Das Monozyten chemotaktische Protein-1 (MCP-1) ist ein CC-Chemokin, das in seiner Rolle als Chemoattraktor auf Monozyten in der Genese von Malignomen eine wesentliche Rolle spielt. Dabei kann es sowohl zur lokalen Tumorabwehr als auch zur Tumorgenese, Tumor-angiogenese und Metastasierung beitragen. Die vorliegende Arbeit untersucht die MCP-1-Inhibition und die Stimulation durch MCP-1 auf die Koloniebildung und die Zytokinexpression von Plattenepithelkarzinomen der Kopf-Hals-Region (HNSCC) im FLAVINO-Assay. Dieser ist ein klonogener, qualitätskontrollierter Ex-vivo-Koloniebildungsassay, der an der Klinik für Hals-Nasen-Ohrenheilkunde der Universität Leipzig etabliert und patentiert wurde und unter flavinschützenden Bedingungen durchgeführt wird. Weiterhin wird die Eignung von MCP-1, Interleukin-6 (IL-6), Interleukin-8 (IL-8) und des Vascular endothelial growth factor (VEGF) als Biomarker in HNSCC, die mithilfe von ELISA in Seren und Kulturüberständen quantifiziert wurden, untersucht. Durch die Stimulation durch MCP-1 und dessen Blockade sowie durch in vivo tolerierbare Konzentrationen von Cisplatin, Docetaxel, Cilengitide und Temsirolimus wurde die Expression der untersuchten Zytokine in den Kulturüberständen der HNSCC unterschiedlich moduliert. Cisplatin und MCP-1 supprimierten die Koloniebildung signifikant, während unter Docetaxel und Temsirolimus eine insignifikante Reduktion und durch Cilengitide eine insignifikante Stimulation der Koloniebildung beobachtet wurde. Die MCP-1-Blockade durch einen Anti-MCP-1-Antikörper führte zu keiner signifikanten Modulation der Koloniebildung. MCP-1 und der Anti-MCP-1-Antikörper senkten die Zytokinexpression, während bis auf Cisplatin alle Zytostatika die Zytokinexpressionen steigerten. Bezüglich der kombinierten Testung der Zytostatika und der MCP-1-Blockade bzw. Stimulation unterschieden sich die Proben, sodass additive, synergistische und antagonistische Effekte resultierten. Da durch MCP-1 gesteuerte tumorassoziierte Makrophagen das Mikromilieu eines Tumors wesentlich beeinflussen, gebührt diesen ebenfalls eine besondere Aufmerksamkeit. In dieser Arbeit wurden unter MCP-1 antitumoröse Effekte beobachtet, sodass weitere klinische Testungen der antitumorösen Wirkung des MCP-1 auf HNSCC lohnenswert erscheinen. Die individuelle Chemoresponse-Testung kann dabei helfen, das biologisch heterogene Verhalten der HNSCC besser zu verstehen. In diesem Sinne wäre die klinische Validierung solcher Testsysteme wertvoll.

