Produção de anticorpos monoclonais para o receptor a de estrógeno humano (hERa)

Orientador : Prof. Dr. Silvio Marques ZanataDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciencias Biológicas (Microbiologia, Parasitologia e Patologia Básica). Defesa: Curitiba, 22/03/2011Inclui referências : f. 54-57Área de concentraçãoResumo: O câncer de mama é um mal que acomete mais de 49.000 mulheres anualmente no Brasil. Na decisão terapêutica para esse tipo de tumor, é importante a definição de alguns parâmetros ligados a proteínas presentes ou não no carcinoma. Dentre essas proteínas, destaca-se o receptor ? de estrógeno (hER?). A detecção do receptor de estrógeno é feita através de imunohistoquímica e para essa técnica são usados anticorpos monoclonais anti hER?. Uma vez que o Brasil ainda é totalmente dependente da importação desse insumo, a obtenção de linhagens de hibridomas secretores desses anticorpos torna-se uma questão de soberania. No presente trabalho, foi elaborado todo o processo e ferramentas necessários para a obtenção dos hibridomas. Partindo-se do RNA total de células produtoras desse receptor, foi produzido cDNA a partir do qual o fragmento codificante para hER? foi clonado em plasmídeo para produção de proteína recombinante em sistema heterólogo em E. coli. Essa proteína foi fusionada a uma cauda de histidina que foi utilizada na sua purificação por cromatografia de afinidade a metal imobilizado. O peptídeo purificado foi utilizado como antígeno na imunização de camundongos Balb/C. Animais que apresentaram uma boa resposta imunológica frente ao antígeno de interesse foram eutanasiados e tiveram o seu baço removido para a realização dos ensaios de fusão. Os esplenócitos dissociados foram fusionados a células de linhagens de mieloma através da utilização de polietilenoglicol com o objetivo de se obter hibridomas secretores de anticorpos monoclonais de interesse. Durante o período do trabalho nenhum hibridoma estável foi obtido, no entanto foram desenvolvidas todas as ferramentas necessárias para a obtenção do insumo. Palavras-chave: hER?, receptor de estrógeno, anticorpos policlonais, anticorpos monoclonais, proteinas recombinantes.Abstract: Breast cancer is a disease that affects more than 49,000 women annually in Brazil. To achieve a therapeutic decision for this type of tumor, it is important to define some parameters linked to proteins present or absent in the carcinoma. Among those proteins, the estrogen receptor ? (hER?) is highlighted. The detection of estrogen receptor ? is made by immunohistochemistry and, for this technique, monoclonal antibodies against hER? are used. Since Brazil is still completely dependent on imports of this material, obtaining hibridoma lines that produce those antibodies becomes an issue of sovereignty. In this study, the hole process and tools necessary to obtain those hybridomas was developed. Starting from the total RNA of cells that produce the receptor, cDNA was produced. The encoding sequence for hER? was cloned into a plasmid from which the recombinant protein was produced in heterologous system in E. coli. This protein was fused to a histidine tag that was used for its purification by Immobilized Metal Affinity Chromatography. The purified peptide was used as antigen to immunize Balb/C mice. Animals that showed a good immune response against the antigen of interest were euthanized and had their spleen removed for fusion assay. The dissociated splenocytes were fused to myeloma cell lines by using polyethyleneglycol in order to obtain hybridomas secreting monoclonal antibodies of interest. During the study, no stable hybridoma was obtained, however have been developed all the tools necessary to obtain the desired material. Key words: hER?, estrogen receptor, polyclonal antibodies, monoclonal antibodies, recombinant proteins

Determinação de Di(2-etilhexil ftalato) liberado no sangue por linhas de PVC durante o procedimento de hemodiálise

