anemia, leucopenia, hipoplasia medular e diminuição na concentração sérica de proteínas, albumina e pré-albumina. A GLUT, in vitro, apresentou capacidade de reduzir a produção de IL-1α e IL-6, bem como a ativação da via do NF-κB. No modelo in vivo a GLUT, em animais estimulados com LPS, alterou a cinética de migração neutrofílica e reduziu a expressão de CD18, bem como diminuiu os níveis de TNFα circulantes. / Malnutrition is a nutritional condition that can affect many aspects of immune responses, affecting cell migration, phagocytosis, bactericidal response and changing free radicals production as nitrogen species and production of proinflammatory cytokines. Therefore, malnourished individuals are more susceptible to infections. Once glutamine is an amino acid of extreme importance to the functionality of various immune cells and those cells exhibit increased use of this amino acid during infectious processes. In this work was investigated, the effects of glutamine in some aspects of mobilization, cell migration and signaling in an experimental model of protein malnutrition. For this purpose, we used BALB/c mice, which received isocaloric diets, normoproteic or hypoproteic, containing respectively, 12% (Control group) and 2% (Malnourished group) of protein for a period of 5 weeks. The animals in both groups, for in vivo evaluations, received intravenous 100 µl of a solution containing 1.25µg of LPS and after 1 hour 0.75mg/kg of L-glutamine (GLUT). After the malnutrition period or the inflammatory process induction, the animals were euthanized and biological samples were collected. Were evaluated blood count, bone marrow, the cytokines IL-10 and TNF-α circulating and expression of CD11b/CD18 in granulocytes from peripheral blood of animals stimulated in vivo. In vitro were evaluated the migratory capacity, the expression of CD11b/CD18 polymorphonuclear bone marrow and peripheral blood, as well as the cytokines synthesis IL-1α, IL-6, IL-10, IL-12 and TNF-α and the expression of NF-κB and IκBα in cultured cells in media with 0; 0.6; 2 and 10 mM GLUT. Malnourished animals presented anemia, leukopenia, marrow hypoplasia and lower serum proteins, albumin and prealbumin. The GLUT in vitro has the capacity to reduce IL-1α and IL-6 as well as the activation of the NF-κB. In in vivo model, the GLUT altered neutrophil migration kinetics and reduced the expression of CD18, as well as decreased levels of circulating TNF-α in animals stimulated with LPS.

A desnutrição é uma condição nutricional que pode afetar muitos aspectos da resposta imunológica, como alterações na migração celular, na fagocitose, na resposta bactericida, mudanças na produção de radicais livres e espécies de nitrogênio e na produção de citocinas pró-inflamatórias. Logo, indivíduos desnutridos apresentam maior susceptibilidade a infecções. Visto que a glutamina é um aminoácido de extrema importância para a funcionalidade de diversas células do sistema imune e que as mesmas apresentam aumento da utilização desse aminoácido durante processos infecciosos, investigou-se, neste trabalho, quais os efeitos da glutamina sobre alguns aspectos da mobilização, migração e sinalização celular em um modelo experimental de desnutrição proteica. Para tanto, utilizou-se camundongos da linhagem BALB/c machos, os quais foram divididos em dois grupos, Controle e Desnutrido, que passaram a receber dietas isocalóricas contendo 12% (normoproteica) e 2% de caseína (hipoproteica), respectivamente, durante 5 semanas. Para as avaliações in vivo, animais de ambos os grupos receberam por via endovenosa 100µL de solução contendo 1,25µg de LPS e após 1 hora 0,75mg/Kg de L-glutamina (GLUT). Após o período de desnutrição ou de indução ao processo inflamatório, os animais foram eutanasiados e as amostras biológicas coletas. Foram avaliados nos animais estimulados in vivo hemograma, mielograma, as citocinas IL-10 e TNF-α circulantes e a expressão de CD11b/CD18 nos granulócitos do sangue periférico. Foi avaliado, in vitro, a capacidade migratória, a expressão de CD11b/CD18 de polimorfonucleados da medula óssea e do sangue periférico, bem como a síntese de citocinas IL-1α, IL-6, IL-10, IL-12 e TNF-α e a expressão de NF-κB e IκBα em células cultivadas em meio com 0; 0,6; 2 e 10 mM de GLUT. Os animais desnutridos apresentaram anemia, leucopenia, hipoplasia medular e diminuição na concentração sérica de proteínas, albumina e pré-albumina. A GLUT, in vitro, apresentou capacidade de reduzir a produção de IL-1α e IL-6, bem como a ativação da via do NF-κB. No modelo in vivo a GLUT, em animais estimulados com LPS, alterou a cinética de