Identification, Selection, and Enrichment of Cardiomyocyte Precursors

The large-scale production of cardiomyocytes is a key step in the development of cell therapy and tissue engineering to treat cardiovascular diseases, particularly those caused by ischemia. the main objective of this study was to establish a procedure for the efficient production of cardiomyocytes by reprogramming mesenchymal stem cells from adipose tissue. First, lentiviral vectors expressing neoR and GFP under the control of promoters expressed specifically during cardiomyogenesis were constructed to monitor cell reprogramming into precardiomyocytes and to select cells for amplification and characterization. Cellular reprogramming was performed using 5'-azacytidine followed by electroporation with plasmid pOKS2a, which expressed Oct4, Sox2, and Klf4. Under these conditions, GFP expression began only after transfection with pOKS2a, and less than 0.015% of cells were GFP(+). These GFP(+) cells were selected for G418 resistance to find molecular markers of cardiomyocytes by RT-PCR and immunocytochemistry. Both genetic and protein markers of cardiomyocytes were present in the selected cells, with some variations among them. Cell doubling time did not change after selection. Together, these results indicate that enrichment with vectors expressing GFP and neoR under cardiomyocyte-specific promoters can produce large numbers of cardiomyocyte precursors (CMPs), which can then be differentiated terminally for cell therapy and tissue engineering.Universidade Federal de São Paulo, Gene Therapy Invest Ctr, Dept Biophys, BR-04044010 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Surg, BR-04044010 São Paulo, BrazilUniversidade Federal de São Paulo, Gene Therapy Invest Ctr, Dept Biophys, BR-04044010 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Surg, BR-04044010 São Paulo, BrazilWeb of Scienc

Zika Virus Outbreak - Should assisted reproduction patients avoid pregnancy?

Objective: To discuss the requirement from the National Health Surveillance Agency(ANVISA), for assisted reproduction treatment patients to undergo laboratory tests for ZIKV detection, and if the public health authorities and government leaders' recommendations to women simply avoid pregnancy is prudent. Methods: This study was performed in a universityaffiliated in vitro fertilization center in Brazil. We present a critical discussion on the risk of microcephaly due to ZIKV infection and the prevalence of other harmful pathogens to vulnerable pregnant women and infants. We assessed, 954 patients undergoing intracytoplasmic sperm injection cycles(ICSI), between April and November of 2016, concerning the results of ZIKV test, according to different regions in Brazil. Results: Patients undergoing ICSI cycles were split into groups, according to their region of origin: 28(3.0%) were from the North, 27(2.8%) were from the Northeast, 40(4.2%) were from the Midwest, 830(87.2%) were from the Southeast, and 29(3.0%) were from the South. Concerning the diagnosis, 112 samples had a positive or inconclusive result for ZIKV, by chromatography immunoassay. These samples were re-analyzed by ELISA and no result was positive. All positive results were from the Southeast region and none from the Northeast or Midwest regions, which are considered endemic regions. Conclusion: ZIKV test before the onset of assisted reproduction treatments does not rule out the risk of the infection during pregnancy. In addition, although ZIKV infection risk is extremely high, the microcephaly risk due to ZIKV is not higher than the risk of miscarriage and birth defects due to other recognized pathogens.Fertil Med Grp, Sao Paulo, SP, BrazilCtr Estudos & Pesquisa Reprod Humana Assistida, Inst Sapientiae, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, Dept Cirurgia, Area Reprod Humana, Disciplina Urol, Sao Paulo, BrazilUniv Fed Sao Paulo UNIFESP, Dept Cirurgia, Area Reprod Humana, Disciplina Urol, Sao Paulo, BrazilWeb of Scienc

Efeito De Citocinas E Toxina Tetânica Na Vacinação Gênica De Tumores Que Expressam Cea Com Scfv6C4