O objetivo deste trabalho é demonstrar a aplicabilidade da Cromatografia Líquida de Alta Eficiência na quan-tificação do Di(2-etilhexil ftalato) (DEHP) liberado pela linha de Policloreto de vinila (PVC) no sangue cir-culante durante o procedimento de hemodiálise. Como a proposta não é a validação do método foram coleta-das amostras de sangue de cinco pacientes voluntários e de três voluntários saudáveis e os plasmas foram isolados, preparados e analisados. A concentração média de DEHP no sangue no plasma dos pacientes foi 0.19À0.12 mg/kg de massa corpórea, enquanto que no plasma dos voluntários saudáveis foi 0.003À0.002 μg/kg de massa corpórea. Os resultados mostraram que o método apresentado é uma alternativa para a de-terminação de DEPH no sangue e que poderá auxiliar na busca por outros materiais para aplicação em equi-pamentos de circulação extracorpórea. O estudo foi realizado de acordo com os padrões nacionais e interna-cionais de ética em pesquisa envolvendo seres humanos (CEP PUCPR Prot. n.5802).Palavras-chave: Diálise renal, Toxicidade crônica, DEHP

Assessment of MMP-9, TIMP-1, and COX-2 in normal tissue and in advanced symptomatic and asymptomatic carotid plaques

<p>Abstract</p> <p>Background</p> <p>Mature carotid plaques are complex structures, and their histological classification is challenging. The carotid plaques of asymptomatic and symptomatic patients could exhibit identical histological components.</p> <p>Objectives</p> <p>To investigate whether matrix metalloproteinase 9 (MMP-9), tissue inhibitor of MMP (TIMP), and cyclooxygenase-2 (COX-2) have different expression levels in advanced symptomatic carotid plaques, asymptomatic carotid plaques, and normal tissue.</p> <p>Methods</p> <p>Thirty patients admitted for carotid endarterectomy were selected. Each patient was assigned preoperatively to one of two groups: group I consisted of symptomatic patients (n = 16, 12 males, mean age 66.7 ± 6.8 years), and group II consisted of asymptomatic patients (n = 14, 8 males, mean age 67.6 ± 6.81 years). Nine normal carotid arteries were used as control. Tissue specimens were analyzed for fibromuscular, lipid and calcium contents. The expressions of MMP-9, TIMP-1 and COX-2 in each plaque were quantified.</p> <p>Results</p> <p>Fifty-eight percent of all carotid plaques were classified as Type VI according to the American Heart Association Committee on Vascular Lesions. The control carotid arteries all were classified as Type III. The median percentage of fibromuscular tissue was significantly greater in group II compared to group I (<it>p </it>< 0.05). The median percentage of lipid tissue had a tendency to be greater in group I than in group II (<it>p </it>= 0.057). The percentages of calcification were similar among the two groups. MMP-9 protein expression levels were significantly higher in group II and in the control group when compared with group I (p < 0.001). TIMP-1 expression levels were significantly higher in the control group and in group II when compared to group I, with statistical difference between control group and group I (p = 0.010). COX-2 expression levels did not differ among groups. There was no statistical correlation between MMP-9, COX-2, and TIMP-1 levels and fibrous tissue.</p> <p>Conclusions</p> <p>MMP-9 and TIMP-1 are present in all stages of atherosclerotic plaque progression, from normal tissue to advanced lesions. When sections of a plaque are analyzed without preselection, MMP-9 concentration is higher in normal tissues and asymptomatic surgical specimens than in symptomatic specimens, and TIMP-1 concentration is higher in normal tissue than in symptomatic specimens.</p

Ativação de macrófagos de frango através da interaçao entre os receptores CD80 e CD86 com proteina CD28 recombinante

