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Einstein (Säo Paulo) ; 18: eAO5105, 2020. tab, graf
Article in English | LILACS | ID: biblio-1090040


ABSTRACT Objective To evaluate the density of anti-galectin-3-immunostained cells, collagen percentage, mast cell density and presence of pathological processes in intestinal muscle biopsies of patients. Methods Thirty-five patients who underwent intestinal biopsy were selected from 1997 to 2015. Patients were divided into three groups: chagasic patients with mucosal lesion (n=13), chagasic patients with intact mucosa (n=12) and non-chagasic patients with no mucosal lesion (n=10). Histological processing of the biopsied fragments and immunohistochemistry for galectin-3 were performed. Additional sections were stained with hematoxylin and eosin to evaluate the general pathological processes, picrosirius for evaluation of collagen and toluidine blue to evaluate the mast cell density. Results Patients of mucosal lesion group had a significantly higher frequency of ganglionitis and myositis when compared to the chagasic patients with intact mucosa and non-chagasic group. The density of anti-galectin-3-immunostained cells was significantly higher in the chagasic patients with intact mucosa group when compared to the non-chagasic group. The group of chagasic patients with intact mucosa presented a higher percentage of collagen in relation to the patients with mucosal lesion and to the non-chagasic group, with a significant difference. There was no significant difference in mast cell density among the three groups. Conclusion The higher density of anti-galectin-3-immunostained cells in patients in the chagasic patients with intact mucosa group suggested the need for greater attention in clinical evaluation of these patients, since this protein is associated with neoplastic transformation and progression.

RESUMO Objetivo Avaliar a densidade de células imunomarcadas por anti-galectina-3, a percentagem de colágeno, a densidade de mastócitos e a presença de processos patológicos na musculatura intestinal de pacientes biopsiados. Métodos Foram selecionados 35 pacientes submetidos à biópsia de intestino entre 1997 a 2015. Os pacientes foram divididos em três grupos: chagásicos com lesão de mucosa (n=13), chagásicos com mucosa íntegra (n=12) e não chagásicos sem lesão de mucosa (n=10). Foram realizados processamento histológico dos fragmentos biopsiados e imunohistoquímica para galectina-3. Cortes adicionais foram corados por hematoxilina e eosina, para avaliar os processos patológicos gerais, pelo picrosírius, para avaliação do colágeno, e pelo azul de toluidina, para avaliar a densidade de mastócitos. Resultados Os pacientes do grupo chagásicos com lesão de mucosa apresentaram frequência significativamente maior de ganglionite e miosite quando comparados aos dos grupos chagásico com mucosa íntegra e não chagásicos. A densidade das células imunomarcadas por anti-galectina-3 foi significativamente maior no grupo chagásicos com mucosa íntegra quando comparada ao grupo não chagásico. O grupo de chagásicos com mucosa íntegra apresentou maior percentagem de colágeno em relação aos grupos chagásicos com mucosa lesada e ao grupo de não chagásicos, com diferença significativa. Não houve diferença significativa com relação à densidade de mastócitos entre os três grupos. Conclusão A maior densidade de células imunomarcadas por anti-galectina-3 nos pacientes do grupo chagásico com mucosa íntegra sugere a necessidade de maior atenção na avaliação clínica desses pacientes, uma vez que essa proteína está associada com transformação e progressão neoplásica.

Humans , Male , Female , Adult , Aged , Aged, 80 and over , Colonoscopy/methods , Chagas Disease/pathology , Galectin 3/analysis , Intestinal Mucosa/pathology , Megacolon/pathology , Antibodies, Monoclonal/analysis , Biopsy , Fibrosis , Immunohistochemistry , Case-Control Studies , Cell Count , Retrospective Studies , Analysis of Variance , Collagen/analysis , Statistics, Nonparametric , Galectin 3/immunology , Mast Cells/pathology , Middle Aged , Myositis/pathology
Clinics ; 74: e701, 2019. tab, graf
Article in English | LILACS | ID: biblio-1001831


OBJECTIVES: Osteosarcoma of the jaw (OSAJ) is fundamentally different in clinical practice from its peripheral counterparts. Studies are difficult to conduct due to low incidence rates. The primary aim of this study was to provide for the first time a comprehensive retrospective analysis of the treatment concepts and outcome data of OSAJ patients treated at the University Hospital Vienna and to compare these with two recently published studies on OSAJ. The clinical study was accompanied by a biomarker study investigating the prognostic relevance of melanoma-associated antigen-A (MAGE-A) in OSAJ specimens. METHOD: Eighteen patients were included, and their outcomes were compared to published data. Immunohistochemistry was performed with mouse monoclonal antibodies against MAGE-A. Survival rates were estimated by the Kaplan-Meyer method. The log-rank test was used to analyze potential prognostic parameters. Fisher's exact test was performed to define the significant differences between the survival rates of the current study and the DOESAK registry. RESULTS: Disease-specific survival was 93.8% after five and 56.3% after ten years. The development of metastases (p=0.033) or relapse (p=0.037) was associated with worsened outcomes in our group as well as in the comparative group. Despite the different treatment concepts of the study groups, survival rates were comparable. MAGE-A failed to show prognostic relevance for OSAJ patients. CONCLUSIONS: Uncertainties about the optimal treatment strategies of OSAJ patients will currently remain. Thus, prospective studies of OSAJ are needed but are only feasible in a multicenter study setting, conducted over a prolonged time period.

Humans , Male , Female , Adult , Middle Aged , Aged , Aged, 80 and over , Bone Neoplasms/therapy , Osteosarcoma/therapy , Prognosis , Austria/epidemiology , Bone Neoplasms/mortality , Bone Neoplasms/pathology , Immunohistochemistry , Biomarkers/analysis , Osteosarcoma/mortality , Osteosarcoma/pathology , Survival Rate , Retrospective Studies , Antibodies, Monoclonal/analysis , Antigens, Neoplasm/analysis
São Paulo; s.n; s.n; 2018. 71 p. graf, tab.
Thesis in Portuguese | LILACS | ID: biblio-996644


Anticorpos são moléculas de grande interesse científico e farmacêutico, principalmente, devido a sua alta especificidade contra antígenos determinados. Atualmente, anticorpos monoclonais estão entre os medicamentos (biofármacos) mais vendidos do mundo. São utilizados para o tratamento das mais diversas doenças, como câncer, retinopatias, doenças inflamatórias e do sistema imune, entre outras. Nos últimos 30 anos, as tecnologias para a obtenção de anticorpos monoclonais evoluíram muito, desde a tecnologia do hibridoma, até os processos de humanização de anticorpos murinos. Entre os métodos mais utilizados para a produção de anticorpos humanos, destaca-se a tecnologia do Phage Display. Nesta técnica, os genes que codificam as regiões variáveis de imunoglobulinas são inseridos no genoma de um bacteriófago, resultando na produção de partículas virais híbridas que contém fragmentos de anticorpos em fusão com uma das proteínas do capsídeo viral. Neste trabalho, desenvolvemos novos vetores para a apresentação de fragmentos ScFv em fusão com duas proteínas das proteínas do capsídeo viral, a pIII e pVIII. Os oligonucleotídeos utilizados para amplificar os genes de imunoglobulinas foram redesenhados e para minimizar a perda do repertório durante a produção da biblioteca, avaliamos em bancos de dados enzimas de restrição que não apresentam sítios de restrição nas sequencias gênicas. Esses sítios de restrição foram utilizados para construir as regiões de clonagem do vetor Phagemid. Outra etapa crítica na produção de bibliotecas de anticorpos é a reação do PCR de overlap, que pode restringir a diversidade de anticorpos e resultar na produção de amplicons codificando anticorpos truncados. Por isso, nossos vetores foram desenhados para permitir a clonagem direta das regiões variáveis das imunoglobulinas humanas ou murinas, sem a necessidade do PCR de overlap. Nossa expectativa, é que estes novos reagentes serão mais efetivos para a produção de novas bibliotecas de anticorpos pelo sistema do Phage Display

