Analysis of cytokine profile and growth factors in platelet-rich plasma obtained by open systems and commercial columns.

OBJECTIVE:: To evaluate growth factors and cytokines in samples of platelet-rich plasma obtained by three different centrifugation methods. METHODS:: Peripheral blood of six individuals with no hematological diseases, aged 18 to 68 years, was drawn to obtain platelet-rich plasma, using the open method and commercial columns by Medtronic and Biomet. The products obtained with the different types of centrifugation were submitted to laboratory analysis, including pro-inflammatory cytokines and chemokines by flow cytometry assays, the concentration of fibroblast growth factors-2 (FGF-2) and transforming growth factor-beta1 (TGF-ß1). RESULTS:: The diverse separation methods generated systematically different profiles regarding number of platelets and leukocytes. The Medtronic system yielded a product with the highest concentration of platelets, and the open method, with the lowest concentration of platelets. The results of cytokine analysis showed that the different types of centrifugation yielded products with high concentrations of interleukin 8, interleukin 1ß. The open system resulted in a product with high levels of interleukin 6. Other cytokines and chemokines measured were similar between systems. The product obtained with the open method showed higher levels of TGF-ß1 in relation to other systems and low FGF-2 levels. CONCLUSION:: The formed elements, growth factors and cytokines in samples of platelet-rich plasma varied according to the centrifugation technique used. OBJETIVO:: Avaliar fatores de crescimento e citocinas em amostras de plasma rico em plaquetas obtidas por três diferentes métodos de centrifugação. MÉTODOS:: Foi coletado sangue periférico de seis indivíduos, sem doença hematológica, com idades entre 18 e 68 anos, para obtenção de plasma rico em plaquetas, utilizando o método aberto e sistemas comerciais das empresas Medtronic e Biomet. Os produtos obtidos com os diferentes tipos de centrifugação foram submetidos às análises laboratoriais, incluindo citocinas próinflamatórias e quimiocinas, por meio de ensaios de citometria de fluxo, concentração do fator de crescimento fibroblástico-2 (FGF-2) e fator de crescimento transformador-beta1 (TGF-ß1). RESULTADOS:: As diferentes centrifugações geraram perfis sistematicamente diferentes referentes ao número de plaquetas e de leucócitos. O sistema da Medtronic originou produto com a maior concentração de plaquetas, e o método aberto com a menor concentração de plaquetas. Os resultados da análise de citocinas demonstraram que os diferentes tipos de centrifugação originaram produtos com elevadas concentrações de interleucina 8 e interleucina 1ß. O sistema aberto resultou em produto com elevados níveis de interleucina 6. As demais citocinas e quimiocinas mensuradas foram similares entre os sistemas. O produto obtido com o método aberto apresentou níveis superiores de TGF-ß1 em relação aos demais sistemas e reduzidos níveis de FGF-2. CONCLUSÃO:: Os elementos figurados, fatores de crescimento e citocinas, em amostras de plasma rico em plaquetas, variaram conforme a técnica de centrifugação utilizada.

Analysis of cytokine profile and growth factors in platelet-rich plasma obtained by open systems and commercial columns / Análise do perfil de citocinas e fatores de crescimento em plasma rico em plaquetas obtido por meio das metodologias do sistema aberto e colunas

ABSTRACT Objective: To evaluate growth factors and cytokines in samples of platelet-rich plasma obtained by three different centrifugation methods. Methods: Peripheral blood of six individuals with no hematological diseases, aged 18 to 68 years, was drawn to obtain platelet-rich plasma, using the open method and commercial columns by Medtronic and Biomet. The products obtained with the different types of centrifugation were submitted to laboratory analysis, including pro-inflammatory cytokines and chemokines by flow cytometry assays, the concentration of fibroblast growth factors-2 (FGF-2) and transforming growth factor-beta1 (TGF-β1). Results: The diverse separation methods generated systematically different profiles regarding number of platelets and leukocytes. The Medtronic system yielded a product with the highest concentration of platelets, and the open method, with the lowest concentration of platelets. The results of cytokine analysis showed that the different types of centrifugation yielded products with high concentrations of interleukin 8, interleukin 1β. The open system resulted in a product with high levels of interleukin 6. Other cytokines and chemokines measured were similar between systems. The product obtained with the open method showed higher levels of TGF-β1 in relation to other systems and low FGF-2 levels. Conclusion: The formed elements, growth factors and cytokines in samples of platelet-rich plasma varied according to the centrifugation technique used.

