Made available in DSpace on 2014-06-11T19:29:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-02Bitstream added on 2014-06-13T18:39:10Z : No. of bitstreams: 1 oliveira_jcv_me_botfmvz.pdf: 876846 bytes, checksum: 0964099d5611eb445b184c0c142d8ec3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A cisticercose é uma das principais causas de perdas econômicas na pecuária de corte brasileira devido às condenações da carne para o controle do complexo teníase-cisticercose. O tratamento quimioterápico pode ser utilizado para o controle da infecção nos animais, mas sua eficácia deve ser monitorada por testes de diagnóstico que detectem parasitas vivos ou seus produtos. Na presente pesquisa, anticorpos monoclonais contra antígenos totais (TAEB) e de fluido vesicular (TAEF) de metacestóides de Taenia saginata foram produzidos através de fusão celular, utilizando-se células de baço de camundongos BALB/c imunizados. Cinco fusões TAEB e quatro TAEF foram realizadas e os sobrenadantes de cultura foram triados por teste ELISA indireto. Dez e nove híbridos pertencentes aos protocolos TAEB e TAEF, respectivamente, foram selecionados e clonados. As linhagens celulares clonadas foram mantidas em meio de cultura para a obtenção dos anticorpos monoclonais, resultando em 18 clones IgG1 e 32 IgM reativos para TAEB e 9 IgG1 e 9 IgM para TAEF. Dentre esses clones, 5 do protocolo TAEB e 5 TAEF foram selecionados para produção de líquido ascítico, através da injeção dos clones em camundongos BALB/c. A avaliação da identificação antigênica dos anticorpos produzidos aos antígenos homólogos foi realizada por meio de western blotting, resultando em reatividade com frações protéicas de baixo peso molecular (< 18kDa), 43, 55, 66 e 100kDa. A imunofluorescência indireta demonstrou que os anticorpos monoclonais avaliados reconhecem antígenos presentes tanto do escólex quanto da membrana vesicular de metacestóides de bovinos naturalmente infectados. Os anticorpos monoclonais produzidos poderão ser utilizados para detecção de antígenos circulantes na avaliação da eficácia do tratamento de bovinos infectados, evitando assim perdas econômicas e danos à saúde pública. / Cysticercosis is a major cause of economic losses in the Brazilian bovine chain production due to meat condemnation for the taeniasis-cysticercosis complex control. Chemotherapy can be used to control infection in cattle but treatment efficacy should be monitored by diagnostic tests that can detect live parasites or their products. Monoclonal antibodies against Taenia saginata metacestode crude (TAEB) and cyst fluid (TAEF) antigens were produced by cell fusion procedures using spleen cells from immunized BALB / c mice. Five TAEB and four TAEF fusions were performed and the culture supernatants from these cell cultures were screened by indirect ELISA assay. Ten TAEB and nine TAEF hybrids were selected and cloned. Cloned cell lines were grown in culture medium to collect the monoclonal antibodies, resulting in 18 IgG1 and 32 IgM clones reactive to TAEB and 9 IgG1 and 9 IgM clones reactive to TAEF. Five TAEB and five TAEB clones were selected for ascitic fluid production by injection in BALB/c mice and further harvesting. Western blotting was performed to test the antigenic identification by the antibodies produced resulting in reactivity to protein fractions of low molecular weight (<18kDa) and to 43, 55, 66 and 100kDa. The indirect immunofluorescence test showed that monoclonal antibodies recognize antigens from both the scolex and the bladder wall of metadestodes from naturally infected bovine. The monoclonal antibodies obtained can be useful for detection of circulant antigens in treatment efficacy evaluation preventing economic losses and public health injury.