Antibodies are molecules of great scientific and pharmaceutical interest, mainly because of their high specificity against certain antigens. Currently, monoclonal antibodies are among the best selling drugs (biopharmaceuticals) in the world. They are used for the treatment of the most diverse disorders, such as cancer, retinopathies, inflammatory and immune system diseases, among others. In the past 30 years, technologies for obtaining monoclonal antibodies has greatly evolved from hybridoma technology to the humanization processes of murine antibodies. Among the methods used for the production of human antibodies, the technology of Phage Display stands out. In this technique, the genes encoding the immunoglobulin variable regions are inserted into the genome of a bacteriophage, resulting in the production of hybrid virus particles which contain fragments of antibodies in fusion with one of the viral capsid proteins. In this work, we developed new vectors for the presentation of ScFv fragments in fusion with two proteins of viral capsid proteins, pIII and pVIII. The oligonucleotides used to amplify the immunoglobulin genes were redesigned and to minimize repertory loss during library production, we evaluated restriction enzymes in databases that lack restriction sites in the gene sequences. These restriction sites were used to construct the cloning regions of the Phagemid vector. Another critical step in the production of antibody libraries is the overlap PCR reaction, which may restrict the diversity of antibodies and result in the production of amplicons encoding truncated antibodies. Therefore, our vectors were designed to allow the direct cloning of human or murine Immunoglobulins variable regions without the need for overlap PCR. Our expectation is that these new reagents will be more effective for the production of new antibody libraries by the Phage Display system

Pharmaceutical Preparations , Cell Surface Display Techniques/instrumentation , Antibodies, Monoclonal/analysis , Immunoglobulins/classification , Single-Chain Antibodies
Braz. J. Pharm. Sci. (Online) ; 54(spe): e01007, 2018. graf, ilus
Article in English | LILACS | ID: biblio-974431


The use of serum containing polyclonal antibodies from animals immunized with toxins marked the beginning of the application of antibody-based therapy in late nineteenth century. Advances in basic research led to the development of the hybridoma technology in 1975. Eleven years later, the first therapeutic monoclonal antibody (mAb) was approved, and since then, driven by technological advances, the development of mAbs has played a prominent role in the pharmaceutical industry. In this review, we present the developments to circumvent problems of safety and efficacy arising from the murine origin of the first mAbs and generate structures more similar to human antibodies. As of October 2017, there are 61 mAbs and 11 Fc-fusion proteins in clinical use. An overview of all mAbs currently approved is provided, showing the development of sophisticated mAbs formats that were engineered based on the challenges posed by therapeutic indications, including antibody-drug conjugates (ADC) and glycoengineered mAbs. In the field of immunotherapy, the use of immunomodulators, bispecific mAbs and CAR-T cells are highlighted. As an example of promising therapy to treat infectious diseases, we discuss the generation of neutralizing monoclonal-oligoclonal antibodies obtained from human B cells. Scientific and technological advances represent mAbs successful translation to the clinic

Animals , Mice , Technological Development/classification , Antibodies , Antibodies, Monoclonal/analysis , Mice, Transgenic/classification , Immunotherapy/adverse effects
Arq. bras. oftalmol ; 79(6): 395-399, Nov.-Dec. 2016. tab, graf
Article in English | LILACS | ID: biblio-838761


ABSTRACT Purpose: The cellular origin of retinoblastoma is uncertain as constituent tumor cells heterogeneously express markers of both immature and mature retinal cells. An immunohistochemical analysis of cellular origin may yield valuable insights into disease progression and treatment options. This study aimed to determine the cellular origin of retinoblastoma in a large case series and correlate these findings with histopathological prognostic factors. Methods: Thirty-nine retinoblastoma cases were histopathologically diagnosed and analyzed by immunohistochemistry using monoclonal antibodies against the immature neural cell marker SRY-box containing gene 2 (SOX-2), the mature neuronal cell marker microtubule-associated protein 2 (MAP2), and the mature glial cell marker glial fibrillary acidic protein (GFAP). Histopathological features were also evaluated, including patterns of growth, differentiation, vitreous seeding, and choroidal/scleral, optic nerve, and anterior chamber invasion. Two retinoblastoma cell lines, WERI-1 and Y79, were studied by immunocytochemistry using the same antibodies. Results: Expression of SOX-2 was strong in 97.4% of retinoblastoma cases, while MAP-2 was expressed in 59% of cases. Immunostaining for GFAP was positive only in reactive stromal astrocytes interspersed amongst tumor cells and in peritumoral tissue. There was no correlation between histopathological prognostic factors and immunohistochemical markers. Retinoblastoma cell lines showed strong positivity for SOX2 (90% of WERI-1 cells and 70% of Y79 cells) and MAP2 (90% of cells in both lines). GFAP was completely negative in both cell lines. Conclusion: The majority of retinoblastomas and both RB cell lines expressed an immature neural and/or a mature neuronal cell marker, but not a glial marker. These results indicate a typical neuroblast or neuronal origin and eliminate astrocyte differentiation from neural stem cells as the source of retinoblastoma.

RESUMO Objetivos: Este estudo visa determinar a origem do retinoblastoma em um número de casos e correlacionar essos achados com fatores prognósticos e histopatológicos conhecidos. Métodos: Trinta e nove casos de retinoblastoma foram diagnosticados e analisados com imuno-histoquímica usando marcadores de anticorpos monoclonais contra as células de retina imaturas (SOX-2: SRY-box containing gene 2), contra as células da retina maturas (MAP2: microtubule -associated protein 2) e contra as células gliais maturas (GFAP: glial fibrillar acidic protein). Foram avaliadas características microscópicas dos casos (grau de diferenciação, presença de semeadura vítrea, invasão de coroide/esclera, nervo óptico e câmara anterior). Duas linhas celulares de retinoblastoma (WERI-1 e Y79) também foram testadas, utilizando os três marcadores. Resultados: A expressão de SOX-2 foi positiva em 97,4% dos casos de retinoblastoma, enquanto MAP2 foi positivo em 59% dos casos. GFAP foi apenas positivo no estroma (astrócitos reativos). Não houve correlação entre preditores histopatológicos e marcadores imunohistoquímicos avaliados. As linhagens celulares mostraram positividade para SOX-2 (90% em WERI-1 e 70% das células Y79). Ambas as linhagens celulares se mostraram fortemente positivas con MAP2 (90%), enquanto não houve expressão de GFAP em nenhuma das linhas celulares estudadas. Conclusões: A maioria das células de retinoblastoma desta série de casos expressa marcadores de células retinianas imaturas, além de marcadores de células maduras. As linhas celulares Y79 e WERI-1 apresentaram imunomarcação para ambos os marcadores neurais em percentagens semelhantes a dos casos avaliados. Portanto, estes resultados confirmam a origem neural do tumor em particular. Alem disso, a ausência de células positivas para GFAP no tumor descarta diferenciação de astrócitos em retinoblastoma.