Orientadores: Cecília Amélia de Carvalho Zavaglia, Aron José Pazin de Andrade / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica / Made available in DSpace on 2018-08-22T16:21:16Z (GMT). No. of bitstreams: 1 Silva_Cibeleda_M.pdf: 8042828 bytes, checksum: f69ba730298b110aecb701c7d8d19cf6 (MD5) Previous issue date: 2013 / Resumo: Neste trabalho foram elaborados os métodos e realizados estudos In Vivo e Avaliação Clínica de uma Bomba de Sangue para Circulação Extracorpórea (CEC), durante Cirurgia Cardíaca, denominada Spiral Pump® (SP). Esses estudos foram realizados com o objetivo de avaliar o desempenho e segurança da bomba, finalizando assim o projeto e avaliando a SP como um produto em sua aplicação rotineira durante as cirurgias cardíacas. A SP utiliza, simultaneamente, dois princípios de bombeamento, o centrífugo e o axial, proporcionados por sua geometria cônica, visando aumentar a eficiência de bombeamento sem o aumento dos índices de destruição dos elementos figurados do sangue. O primeiro passo para avaliação In Vivo foi à elaboração de um protocolo para avaliação In Vivo animal e sua submissão a Comissão de Ética para Uso de Animais (CEUA) do Instituto Dante Pazzanese de Cardiologia (IDPC). As avaliações In Vivo consistiram em instalação normal da CEC, em animais suínos, utilizando a SP conectada a um módulo de acionamento e, para fins de comparação, foi realizado o mesmo procedimento com a Bio-Pump®. Foram realizados seis experimentos com a SP, sendo o primeiro considerado piloto, e três experimentos com a Bio-Pump®, com duração de seis horas cada. As observações realizadas pela equipe médica e pela perfusionista demonstraram grande semelhança de funcionamento entre as duas bombas, inclusive em relação a vibrações, ruídos e facilidade de utilização. As bombas foram comparadas em relação à medição do trauma, analisado a partir da evolução da hemoglobina livre no plasma (PFH). Analisando os resultados laboratoriais e de hemólise obtidos com a SP e a Bio-Pump®, pode-se verificar que não existem diferenças significativas entre elas. Com resultados satisfatórios nos ensaios In Vivo, a SP foi aprovada para Avaliação Clínica, realizada de acordo com a legislação vigente. Foi elaborado um Protocolo de Pesquisa Clínica que seguiu a Resolução ANVISA número 39 de 05 de junho de 2008, que estabelece requisitos para realização de pesquisa clínica no Brasil. Esse protocolo foi submetido ao Comitê de Ética em Pesquisa (CEP) do IDPC e ao Conselho Nacional de Ética em Pesquisa (CONEP). A pesquisa clínica foi realizada no Centro Cirúrgico do IDPC em um grupo de quarenta pacientes com indicação de cirurgia cardíaca com CEC, com ou sem cardioplegia, ambos os sexos, adultos, peso corporal mínimo de 50 kg. Durante a CEC, a SP substituiu as convencionais bombas de roletes no circuito de CEC. Todos os pacientes foram entrevistados e autorizaram a realização do estudo, assinando do Termo de Consentimento Livre e Esclarecido. Foram coletados dados comumente monitorados durante uma cirurgia cardíaca e foram realizados, nos períodos de pré e pós CEC, exames de Desidrogenase Láctica (U/L), Plaquetas (mil/mm3), Fibrinogênio (mg/dL), além de monitoração da hemólise através de fita de urianálise. Os procedimentos transcorreram de forma habitual, e os parâmetros mantiveram-se dentro do esperado para o estudo, demonstrando a eficiência e segurança da SP como bomba para circulação extracorpórea durante cirurgia cardíaca / Abstract: Were developed methods and studies In Vivo and clinical evaluation were conducted with a blood pump for cardiopulmonary bypass (CPB), during cardiac surgery, the Spiral Pump® (SP). These studies are designed to assess the performance and safety of SP, finalizing the project and evaluating the SP as a product in its routine application during heart surgeries. The SP uses simultaneously, two principles of pumping, axial and centrifugal, provided by its conical geometry, in order to increase pumping efficiency without increased levels of destruction of the figured elements from blood. First step for In Vivo evaluation was the development of a protocol for In Vivo experiments and its submission to the "Ethic Committee for Animal Use" of Institute Dante Pazzanese of Cardiology (IDPC). The In Vivo assessments consisted of normal installation of the CPB in pigs, using the SP connected to a driver console and, for comparison purposes, the same procedure was performed with Bio-Pump®. Six experiments were performed with SP and three experiments with Bio-Pump®, lasting six hours each. Observations made by medical team showed great similarity between two pumps, including characteristics of vibration and noise. Two pumps were compared concerning to measurement of trauma, through the evolution of plasma free hemoglobin (PFH) and