No útero gestante dos animais com placentação do tipo hemocorial, ocorre uma migração e acúmulo transitório de linfócitos natural killer (NK) , cuja atuação na gestação não está totalmente elucidada. Estas células NK do ambiente uterino (uNK) apresentam comportamento distinto daquelas encontradas no sangue circulante (cNK), constituindo uma sub-população das células NK com expressão gênica específica ditada pelo ambiente uterino gestante. De fato, se estas células isoladas do útero de camundongos prenhes forem inoculadas em machos da mesma espécie eram capazes de induzir a resposta imunológica com produção de anticorpos que reagem especificamente com as células uNK. No presente trabalho foi utilizado um destes anticorpos monoclonais denominados de \"mouse anti-mouse uterine natural killer cell clone 1 (mam-uNK1)\" obtidos anteriormente em nosso laboratório para avaliar a especificidade deste anticorpo e a localização da molécula antigênica correspondente. Para tanto, foram utilizados cortes histológicos do útero no 9º dia de gestação, dos órgãos linfóides (baço, timo e linfonodo), do cérebro, do fígado e do coração submetidos à reações imunocitoquímicas com o anticorpo mam-uNK1 em nível da microscopia de luz e, pela imunomicroscopia eletrônica nas células uNK do útero gestante e células estriadas cardíacas do miocárdio. Foram obtidos homogenados teciduais dos mesmos órgãos avaliados pela imunocitoquímica para realização do SDS-PAGE e Western-blot com o intuído de identificar as frações protéicas reativas e homologia entre os diversos órgãos. Os padrões de imunomarcação com o mam-uNK1 foram comparadas com o padrão de reatividade da lectina DBA (Dolichos biflorus) tanto nos cortes histológicos quanto nos homogenados submetidos ao Western-blot. Os resultados demonstraram reação positiva distribuída difusamente no citoplasma, com maior intensidade no perímetro das células uNK, e marcação difusa no citoplasma das células deciduais e das células musculares lisas do miométrio. Reações positivas foram encontradas também no citoplasma das células musculares cardíacas, no citoplasma das células reticulares dos órgãos linfóides, nos feixes de nervos do sistema nervoso central e no citoplasma dos hepatócitos. Pela imunomicroscopia eletrônica foram observadas partículas de ouro coloidal em maior número no citoplasma que preenchem os prolongamentos citoplasmáticos tipo microvilosidades e no citoplasma marginal abaixo da membrana plasmática nas células uNK. Nas células musculares cardíacas as marcações mais intensas foram constatadas no citoplasma da extremidade destas células onde as miofibrilas eram menos organizadas e se ancoravam à membrana plasmática. Pelo Western-blot, foram identificadas duas bandas reativas ao mam-uNK1 com peso molecular de 52 e 54 kDa comuns a todos os órgãos analisados. Estes dados demonstram que a molécula reconhecida pelo anticorpo mam-uNK1 tem ampla distribuição em diversos tipos celulares não sendo específica para as células uNK, porém apresentam uma localização peculiar nestas células e nas células musculares cardíacas. Pelo padrão de localização identificado em imunomicroscopia eletrônica, presume-se que estas moléculas estejam associadas com a modulação do citoesqueleto nas diversas atividades que estes componentes estruturais desempenham nas células, sendo particularmente interessante a relação com a motilidade celular. / In the pregnant uterus of animals developing hemochorial type placentation occurs a transient migration and accumulation of natural killer lymphocytes (NK) which activity in the pregnancy has not been fully elucidated. These NK cells from uterine environment (uNK) present a distinct behavior from those found in the peripheral circulating blood (cNK), composing a subset of NK cells with specific gene expression that is regulated by pregnant uterus. Actually, these cells isolated from pregnant mice uteri were inoculated in the male mice of same strains, they induced immune response with production of antibodies reactivity specifically to uNK cells. In the present work it was used one of these mouse anti-mouse uterine natural killer cells clone 1 (mam-uNK1) monoclonal antibody that was obtained previously in our laboratory, in the aim to evaluate the specificity of this antibody and localization of corresponding antigenic molecule. It was used histological sections of uteri on 9º gestational day, lymphoid organs (spleen, thymus and lymph node), brain, liver and heart processed for immunocytochemistry with mam-uNK antibody at light microscopy and immunoelectron microscopy for uNK cells in pregnant uterus and cardiac muscle cells. Tissue homogenates from the same organs that were evaluated by immunocytochemistry were obtained to perform SDS-PAGE and Western-blot primary to identify the proteins fractions reactive with mam-uNK antibody and possible homology among the tissues. The immunolabeling pattern using mam-uNK both, in histological sections and Western-blot were compared with pattern of Dolichos biflorus (DBA) lectin reactions. The results showed diffuse positive reaction distributed in the cytoplasm with higher intensity on perimeter of uNK cells and diffuse labeling in the cytoplasm of decidual cells and, in smooth muscle cells of miometrium. Positive reaction was also found in the cytoplasm of cardiac muscle cells, reticular cells of lymphoid organs, hepatocytes and in the axon bundles of the brain. By immunelectron microscopy, were observed higher number of gold particles in the cytoplasm of microvillous-like cell processes and in the marginal cytoplasm near the plasma membrane of uNK cells. In the cardiac muscle cells the most conspicuous labeling was seen in the cytoplasm of muscle cells at the end portions, that is, where the myofibrills were less organized and anchoring to plasma membrane. The Western-blot identified two bands with 52 and 54 kDa reactive do mam-uNK constantly found in all organs analyzed. These data show that the molecule that is recognized by mam-uNK1 antibody is widely distributed in several cell types, not specific for mouse uNK cells, but has a very peculiar localization in these cells and in the cardiac muscle cells. To the localization pattern that was identified by immunelectron microscopy it was suggested that, these molecules were associated to the modulation of the cytoskeleton in many activities that these structural component potentially could carry out in the cells, being particularly attractive those related to cell motility.

