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Hematopoietic cells of the myelomonocytic lineage play a central role in orchestrating both innate and adaptive immunity. They are important in the control of infectious agents and in the pathogenesis of diseases characterized by dysregulated immune response. Like allergic asthma in human patients, recurrent airway obstruction (RAO) of horses is a disease exemplified by chronic airway inflammation in the absence of infectious agents. However, unlike allergic asthma, RAO is marked by preponderance of neutrophils rather than eosinophils in the airways. Attempts to understand the immunological basis of RAO by studying lymphocytes produced equivocal results. This thesis examined the possible role of alveolar macrophages (AM) recovered from bronchoalveolar lavage fluid (BALF) in RAO. Since macrophages are predominantly derived from circulating monocytes, the thesis investigated first the phenotypic characteristics of circulating monocytes, second those of macrophages in vitro derived from monocytes, and finally attributes of AM derived in vivo. Flow cytometric analysis following antibody staining of monocytes from 61 horses showed that the clustering pattern of human leukocytes may not always be extrapolated to horses when using this technique since clusters of granulocytes often spill over into the monocyte population. The study showed that DH24A, a monoclonal antibody directed against CD90, which recognizes T cells in other species, will specifically recognize granulocytes in horses and was therefore used to separate neutrophils from monocytes during analysis. In addition, investigation of circulating monocytes showed that expression of the hemoglobin-haptoglobin receptor CD163 on circulating monocytes is significantly increased in horses with systemic inflammation when compared with healthy horses. Evaluating cytokine and chemokine production by macrophages, it was demonstrated that CD163+ macrophages preferentially expressed IL10 while CD163- macrophages showed predominant expression of CCL17. It was, therefore, concluded that CD163+ IL10-producing macrophages of horses are homologues of the alternatively activated anti-inflammatory macrophage subset of humans. Finally, probing of alveolar macrophages for CD163 and CD206 expression showed a significant reduction in the proportion of CD163+ macrophages in horses with RAO. These findings suggest that RAO is associated with a reduction in anti-inflammatory macrophages, an observation that may in part explain the chronic airway inflammation associated with this disease.

The kinetics and mechanism of accumulation for liposomal ciprofloxacin (Lipo-CPFX) into the rat alveolar macrophage NR8383 cells were studied in vitro, in comparison to unformulated ciprofloxacin (CPFX). Upon incubation with CPFX or Lipo-CPFX, cellular drug accumulation was determined from the cell lysates or efflux was from the extracellular media by fluorescence-HPLC. The accumulation for Lipo-CPFX reached the asymptotic values at ≥ 2 hours, which was a result of uptake and efflux. The uptake appeared to be due to liposomes, mediated via cellular energy-independent mechanism like lipid fusion. In contrast, the efflux appeared to be due to ciprofloxacin, partly cellular energy-dependent, and involve probenecid-sensitive multidrug resistance proteins (MRPs). Overall, Lipo-CPFX enabled greater drug accumulation into the NR8383 cells than CPFX. This logically suggests a greater potential to treat respiratory infections especially caused by bacteria resistant to phagocytic killing.

Pneumonia is considered to be one of the leading causes of death worldwide. The outcome depends on both, proper antibiotic treatment and the effectivity of the immune response of the host. However, due to the complexity of the immunologic cascade initiated during infection, the latter cannot be predicted easily. We construct a biomathematical model of the murine immune response during infection with pneumococcus aiming at predicting the outcome of antibiotic treatment. The model consists of a number of non-linear ordinary differential equations describing dynamics of pneumococcal population, the inflammatory cytokine IL-6, neutrophils and macrophages fighting the infection and destruction of alveolar tissue due to pneumococcus. Equations were derived by translating known biological mechanisms and assuming certain response kinetics. Antibiotic therapy is modelled by a transient depletion of bacteria. Unknown model parameters were determined by fitting the predictions of the model to data sets derived from mice experiments of pneumococcal lung infection with and without antibiotic treatment. Time series of pneumococcal population, debris, neutrophils, activated epithelial cells, macrophages, monocytes and IL-6 serum concentrations were available for this purpose. The antibiotics Ampicillin and Moxifloxacin were considered. Parameter fittings resulted in a good agreement of model and data for all experimental scenarios. Identifiability of parameters is also estimated. The model can be used to predict the performance of alternative schedules of antibiotic treatment. We conclude that we established a biomathematical model of pneumococcal lung infection in mice allowing predictions regarding the outcome of different schedules of antibiotic treatment. We aim at translating the model to the human situation in the near future.