Humans , Male , Female , Infant , Child, Preschool , Child , Retinoblastoma/metabolism , Neuroglia/metabolism , Retinal Neoplasms/metabolism , Neural Stem Cells/pathology , Phenotype , Prognosis , Retinoblastoma/pathology , Immunohistochemistry , Biomarkers/metabolism , Neuroglia/pathology , Astrocytes/metabolism , Astrocytes/pathology , SOXB1 Transcription Factors/metabolism , Neural Stem Cells/metabolism , Glial Fibrillary Acidic Protein/metabolism , Microtubule-Associated Proteins/metabolism , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/metabolism
Pesqui. vet. bras ; 36(4): 339-344, graf
Article in Portuguese | LILACS | ID: lil-787567


O objetivo detse artigo é de descrever um protocolo de isolamento das células mononucleares da medula óssea de coelhos, seguido de purificação celular por depleção negativa com o anticorpo monoclonal CD45 e posterior expansão em meio de cultura MesenCult®. Dez coelhos machos adultos, da raça Nova Zelândia, com idade média de 1,0±0,2 anos e peso médio 3,5±0,24kg, foram utilizados para padronização da metodologia. O isolamento das células mononuclares da medula óssea foi realizado pelo gradiente de densidade Ficoll-paque® e a purificação e obtenção das células- pela depleção negativa com o anticorpo monoclonal CD45 em base imunomagnética. A população celular obtida foi expandida posteriormente em meio de cultura MesenCult®. No isolamento pelo gradiente de icoll-Paque® foi obtido um rendimento médio de 7,31x106 células/mL. Após purificação e obtenção das possíveis células-tronco mesenquimais pela base imunomagnética, houve um decréscimo do rendimento para 2,28x106 células/mL, mas o processo de expansão foi incrementado pelo cultivo celular. Os resultados indicaram que as células obtidas da fração mononuclear da medula óssea, cultivadas in vitro foram capazes de gerar células aderentes 24 horas após o cultivo, com predominância de células fibroblastóides sugestivas de células-tronco mesenquimais. Concluiu-se que a obtenção de células-tronco mesenquimais pode ser alcançada após purificação das células mononucleares da medula óssea de coelhos pelo método imunomagético, o meio de cultura MesenCult® proporciona um ambiente adequado para a rápida expansão in vitro e o número de passagens exerce influência negativa sobre as características morfológicas das células.

The objective of this study was to describe guidelines for the isolation of bone marrow mononuclear cells from rabbits, followed by cell purification by negative depletion with CD45 monoclonal antibody, and further expansion in MesenCult® medium. Ten adult male New Zealand White rabbits, age average of 1.0±0.2 years and weighting 3.5±0.24kg, were used to obtain a standardized method. The mononuclear cells of the bone marrow were isolated with Ficoll-paque® density gradient centrifugation, and the cell purification and acquisition was completed by negative depletion with CD45 monoclonal antibody in immunomagnetic base. The cell population obtained was expanded in MesenCult® medium. Through isolation with Ficoll-paque® density gradient was possible to obtain an average yield of 7.31x106 cells/mL. After purification and acquisiton of potential mesenchymal stem cells by the immunomagnetic base, there was a yield decrease to 2.28x106 cells/mL; however the expansion process was increased in cell culture. The results indicated that cells obtained from the mononuclear fraction of bone marrow and cultivated in vitro were capable to generate adherent cells 24 hours after culture, with predominance of fibroblastoid cells suggestive of mesenchymal stem cells. It can be concluded that mesenchymal stem cells can be achieved with purified rabbit bone marrow mononuclear cells through the immunomagnetic method, as the MesenCult® medium provides a suitable environment for a quick in vitro expansion, and the number of passages exerts negative influence on the morphological characteristics.

Animals , Male , Rabbits , Adult Stem Cells , Antibodies, Monoclonal/analysis , Bone Marrow Cells , Cell Separation/veterinary , Lagomorpha , Immunomagnetic Separation/veterinary , In Vitro Techniques/veterinary
Rev. panam. salud pública ; 39(3): 149-156, Mar. 2016. tab, graf
Article in Portuguese | LILACS | ID: lil-783949


RESUMO Objetivo Analisar o percurso dos ensaios clínicos com anticorpos monoclonais e biomedicamentos oncológicos realizados em instituições brasileiras de 2003 a 2012. Método Neste estudo retrospectivo e descritivo, realizou-se um levantamento junto aos repositórios e ReBEC. Foram incluídos ensaios de fase II ou III com participação do Brasil e registro em pelo menos um dos repositórios. Após a seleção dos ensaios, foi investigada a trajetória dos anticorpos monoclonais e biomedicamentos desde a pesquisa até o registro sanitário junto a Agência Nacional de Vigilância Sanitária (Anvisa), Food and Drug Administration (FDA) e European Medicines Agency (EMA). Resultado Nove ensaios foram selecionados, todos randomizados controlados. Oito eram de fase III e um de fase II. Dois ensaios utilizaram cegamento duplo e sete eram abertos. Todos apresentaram recrutamento para ambos os sexos, com idade mínima de 18 anos. A média de recrutamento foi de 985,2 pacientes. Sete ensaios estavam finalizados e dois haviam sido encerrados prematuramente. Todos os ensaios foram financiados por indústrias farmacêuticas não brasileiras e enfocaram câncer renal, colorretal, gástrico, de pulmão (não pequenas células), linfoma não-Hodgkin e melanoma, envolvendo a utilização de cetuximabe, figitumumabe, ipilimumabe, rituximabe, bevacizumabe e interferon alfa-2a. A FDA foi a primeira agência a registrar os medicamentos, seguida pela EMA, a não ser no caso do interferon alfa-2a, recusado pela EMA. Não foi possível avaliar o ano de aprovação no Brasil pela Anvisa. Conclusão A participação do Brasil nos ensaios clínicos com anticorpos monoclonais e biomedicamentos oncológicos é insuficiente. A limitação do conteúdo disponível sobre os estudos, histórico de registro e outros elementos relevantes é uma fragilidade importante das fontes consultadas.