migração neutrofílica e reduziu a expressão de CD18, bem como diminuiu os níveis de TNFα circulantes. / Malnutrition is a nutritional condition that can affect many aspects of immune responses, affecting cell migration, phagocytosis, bactericidal response and changing free radicals production as nitrogen species and production of proinflammatory cytokines. Therefore, malnourished individuals are more susceptible to infections. Once glutamine is an amino acid of extreme importance to the functionality of various immune cells and those cells exhibit increased use of this amino acid during infectious processes. In this work was investigated, the effects of glutamine in some aspects of mobilization, cell migration and signaling in an experimental model of protein malnutrition. For this purpose, we used BALB/c mice, which received isocaloric diets, normoproteic or hypoproteic, containing respectively, 12% (Control group) and 2% (Malnourished group) of protein for a period of 5 weeks. The animals in both groups, for in vivo evaluations, received intravenous 100 µl of a solution containing 1.25µg of LPS and after 1 hour 0.75mg/kg of L-glutamine (GLUT). After the malnutrition period or the inflammatory process induction, the animals were euthanized and biological samples were collected. Were evaluated blood count, bone marrow, the cytokines IL-10 and TNF-α circulating and expression of CD11b/CD18 in granulocytes from peripheral blood of animals stimulated in vivo. In vitro were evaluated the migratory capacity, the expression of CD11b/CD18 polymorphonuclear bone marrow and peripheral blood, as well as the cytokines synthesis IL-1α, IL-6, IL-10, IL-12 and TNF-α and the expression of NF-κB and IκBα in cultured cells in media with 0; 0.6; 2 and 10 mM GLUT. Malnourished animals presented anemia, leukopenia, marrow hypoplasia and lower serum proteins, albumin and prealbumin. The GLUT in vitro has the capacity to reduce IL-1α and IL-6 as well as the activation of the NF-κB. In in vivo model, the GLUT altered neutrophil migration kinetics and reduced the expression of CD18, as well as decreased levels of circulating TNF-α in animals stimulated with LPS.

Small diameter synthetic vascular grafts have yet to be clinically successful due to luminal narrowing from thrombosis and intimal hyperplasia. Current attempts to address this issue include the development of materials that support endothelialisation and protein modification to the material surfaces that reduce thrombosis. The extracellular matrix protein elastin has been found to be one of the least thrombogenic components of blood vessels, and its purified and recombinant forms have shown reduced thrombogenicity in both in vitro and in vivo models. Biomaterial coatings of elastin-like polypeptides (ELPs) recombinantly produced in the Woodhouse laboratory showed reduced fibrinogen adsorption, platelet adhesion, and platelet activity. However, the reason for their relative non-thrombogenicity is still not fully understood. In this work, the leukocyte response to ELP-coated materials was investigated. In particular, ELP1 and ELP4, which differ in molecular weight and sequence length, were physically adsorbed to a polyethylene terephthalate surface (MylarTM), yielding 0.22 ± 0.13 μg/cm2 and 0.37 ± 0.19 μg/cm2 surface coverage, respectively, as determined by the colorimetric assay, FastinTM Elastin. These surfaces were exposed to flowing citrated whole blood for surface and bulk evaluation of leukocyte activity using scanning electron microscopy and flow cytometry, respectively. Little leukocyte activation was observed on the surface of the controls, low-density polyethylene and uncoated MylarTM. In the bulk, tissue factor (TF) expression (monocytes: ELP1 = 38.6 ± 16.3 %, ELP4 = 33.9 ± 18.1 %) and platelet-leukocyte aggregates determined by CD61 (monocytes: ELP1 = 63.1 ± 17.1 %, ELP4 = 61.8 ± 16.8 %; granulocytes: ELP1 = 62.7 ± 17.0 %, ELP4 = 60.5 ± 20.1 %) were both decreased compared to uncoated MylarTM, while CD11b upregulation (monocytes: ELP1 = 18.7 ± 2.2 %, ELP4 = 19.7 ± 2.7 %; granulocytes: ELP = 21.4 ± 3.7 %, ELP4 = 22.0 ± 3.2 %) was increased. The statistical dependence of TF expression and platelet-monocyte aggregates was tested; however, no correlation was found. Overall, platelet-leukocyte aggregate formation was reduced and there were conflicting results with regards to the reduction of leukocyte activation for the ELP coatings on MylarTM. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-10-10 15:34:51.802

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 14, 2010). Includes bibliographical references.

