ANCA-associated vasculitis is a group of devastating autoimmune diseases that predominantly target and destroy small blood vessels. The interaction of neutrophils and monocytes with the endothelial cell lining of blood vessels is imperative to understanding the pathophysiology of the disease. The nature and temporal dynamics of these interactions are mostly unknown and could provide a currently unmet clinical need for more reliable biomarkers of disease activity. This study describes the development of a 4-dimensional in vitro live cell imaging model allowing the interactions of leukocytes with endothelial cells to be analysed in the context of health and disease. Monocytes and neutrophils were isolated from peripheral venous blood of AAV patients and healthy donors. Cells were fluorescently labelled and imaged on a monolayer of human umbilical vein endothelial cells (HUVEC) using a spinning disc confocal microscope. Leukocyte migration, partial and full transmigration, route of transmigration, degranulation and the presence of leukocyte-derived particles inside endothelial cells were measured and the influence of ANCA or BVAS status considered. Following a series of preliminary experiments, it was determined that neutrophil degranulation and partial transmigration indicated early promise as potential biomarkers of disease activity. Several circulating serum analytes correlated with in vitro leukocyte functions, complementing these findings but also highlighting the prevalent immune dysfunction in the pathophysiology and development of the disease. Fatigue is a common symptom within the AAV community and the complex relationship between autoimmune fatigue and leukocyte functions was examined. The data in this thesis describes the development of a novel in vitro live cell imaging platform which can be used to investigate leukocyte functions as potential markers of disease activity as well as understanding their role in the pathophysiology of AAV.

Bibliography: leaves 87-117. / vii, 117, [64] leaves, [8] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Indicates that PGE's and CsA may have similar modes of action. Findings suggest that therapies that selectively subpopulations of leucocytes, and manipulate the inflammatory mediators they produce, may be effective in the treatment of chronic immuno-inflammatory diseases similar to rheumatioid arthritis. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pathology, 1994?

No description available.

Thesis (M.S. in Bio. E.)--School of Biomedical Engineering, Georgia Institute of Technology, 2004. Directed by Cheng Zhu. / Includes bibliographical references (leaves 79-84).

Thesis (Ph.D.)--Ohio University, November, 2007. / Title from PDF t.p. Includes bibliographical references.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Garcia, Andres; Committee Member: Babensee, Julia; Committee Member: Bellamkonda, Ravi; Committee Member: Lyon, Andrew; Committee Member: Temenoff, Johnna. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Made available in DSpace on 2014-06-11T19:27:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-01-28Bitstream added on 2014-06-13T18:56:56Z : No. of bitstreams: 1 soares_lk_me_jabo.pdf: 616158 bytes, checksum: 2557b02f58af18928da051514b8e5c20 (MD5) / Na tentativa de minimizar os possíveis efeitos colaterais causados pelos glicocorticóides, a imunoterapia tem sido estudada e aplicada por intermédio da estimulação do sistema imunológico. Dentre as alternativas o Saccharomyces cerevisiae tem sido estudado amplamente quanto aos seus efeitos imunomodulatórios, bem como o emprego da β-glucana tendo em vista sua habilidade de ativar mecanismos de defesa no hospedeiro, estimulando o sistema hematopoiético. Nesse sentido o intuito deste ensaio foi avaliar a capacidade imunomoduladora da β- Glucana e do Saccharomyces cerevisae por meio de parâmetros hematológicos, avaliação da toxicidade pela análise bioquímica e avaliação do perfil eletroforético em Ratus da linhagem Wistar imunossuprimidos após a administração experimental de Dexametasona. Foi possivél constatar o aumento dos leucócitos e estimulação das proteínas de fase aguda nos grupos tratados com as suplementações, de forma benéfica, após indução do quadro clinico de leucopenia induzida / Attempting to minimize possible side effects caused by glicocorticosteroids, immunotherapy has been studied and applied through immune system stimulation. Among alternatives the Sacharomyces cerevisiae has been widely studied regarding its immunomodulatory effects, as well as the use of β-glucane because of its ability to activate defense mechanisms in the host, stimulating the hematopoietic system. Therefore the reason of this test is to evaluate the β –glucane and Sacharomyces cervisiae immunomodulatory capacity through hematologic parameters, toxicity evaluation by biochemical analysis and electrophoretic profile evaluation in Ratus (Wistar bloodline) immunosuppressed after experimental administration of Dexametasona. It has been possible to verify the leucocite growing and high phase protein stimulation in the groups treated with supplementation, beneficially, after inducing clinical condition of leukopenia