ABSTRACT Objective To analyze the pathway of clinical trials of monoclonal antibodies and biological medicines for cancer treatment involving Brazilian institutions from 2003 to 2012. Method This retrospective, descriptive study was based on review of two clinical trial registries, and the Brazilian registry ReBEC. Phase II or III studies with participation from Brazilian institutions listed in at least one of the registries were included. Following selection of the trials, the pathway of monoclonal antibodies and biological medicines was investigated from the research stage until licensing by the Brazilian Agency for Sanitary Surveillance (Anvisa), Food and Drug Administration (FDA), and European Medicines Agency (EMA). Results Nine trials (eight phase III and one phase II) were selected. All had a randomized, controlled design. Two trials were double-blind and seven were open-label, and all recruited adults (≥ 18 years of age) of both sexes. The mean number of patients recruited per trial was 985.2. Seven trials had been completed and two had been terminated early. All trials were sponsored by non-Brazilian pharmaceutical companies and focused on renal, colorectal, gastric, and lung (non-small cell) cancer, non-Hodgkin lymphoma, and melanoma, and involved the use of cetuximab, figitumumab, ipilimumab, rituximab, bevacizumab and interferon alfa-2a. The FDA was the first agency to license the drugs, followed by EMA, except in the case of interferon alfa-2a, which was not approved by EMA. We were unable to determine the year of drug licensing by Anvisa in Brazil. Conclusions The participation of Brazil in clinical trials of monoclonal antibodies and biological medications for cancer treatment is insufficient. The quality of the available information on trials, history of licensing, and other relevant elements is a major weakness of the sources reviewed.

Biomedical Research/methods , Medical Oncology/trends , Antibodies, Monoclonal/analysis , Brazil
São Paulo; s.n; s.n; 2016. 145 p. tab, graf, ilus.
Thesis in Portuguese | LILACS | ID: biblio-847514


A família proteína quinases C (PKC) é composta por dez isoenzimas, as quais são capazes de fosforilar resíduos de serina e treonina. A ativação dessas quinases envolve mudanças conformacionais, como a remoção do pseudo-substrato do sítio ativo e associação dessas enzimas com lipídeos em membranas biológicas. Além disso, três fosforilações são importantes para a maturação/ enovelamento da enzima e não estão associadas com o estado de ativação das cPKCs. Apesar dessas quinases estarem envolvidas em vários processos patológicos, como carcinogênese e doenças cardiovasculares, ainda não se estabeleceu a relação entre estado de ativação das PKCs com essas doenças. Isso se deve, em parte, à ausência de ferramentas que possibilitam a distinção das formas ativas e inativas das PKCs. Na presente tese, baseando-se em mudanças conformacionais sofridas pelas PKCs durante o processo de ativação, dois anticorpos contra cPKCs ativas foram racionalmente desenvolvidos, sendo um anticorpo policlonal (anti-C2Cat) e outro monoclonal (4.8E). O anticorpo anti-C2Cat foi desenvolvido a partir de imunização de coelhos com um peptídeo localizado na região de interação entre os domínios C2 e catalítico na PKC inativa. Já o anticorpo monoclonal 4.8E foi produzido após a imunização de camundongos Balb/ C com extrato de proteínas proveniente de células HEK293T superexpressando formas constitutivamente ativas da PKCßI. A seletividade de anti-C2Cat e 4.8E por cPKCs ativas foi demonstrada por ensaios de ELISA e de imunoprecipitação, sendo que os anticorpos sempre apresentaram maior afinidade por cPKCs ativas purificadas, superexpressas ou mesmo as endógenas. O anticorpo anti-C2Cat foi capaz de monitorar a dinâmica espaço-temporal da ativação das cPKCs em linhagens de neuroblastoma (Neuro-2A e SK-N-SH) estimuladas com PMA, morfina, ATP ou glutamato por diferentes tempos. Ainda, um maior conteúdo de cPKCs ativas foi detectado por anti-C2Cat na linhagem de câncer de mama MDA-MB-231 (triplo- negativa) do que em células MCF-7 (ER+). Em acordo com esses dados, anti-C2Cat identificou uma maior ativação de cPKCs em tumores mais agressivos de câncer de mama (subtipo triplo-negativo) do que em tumores menos agressivos (ER+, subtipo luminal). Os anticorpos conformacionais anti-C2Cat e 4.8E foram aplicados para elucidar vias de sinalização que levam à carcinogênese em células MDA-MB-231, por meio da realização de ensaios de co-imunoprecipitação, seguida pela identificação das proteínas por espectrometria de massas. Usando essa abordagem, os resultados sugerem que as cPKCs ativas possam estar envolvidas com a tradução de proteínas envolvidas na migração celular, como actina. Em conjunto, os resultado obtidos na presente tese demonstram duas formas racionais de desenvolver anticorpos contra cPKCs ativas, sendo que algumas aplicações para estas ferramentas foram demonstradas. Estratégias baseadas em mudanças conformacionais, similares às apresentadas aqui, poderão ser utilizadas para a produção racional de anticorpos contra outras quinases ou proteínas

The protein kinase C family (PKC) is composed of ten isoenzymes, which are capable of phosphorylating serine and threonine amino acid residues. PKC activation involves conformational changes, such as removing the pseudo-substrate from the active site and binding of the enzyme to lipids in biological membranes. In addition, PKC undergoes three phosphorylations that are important for the maturation/ folding of the enzyme and are not linked with activation status. Despite the fact that these kinases are involved in various pathological processes, such as carcinogenesis and cardiovascular disease, a relationship between PKC activation status with these diseases has not yet been established. This is partly due to the lack of tools to detect active PKC in tissue samples. In this thesis, based on conformational changes suffered by PKC during its activation, two antibodies against active cPKCs were rationally developed; a polyclonal antibody (anti-C2Cat) and a monoclonal (4.8E). Anti-C2Cat was produced after immunization of rabbits with a peptide located at the interface between the C2 and catalytic domains of cPKCs in an inactive PKC. The monoclonal antibody 4.8E was produced after immunization of Balb/C mice with total lysates from HEK293T cells overexpressing constitutively active forms of PKCßI. The anti-C2Cat and 4.8E specificity by active cPKCs was demonstrated by ELISA and immunoprecipitation assays, where the antibodies always showed higher affinity to active cPKCs. Anti-C2Cat was able to detect the temporal and spatial dynamics of cPKC activation upon receptor (morphine, ATP or glutamate) or phorbol ester stimulation in neuroblastoma lines (Neuro-2A and SK-N-SH). Futhermore, anti-C2Cat is able to detect active PKC in human tissues. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Also, both antibodies were applied to study signaling pathways that lead to carcinogenesis in MDA-MB-231 cells by performing co-immunoprecipitation and mass spectrometry. Using this approach, the results suggest that active cPKCs may be involved in translation of proteins involved in cell migration, such as actin. Taken together, the results obtained in this thesis showed two rational ways to develop antibodies against active cPKCs and some applications for these tools were demonstrated. Strategies based on conformational changes, similar to those presented herein may be used for rational production of antibodies against other kinases and proteins

Animals , Male , Female , Mice , Rabbits , Antibodies, Monoclonal/analysis , Antibodies/analysis , Protein Kinase C/adverse effects , Receptor-Interacting Protein Serine-Threonine Kinases , Breast Neoplasms/complications , Cell Line, Tumor/metabolism , Enzyme-Linked Immunosorbent Assay/methods , Fluorescent Antibody Technique/methods , Hybridomas , Immunoprecipitation/methods , Mass Spectrometry/methods
Article in English | WPRIM | ID: wpr-169634