Η Οξεία λευχαιμία (ΟΛ) αποτελεί νεοπλασματικό, αιμοποιητικό νόσημα, που οφείλεται στον πολλαπλασιασμό και την επέκταση κυττάρων, που προέρχονται από τους λευχαιμικούς βλάστες. Η φυσική εξέλιξη του νοσήματος είναι η αντικατάσταση των φυσιολογικών κυττάρων του αιμοποιητικού ιστού από τους απόγονους των λευχαιμικών βλαστών και θάνατος, λόγω των επιπλοκών της έλλειψης των ώριμων αιμοποιητικών κυττάρων, όπως λοιμώξεις, αναιμία και αιμορραγία. Θεραπευτικά για την αντιμετώπιση της ΟΛ χρησιμοποιούνται σχήματα χημειοθεραπείας και ακτινοθεραπείας, που έχουν σαν σκοπό την καταστροφή των λευχαιμικών βλαστών και την αποκατάσταση της φυσιολογικής αιμοποίησης. Συχνά η ΟΛ, ιδιαίτερα σε ασθενείς μεγάλης ηλικίας, εμφανίζεται ταυτόχρονα με δυσπλαστικές διαταραχές των αιμοποιητικών κυττάρων που ωριμάζουν ή εξελίσσεται σε μυελοδυσπλαστικό σύνδρομο μετά από χημειοθεραπεία. Από την βιβλιογραφία είναι γνωστό ότι, τόσο η ΟΛ μπορεί να είναι στάδιο εξέλιξης των μυελοδυσπλαστικών συνδρόμων και κατά συνέπεια μετά τη θεραπεία της ΟΛ επανέρχεται η δυσπλαστική κατάσταση της αιμοποίησης, όσο ότι η χημειοθεραπεία καθαυτή μπορεί να προκαλέσει μυελοδυσπλασία. Είναι σημαντική η διάγνωση των πρωτοπαθών μυελοδυσπλαστικών συνδρόμων που εξελίσσονται σε ΟΛ, ως προς την πρόγνωση των ασθενών, αλλά και τη θεραπευτική τους αντιμετώπιση. Επίσης, είναι σημαντική η διάκριση ομάδων ασθενών που θα αναπτύξουν δυσπλασία μετά από χημειοθεραπεία, σε σχέση με αυτούς που δεν θα αναπτύξουν και ως προς την πρόγνωση και ως προς τη θεραπευτική αντιμετώπιση. Μέχρι σήμερα, κατά την εμφάνιση της ΟΛ η διάγνωση υποκείμενης μυελοδυσπλασίας είναι δύσκολη και στηρίζεται σε μορφολογικά χαρακτηριστικά των ώριμων κυττάρων και στην παρουσία ορισμένων κυτταρογενετικών διαταραχών. Η διάκριση ομάδων που θα αναπτύξουν δυσπλασία μετά από χημειοθεραπεία είναι αδύνατη. Σκοπός της μελέτης ήταν να συμβάλλει στην ανάπτυξη νέων πρωτοκόλλων στο λογισμικό της κυτταρομετρίας ροής, που θα διευκολύνουν τη διάγνωση πρωτοπαθών δυσπλαστικών συνδρόμων κατά την εμφάνιση της ΟΛ, αλλά και θα διακρίνουν τις ομάδες που μπορούν να αναπτύξουν δυσπλασία μετά από θεραπεία. Για τον σκοπό αυτό, παρακολουθήσαμε την έκφραση χαρακτηριστικών αντιγονικών συνδυασμών, που εκφράζονται σε διαφορετικά στάδια ωρίμανσης των φυσιολογικών κυττάρων, παράλληλα με την έκφραση των αντιγόνων των βλαστών. Τα δεδομένα αυτά μελετήθηκαν, τόσο κατά την εμφάνιση της ΟΛ, όσο και κατά τη παρακολούθηση της. Τα δεδομένα αναλύθηκαν με συστήματα ταυτόχρονης ανάλυσης και συσχέτισης 15-20 