the PFH variation (?PFH). Analyzing laboratory results and hemolysis, from In Vivo assays with SP and the Bio-Pump®, we can observe no significant differences between them. With satisfactory results from In Vivo assays, SP was approved for clinical evaluation, carried out in accordance with current legislation. A Clinical Research Protocol was elaborated following ANVISA (National Health Surveillance Agency) resolution number 39 of 2008, which establishes requirements for conducting clinical research in Brazil. This Protocol was submitted to the Ethic Committee Research of IDPC for review and subsequently to National Council of Ethics in Research. Clinical research was conducted at operating room of IDPC in a group of forty patients under cardiac surgery with CPB, both sexes, adults, minimum of 50 kg of body weight. During CPB, SP replaced conventional CPB roller pump. All patients were interviewed and had authorized this study, signing of consent form. Usually monitored data were collected during a heart surgery and additional examinations were carried out at periods of pre and post CEC such as: Lactic dehydrogenase exams (U/L), platelets (1000/mm3), Fibrinogen (mg/dL), as well as monitoring of hemolysis by urianalysis tape. All procedures were as usual way, and all parameters remained as expected for this study, demonstrating the performance and safety of SP as pump for CPB during heart surgery / Mestrado / Materiais e Processos de Fabricação / Mestra em Engenharia Mecânica

Orientadoresr: Daniele Ribeiro de Araujo, Eneida de Paula / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T08:56:23Z (GMT). No. of bitstreams: 1 Guilherme_VivianeAparecida_M.pdf: 3020599 bytes, checksum: ce06e2f6da5344128ba90d18a9b569f2 (MD5) Previous issue date: 2012 / Resumo: Compostos anti-inflamatórios não esteroídais (AINEs) são amplamente utilizados para o combate da inflamação, mas frequentemente acarretam efeitos adversos que impedem a continuação do tratamento. Atualmente, a terapia a base de plantas tem sido bastante empregada a fim de desenvolver novos fármacos que apresentem eficácia analgésica e anti-inflamatória. Nesse contexto, a utilização de sistemas carreadores, como as nanopartículas de hidróxidos duplos lamelares (HDL), visa melhorar propriedades biofarmacêuticas e farmacológicas dos compostos nelas intercalados. Dessa forma, este estudo teve como objetivo caracterizar a interação entre o ácido coumárico (AC), um composto fenólico extraído de plantas, e hidróxido duplo lamelares (HDL), nanopartículas inorgânicas, considerando parâmetros como a cinética de liberação in vitro, a citotoxicidade e as atividades analgésica-antiinflamatória em relação ao fármaco não intercalado. Os ensaios de liberação in vitro foram realizados utilizando uma célula de difusão vertical (com membrana de acetato de celulose, MWCO 1000 Da) e a viabilidade celular avaliada em células 3T3 pelo teste de incorporação do vermelho neutro (VN). Os testes farmacológicos realizados em camundongos swiss foram: a determinação do número de contorções abdominais, teste tail-flick, ensaios de peritonite e teste de formalina para o composto livre e intercalado nas concentrações de 10, 20 ou 30 mg/kg. Os ensaios de liberação in vitro mostraram que a intercalação reduziu significativamente a constante de liberação (Klib) do AC, em relação ao fármaco livre, sendo os valores de Klib de 41,6 ± 1,5 %.h-1 e 32,4 ± 1,5 %.h-1 , para o AC e AC-HDL, respectivamente. A viabilidade celular foi reduzida apenas em concentrações mais elevadas de AC e AC-HDL (10 e 12,5 mM). Porém, mesmo nestas concentrações foi observada porcentagem de viabilidade celular maior que 50%. Por fim, a avaliação farmacológica apontou o AC-HDL como um sistema de liberação com atividades analgésica e anti-inflamatória mais pronunciadas do que as observadas para anti-inflamatórios não esteroidais como a indometacina (p< 0,001). Esses efeitos foram obtidos para teste de tail-flick, quando o AC foi intercalado em HDL aumentou a duração da analgesia (~ 1,7 vezes) quando comparado com o grupo de controle indometacina. Assim, os resultados indicam que o AC intercalado em nanopartículas inorgânicas de HDL apresentou uma taxa de liberação lenta e também induziu uma atividade analgésica - anti-inflamatória, possivelmente, por um mecanismo semelhante ao observado para um anti-inflamatório não esteroidais como a indometacina / Abstract: Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to avoid inflammation, but these compounds can evoke some side effects, considering these as an important limitation to the treatment. Currently, the plant-based therapy has been widely employed to develop new drugs which have analgesic and anti-inflammatory pharmacological activities. In this context, the use of inorganic nanoparticles