Monoclonal antibody (mAb, NVRQS-DON) against deoxynivalenol (DON) was prepared. DON-Ag coated enzyme linked immunosorbent assay (ELISA) and DON-Ab coated ELISA were prepared by coating the DON-BSA and DON mAb. Quantitative DON calculation ranged from 50 to 4,000 ng/mL for DON-Ab coated ELISA and from 25 to 500 ng/mL for DON-Ag coated ELISA. 50% of inhibitory concentration values of DON, HT-2, 15-acetyl-DON, and nivalenol were 23.44, 22,545, 5,518 and 5,976 ng/mL based on the DON-Ab coated ELISA. Cross-reactivity levels of the mAb to HT-2, 15-acetyl-DON, and nivalenol were 0.1, 0.42, and 0.40%. The intra- and interassay precision coefficient variation (CV) were both <10%. In the mAb-coated ELISA, mean DON recovery rates in animal feed (0 to 1,000 microg/kg) ranged from 68.34 to 95.49% (CV; 4.10 to 13.38%). DON in a buffer solution (250, 500 and 1,000 ng/mL) was isolated using 300 microg of NVRQS-DON and 3 mg of magnetic nanoparticles (MNPs). The mean recovery rates of DON using this mAb-MNP system were 75.2, 96.9, and 88.1% in a buffer solution spiked with DON (250, 500, and 1,000 ng/mL). Conclusively we developed competitive ELISAs for detecting DON in animal feed and created a new tool for DON extraction using mAb-coupled MNPs.

Animal Feed/analysis , Animals , Antibodies, Fungal/analysis , Antibodies, Monoclonal/analysis , Chemistry Techniques, Analytical/methods , Enzyme-Linked Immunosorbent Assay/methods , Female , Food Contamination/analysis , Fusarium/immunology , Imidazoles/chemistry , Magnetics/methods , Mice , Mice, Inbred BALB C , Mycotoxins/analysis , Nanoparticles/chemistry , Ovalbumin/chemistry , Trichothecenes/analysis
Rio de Janeiro; s.n; 2012. xiii,73 p. ilus, graf, tab, mapas.
Thesis in Portuguese | LILACS | ID: lil-653102


As leishmanioses constituem um grave problema de saúde. São causadas por protozoários do gênero Leishmania e sua transmissão ao hospedeiro vertebrado se dá pela picada de fêmeas infectadas do gênero Lutzomyia (Novo Mundo) e Phlebotominae (Velho Mundo). Na alimentação, as leishmanias são ingeridas pelo flebotomíneo junto com sangue e uma etapa fundamental para a manutenção da infecção no vetor refere-se à adesão dos parasitos ao epitélio intestinal com o término da digestão. Para isso, o flagelo das formas promastigotas desempenha papel essencial no processo de fixação dos parasitos às células intestinais. Estudos prévios do nosso grupo identificaram um gene codificante para uma proteína flagelar denominada FLAG que pode estar diretamente envolvida na adesão do parasito ao tubo digestivo do flebotomíneo. Visando a melhor caracterização do gene, realizou-se no presente trabalho a análise da transcrição do gene FLAG nos diferentes estágios de desenvolvimento de Leishmania infantum chagasi e Leishmania pifanoi por PCR em tempo real. Tanto L. i. chagasi quanto L. pifanoi não apresentaram diferença de expressão entre promastigota de fase exponencial de crescimento (procíclica) e de fase estacionária (metacíclica). Entretanto, em L.pifanoi, as promastigotas apresentaram uma maior expressão de FLAG do que as amastigotas que têm um flagelo interno curto. Com o intuito de verificarmos o papel de FLAG na adesão ao tubo digestivo do vetor, realizamos ensaios de inibição de adesão ex vivo e in vivo utilizando anticorpo monoclonal anti-FLAG. Os ensaios foram feitos com os pares L. i. chagasi X Lutzomyia longipalpis, Leishmania amazonensis X L. longipalpis e Leishmania major X Phlebotomus papatasi, incubando previamente os parasitos com o anticorpo anti-FLAG. Os resultados não mostraram diferenças significativas no número de parasitos aderidos entre os grupos teste e controle, nos experimentos com espécies do Novo Mundo. Entretanto, com o par natural L. major e P. papatasi, foi possível observar uma redução significativa de parasitos presentes no tubo digestivo quando os mesmos foram pré-incubados com o anticorpo. Logo, em espécies do Novo Mundo, a proteína FLAG não parece estar relacionada com a adesão de Leishmania ao tubo digestivo do flebotomíneo enquanto que no par do Velho Mundo L. major e P. papatasi, ela parece ter um papel na interação. Em paralelo foram feitos experimentos de “pull down” para a identificação de moléculas potencialmente capazes de interagir com FLAG no intestino de L. longipalpis. Para tal, foram usados extratos protéicos de células embrionárias LL5 de L. longipalpis. Observamos um grande número de ligações inespecíficas à resina ou à proteína de fusão, mas nenhuma banda pôde ser detectada ligando-se especificamente a FLAG. Isto está de acordo com os resulltados de inibição de interação ex vivo e in vivo. Os insetos são organismos que conseguem se adaptar a diversas condições ambientais. Essa capacidade de sobrevivência está apoiada em um sistema de resposta imune inata bem desenvolvido. Em nosso laboratório, uma sequência de cDNA similar a TGF-beta (Transforming Growth Factor – beta) foi identificada em fêmeas de L. longipalpis infectadas com L. i. chagasi através de experimentos de Differential Display RT-PCR (DDRT-PCR). A molécula foi seqüenciada e classificada como pertencente à família das ativinas. As citocinas da super-familia das TGF-beta são responsáveis pela sinalização e regulação de uma série de processos biológicos em diversos organismos. Essas proteínas são agrupadas em diferentes sub-familias, dentre elas a da ativina/inibina está relacionada a processo de desenvolvimento neuronal e processos de diferenciação. A análise da expressão do RNA de TGF-beta revelou um aumento significativo 72 horas após a infecção com L. i. chagasi quando comparado com fêmeas alimentadas apenas com sangue. Para avaliar o possível papel de TGF-beta na infecção por Leishmania, realizamos experimentos de infecção de L. longipalpis com L. i. infantum contendo ou não anticorpo anti-TGF-beta. Observamos um aumento de parasitos no grupo alimentado com anticorpo, indicando um possível papel de TGF-beta na ativação da resposta imune do vetor contra a Leishmania. A interação entre parasitos e seus vetores é uma etapa fundamental do ciclo infectivo. Por isso, o estudo de moléculas – chave para o desenvolvimento do ciclo de transmissão torna-se cada vez mais importante.