Orientador: Laura Sterian Ward / Dissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T07:53:42Z (GMT). No. of bitstreams: 1 Santos_AnaPaulaCarneiroDos_M.pdf: 2998875 bytes, checksum: 9fd6ce397ec8eae4d85db8afcd041e9e (MD5) Previous issue date: 2010 / Resumo: Fragmentos de anticorpos recombinantes têm se tornado ferramentas importantes em diversas áreas, tais como: Biologia Molecular, Farmacêutica e pesquisa Médica. Avanços recentes estão relacionados à aplicação desses anticorpos na oncologia, com estratégias diagnósticas e terapêuticas para diferentes carcinomas. Neste estudo, uma biblioteca de fragmentos de anticorpos monoclonais scFv foi construída utilizando RNA total de sangue periférico de 25 pacientes com Carcinoma Diferenciado da Tireóide. Essa biblioteca scFv foi selecionada utilizando os métodos Bioppaning and Rapid Analysis of Selective Interactive Ligands (BRASIL) e Phage display contra células tumorais de tireóide, com o objetivo de encontrar ligantes específicos a superfície celular tumoral. Os clones selecionados foram identificados por Dot blotting, e a reatividade contra proteínas de tumor, adenoma e bócio foi analisada por Elisa. O clone scFv-C1 apresentou melhor reatividade pelas proteínas tumorais e foi escolhido para a imunoistoquímica. Esta foi realizada com lâmina de Micro-arranjo de tecido (TMA) com duzentos e vinte nove casos de tireóide, sendo 110 Carcinomas, 52 Adenomas Foliculares, 49 Bócios e 18 tecidos normais de tireóide. O anticorpo scFv-C1 reagiu especificamente aos tecidos de câncer, com reatividade ao citoplasma das células tumorais, foi capaz de distinguir o Grupo Câncer do Controle (Bócio, Adenoma e tireóide normal) com significância estatística (p<0,0001) e entre os carcinomas reagiu melhor com os tumores pequenos (TNM 1 e 2) e com pouca agressividade (p=0,050). O fragmento de anticorpo scFv-C1 pode ser um potencial candidato a biomarcador para o diagnóstico do Câncer de tireóide / Abstract: Recombinant antibody fragments have become important tools in several fields, including molecular biology, pharmaceutical and medical research. In this study, a human single-chain variable fragment (scFv) antibody library was constructed using total RNA of leukocyte cells obtained from blood of patients with well differentiated thyroid carcinoma. This scFv antibody library was selected using the Biopanning and Rapid Analysis of Selective Interactive Ligands method (BRASIL) and Phage display technology against tumor thyroid cells, aiming to find specific cell-surface binders. The selected clones were identified by dot blot and ELISA assays and their reactivity analyzed against tumor, goiter and adenoma proteins. One clone (scFv-C1) presented the highest reactivity ratio between cancer and the control group (goiter and adenoma) and was chosen for further analysis. Immunohistochemistry was performed by means of Tissue Microarray with two hundred and twenty-nine thyroid cases (110 carcinomas, 52 follicular adenomas, 49 goiters and 18 normal tissues) including 38 papillary, 42 follicular and 30 variant follicular in the carcinoma group. The scFv-C1 reacted specifically to cancer tissues sections, showed strong reactivity with cytoplasm and was able to distinguish cancer to control groups (goiter, adenoma and normal thyroid) with_statistically significance (p<0,0001). The scFv-C1 fragment antibody described here may be a potential biomarker candidate for diagnostics and prognostics of thyroid cancer / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Human fungal infections among severely immunocompromised individuals have increased dramatically over the last 30 years and that coincidence with an expanding patient numbers of bone marrow and solid organ transplantation and those receiving aggressive cytotoxic chemotherapy for neoplastic diseases or immunosuppressive drugs. In recent years, many of opportunistic fungi have emerged as serious human pathogens and causing life-threatening infections of humans such as Fusarium species. Due to lack of a highly accurate diagnostic test for tracking the pathogenic Fusarium species, fusariosis is frequently misdiagnosed as aspergillosis. Delays in identification and differentiation of Fusarium spp. from other causative agents of hyalohyphomycetes associated with high morbidity and mortality rate among immunocompromised patients. This research aimed to develop a highly specific monoclonal antibody (mAb) using hybridoma technology to produce a highly genus-specific murine mAb ED7 that could be used for tracking and early detecting circulating Fusarium species antigens from other opportunistic pathogens. At present, a very little is known about the pathogenicity and interaction of human pathogenic F. solani and cells of the innate immune system like alveolar macrophages (AMØ), the residential innate immune cells of alveoli. For this reason, F. solani was genetically transformed with GFP gene and a