Objetivou-se caracterizar clinicamente a Doença Inflamatória de Vias Aéreas (DIVA) assim como verificar o comportamento da atividade dos macrófagos alveolares destes animais. Utilizaram-se 17 eqüinos adultos machos e fêmeas, com idade variando entre 11 e 24 anos, sendo oito do Regimento Escola de Cavalaria Andrade Neves (grupo Controle) e nove da Polícia Militar do Rio de Janeiro (grupo Doente, com DIVA). Estes animais não apresentavam história pregressa recente (dois meses) compatível com doença respiratória, nesse período não foram submetidos a nenhum tipo de tratamento e apresentavam resultados de leucogramas e determinações de fibrinogênio plasmático dentro da normalidade. Realizaram-se exames físicos, endoscópicos, mensurações da diferença de pressão intrapleural máxima (Ventigrafia), e citologia broncoalveolar. Para o grupo Controle os animais foram selecionados levando-se em conta os resultados de normalidade nos exames executados. O grupo Doente foi selecionado considerando-se achados de anormalidade compatíveis com alterações inflamatórias pulmonares no mínimo em dois exames, sendo obrigatórios os exames endoscópicos e de citologia broncoalveolar. Para a avaliação da atividade macrofágica, macrófagos alveolares obtidos através de lavagem broncoalveolar foram cultivados a 37ºC em atmosfera de 7% CO2, aderidos em lamínulas de vidro estéril, de 13 mm de diâmetro em placas de 24 poços contendo 300 µL de RPMI 1640 enriquecido com 10% de Soro Fetal Bovino inativado pelo calor. Realizaram-se testes de fagocitose de Zymosan (por uma hora) e de interação com Promastigotas de Leishmania braziliensis cepa 3456 (por uma até 48 horas pós infecção). A análise das funções vitais e das mensurações da diferença de pressão intrapleural máxima revelou, ainda que dentro de valores fisiológicos, diferenças significativas apenas para a temperatura corpórea e freqüência cardíaca. Para os achados do exame do exame físico, não foi detectada diferença estatística entre os grupos. Com relação ao lavado broncoalveolar, o grupo Doente apresentou aumento na contagem celular total, moderado aumento na porcentagem de neutrófilos, discreta redução na porcentagem de macrófagos, porém com aumento no número de macrófagos espumosos e de células epiteliais além de discreto aumento na porcentagem de eosinófilos. Não observou-se diferença, entre os grupos, na capacidade fagocítica com relação ao Zymosan. Porém, uma diferença significativa na capacidade fagocítica após uma hora de interação e redução do índice de sobrevivência de L. braziliensis após 48 horas de cultivo no grupo Doente foi observada. Os resultados demonstraram que a DIVA nos animais de policiamento apresentou caráter assintomático e que os macrófagos alveolares provenientes destes animais, quando comparados aos do grupo Controle, não apresentaram alteração em sua capacidade fagocítica para Zymosan, mas sim para L. braziliensis e um maior estado de ativação, caracterizado pela maior redução no índice de sobrevivência deste parasita após 48 horas de cultivo. / The objectives were to characterize clinically the inflammatory airway disease (IAD), as well as to verify the behavior of the alveolar macrophages activity in the horses. Seventeen adult horses, from both sexes, from 11 to 24 years old, were used, eight from the Regimento Escola de Cavalaria Andrade Neves (Control group) and nine from the Polícia Militar do Rio de Janeiro (Diseased group, with IAD). These animals did not present recente previous history (two months) compatible with respiratory disease. In this period the horses did not undergo any kind of treatment and presented leucogram and plasmatic fibrinogen determination results under normality. Physical and endoscopic examination, measurement of the maximal intrapleural pressure (Ventigraphy) and bronchoalveolar cytology were done. For the Control group, the animals were selected considering the normal results of the exams. The Diseased group was selected considering abnormal findings compatible with inflammatory