Antibodies, Monoclonal/analysis , Leishmania , Leishmaniasis , Psychodidae , Polymerase Chain Reaction , Transforming Growth Factor beta
Braz. j. med. biol. res ; 43(12): 1215-1224, Dec. 2010. ilus, tab
Article in English | LILACS | ID: lil-568996


Rubinstein-Taybi syndrome (RTS) is a rare developmental disorder characterized by craniofacial dysmorphisms, broad thumbs and toes, mental and growth deficiency, and recurrent respiratory infections. RTS has been associated with CREBBP gene mutations, but EP300 gene mutations have recently been reported in 6 individuals. In the present study, the humoral immune response in 16 RTS patients with recurrent respiratory infections of possible bacterial etiology was evaluated. No significant differences between patients and 16 healthy controls were detected to explain the high susceptibility to respiratory infections: normal or elevated serum immunoglobulin levels, normal salivary IgA levels, and a good antibody response to both polysaccharide and protein antigens were observed. However, most patients presented high serum IgM levels, a high number of total B cell and B subsets, and also high percentiles of apoptosis, suggesting that they could present B dysregulation. The CREBBP/p300 family gene is extremely important for B-cell regulation, and RTS may represent an interesting human model for studying the molecular mechanisms involved in B-cell development.

Adolescent , Child , Child, Preschool , Female , Humans , Male , Young Adult , Antibodies, Monoclonal/analysis , B-Lymphocytes/immunology , Immunity, Humoral/immunology , Immunoglobulins/analysis , Respiratory Tract Infections/immunology , Rubinstein-Taybi Syndrome/immunology , Antibodies, Monoclonal/immunology , Case-Control Studies , CREB-Binding Protein/genetics , Immunity, Humoral/genetics , Immunoglobulins/immunology , Recurrence
Medicina (B.Aires) ; 70(2): 163-165, Apr. 2010. graf, tab
Article in Spanish | LILACS | ID: lil-633736


La coexistencia de enfermedades mieloproliferativas y linfoproliferativas en el mismo paciente no es común. La mayoría de los casos corresponden a pacientes que desarrollan leucemia aguda durante el curso evolutivo de una leucemia linfática crónica tratada con drogas quimioterápicas. Se presenta un caso de leucemia mielomonocítica aguda y leucemia linfática crónica B diagnosticadas simultáneamente en un paciente en el cual, el análisis por citometría de flujo utilizando un amplio panel de anticuerpos monoclonales, permitió identificar las diferentes poblaciones patológicas y determinar su inmunofenotipo característico. Una revisión de la bibliografía muestra solamente la descripción de casos aislados sin encontrar datos sobre la incidencia de esta asociación. Destacamos la utilidad de la técnica de citometría de flujo para identificar las células anormales que nos llevan al diagnóstico de estas dos enfermedades.

The coexistence of acute myeloid leukemia and chronic lymphocytic leukemia in the same patient is rare. The majority of the cases correspond to patients that developed acute leukemia during the evolutionary course of a chronic lymphatic leukemia following treatment with chemotherapy drugs. We report a case of acute myelomonocytic leukemia concurrent with untreated B-cell chronic lymphocytic leukemia in which the use of flow cytometry analysis with a large panel of monoclonal antibodies, allowed the demonstration of different pathological populations and determine immunophenotyping patterns. Published cases of simultaneous chronic lymphocytic leukemia and acute leukemia are reviewed. The use of multiparametric flow cytometry to differentiate the populations demonstrates the utility of this technology in the diagnosis of these hematological malignancies.

Aged , Humans , Male , Antibodies, Monoclonal/analysis , B-Lymphocytes/pathology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Leukemia, Myelomonocytic, Acute/pathology , Neoplasms, Multiple Primary/pathology , B-Lymphocytes/immunology , Clone Cells , Flow Cytometry , Immunophenotyping , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Myelomonocytic, Acute/immunology , Neoplasms, Multiple Primary/immunology
Rev. méd. Chile ; 137(11): 1474-1477, nov. 2009. ilus
Article in Spanish | LILACS | ID: lil-537011


Lymphangioleiomyomatosis (LAM) is a rare interstitial lung disease, of unknown etiology, affecting almost exclusively women. Microscopically LAM consists of a diffuse proliferation of smooth muscle cells. LAM can occur without evidence of other diseases (sporadic LAM) or in conjunction with tuberous sclerosis complex (TSC). It presents with progressive breathlessness or with recurrent pneumothorax or chylothorax. We report a 33 year-old woman with a history of recurrent pneumothorax. Computed tomography (CT) scans showed numerous thin-walled cysts throughout the lungs, a characteristic finding in LAM. A pulmonary biopsy was compatible with the diagnosis and HMB-45 monoclonal antibodies were positive. Treatment with Sirolimus was started.

Adult , Female , Humans , Lung Diseases, Interstitial/pathology , Lung Neoplasms/pathology , Lymphangioleiomyomatosis/pathology , Antibodies, Monoclonal/analysis , Lung Neoplasms/complications , Lymphangioleiomyomatosis/complications , Pneumothorax/etiology , Recurrence
Braz. j. med. biol. res ; 42(7): 593-598, July 2009. ilus, tab
Article in English | LILACS | ID: lil-517801


Blood and lymphatic vessel proliferation is essential for tumor growth and progression. Most colorectal carcinomas develop from adenomas (adenoma-carcinoma sequence) in a process due to accumulation of molecular genetic alterations. About 5% of adenomatous polyps are expected to become malignant, but data on the differential angiogenic patterns of these lesions in patients with and without concomitant cancer are missing. The aim of the present study is to compare the angiogenic and lymphatic patterns of adenomatous polyps from patients with and without sporadic cancer. Thirty adenomatous polyps (15 from patients with another principal malignant lesion, and 15 from patients without cancer) were submitted to immunohistochemical staining for CD105 (marker for neoangiogenesis) and D2-40 (marker for lymphatic endothelium). Microvessel density and total vascular area were determined by computer image analysis to quantify the immunostained and total areas, and to assess the number of microvessels. Adenomas from patients with carcinoma showed significantly higher values of total vascular area determined by immunostaining for CD105 (cutoff value = 4386 µm²; P = 0.019) and of lymphatic microvessel density determined by immunostaining with D2-40 (cutoff value = 11.5; P = 0.041) when compared with those from patients without cancer. The present data indicate a significant increase in blood microvascular area and in lymphatic microvascular counts in adenomas removed from patients with cancer.

Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Adenomatous Polyps/pathology , Colorectal Neoplasms/pathology , Lymphangiogenesis/physiology , Neovascularization, Pathologic/pathology , Adenomatous Polyps/blood supply , Adenomatous Polyps/chemistry , Antibodies, Monoclonal/analysis , Antigens, CD/analysis , Biomarkers/analysis , Colorectal Neoplasms/blood supply , Colorectal Neoplasms/chemistry , Immunohistochemistry , Lymphatic Vessels/chemistry , Lymphatic Vessels/pathology , Microcirculation , Retrospective Studies , Receptors, Cell Surface/analysis
São Paulo; s.n; 2009. 127 p. ilus, tab.
Thesis in Portuguese | LILACS | ID: lil-568920