Orientador: Áureo Evangelista Santana / Banca: Sabryna Gouveia Calazans / Banca: Márcia Ferreira da Rosa Sobreira / Resumo: Na tentativa de minimizar os possíveis efeitos colaterais causados pelos glicocorticóides, a imunoterapia tem sido estudada e aplicada por intermédio da estimulação do sistema imunológico. Dentre as alternativas o Saccharomyces cerevisiae tem sido estudado amplamente quanto aos seus efeitos imunomodulatórios, bem como o emprego da β-glucana tendo em vista sua habilidade de ativar mecanismos de defesa no hospedeiro, estimulando o sistema hematopoiético. Nesse sentido o intuito deste ensaio foi avaliar a capacidade imunomoduladora da β- Glucana e do Saccharomyces cerevisae por meio de parâmetros hematológicos, avaliação da toxicidade pela análise bioquímica e avaliação do perfil eletroforético em Ratus da linhagem Wistar imunossuprimidos após a administração experimental de Dexametasona. Foi possivél constatar o aumento dos leucócitos e estimulação das proteínas de fase aguda nos grupos tratados com as suplementações, de forma benéfica, após indução do quadro clinico de leucopenia induzida / Abstract: Attempting to minimize possible side effects caused by glicocorticosteroids, immunotherapy has been studied and applied through immune system stimulation. Among alternatives the Sacharomyces cerevisiae has been widely studied regarding its immunomodulatory effects, as well as the use of β-glucane because of its ability to activate defense mechanisms in the host, stimulating the hematopoietic system. Therefore the reason of this test is to evaluate the β -glucane and Sacharomyces cervisiae immunomodulatory capacity through hematologic parameters, toxicity evaluation by biochemical analysis and electrophoretic profile evaluation in Ratus (Wistar bloodline) immunosuppressed after experimental administration of Dexametasona. It has been possible to verify the leucocite growing and high phase protein stimulation in the groups treated with supplementation, beneficially, after inducing clinical condition of leukopenia / Mestre

