SERGIO PAULO BYDLOWSKI
Projetos de Pesquisa
Unidades Organizacionais
Departamento de Clínica Médica, Faculdade de Medicina - Docente
LIM/19 - Laboratório de Histocompatibilidade e Imunidade Celular, Hospital das Clínicas, Faculdade de Medicina - Líder
LIM/19 - Laboratório de Histocompatibilidade e Imunidade Celular, Hospital das Clínicas, Faculdade de Medicina - Líder
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conferenceObject Effects of Human Amniotic Fluid Stem Cells in a Model of Aorta Allograft Vasculopathy(2012) SANTANA, A. C.; DELLE, H.; CAVAGLIERI, R. C.; LOPES, M. A. B.; FRANCISCO, R. P. V.; ZUGAIB, M.; BYDLOWSKI, S. P.; NORONHA, I. L.Chronic allograft vasculopathy (CAV) is an important cause of graft loss. Considering the immune-in flammatory events involved in the development of CAV, therapeutic approaches to target this process are of relevance. Human amniotic fluid derived stem cells (hAFSC), a class of fetal, pluripotent stem cells with intermediate characteristics between embryonic and adult stem cells display immunomodulatory properties. hAFSC express mesenchymal and embryonic markers, show high proliferation rates, but do not induce tumor formation and their use does not raise ethical issues. Thus, we sought to investigate the effect of hAFSC on CAV in a model of aorta transplantation. Orthotopic aorta transplantation was performed using Fisher (F344) rats as donors and Lewis rats as recipients. Rats were divided into 3 groups: syngeneic (SYNG), untreated F344 receiving aorta from F344 (n=8); allogeneic (ALLO), Lewis rats receiving allogeneic aorta from F344 (n=8); and ALLO+hAFSC, ALLO rats treated with hAFSC (106 cells) (n=8). Histological analysis and immunohistochemistry were performed 30 days post transplantation. ALLO developed a robust aortic neointimal formation, accompanied by a high number of ED1+ and CD43+ cells, and enhanced expression of α-SMA in theneointima. Treatment with hAFSC diminished neointimal thickness and induced a significant decrease of ED1+, CD43+ cells and α-SMA expression in the neointima. Comparative analyses in the differents groups PARAMETERS SYNG ALLO ALLO+hAFSC Neointima thickness (μm) 0±0 208.7±25.4* 180.7±23.7* ED-1+ (cells/mm2) 0±0 4.845±841* 1.100±276*, #CD43+ (cells/mm2) 0±0 4.064±563* 1.080±309*,#α-SMA (%) 0±0 25±6* 8±3*, #*p<0.05 vs. SYNG; #P<0.05 vs. ALLO These preliminary results showed that hAFSC suppressed inflammation and myofibroblast migration to the intima, which may contribute to ameliorate vascular changes in CAV.- Evaluation of Distinct Freezing Methods and Cryoprotectants for Human Amniotic Fluid Stem Cells Cryopreservation(2012) JANZ, Felipe de Lara; DEBES, Adriana de Aguiar; CAVAGLIERI, Rita de Cassia; DUARTE, Sergio Aloisio; ROMAO, Carolina Martinez; MORON, Antonio Fernandes; ZUGAIB, Marcelo; BYDLOWSKI, Sergio PauloAmniotic fluid (AF) was described as a potential source of mesenchymal stem cells (MSCs) for biomedicine purposes. Therefore, evaluation of alternative cryoprotectants and freezing protocols capable to maintain the viability and stemness of these cells after cooling is still needed. AF stem cells (AFSCs) were tested for different freezing methods and cryoprotectants. Cell viability, gene expression, surface markers, and plasticity were evaluated after thawing. AFSCs expressed undifferentiated genes Oct4 and Nanog; presented typical markers (CD29, CD44, CD90, and CD105) and were able to differentiate into mesenchymal lineages. All tested cryoprotectants preserved the features of AFSCs however, variations in cell viability were observed. In this concern, dimethyl sulfoxide (Me2SO) showed the best results. The freezing protocols tested did not promote significant changes in the AFSCs viability. Time programmed and nonprogrammed freezing methods could be used for successful AFSCs cryopreservation for 6 months. Although tested cryoprotectants maintained undifferentiated gene expression, typical markers, and plasticity of AFSCs, only Me2SO and glycerol presented workable viability ratios.
- 7-Ketocholesterol overcomes drug resistance in chronic myeloid leukemia cell lines beyond MDRI mechanism(2017) FERNANDES, Livia Rosa; STERN, Ana Carolina Bassi; CAVAGLIERI, Rita de Cassia; NOGUEIRA, Fabio Cesar Sousa; DOMONT, Gilberto; PALMISANO, Giuseppe; BYDLOWSKI, Sergio PauloChronic myeloid leukemia (CML) is a myeloproliferative disease with a characteristic BCR-ABL tyrosine kinase (TK) fusion protein. Despite the clinical efficacy accomplished by TKIs therapies, disease progression may affect patient response rate to these inhibitors due to a multitude of factors that could lead to development of a mechanism known as multidrug resistance (MDR). 7-Ketocholesterol (7KC) is an oxidized cholesterol derivative that has been extensively reported to cause cell death in a variety of cancer models. In this study, we showed the in vitro efficacy of 7KC against MDR leukemia cell line, Lucena. 7KC treatment induced reduction in cell viability, together with apoptosis-mediated cell death. Moreover, downregulation of MDR protein caused intracellular drug accumulation and 7KC co-incubation with either Daunorubicin or Vincristine reduced cell viability compared to the use of each drug alone. Additionally, quantitative label-free mass spectrometry-based protein quantification showed alteration of different molecular pathways involved in cell cycle arrest, induction of apoptosis and misfolded protein response. Conclusively, this study highlights the effect of 7KC as a sensitizing agent of multi drug resistance CML and elucidates its molecular mechanisms. Significance: CML patients treated with tyrosine kinase inhibitors (TKIs) have showed a 5-year estimated overall survival of 89%, with cumulative complete cytogenetic response of 87%. However, development of drug resistance is a common feature of the disease progression. This study aimed at showing the effect of 7KC as a cytotoxic and sensitizing agent of multidrug resistance CML cell lines. The cellular and molecular basis of this compound were elucidated using a comprehensive strategy based on quantitative proteomic and cell biology assays. We showed that 7KC induced cell death and overcomes drug resistance in CML through mechanisms that go beyond the classical MDR1 pathways.