Intimina é o principal fator de virulência na patogênese de Escherichia coli enteropatogênica (EPEC) e de Escherichia coli enterohemorrágica (EHEC). A detecçãode EHEC e EPEC típica ou atípica é de fundamental impotância na definição da conduta terapêutica das infecções promovidas por E. coli, que ainda são a principal causa de diarreia aguda em crianças e adultos em muitos paises desenvolvidos e em desenvolvimento. Anticorpos são ferramentas importantes na detecção de diversos patógenos. Neste trabalho avaliou-se a sensibilidade e especificidade dos anticorpos policlonal e monoclonal anti-intimina frente a isolados de EPEC e EHEC por immunoblotting. Os anticorpos apresentam 100% de especificidade e a sensibilidade foi de 97%, 92% e 78%, quando se utilizou a fração enriquecida em IgG do soro de coelho, antissoro de rato e anticorpo monoclonal, respectivamente. Esse anticorpo monoclonal anti-intimina foi caracterizado como IgG2b e 1 μg desse corpo reconheceu 0,6 μg de intimina purificada com uma constante de dissociação de 1.3 x 10&sup-8; M. A menor reatividade do anticorpo monoclonal em relação aos anticorpos policlonais levou-nos à clonagem e expressão do fragmento variável de cadeia simples desse anticorpo (scFv). Para isto, o mRNA do hibridoma anti-intimina foi extraído, reversamente transcrito para cDNA e amplicadas as cadeias leve e pesada da fração variável do anticorpo, utilizando iniciadores aleatórios comerciais. As cadeias amplificadas foram ligadas ao vetor pGEM-T Easy e sequenciadas. Iniciadores específicos foram desenhados e utilizados em uma estratégia de amplificação e união das cadeias, formando o scFv, que por sua vez foi clonado no vetor de expressao pAE. Linhagem E. coli BL 21(DE3)pLys foi transformada com o plasmídeo pAE-scFv anti-intimina e submetida a indução proteíca. O scFv anti-intimina foi expresso de forma insolúvel, solubilizado, purificado e submetido ao ensaio de refolding. O rendimento obtido foi de 1 mg de proteina por 100mL...

Intimin is the major virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli(EPEC) and enterohemorrhagic Escherichia coli(EHEC). The detection of EHEC and typical or atypical EPEC has fundamental importance in defining the therapeutic management of infections causes by E. Coli, which are still the leading cause of acute diarrhea in children and adults in many developed and developing countries. Antibodies are important tools in the detection of several pathogens. In this study it was evaluated the sensitivity and specificityit of polyclonal and monoclonal antibodies against intimin in the detection of EPEC AND EHEC by immunobloting. All employed antibodies showed 100% specifity and sensitivity was 97%, 92% and 78% for rabbit anti-intimin IgG-enriched fraction, rat antisera and monoclonal antibody, repectively. This anti-intimin monoclonal was characterized as IgG2b and 1 μg recognized 0.6 μg of purified intimin with a dissociation constant of 1.3 x 10&sup-8;M. The less extend reactivity of monoclonal led us to clone and express the single chain fragment variable of this antibody (scFV). Thus, the anti-intimin hybridoma mRNA was extrated, reverse transcribed to cDNA and the light and heavy chains of variable fragment of the antibody were amplified using commercial random primers. The chains were amplified, ligated to the pGEM-T Easy vector and the insert was sequenced. Specific primers were designed and used in strategy to amplify and link the chains, obtaining the scFv, wich was cloned into the pAE expression vector. E. coli BL21(DE3)plys was transformed with the pAE-scFv anti-intimin plasmid and subjected to induction to protein expression. The scFv anti-intimin, expressed in the insoluble fraction, was purified and submitted to refolding. The yield was 1 mg of protein per 100 mL of bacterial culture. To test the functionalityof scFv, ELISA and immunofluorescence assays were performed. The results showed 275 ng of scFv...

Antibodies, Monoclonal/analysis , Antibodies/analysis , Enterohemorrhagic Escherichia coli , Enteropathogenic Escherichia coli/chemistry , Reactivity-Stability , Enzyme-Linked Immunosorbent Assay
Rev. méd. hondur ; 76(3): 101-107, jul.-sept. 2008. ilus
Article in Spanish | LILACS | ID: lil-505104


OBJETIVO: Caracterizar los pacientes con Leishmaniasis cutánea atípica atendidos en el Servicio de Dermatología del Hospital Escuela de Tegucigalpa. PACIENTES Y METODO: Estudio descriptivo. Se estudiaron 27 pacientes con diagnóstico de Leishmaniasis Cutánea Atípica, en el periodo comprendido de Septiembre 1999 a Marzo del 2000, en quienes se realizó el diagnóstico mediante frotis, cultivo de las lesiones y cuantificación de anticuerpos monoclonales; a un subgrupo se le realizó además análisis isoenzimático y test de Montenegro. RESULTADOS: la edad promedio fue 9.4 años, 17 eran del sexo masculino, 17 provenían de la región sur del país. En 23 (85) las lesiones estaban en la cara, constituidas por pápulas, nódulos y placas no ulceradas, asintomáticas y de evolución crónica. En 15 (56) pacientes se encontró desnutrición crónica. Las especies identificadas fueron Leishmania chagasi en 10 casos y Leishmania mexicana en 1 caso. CONCLUSIONES: El presente estudio es consistente con otras publicaciones que identifican a la zona sur de Honduras como una zona endémica de Leishmaniasis cutánea atípica, siendo L. Chagasi el agente causal predominante de esta enfermedad. La mayoría eran pacientes masculinos, mayores de cinco años de edad, con desnutrición...

Humans , Male , Child , Adolescent , Atypical Bacterial Forms , Leishmaniasis, Cutaneous/diagnosis , Skin Diseases , Antibodies, Monoclonal/analysis , Leishmaniasis, Visceral/diagnosis
Rev. Assoc. Med. Bras. (1992) ; 54(3): 203-207, maio-jun. 2008. tab
Article in Portuguese | LILACS | ID: lil-485601


OBJETIVO: Avaliar e relacionar fatores morfológicos e moleculares de câncer de mama preditivos de metástases em linfonodos axilares. MÉTODOS: Selecionamos 123 casos de carcinomas mamários invasores subdivididos em três grupos de acordo com o status axilar (pacientes com macrometástases, com micrometástases e linfonodo-negativas). Avaliamos e correlacionamos a presença de metástases axilares com fatores morfológicos (tamanho do tumor, tipo e grau histológicos, invasão linfática e sangüínea em lâminas coradas pela hematoxilina e eosina) e moleculares do tumor primário (receptores de estrógeno e progesterona, Ki67, p53, E-caderina, Her2, e invasão linfática e sangüínea em lâminas coradas pela imunoistoquímica, para D2-40 e CD31). RESULTADOS: A ocorrência de metástases axilares esteve positivamente relacionada à embolização neoplásica em vasos linfáticos em lâminas coradas pela hematoxilina e eosina (HE), quando analisamos os casos com metástases e sem metástases (p=0,04), e, quando eles eram analisados em três subgrupos (p=0,002). Também identificamos relação positiva e estatisticamente significativa entre a presença de metástases axilares e invasão de vasos sangüíneos em lâminas coradas pelo CD31 (p=0,02). As demais variáveis moleculares e morfológicas não mostraram relação estatisticamente significativa com a presença de metástases. CONCLUSÃO: A invasão neoplásica em vasos linfáticos e sangüíneos identificadas em cortes histológicos corados pela HE e por marcadores imunoistoquímicos relaciona-se positivamente com a ocorrência de metástases, e é preditivo de metástases em linfonodos axilares em câncer de mama.

