Obtenção e caracterização de anticorpo monoclonal murino anti-fator VIII da coagulação sangüínea Attainment and characterization of murine monoclonal anti-factor VIII antibodies

Entre os avanços da engenharia celular e biotecnologia nas últimas décadas, destaca-se a produção de anticorpos monoclonais murinos (AcMm) utilizados no aprimoramento diagnóstico nas rotinas laboratoriais. A produção de fator VIII de alta pureza sempre foi o desejo e a preocupação das indústrias de hemoderivados para tratamento de pacientes portadores de hemofilia A, porém este produto inexiste no Brasil, sendo necessária sua obtenção no mercado internacional a custos elevados. O trabalho tem por objetivo a produção de AcMm anti-fator VIII humano (FVIII H) através da expansão dos clones e caracterização imunoquímica do anticorpo. Camundongos Balb/c foram imunizados com FVIII H purificado como também proveniente de crioprecipitado e as células esplênicas dos animais foram fusionadas com células mielomatosas murinas segundo o método descrito por Kohler e Milstein para produção de híbridos em cultura. Foram testados 1.983 híbridos dos quais 105 foram submetidos à clonagem. Destes, 39 obtiveram monoclonalidade e 7 destes clones foram caracterizados através de técnicas de immunoblotting. Foram submetidas à purificação por cromatografia três imunoglobulinas de diferentes classes pertencentes aos clones LAMB1-10A1A4, LAMB1-17A1A1 e LAMB1-24A2A1. A imunoglobulina purificada pertencente ao clone LAMB1-10A1A4 foi adsorvida em coluna de imunoafinidade para purificação de concentrado de FVIII proveniente de crioprecipitado plasmático.<br>Among the advances in cellular engineering and biotechnology over the last decades, the production of murine monoclonal antibodies (AcMm), used to improve laboratory diagnoses, stands out. The production of very pure factor VIII has always been a concern of suppliers of blood products to treat patients with hemophilia A and this product is still not produced in Brazil. Hence, it can only be attained on the international market at a high cost. The aim of this work was to produce AcMm anti-factor VIII human (FVIIIh) by expanding clones and characterizing the antibodies by immunochemistry. Balb/c mice were immunized with FVIII both purified and from cryoprecipitation and the splenic cells of the animals were fused with myelomatous murine cells according to the method described by Kohler and Milstein to produce hybrids in a culture. A total of 1983 hybrids were tested and 105 were selected for cloning. Of these, 39 developed monoclonality and 7 of these clones were characterized through immunoblotting techniques. Three immunoglobulins from different classes, LAMB1-10A1A4, LAMB1-17A1A1 and LAMB1-24A2A1, were submitted to chromatography for purification. The purified immunoglobulin from the LAMB1-10A1A4 clone was adsorbed in the immunoaffinity column to purify the factor VIII concentrate coming from the plasmatic cryoprecipitate

Treatment of osteochondral injuries with platelet gel

OBJECTIVES: Treatments for injured articular cartilage have not advanced to the point that efficient regeneration is possible. However, there has been an increase in the use of platelet-rich plasma for the treatment of several orthopedic disorders, including chondral injuries. Our hypothesis is that the treatment of chondral injuries with platelet gel results in higher-quality repair tissue after 180 days compared with chondral injuries not treated with gel. METHODS: A controlled experimental laboratory study was performed on 30 male rabbits to evaluate osteochondral injury repair after treatment with or without platelet gel. Osteochondral injuries were surgically induced in both knees of each rabbit at the medial femoral condyle. The left knee injury was filled with the platelet gel, and the right knee was not treated. Microscopic analysis of both knee samples was performed after 180 days using a histological grading scale. RESULTS: The only histological evaluation criterion that was not significantly different between treatments was metachromasia. The group that was treated with platelet gel exhibited superior results in all other criteria (cell morphology, surface regularity, chondral thickness and repair tissue integration) and in the total score. CONCLUSION: The repair tissue was histologically superior after 180 days in the study group treated with platelet gel compared with the group of untreated injuries

Finasteride Inhibits Human Prostate Cancer Cell Invasion through MMP2 and MMP9 Downregulation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Introduction: The use of the 5-alpha reductase inhibitors (5-ARIs) finasteride and dutasteride for prostate cancer prevention is still under debate. The FDA recently concluded that the increased prevalence of high-grade tumors among 5-ARI-treated patients must not be neglected, and they decided to disallow the use of 5-ARIs for prostate cancer prevention. This study was conducted to verify the effects of finasteride on prostate cell migration and invasion and the related enzymes/proteins in normal human and tumoral prostatic cell lines. Materials and Methods: RWPE-1, LNCaP, PC3 and DU145 cells were cultivated to 60% confluence and exposed for different periods to either 10 mu M or 50 mu M finasteride that was diluted in culture medium. The conditioned media were collected and concentrated, and MMP2 and MMP9 activities and TIMP-1 and TIMP-2 protein expression were determined. Cell viability, migration and invasion were analyzed, and the remaining cell extracts were submitted to androgen receptor (AR) detection by western blotting techniques. Experiments were carried out in triplicate. Results: Cell viability was not significantly affected by finasteride exposure. Finasteride significantly downregulated MMP2 and MMP9 activities in RWPE-1 and PC3 cells and MMP2 in DU145 cells. TIMP-2 expression in RWPE-1 cells was upregulated after exposure. The cell invasion of all four tested cell lines was inhibited by exposure to 50 mM of finasteride, and migration inhibition only occurred for RWPE-1 and LNCaP cells. AR was expressed by LNCaP, RWPE-1 and PC3 cells. Conclusions: Although the debate on the higher incidence of high-grade prostate cancer among 5-ARI-treated patients remains, our findings indicate that finasteride may attenuate tumor aggressiveness and invasion, which could vary depending on the androgen responsiveness of a patient's prostate cells.812Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FAPESP [2010/16671-3