RESUMO Objetivo: Avaliar fatores de crescimento e citocinas em amostras de plasma rico em plaquetas obtidas por três diferentes métodos de centrifugação. Métodos: Foi coletado sangue periférico de seis indivíduos, sem doença hematológica, com idades entre 18 e 68 anos, para obtenção de plasma rico em plaquetas, utilizando o método aberto e sistemas comerciais das empresas Medtronic e Biomet. Os produtos obtidos com os diferentes tipos de centrifugação foram submetidos às análises laboratoriais, incluindo citocinas próinflamatórias e quimiocinas, por meio de ensaios de citometria de fluxo, concentração do fator de crescimento fibroblástico-2 (FGF-2) e fator de crescimento transformador-beta1 (TGF-β1). Resultados: As diferentes centrifugações geraram perfis sistematicamente diferentes referentes ao número de plaquetas e de leucócitos. O sistema da Medtronic originou produto com a maior concentração de plaquetas, e o método aberto com a menor concentração de plaquetas. Os resultados da análise de citocinas demonstraram que os diferentes tipos de centrifugação originaram produtos com elevadas concentrações de interleucina 8 e interleucina 1β. O sistema aberto resultou em produto com elevados níveis de interleucina 6. As demais citocinas e quimiocinas mensuradas foram similares entre os sistemas. O produto obtido com o método aberto apresentou níveis superiores de TGF-β1 em relação aos demais sistemas e reduzidos níveis de FGF-2. Conclusão: Os elementos figurados, fatores de crescimento e citocinas, em amostras de plasma rico em plaquetas, variaram conforme a técnica de centrifugação utilizada.

The liver plays a major role in clearance and destruction of blood trypomastigotes in Trypanosoma cruzi chronically infected mice.

Intravenous challenge with Trypanosoma cruzi can be used to investigate the process and consequences of blood parasite clearance in experimental Chagas disease. One hour after intravenous challenge of chronically infected mice with 5x10(6) trypomastigotes, the liver constituted a major site of parasite accumulation, as revealed by PCR. Intact parasites and/or parasite remnants were visualized at this time point scattered in the liver parenchyma. Moreover, at this time, many of liver-cleared parasites were viable, as estimated by the frequency of positive cultures, which considerably diminished after 48 h. Following clearance, the number of infiltrating cells in the hepatic tissue notably increased: initially (at 24 h) as diffuse infiltrates affecting the whole parenchyma, and at 48 h, in the form of large focal infiltrates in both the parenchyma and perivascular spaces. Phenotypic characterization of liver-infiltrating cells 24 h after challenge revealed an increase in Mac1(+), CD8(+) and CD4(+) cells, followed by natural killer (NK) cells. As evidence that liver-infiltrating CD4(+) and CD8(+) cells were activated, increased frequencies of CD69(+)CD8(+), CD69(+)CD4(+) and CD25(+)CD122(+)CD4(+) cells were observed at 24 and 48 h after challenge, and of CD25(-)CD122(+)CD4(+) cells at 48 h. The major role of CD4(+) cells in liver protection was suggested by data showing a very high frequency of interferon (IFN)-gamma-producing CD4(+) cells 24 h after challenge. In contrast, liver CD8(+) cells produced little IFN-gamma, even though they showed an enhanced potential for secreting this cytokine, as revealed by in vitro T cell receptor (TCR) stimulation. Confirming the effectiveness of the liver immune response in blood parasite control during the chronic phase of infection, no live parasites were detected in this organ 7 days after challenge.