OBJECTIVE: The aim of our study was to analyze morphologic and molecular markers of breast cancer relating them to the presence of metastases in axillary lymph nodes. METHODS: We selected 123 cases of invasive mammary carcinomas stratified into three subgroups: with macrometastases, with micrometastases, and lymph node negative. Presence of metastases was evaluated relating them with morphologic factors (size of primary tumor, type and grade, presence of lymphatic and blood vessel invasion in hematoxylin and eosin-stained slides) and molecular factors of primary tumor (estrogen and progesterone receptors, E-cadherin, Ki67, p53, Her2 expression, and the presence of lymphatic and blood vessel invasion in immunostained sections for D2-40 and CD31). RESULTS: Axillary lymph node metastases were positively related to the presence of lymphatic vessel invasion in hematoxylin and eosin (H&E)-stained slides, when analyzed with or without metastases (p=0.04) and when analyzed in the three subgroups (p=0.002). Lymph node metastases were also positively related to presence of blood vessel invasion identified by immunohistochemistry (IHC) for CD31 (p=0.02). However other morphologic and molecular factors were not related to the presence of axillary node metastases. CONCLUSION: Lymphatic and blood vessel invasion identified in H&E and IHC-stained slides are positively related to the rmetastatic status of axillary lymph nodes and are predictive of axillary lymph node metastases in breast cancer.

Adult , Aged , Aged, 80 and over , Female , Humans , Middle Aged , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/secondary , Lymph Nodes/pathology , Lymphatic Vessels/pathology , Biomarkers, Tumor/analysis , Axilla , Antibodies, Monoclonal/analysis , /analysis , Blood Vessels/pathology , Breast Neoplasms/chemistry , Epidemiologic Methods , Neoplasm Invasiveness , Prognosis
Arq. bras. oftalmol ; 70(6): 953-960, nov.-dez. 2007. ilus, graf, tab
Article in Portuguese | LILACS | ID: lil-474101


OBJETIVO: Avaliar a cinética celular do epitélio corneano de coelhas em três situações: controle, hiperproliferação e hipoproliferação celular, com a utilização dos marcadores de proliferação celular BrdU, Ki-67/MIB-1 e AgNOR. MÉTODOS: Foram utilizadas quinze coelhas albinas que tiveram seus olhos aleatoriamente divididos em 3 grupos (A, B e C). O grupo A incluiu olhos que foram submetidos à instilação de tampão fosfato (total de 10 olhos); o grupo B, instilação de tampão fosfato após a remoção de uma área central do epitélio corneano de 10 mm (total de 10 olhos) e o grupo C, instilação de 5-fluoruracil em superfície ocular íntegra (total de 10 olhos). RESULTADOS: Os resultados da média e desvio-padrão do número de células marcadas pela BrdU nos grupos A, B e C foram, respectivamente, de 7,17 ± 0,74; 35,00 ± 3,01 e 0,22 ± 0,1 células marcadas por 100 células basais. As diferenças entre os grupos foram estatisticamente significantes. A média e o desvio padrão do número de células marcadas utilizando o Ki-67 foram de 7,55 ± 1,22 no grupo A; 35,55 ± 3,84 no grupo B e 0,34 ± 0,14 no grupo C. As diferenças entre os grupos foram estatisticamente significantes. A média e o desvio-padrão da medida da área das NORs no grupo A foram de 1,92 ± 0,24, no grupo B foram de 3,61 ± 0,27 e no grupo C foram de 1,71 ± 0,26. CONCLUSÕES: Os marcadores BrdU, Ki-67 e AgNOR apresentaram uma correlação positiva e estatisticamente significante nas situações de proliferação celular avaliadas (controle, hiperproliferação e hipoproliferação); o emprego do AgNOR não permitiu identificar diferenças na proliferação celular nas situações controle e hipoproliferação e houve maior concordância de resultados entre a BrdU e o Ki-67 nas três situações de proliferação celular.

PURPOSE: In order to maintain its clear and uniform structure, the corneal epithelium needs constant equilibrium between production (division) and desquamation of its epithelial cells. The author aimed to evaluate the cell kinetics of corneal epithelium of rabbits in three situations (control, hypoproliferation and hyperproliferation) using BrdU, Ki-67/MIB-1 and AgNORs proliferation markers. METHODS: Fifteen white female rabbits had their eyes randomly divided into three groups (A, B and. C). Group A included eyes submitted to phosphate buffer saline instillation (total 10 eyes); group B, instillation of phosphate buffer saline after removing a 10 mm central area of the corneal epithelium (total 10 eyes) and group C, instillation of 5-fluorouracil in normal epithelium (total 10 eyes). RESULTS: The results of the mean number and standard deviation of the marked cells using BrdU in groups A, B e C were, respectively, 7.17 ± 0.74; 35.00 ± 3.01 e 0.22 ± 0.1 marked cells per 100 basal cells. Differences among groups were statistically significant. The mean number and standard deviation of the labelled cells using Ki-67 were 7.55 ± 1.22 in group A; 35.55 ± 3.84 in group B and 0.34 ± 0.14 in group C. Differences among groups were statistically significant. The mean area and standard deviation of NORs in group A were 1.92 ± 0.24, in group B, 3.61 ± 0.27 and in group C, 1.71 ± 0.26. CONCLUSIONS: The markers BrdU, Ki-67 and AgNOR showed a positive correlation with statistical significance among the cellular proliferation situations studied (control, hypoproliferation and hyperproliferation); the AgNOR did not show statistically significant differences among the control and hypoproliferation situations and there was more agreement in the results among markers BrdU and Ki-67 in three cell proliferation situations.

Animals , Female , Rabbits , Cell Division/physiology , Cornea/cytology , Epithelial Cells/metabolism , Antibodies, Antinuclear/analysis , Antibodies, Monoclonal/analysis , Biomarkers/analysis , Bromodeoxyuridine/analysis , Cell Division/drug effects , Cell Proliferation/drug effects , Cornea/drug effects , Cornea/injuries , Cornea/metabolism , Epithelial Cells/drug effects , Fluorouracil/administration & dosage , Kinetics , /analysis , Nucleolus Organizer Region/chemistry , Phosphates/administration & dosage , Random Allocation , Silver Staining , Statistics, Nonparametric , Sodium Chloride/administration & dosage
Article in English | WPRIM | ID: wpr-219746


Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.

Alpha-Amanitin/pharmacology , Animals , Antibodies, Monoclonal/analysis , Antibodies, Protozoan/analysis , Dactinomycin/pharmacology , Fluorescent Antibody Technique, Direct , Gene Expression/physiology , Green Fluorescent Proteins/genetics , HeLa Cells , Humans , Mice , Mice, Inbred BALB C , Nucleic Acid Synthesis Inhibitors/pharmacology , Nucleolus Organizer Region/drug effects , Pol1 Transcription Initiation Complex Proteins/metabolism , Protein Sorting Signals/physiology , Protozoan Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Toxoplasma/physiology , Transfection