pulmonary alterations at least in two exams, obligatory the endoscopic examination and bronchoalveolar cytology. To evaluate the macrophagic activity, alveolar macrophages obtained through bronchoalveolar lavage were cultured under 37oC in a 7% CO2 atmosphere, adhered to 13 mm diameter sterile glass cover slips , in 24 wells polystyrene plates containing 300µL of RPMI 1640 enriched with 10% Foetal Calf Serum inactivated by heat. Assays for phagocytosis of Zymosan (for 1h) and for promastigotes of Leishmania braziliensis strain 3456 binding (for one until 48 hours post-infection) were done. The vital functions and the measurement of the maximal intrapleural pressure measurement analyses showed, even under physiological values, significant differences for body temperature and heart rate. There was not statistical difference between the groups for the physical examination findings. In relation to the bronchoalveolar lavage, the Diseased group presented an increase in total cell count, a moderate increase in neutrophil percentage, a discrete reduction in macrophage percentage, but with a higher number of foamy macrophages and epithelial cells, and also a discrete increase of eosinophil percentage. It was not observed any difference between groups in the phagocytic capability in relation to Zymosan. However, a significant difference in the phagocytic capability after one hour interaction and a reduction of the survival index of L. braziliensis after 48 hours of culture in the Diseased group was observed. The results demonstrated that IAD in the police horses presented an asymptomatic characterization and that the alveolar macrophages from these horses, when compared to the Control group, did not present alteration in their phagocytic capacity for Zymosan, but did for L. braziliensis, and a greater activation status, characterized by a greater reduction of the survival index of L. braziliensis after 48 hours of culture.

Made available in DSpace on 2014-06-11T19:27:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-01Bitstream added on 2014-06-13T20:35:40Z : No. of bitstreams: 1 voltan_ar_me_arafcf.pdf: 3523715 bytes, checksum: a45dbb04885b30d3dab365502064fdcf (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / Paracoccidioides brasiliensis é um fungo dimórfico, agente de doença granulomatosa crônica denominada de paracoccidioidomicose (PCM), com grande variedade de manifestações clínicas, desde formas localizadas até disseminadas evoluindo para letalidade. O fungo tem capacidade de aderir, extravasar e invadir barreiras impostas pelos tecidos do hospedeiro. Em ensaios in vitro verificou-se que P. brasiliensis pode aderir e invadir células epiteliais, aparecendo algumas vezes internalizado dentro de um vacúolo. A identificação do mecanismo pelo qual este fungo adere, invade e sobrevive no interior da célula hospedeira é campo fértil para a descoberta de sua patogênese. Para tanto, a análise de proteínas presentes no microrganismo e nas células do hospedeiro e também os marcadores da via endocítica para co-localizar os microrganismos no interior das mesmas é o objetivo deste estudo. Assim, foi realizada a infecção de células epiteliais A549 e macrófagos AMJ2-C11com P. brasiliensis (Pb18) nos períodos de 3, 5, 8, 24 e 3, 5, 10 e 24h, respectivamente e feitas análises por técnicas de imunofluorescência para as proteínas de junções compactas, junções aderentes, junções tipo fenda e proteínas de ancoramento intracelular, bem como as proteínas da via endocítica. Entres estas, foram ensaiadas a expressão de clatrina, Rab7 e LAMP-1 por imunofluorescência e EEA1, Rab7 e LAMP-1 por imunoblot. As proteínas de junções foram expressas nas células, bem como na parede do fungo. A clatrina foi evidente na célula e aparentemente mais expressa na parede do fungo. Nas células A549 não houve alteração na expressão de Rab7 e LAMP-1, tanto na imunofluorescência quanto no imunoblot, sendo que para EEA1 e Rab5 não foi evidenciada alteração na expressão pela técnica de imunoblot. Rab7 