model of immunoassay was developed to investigate the interactions of F. solani with AMØ that would allow studying the fungal pathogenicity, visualising and quantification of the pathogen during the process of macrophage phagocytosis. In addition, this model can be used to evaluate the effect of a mAb on fungal uptake by AMØ. Habitates providing direct human exposure to infectious propargules are largely unknown, but there is growing evidence that plumbing systems are sources of human pathogenic strains in the F. solani and F. oxysporum species complexes, the most common groups infecting humans. Using mAb ED7 specific to the Fusarium species, this work demonstrates how the mAb can be used as a powerful immunodiagnostic tool for accurately tracking the Fusarium species antigens in sink drain biofilms and water system samples containing mixed populations of human opportunistic filamentous and yeasts pathogenic fungi across a University campus and a tertiary care hospital. Specificity of the ELISA was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of culturable yeasts and moulds that were recovered using mycological culture, while translation elongation factor (TEF)-1 analysis of Fusarium isolates included FSSC 1-a, FOSC 33 and FDSC ET-gr, the most common clinical pathotypes in each group. The mAb ED7 is, therefore, suitable to be carried forward for use in diagnostic assays, such as the lateral flow device.

Subcutaneous (SC) injection is a safe and effective way to administrate monoclonal antibodies (mAbs) and very often valued by patients and healthcare providers alike. A SC injection offers noteworthy advantages over intravenous (IV) injections, such as lowering hospital and clinical costs. Additionally, SC injections requires a lower number administration and allows for self-administrated or caregiver-supported dosing at home, thus a reduced healthcare resource utilization and healthcare provider time. In recent years biotherapeutics have been one of the fastest growing class of drugs in the pharmaceutical industry due to effective therapeutics with target specificity and potency. Biopharmaceuticals are usually administrated through IV, intramuscular (IM) or SC injection. However, patients are not administrated drugs, they are administrated formulations containing a drug. It is also important to recognize that biopharmaceuticals that are meant for SC injection are usually formulated at an acidic (4-6) pH, which can be attained by using a variety of excipients and stabilising agents. Excipients such as sugar, salt and surfactant are commonly found in biopharmaceuticals and can be used to attain a multi-year shelf life. Additionally, biopharmaceuticals can form aggregates and this occurrence happens due to loss of a stabilizing excipient. Hence the importance of understanding how and why different excipients are or can be used in a formulation.

In meeting the high demand for monoclonal antibodies, the chicken immunoglobulin system was exploited to generate recombinant antibodies against multiple target antigens. Following simultaneous immunisation of two chickens with a mixture of Plasmodium falciparum recombinant lactate dehydrogenase (LDH), histidine rich protein II (HRPII) and aldolase (ALDO), recombinant trypanosome variable surface glycoprotein (VSG) and malignant catarrhal fever virus (MCFV) each chicken produced egg yolk antibodies (IgY) against four of the five antigens. Using phage display technology, two single-chain variable fragment (scFv) antibody libraries, one with the immunoglobulin VH and VL chain regions joined by a single amino acid (G) and the other with a 15 amino acid flexible linker [(G4S) 3] were constructed using pooled splenic RNA. The single amino acid-linked scFv repertoire was evaluated as a source of highly specific diagnostic antibodies by panning against each of the five different antigens. After two rounds of panning, polyclonal phage ELISA showed the presence of antigen-specific phage antibodies against three (LDH, HRPII and VSG) of the five antigens. Five different anti-LDH and six different anti-HRPII scFvs were identified by sequence analysis. Evidence of high levels of antigen-driven gene conversion events was found in the framework and complementary determining regions and the VL chain pseudogene donors were identified. Stability of the selected scFvs was determined by incubation at different times and at different temperatures. The specificity and potential use of an LDH-specific scFv as a diagnostic reagent was shown in sandwich and competitive inhibition ELISAs. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2007. / Veterinary Tropical Diseases / unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21072号 / 工博第4436号 / 新制||工||1689(附属図書館) / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 大塚 浩二, 教授 松原 誠二郎, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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