O presente estudo teve por objetivo avaliar quantitativa e qualitativamente os fagócitos presentes no leite de búfalas (Bubalus bubalis) hígidas criadas nos Estado de São Paulo. Para tal, o experimento foi subdividido em três etapas, designadas capítulos, conforme os objetivos: (1) determinar e estudar a celularidade do leite de búfalas hígidas; (2) estabelecer metodologia adequada para a recuperação das células somáticas do leite de búfalas, possibilitando a avaliação in vitro; (3) descrever e avaliar as provas de atividade fagocítica das células presentes no leite de búfalas hígidas, através de testes de mensuração direta (espraiamento e fagocitose de partículas de Zymosan) e por testes indiretos, que mensuram os metabólitos de oxigênio gerados na “explosão respiratória”. Foram colhidas 132 amostras de leite de 35 búfalas hígidas (sem alterações ao exame físico da glândula mamária, bem como resultado da prova de CMT e exame microbiológico negativos). Após as avaliações do CMT, da CCS, da contagem diferencial, da viabilidade celular, do exame bacteriológico, dos testes de fagocitose, de espraiamento e da liberação de peróxido de hidrogênio, concluiu-se que: (1) os resultados das contagens de células somáticas, microscópica e automática, foram semelhantes, mas a predominância celular diferiu conforme a técnica empregada, sendo identificadas percentualmente mais células mononucleares na lâmina de suspensão celular citocentrifugada, e mais polimorfonucleares no leite submetido à técnica de Prescott e Breed; o leite diluído e citocentrifugado permitiu melhor avaliação da morfologia celular sendo identificados: 61,1% de monócitos e macrófagos; 32,9% de neutrófilos; 5,3% de linfócitos e 0,7% de eosinófilos; os fagócitos mononucleares apresentaram uma acentuada plasticidade na estrutura, com variados padrões morfológicos; (2) a baixa celularidade do leite necessitou maiores volumes da amostra mas, o aumento do volume da amostra aumenta a concentração celular obtida, mas não de forma ilimitada pois pode passar a comprometer a viabilidade das mesmas pelo excesso de manipulação e tempo exigidos pelas amostras mais volumosas; não foi necessária a elicitação, sendo possível a recuperação de 2 x 106 células viáveis/mL de leite de búfalas hígidas com 500 mL de leite, no mínimo; (3) os macrófagos aderidos espraiaram significativamente, além de apresentarem correlação com outro marcador de ativação celular, no caso, a liberação de peróxido de hidrogênio; mais da metade dos macrófagos aderidos fagocitaram partículas de Zymosan; os fagócitos mantêm sua capacidade de liberar peróxido de hidrogênio, espontaneamente ou não, em grau máximo, com uma significativa variação entre amostras / The aim of this study was to make a quantitative and a qualitative evaluation of phagocytes from healthy buffaloes (Bubalus bubalis) milk bred in Sao Paulo state. For this purpose, the experiment was divided in three parts, called chapters, as specific objectives: (1) establish and study the cellularity of healthy buffaloes milk; (2) establish an adequate method for the recuperation of somatic cells from buffaloes milk, making possible an in vitro evaluation; (3) standardize and evaluate the phagocytic activity tests of cells from healthy buffaloes milk, through direct measurement methods (spreading and phagocytosis of Zymosan particles) and indirect methods, which measure the oxygen metabolites from the respiratory burst. Milk samples (n=132) of healthy 35 buffaloes (without alterations on the physical examination of the mammary gland, and also negative results on CMT and microbiologic exams) were obtained. After the evaluations of CMT, SCC, differential cells count, cellular viability, bacteriologic exam, phagocytic tests, spreading and hydrogen peroxide liberation, the conclusions were: (1) the results of somatic cells count, microscopic and automatic, were similar but the cellular predominance differed with the utilized method, being percentually identified more mononuclear cells on slide of cytocentrifugation cellular suspension and more polymorphonuclear on milk submitted to the Prescott and Breed methods; the diluted and cytology by cytocentrifugation of milk permitted a best evaluation of the cellular morphology, being identified: 61.1% of monocytes and macrophages; 32.9% of neutrophils; 5.3% of lymphocytes and 0.7% of eosinophils; mononuclear phagocytes showed a high plasticity on structure with many morphological patterns; (2) a lower cellularity on milk requires higher quantities of the samples but an increase on sample volume increases cellular concentrations obtained, not on an unlimited way because it can compromise the cells viability with the excess of manipulation and time required by the biggest samples; it was not necessary the elicitation, being possible the recuperation of 2 x 106 viable cells/mL on milk of healthy buffaloes with 500 mL of milk minimum (3) adherent macrophages spreaded and were correlated with hydrogen peroxide release; majority of adherent macrophages phagocyted Zymosan particles; milk phagocytes showered a high oxidative metabolism, undependably of PMA stimulation, but with a great individual variation.