παραμέτρων, με σκοπό τον καθορισμό συσχετισμών που θα έχουν διαγνωστική και προγνωστική σημασία για τους ασθενείς με ΟΛ. Τα αποτελέσματα του ανοσοφαινοτύπου αναλύθηκαν, επιπλέον, με το λογισμικό πακέτο στατιστικής ανάλυσης SPSS 16.0. Για τους σκοπούς της μελέτης αναλύθηκαν αναδρομικά τα αποτελέσματα της κυτταρομετρίας ροής στο μυελό των οστών 148 ασθενών με ΟΜΛ κατά την εμφάνιση της νόσου, κατά την διάρκεια και μετά από θεραπεία. Αναλύθηκε η έκφραση των αντιγόνων CD11b/CD16/CD13 σε όλα τα στάδια ωρίμανσης της μυελικής σειράς, ενώ η έκφραση των αντιγόνων CD34/CD117 μόνο στα άωρα κύτταρα. Τα ευρήματα του ανοσοφαινοτύπου συγκρίθηκαν με τα μορφολογικά χαρακτηριστικά των αντίστοιχων μυελών των οστών. Συμπερασματικά, τα αποτελέσματα έδειξαν ότι, η έκφραση των αντιγόνων CD11b και CD13 στα μεταμυελοκύτταρα και ουδετερόφιλα των ασθενών διακρίνει αποτελεσματικά τους ασθενείς με de novo ΟΜΛ σε σχέση με αυτούς που εμφάνισαν ΟΜΛ μετά από ΜΔΣ. Στους ασθενείς με de novo ΟΜΛ η έκφραση των αντιγόνων CD11b, CD13, CD16 δεν διέφερε κατά την εμφάνιση της ΟΛ στους υποπληθυσμούς των προμυελοκυττάρων, μυελοκυττάρων, μεταμυελοκυττάρων και ουδετερόφιλων μεταξύ των ασθενών που κατά ή μετά την θεραπεία εμφάνισαν μυελοδυσπλασία, σε σχέση με αυτούς που δεν εμφάνισαν. Επίσης, η θετική συν- έκφραση των αντιγόνων CD34/CD117 στους λευχαιμικούς βλάστες κατά την εμφάνιση δεν συσχετίζονταν με την εμφάνιση ΜΔΣ μετά την θεραπεία. Αντίθετα, η υψηλή έκφραση του λευχαιμικού φαινοτύπου CD34+/CD117- στα άωρα κύτταρα της μυελικής σειράς κατά την εμφάνιση της ΟΜΛ, έδειξε ότι σχετίζεται με την εμφάνιση μυελοδυσπλαστικών χαρακτηριστικών μετά τη θεραπεία. Η ανάλυση των ανοσοφαινοτύπων του μυελού των οστών κατά ή μετά την θεραπεία έδειξε παθολογική έκφραση CD11b, CD13, CD16 στα μεταμυελοκύτταρα και ουδετερόφιλα των ασθενών που εμφάνισαν ΜΔΣ μετά θεραπεία για de novo ΟΜΛ και ασθενών (5/17) που δεν εμφάνισαν ΜΔΣ. Συμπερασματικά, η έκφραση των αντιγόνων CD11b, CD16 και CD13 στα ώριμα κύτταρα της μυελικής σειράς κατά την εμφάνιση της ΟΜΛ διαχωρίζει αποτελεσματικά την de novo ΟΜΛ από την δευτεροπαθή μετά ΜΔΣ. Η έκφραση αυτών των αντιγόνων κατά την εμφάνιση της ΟΜΛ δεν μπορεί, όμως, να προβλέψει την εξέλιξη της de novo ΟΜΛ και την εμφάνιση δυσπλαστικών χαρακτηριστικών μετά τη θεραπεία. Αντιθέτως, η μελέτη των λευχαιμικών φαινοτύπων CD34+/CD117- και CD34+/CD117+ μπορεί να παίξει καθοριστικό ρόλο στην πρόγνωση της εμφάνισης μυελοδυσπλαστικών χαρακτηριστικών κατά τη διάρκεια ή μετά τη θεραπεία του ασθενούς. / --