foi expressa nos macrófagos, localizada na periferia das células e também no... / Paracoccidioides brasiliensis is a thermally dimorphic fungus, the etiologic agent of paracoccidioidomycosis (PCM). PCM is a polymorphic disease manifested by an array of diverse clinical manifestations and varying organ involvement. P. brasiliensis may actively penetrate the mucocutaneous surface and parasitize epithelial cells, thus evading the host defenses and reaching deeper tissues. The ability of this fungus to adhere to and invade non-professional phagocyte cells has been observed. In vitro assays verify that P. brasiliensis can adhere and could appear internalized within vacuole. The identification of the mechanism by which this fungus adheres, invades and survivor inside the host cell is a fertile area for discover their pathogenesis. The aim of this study was analyzed the junctions proteins in the fungus and the host cells, as well as the endocytic markers to colocate the microorganisms inside the cells. Thus, the infection was performed in A549 epithelial cells and macrophages AMJ2-C11 with P. brasiliensis (PB18) for periods of 3, 5, 8, 24 and 3, 5, 10 and 24 h, respectively, and the immunofluorescence analysis was developed for the proteins claudin-1, claudin-4, E-cadherin, conexin 43, talin e vinculin, and the endocytic proteins. The macrophages and the cells A549 have been blemish along anti-clatrin, anti-Rab7 and anti-LAMP-1. The junctions’ proteins have been expressed at the cells, as well as on the wall from the fungus. The clathrin was present at the cells and apparently more expressed on the fungus wall. Rab7 and LAMP-1 were not expressed in the cells A549 as demonstrated by the imunofluorescence and the imunoblot. On the contrary Rab7 was expressed in macrophages located at the periphery of the cells and also in the own fungus. The expression of LAMP-1 apparently not presented alteration both at the imunofluorescence and at the imunoblot. The expression of EEA1 was... (Complete abstract click electronic access below)

Orientador: Maria José Soares Mendes Giannini / Banca: Henrique Leonel Lenzi / Banca: Christiane Pienna Soares / Banca: Maria José Soares Mendes Giannini / Resumo: Paracoccidioides brasiliensis é um fungo dimórfico, agente de doença granulomatosa crônica denominada de paracoccidioidomicose (PCM), com grande variedade de manifestações clínicas, desde formas localizadas até disseminadas evoluindo para letalidade. O fungo tem capacidade de aderir, extravasar e invadir barreiras impostas pelos tecidos do hospedeiro. Em ensaios in vitro verificou-se que P. brasiliensis pode aderir e invadir células epiteliais, aparecendo algumas vezes internalizado dentro de um vacúolo. A identificação do mecanismo pelo qual este fungo adere, invade e sobrevive no interior da célula hospedeira é campo fértil para a descoberta de sua patogênese. Para tanto, a análise de proteínas presentes no microrganismo e nas células do hospedeiro e também os marcadores da via endocítica para co-localizar os microrganismos no interior das mesmas é o objetivo deste estudo. Assim, foi realizada a infecção de células epiteliais A549 e macrófagos AMJ2-C11com P. brasiliensis (Pb18) nos períodos de 3, 5, 8, 24 e 3, 5, 10 e 24h, respectivamente e feitas análises por técnicas de imunofluorescência para as proteínas de junções compactas, junções aderentes, junções tipo fenda e proteínas de ancoramento intracelular, bem como as proteínas da via endocítica. Entres estas, foram ensaiadas a expressão de clatrina, Rab7 e LAMP-1 por imunofluorescência e EEA1, Rab7 e LAMP-1 por imunoblot. As proteínas de junções foram expressas nas células, bem como na parede do fungo. A clatrina foi evidente na célula e aparentemente mais expressa na parede do fungo. Nas células A549 não houve alteração na expressão de Rab7 e LAMP-1, tanto na imunofluorescência quanto no imunoblot, sendo que para EEA1 e Rab5 não foi evidenciada alteração na expressão pela técnica de imunoblot. Rab7 foi expressa nos macrófagos, localizada na periferia das células e também no... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Paracoccidioides brasiliensis is a thermally dimorphic fungus, the etiologic agent of paracoccidioidomycosis (PCM). PCM is a polymorphic disease manifested by an array of diverse clinical manifestations and varying organ involvement. P. brasiliensis may actively penetrate the mucocutaneous surface and parasitize epithelial cells, thus evading the host defenses and reaching deeper tissues. The ability of this fungus to adhere to and invade non-professional phagocyte cells has been observed. In vitro assays verify that P. brasiliensis can adhere and could appear internalized within vacuole. The identification of the mechanism by which this fungus adheres, invades and survivor inside the host cell is a fertile area for discover their pathogenesis. The aim of this study was analyzed the junctions proteins in the fungus and the host cells, as well as the endocytic markers to colocate the microorganisms inside the cells. Thus, the infection was performed in A549 epithelial cells and macrophages AMJ2-C11 with P. brasiliensis (PB18) for periods of 3, 5, 8, 24 and 3, 5, 10 and 24 h, respectively, and the immunofluorescence analysis was developed for the proteins claudin-1, claudin-4, E-cadherin, conexin 43, talin e vinculin, and the endocytic proteins. The macrophages and the cells A549 have been blemish along anti-clatrin, anti-Rab7 and anti-LAMP-1. The junctions' proteins have been expressed at the cells, as well as on the wall from the fungus. The clathrin was present at the cells and apparently more expressed on the fungus wall. Rab7 and LAMP-1 were not expressed in the cells A549 as demonstrated by the imunofluorescence and the imunoblot. On the contrary Rab7 was expressed in macrophages located at the periphery of the cells and also in the own fungus. The expression of LAMP-1 apparently not presented alteration both at the imunofluorescence and at the imunoblot. The expression of EEA1 was... (Complete abstract click electronic access below) / Mestre

Objetivou-se caracterizar clinicamente a Doença Inflamatória de Vias Aéreas (DIVA) assim como verificar o comportamento da atividade dos macrófagos alveolares destes animais. Utilizaram-se 17 eqüinos adultos machos e fêmeas, com idade variando entre 11 e 24 anos, sendo oito do Regimento Escola de Cavalaria Andrade Neves (grupo Controle) e nove da Polícia Militar do Rio de Janeiro (grupo Doente, com DIVA). Estes animais não apresentavam história pregressa recente (dois meses) compatível com doença respiratória, nesse período não foram submetidos a nenhum tipo de tratamento e apresentavam resultados de leucogramas e determinações de fibrinogênio plasmático dentro da normalidade. Realizaram-se exames físicos, endoscópicos, mensurações da diferença de pressão intrapleural máxima (Ventigrafia), e citologia broncoalveolar. Para o grupo Controle os animais foram selecionados levando-se em conta os resultados de normalidade nos exames executados. O grupo Doente foi selecionado considerando-se achados de anormalidade compatíveis com alterações inflamatórias pulmonares no mínimo em dois exames, sendo obrigatórios os exames endoscópicos e de citologia broncoalveolar. Para a avaliação da atividade macrofágica, macrófagos alveolares obtidos através de lavagem broncoalveolar foram cultivados a 37ºC em atmosfera de 7% CO2, aderidos em lamínulas de vidro estéril, de 13 mm de diâmetro em placas de 24 poços contendo 300 µL de RPMI 1640 enriquecido com 10% de Soro Fetal Bovino inativado pelo calor. Realizaram-se testes de fagocitose de Zymosan (por uma hora) e de interação com Promastigotas de Leishmania braziliensis cepa 3456 (por uma até 48 horas pós infecção). A análise das funções vitais e das mensurações da diferença de pressão intrapleural máxima revelou, ainda que dentro de valores fisiológicos, diferenças significativas apenas para a temperatura corpórea e freqüência cardíaca. Para os achados do exame do exame físico, não foi