A presente investigação teve o objetivo de avaliar quantitativa e qualitativamente a celularidade de cabras hígidas criadas no Estado de São Paulo, assim como correlacionar as células somáticas do leite com os leucócitos presentes no sangue, informações que, além de raras em relação à glândula mamária de cabras sadias, são extremamente conflitantes. Foram colhidas amostras de cada metade mamária, provenientes de 51 cabras hígidas das raças Saanen, Alpina e Toggenburg, em diferentes fases de lactação, pertencentes a diferentes criatórios do Estado de São Paulo. As amostras de leite foram submetidas ao California Mastitis Test (CMT), a contagem eletrônica de células somáticas e microscopia direta, utilizando o corante de verde de metil e pironina-Y, além da citocentrifugação para a avaliação dos leucócitos; com as amostras de sangue foi realizado o leucograma. Associando-se os escores do CMT com a contagem celular eletrônica foram encontrados os valores medianos de 181.000, 578.000, 628.000, 1.421.500 e 5.542.000, respectivamente. Já na contagem celular por microscopia direta os valores medianos obtidos foram de 74.991, 271.396, 71.420, 640.995 e 5.049.394, correspondendo, respectivamente aos escores negativo, traços, 1+, 2+ e 3+. Relacionando-se as contagens celulares de acordo com as fases da lactação, os valores medianos obtidos não apresentaram diferenças significativas entre as fases inicial, intermediária e final da lactação tanto para a contagem eletrônica quanto para a contagem microscópica direta, respectivamente. A correlação entre a contagem celular automática e microscópica direta foi de 88%. Não foram encontradas diferenças significativas quando se comparou a contagem de células somáticas nas glândulas direita e esquerda, tanto na contagem automática quanto na microscópica direta. Os valores relativos dos leucócitos presentes no leite foram determinados, obtendo-se os valores médios e desvios-padrão de 77,0 ± 30,5%; 76,5 ± 20,9% e 65,3 ± 25,6% de neutrófilos, 2,24 ± 3,26%; 2,04 ± 2,33% e 2,14 ± 2,81% de linfócitos e 22,6 ± 20,2%, 21,4 ± 20,0% e 23,0 ± 16,2% de monócitos, correspondendo às fases inicial, intermediária e final da lactação, respectivamente. No sangue, determinou-se o valor total de leucócitos, obtendo-se o valor médio de 8525 ± 3244 células/mL. Os valores relativos médios dos diferentes tipos de leucócitos presentes no sangue de cabras sadias foram de 56,8 ± 11,8% de neutrófilos, 38,5 ± 11,8% de linfócitos, 2,8 ± 1,7% de monócitos e 1,9 ± 1,6% de eosinófilos. Correlacionando-se os leucócitos presentes no sangue com os presentes no leite, não foram observadas correlações significativas, porém, quando se analisou os leucócitos do sangue com as fases da lactação, foram encontradas diferenças significativas nos valores relativos médios de neutrófilos e linfócitos, respectivamente, na fase final da lactação. Os resultados obtidos permitiram concluir que existiu diferença entre a contagem celular automática e a microscópica direta na determinação do número de células somáticas no leite caprino; os valores celulares aumentam conforme o escore do CMT, o leucócito predominante no leite e sangue de cabras hígidas é o neutrófilo e existe relação íntima entre o neutrófilo e o linfócito no sangue de cabras, sendo estes influenciados pela fase da lactação. / The present investigation had the objective to evaluate quantitatively and qualitatively the celularity of created healthy goats in Sao Paulo state, as well as correlating the somatic cells of milk with the leukocytes in the blood, information that, beyond rare in relation to the mammary gland of healthy goats, are extremely conflicting. Samples of each mammary half had been harvested, proceeding from 51 Saanen, Alpina and Toggenburg healthy goats, in different phases of lactation, from the different breeders of Sao Paulo state. The milk samples had been submitted to California Mastitis Test (CMT), the electronic counting of somatic cells and direct microscopy, using the pyronin Y-methyl green stain, beyond the cytospin centrifuge for the evaluation of leukocytes; with the samples of blood the leucogram was carried through. Associating you prop up of the CMT with electronic the cellular counting had been found the medium values of 181.000, 578.000, 628.000, 1.421.500 and 5.542.000, respectively. Already in the cellular counting for direct microscopy the gotten medium values had been of 74.991, 271,396, 71,420, 640,995 and 5.049.394, corresponding, respectively to negative, traces, 1+, 2+ and 3+ scores. Becoming related the cellular counting in accordance with the phases of the lactation; the gotten medium values had not presented significant differences between the phases, initial, intermediate and final of the lactation in such for the electronic counting how much for the direct microscopically counting, respectively. The correlation between the automatic and direct microscopically counting cellular was of 88%. Significant differences had not been found when the counting of somatic cells in the glands was compared right and left, as much in the microscopically automatic counting in the direct one. The relative values of the leucocytes gifts in milk had been determined, getting the average values and 77,0 ± 30,5%; 76,5 ± 20.9% and 65,3 ± 25.6% of neutrophils, 2,24 ± 3,26%; 2,04 ± 2.33% and 2,14 ± 2.81% of lymphocytes and 22,6 ± 20.2%, 21,4 ± 20.0% and 23,0 ± 16.2% of monocytes, corresponding the phases initial, intermediate and final of the lactation, respectively. In the blood, the total value of leucocytes was determined, getting itself the average value of 8525 ± 3244 céls/mL. The average relative values of the different types of leukocytes gifts in the blood of healthy goats had been of 56,8 ± 11.8% of neutrophils, 38,5 ± 11.8% of lymphocytes, 2,8 ± 1.7% of monocytes and 1,9 ± 1.6% of eosinophils. Correlating the leukocytes gifts in the blood with the gifts in milk, significant correlation had not been observed, however, when it analyzed the leucocytes of the blood with the phases of the lactation, significant differences in the average relative values of neutrophils and lymphocytes had been found, respectively, in the final phase of lactation. The gotten results allow to conclude that difference between the automatic cellular counting and the direct microscopically one in the determination of the somatic frame number in goat milk exists, the cellular values increase as prop up it of the CMT, the predominant leucocyte in milk and blood of healthy goats is neutrophil, exists close relation between neutrophil and lymphocytes in the blood of goats, being these influenced by the phase of the lactation.