Orientador : Prof. Dr. Silvio Marques ZanataTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciencias Biológicas (Microbiologia, Parasitologia e Patologia Básica). Defesa: Curitiba, 14/08/2015Inclui referências : f. 81-95Área de concentraçãoResumo: Em um ramo novo da ciência como a imunologia, poucos modelos são capazes de resistir por tanto tempo quanto o modelo de sinalização dupla, proposto por Bretcher e Cohn em 1970. Atualmente, entende-se que a dupla sinalização atua de forma sinérgica para a ativação dos linfócitos T e que a via do CD28 é apenas uma parte de uma grande rede de controle incluindo diversas outras vias que atuam em conjunto para controlar a ativação de linfócitos. Tanto do ponto de vista funcional, assim como do ponto de vista estrutural, a abordagem da via de CD28-CD80/86 na maioria dos casos tem um enfoque sobre a via de ativação de linfócitos, a qual é bastante descrita enquanto as APCs aparecem apenas como coadjuvantes nessa interação. Essa perspectiva começou a mudar em 2004 quando foi descrito que células dendríticas de camundongo, quando estimuladas com LPS e CD28 sofrem uma ativação e iniciam a secreção de interleucinas. Num contexto no qual o efeito das vias de sinalização sobre os APCs começa a ser compreendido, torna-se importante o estabelecimento de modelos adequados a esse estudo. Frangos são um modelo clássico para o estudo em imunologia, desde o início do desenvolvimento desse campo de conhecimento. Não foi descrita na literatura, até o momento, a sinalização CD28-CD80/86 em macrófagos de aves, as quais podem apresentar diferenças fisiológicas e funcionais quando comparadas às de mamíferos. Desta forma, este estudo pretende estabelecer se os mecanismos de sinalização reversa presentes em mamíferos e aves foi conservado durante a evolução, avaliando ainda a hipótese de que macrófagos possam ser artífices da sinalização reversa, fato observado apenas para células dendríticas. Este trabalho tem como objetivo avaliar a ativação de macrófagos de frango frente ao estímulo com CD28 recombinante. Às células previamente estimuladas com LPS, foram adicionados meios de cultivo com LPS, CD28 e CD28 deletada (com uma deleção em seu sítio MYPPPY de ligação com CD80/86) e os estímulos mensurados através de qPCR para diversas citocinas em diferentes tempos experimentais. Para o primeiro estimulo com LPS, houve uma redução na produção de IL-4, IL-2 e IFN-? e uma alteração sugerida para a expressão reduzida de IL-6, IL-12, IL-1? e aumentada de TNF?. Na segunda estimulação apenas com LPS, pode-se concluir que o ambiente resultante é pró-inflamatório, uma vez que a expressão das interleucinas com características anti-inflamatórias, IL-4 e IL-10, está diminuída ou ausente, enquanto IL-2, IL-18 e IL-12 estão aumentadas. Sugere-se que o estimulo com CD28 produz uma ativação de macrófagos que tem característica pró-inflamatória, porém sem direcionamento M1 ou M2. Essa polarização provavelmente advém de uma interação com os outros atores do sistema imune por diversos mecanismos de troca de informação, como citocinas, quimiocinas e outras moléculas sinalizadoras. Este trabalho mostrou pela primeira vez o mecanismo de sinalização reversa mediado por células apresentadoras de antígeno profissionais em macrófagos de aves. Palavras-chave: imunologia; aves; gallus; macrófago; CD28; sinalização reversa; citocinas.Abstract: In a new branch of science such as immunology, just a few models are able to resist for as long as the dual signaling model proposed by Bretcher and Cohn in 1970. Currently, it is known that the double signaling acts synergistically to activate T lymphocytes and that the path of CD28 is only one part of a large network control including several other ways that work together to control the activation of lymphocytes. In a functional point of view as well as from a structural point of view, the approach of CD28-CD80 / 86 in most cases has a focus on the activation pathway of lymphocytes, which is detailed described as the APCs appear only with a secondary role in this interaction. This perspective started to change in 2004 when it was reported that mouse dendritic cells when stimulated with LPS and CD28 undergoes activation and start to secrete interleukins. In a context that the effect of signaling pathways on APCs begins to be understood, it is important to establish suitable models in this study. Chickens are a classic model for the study in immunology, since the beginning of development of this field of knowledge. It has not been described in so far, CD28-CD80/86 signaling in avian macrophage, which may have physiological and functional differences compared to mammal ones. Thus, this study aims to establish if the mechanisms of reverse signaling present in mammals has been conserved during evolution and is similar in birds, also evaluating the hypothesis that macrophages can be passive of reverse signaling, which was observed only for dendritic cells. This project aims to evaluate the activation of chicken macrophages front of the stimulation with recombinant CD28. The cells previously stimulated with LPS, received culture media containing LPS, CD28 and deleted CD28 (with a deletion on the MYPPY CD80/86 binding site) the stimuli was measured by qPCR for different cytokines under different experimental times. For the first stimulation with LPS, there was a reduction in the production of IL4, IL2 and IFN-? and a modification on the expression was suggested with an increase for IL6, IL12, IL-1? and decreased for TNF. In the second stimulation with LPS, one may conclude that the resulting environment was pro-inflammatory, since the expression of interleukins with anti-inflammatory characteristics, IL-4 and IL-10, is decreased or absent, while IL-2 IL-18 and IL-12 are increased. It is suggested that CD28 stimulation generate an activation of macrophages that has pro-inflammatory characteristic but without polarization M1 or M2. This bias probably comes from an interaction with other actors of the immune system through several mechanisms of signaling such as cytokines, chemokines, and other signaling molecules. This study showed for the first time the reverse signaling mechanism mediated by professional antigen presenting cells in avian macrophage. Key words: immunology; birds; gallus; macrophage; CD28; reverse signaling; cytokines