- The potential use of stem cells derived from human amniotic fluid in renal diseases(2011) NORONHA, Irene L.; CAVAGLIERI, Rita C.; JANZ, Felipe L.; DUARTE, Sergio A.; LOPES, Marco A. B.; ZUGAIB, Marcelo; BYDLOWSKI, Sergio P.Amniotic fluid (AF) contains a variety of cell types derived from fetal tissues that can easily grow in culture. These cells can be obtained during amniocentesis for prenatal screening of fetal genetic diseases, usually performed during the second trimester of pregnancy. Of particular interest, some expanded sub-populations derived from AF cells are capable of extensive self-renewal and maintain prolonged undifferentiated proliferation, which are defining properties of stem cells. These human AF stem cells (hAFSCs) exhibit multilineage potential and can differentiate into the three germ layers. They have high proliferation rates and express mesenchymal and embryonic markers, but do not induce tumor formation. In this study, hAFSCs derived from amniocentesis performed at 16-20 weeks of pregnancy were isolated, grown in culture, and characterized by flow cytometry and by their potential ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages. After 4-7 passages, 5 x 10(5) hAFSCs were inoculated under the kidney capsule of Wistar rats that were subjected to an experimental model of chronic renal disease, the 5/6 nephrectomy model (Nx). After 30 days, Nx rats treated with hAFSCs displayed significant reductions in blood pressure, proteinuria, macrophages, and a-smooth muscle actin expression compared with Nx animals. These preliminary results suggest that hAFSCs isolated and expanded from AF obtained by routine amniocentesis can promote renoprotection in the Nx model. Considering that the AF cells not used for fetal karyotyping are usually discarded, and that their use does not raise ethical issues, they may represent an alternative source of stem cells for cell therapy and regenerative medicine.
conferenceObject Human Amniotic Fluid Stem Cells Reduce Neointimal Formation and Inflammation in a Model of Aortic Allograft Vasculopathy(2012) SANTANA, A. C.; CAVAGLIERI, R. C.; DELLE, H.; LOPES, M. A. B.; V, R. P. Francisco; ZUGAIB, M.; BYDLOWSKI, S. P.; NORONHA, I. L.- Protective Effects of Human Amniotic Fluid Stem Cells in a Model of Aorta Allograft Vasculopathy in Rats(2012) SANTANA, A. C.; DELLE, H.; CAVAGLIERI, R. C.; LOPES, M. A. B.; FRANCISCO, R. P. V.; ZUGAIB, M.; BYDLOWSKI, S. P.; NORONHA, I. L.Background. Chronic allograft vasculopathy (CAV) is an important cause of graft loss. Considering the immune inflammatory events involved in the development of CAV, therapeutic approaches to target this process are of relevance. Human amniotic fluid derived stem cells (hAFSCs), a class of fetal, pluripotent stem cells with intermediate characteristics between embryonic and adult stem cells, display immunomodulatory properties. hAFSCs express mesenchymal and embryonic markers, show high proliferation rates; however, they do not induce tumor formation, and their use does not raise ethical issues. Thus, we sought to investigate the effect of hAFSC on CAV in a model of aorta transplantation. Methods. Orthotopic aorta transplantation was performed using Fisher (F344) rats as donors and Lewis rats as recipients. Rats were divided into three groups: syngeneic (SYNG), untreated F344 receiving aorta from F344 (n = 8); allogeneic (ALLO), Lewis rats receiving allogeneic aorta from F344 (n = 8); and ALLO + hAFSC, ALLO rats treated with hAFSC (10(6) cells; n = 8). Histological analysis and immunohistochemistry were performed 30 days posttransplantation. Results. The ALLO group developed a robust aortic neointimal formation (208.7 +/- 25.4 gm) accompanied by a significant high number of ED1(+) (4845 +/- 841 cells/mm(2)) and CD43(+) cells (4064 +/- 563 cells/mm(2)), and enhanced expression of a-smooth muscle actin in the neointima (25 +/- 6%). Treatment with hAFSC diminished neointimal thickness (180.7 +/- 23.7 mu m) and induced a significant decrease of ED1(+) (1100 +/- 276 cells/mm(2)), CD43(+) cells (1080 +/- 309 cells/mu m(2)), and alpha-smooth muscle actin expression 8 +/- 3% in the neointima. Conclusions. These preliminary results showed that hAFSC suppressed inflammation and myofibroblast migration to the intima, which may contribute to ameliorate vascular changes in CAV.