detectada diferença estatística entre os grupos. Com relação ao lavado broncoalveolar, o grupo Doente apresentou aumento na contagem celular total, moderado aumento na porcentagem de neutrófilos, discreta redução na porcentagem de macrófagos, porém com aumento no número de macrófagos espumosos e de células epiteliais além de discreto aumento na porcentagem de eosinófilos. Não observou-se diferença, entre os grupos, na capacidade fagocítica com relação ao Zymosan. Porém, uma diferença significativa na capacidade fagocítica após uma hora de interação e redução do índice de sobrevivência de L. braziliensis após 48 horas de cultivo no grupo Doente foi observada. Os resultados demonstraram que a DIVA nos animais de policiamento apresentou caráter assintomático e que os macrófagos alveolares provenientes destes animais, quando comparados aos do grupo Controle, não apresentaram alteração em sua capacidade fagocítica para Zymosan, mas sim para L. braziliensis e um maior estado de ativação, caracterizado pela maior redução no índice de sobrevivência deste parasita após 48 horas de cultivo. / The objectives were to characterize clinically the inflammatory airway disease (IAD), as well as to verify the behavior of the alveolar macrophages activity in the horses. Seventeen adult horses, from both sexes, from 11 to 24 years old, were used, eight from the Regimento Escola de Cavalaria Andrade Neves (Control group) and nine from the Polícia Militar do Rio de Janeiro (Diseased group, with IAD). These animals did not present recente previous history (two months) compatible with respiratory disease. In this period the horses did not undergo any kind of treatment and presented leucogram and plasmatic fibrinogen determination results under normality. Physical and endoscopic examination, measurement of the maximal intrapleural pressure (Ventigraphy) and bronchoalveolar cytology were done. For the Control group, the animals were selected considering the normal results of the exams. The Diseased group was selected considering abnormal findings compatible with inflammatory pulmonary alterations at least in two exams, obligatory the endoscopic examination and bronchoalveolar cytology. To evaluate the macrophagic activity, alveolar macrophages obtained through bronchoalveolar lavage were cultured under 37oC in a 7% CO2 atmosphere, adhered to 13 mm diameter sterile glass cover slips , in 24 wells polystyrene plates containing 300µL of RPMI 1640 enriched with 10% Foetal Calf Serum inactivated by heat. Assays for phagocytosis of Zymosan (for 1h) and for promastigotes of Leishmania braziliensis strain 3456 binding (for one until 48 hours post-infection) were done. The vital functions and the measurement of the maximal intrapleural pressure measurement analyses showed, even under physiological values, significant differences for body temperature and heart rate. There was not statistical difference between the groups for the physical examination findings. In relation to the bronchoalveolar lavage, the Diseased group presented an increase in total cell count, a moderate increase in neutrophil percentage, a discrete reduction in macrophage percentage, but with a higher number of foamy macrophages and epithelial cells, and also a discrete increase of eosinophil percentage. It was not observed any difference between groups in the phagocytic capability in relation to Zymosan. However, a significant difference in the phagocytic capability after one hour interaction and a reduction of the survival index of L. braziliensis after 48 hours of culture in the Diseased group was observed. The results demonstrated that IAD in the police horses presented an asymptomatic characterization and that the alveolar macrophages from these horses, when compared to the Control group, did not present alteration in their phagocytic capacity for Zymosan, but did for L. braziliensis, and a greater activation status, characterized by a greater reduction of the survival index of L. braziliensis after 48 hours of culture.