Recombinant human interferon‐α14 for the treatment of canine allergic pruritic disease in eight dogs

Background Allergic pruritic diseases are increasingly common in dogs. This group of conditions hampers life quality as pruritus progressively interferes with normal behaviours. Therefore, new treatment modalities for canine allergic pruritic diseases are necessary. While novel drugs have recently reached the market, there is still the need for other therapeutic approaches. Some dogs are refractory even to the newer compounds, and cost is also an important issue for these. Older therapeutic modalities are only moderately successful or have considerable secondary effects, as is the case with glucocorticoids. Objectives Report on the use of recombinant human interferon-alpha 14 (rhIFN-alpha 14) for the treatment of canine allergic pruritus. Following the experience with a similar compound in the Japanese market, it was expected that rhIFN-alpha 14 could alter the Th1/Th2 disbalance that drives these diseases. Methods Here, we present an uncontrolled trial in which eight dogs with clinical diagnosis of allergic pruritus were treated with rhIFN-alpha 14, either orally or via subcutaneous injections. Skin condition, microbiota and anti-interferon antibody levels were assessed. Results The parenteral use of interferon induced hypersensitivity in two of the three dogs in which it was used. The oral administration was consistently safe and could reduce signs of the allergic condition in three of the five treated animals. Treatment also altered the skin microbiota, as verified by next-generation sequencing. Conclusion The present results indicate that rhIFN-alpha 14 is a viable candidate for the treatment of canine allergic pruritus. Future controlled studies are needed, and the oral route is indicated for further trials

Monoclonal antibody DL11C8 identifies ADAM23 as a component of lipid raft microdomains

A disintegrin and metalloprotease protein 23 (ADAM23) is a transmembrane type I glycoprotein involved with the development and maintenance of the nervous system, including neurite outgrowth, neuronal adhesion and differentiation and regulation of synaptic transmission. In addition, ADAM23 seems to participate in immune response and tumor establishment through interaction with different members of integrin receptors. Here, we describe a novel monoclonal antibody (DL11C8) that specifically recognizes the cysteine-rich domain of both pre-protein (100 kDa) and mature (70 kDa) forms of ADAM23 from different species, including human, rodents and avian orthologs. Using this antibody, we detected both forms of ADAM23 on the cell surface of three neuronal cell lineages (Neuro-2a, SH-SY5Y and CHLA-20), with a higher relative content of ADAM23. Furthermore, we demonstrate for the first time that a catalytically inactive member of the ADAM family is present in the membrane signaling platforms, namely lipid rafts. Indeed, the mature ADAM23 partitions between raft and non-raft membrane domains, while the pro-protein ADAM23 is mainly expressed in non-raft domains. These membranous distributions were observed in both different brain regions homogenates and primary cultured neurons lysates from mouse cortex and cerebellum. Taken together, these findings point out ADAM23 as a lipid raft molecular component