is important to improve biopharmaceutical and pharmacological properties of the intercalated molecules. Thus, this study aimed to characterize the interaction between coumaric acid (CA), a phenolic compound extracted from plants, and layered double hydroxide (LDH), inorganic nanoparticles, considering parameters such as the in vitro release kinetics cytotoxicity and analgesic-antiinflammatory activities compared to the non-intercalated-CA. In vitro release tests were performed using a vertical diffusion cell (with cellulose acetate membrane, MWCO 1000 Da) and cell viability assessed in 3T3 cells by the neutral red (NR) uptake test. Pharmacological assays were carried in Swiss mice out in order to determine the number of writhings, tail-flick test, peritonitis test and formalin test for the free compound at three different concentrations (10, 20 or 30 mg/kg). In vitro release tests showed that the release constant (Krel) value was significantly reduced when compared to the non-intercalated CA (Krel values of 41.6 ± 1.5 %.h-1 and 32.4 ± 1.5 %.h-1 for the free CA and CA-HDL, respectively). Cell viability was reduced only at higher concentrations (10 and 12.5 mM) of CA and CA-HDL. However, even at these concentrations the percentage of cell viability was more than 50%. Finally, the pharmacological evaluation reveal the CA-HDL as a drug-release system with more pronounced analgesic-antiinflammatory effects than those observed for classic NSAIDs, such as indomethacin (p <0.001). Those effects were obtained, specially, for tail-flick test, when the treatment with CA-LDH increased the duration of analgesia (~ 1.7 times), when compared with the control group (indomethacin). Thus, the results pointed out that the system CA-LDH showed a slow release rate and also induced in vivo analgesic-anti-inflammatory activities, possibly using similar mechanisms to that observed for classic non-steroidal anti-inflammatory drugs, such as indomethacin / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Coccidiosis in chicken causes great economic losses. Increasing resistance of Eimeria species to anticoccidials has forced the search for alternative methods of control. The present study evaluates the anticoccidial activity of some anti-Eimeria tenella antibody fragments expressed in pea plants. Both in vitro and in vivo infection assays including indirect immunofluorescence, in vivo evaluation of antibody neutralization and cell culture invasion-inhibition assays were used to study the inhibitory effect of these antibody fragments on E. tenella sporozoites. Seven of nine antibody fragments (Ab1, Ab4, Ab5, Ab6, Ab7, Ab8 and Ab9) showed binding to sporozoites of E. tenella in an indirect immunofluorescence test. Only two antibodies (Ab4 and Ab5) cross reacted with sporozoites of E. maxima, E. acervulina and E. brunetti. The localization of specific fluorescence differed between species. Ab binding with sporozoites was seen in the area of both anterior and posterior refractile bodies in case of E. tenella, E. brunetti, and E. maxima but was only observed in the posterior refractile body in case of E. acervulina. No antibody binding was observed on merozoites. The suitability of antibody fragments to alter the infectivity of E. tenella sporozoites to Madin Darby Bovine Kidney cells (MDBK) was examined in vitro and the invasion-inhibition rates were quantified by flow cytometry. To assess the inhibitory effect on parasite reproduction, the in vivo antibody neutralization assay was done by retrograde infection of chicken with sporozoites previously incubated with antibody fragments. In vitro invasion rates were reduced by incubation with antibody fragments by approximately 24 to 45 %, with Ab6 and Ab7 showing the most distinct effect. However, proliferation rates (PR) of the respective MDBK cultures were also clearly reduced by 15 to 26 %. PR of MDBK cells treated with 1:1000, 1:100, 1:10 and undiluted mixed antibody fragments were reduced by 1%, 10%, 16%, and 26% with a reduction of invasion rates by 0%, 9%, 15% and 18%, respectively. Immune sera reduced the invasion rates by 16% to 70% and increased PR of the host cells. It appeared that the preparations of the antibody fragments contained compounds cytotoxic to MDBK cells and thus invasion inhibition could not be unequivocally evaluated in vitro. However, after incubation with antibody fragments sporozoites displayed a reduced ability to reproduce after intracloacal application to chicken (especially Ab1, Ab3, Ab5 and Ab9). Other antibody fragments (Ab2, Ab4, Ab6, Ab7 and Ab8) were less capable to reduce sporozoite infectivity and reproduction. More investigations are still required to study the possible use of antibody fragments and their application to infected chicken exposed to coccidiosis.