Introduction: Colon and rectum cancers are highly prevalent among men and women, and prevention and treatment are major medical and scientific challenges. Carcinoembryonic antigen (CEA) is the main tumor associated antigen of these cancers. Previously, our group developed a DNA vaccine against CEA-expressing tumors using a CEA surrogate, scFv6.C4, and its efficacy, evaluated in transgenic mice for CEA, showed 40% tumor-free animais by more than 100 days and in the others the survival increase was between 30 and 70% in relation to the non-vaccinated group. Objective: To evaluate the adjuvant effect of IFNy (Interferon Gamma), GM-CSF (Granulocyte Macrophage Colony-Stimulating Factor), FrC (Fragment C of Tetanus Toxin) and IDUA (Alpha-L-Iduronidase) in gene vaccination with scFv6.C4. Methods: C57BU6J-CEA2682 mice were immunized 4 times by intramuscular electroporation with the plasmid uP-PS/scFv6.C4 alone, or in combination with adjuvant vectors expressing FrC, GM-CSF, IFNy or IDUA. Vaccinated animais were challenged by subcutaneous injection of murine colon adenocarcinoma cells, MC38-CEA, and tumor growth was monitored. The humoral andcellular immune responses were accessed by ELlSA, immunocytochemistry, ELlSPOT, cell proliferation and cYt~toxicity assays. Results: Immunization with scFv6.C4 induced anti-CEA antibodies, with titre about 4- fold higher than preimmune serum. When challenged with MC38-CEA cells, approximately half of the immunized animais did not develop tumor during 80 days of observation, and the others had varying degrees of retardation in tumor growth. The adjuvants tested did not lead to a significant increase in antibody titer, however, animais immunized with scFv6.C4 and FrC or IFNy had increased survival. Cellular response assays showed a significant increase in cytotoxic cell response, especially in the animais vaccinated with FrC. Conclusions: Immunization with scFv6.C4 in combination with adjuvants FrC or IFNy elevated antitumor effect via increased cytotoxic cell response.Introdução: Cânceres do cólon e reto têm alta incidência entre homens e mulheres e a prevenção e o tratamento são grandes desafios médicos e científicos. O antígeno carcinoembrionário (CEA) é o principal marcador tumoral destes cânceres. Previamente, o nosso grupo desenvolveu uma vacina de DNA contra tumores que expressam CEA, usando um mimético a CEA, a scFv6.C4, e sua eficácia, avaliada em camundongos transgênicos para CEA, mostrou 40 % de animais livre de tumor por mais de 100 dias e no restante o aumento de sobrevida foi entre 30 e 70% em relação ao grupo não vacinado. Objetivo: Avaliar o efeito adjuvante do IFNy (Interferon Gama), GM-CSF (Fator Estimulador da Colônias de Macrófagos e Granulócitos), FrC (Fragmento C da Toxina Tetânica) e IDUA (Alpha-L-Iduronidase) na vacinação gênica com scFv6.C4. Métodos: Camundongos C57BU6J-CEA2682 foram imunizados 4 vezes por eletroporação intramuscular com o vetor plasmidial uPPS/ scFv6.C4 sozinho, ou em combinação com vetares adjuvantes que expressam FrC, GM-CSF, IFNy ou IDUA. Os animais vacinados foram desafiados pela injeção subcutânea de células de adenocarcinoma de cólon murino, MC38-CEA, e o T"" crescimento tumoral foi monitorado. As respostas imunes humoral e celular foram acessadas por ELlSA, imunocitoquímica, ELlSPOT, ensaio de proliferação celular e de citotoxicidade. Resultados: A imunização com scFv6.C4 induziu a geração de anticorpos anti-CEA, com título cerca de 4 vezes maior em relação ao soro pré-imune. Quando desafiados com as células MC38-CEA,' aproximadamente metade dos animais imunizados não desenvolveram tumor durante 80 dias de observação, e os demais apresentaram graus variados de retardo de crescimento tumoral. Os adjuvantes testados não levaram a aumento significativo no título de anticarpos, porém, os animais imunizados com scFv6.C4 e FrC ou IFNy tiveram aumento na sobrevida. Ensaios de resposta celulares mostraram um aumento significativo de resposta celular citotóxica, principalmente nos animais vacinados com o FrC. Conclusões: Imunização com scFv6.C4 em associação com adjuvantes FrC ou IFNy elevou efeito antitumoral via aumento de resposta celular citotóxica.Dados abertos - Sucupira - Teses e dissertações (2017

Reprogramming of adipose derived stem cells to the formation and enrichment of cardiomyocytes

A producao em larga escala de cardiomiocitos e um passo fundamental na terapia celular e engenharia tecidual para tratar doencas cardiovasculares, principalmente as causadas pela isquemia. Em vista disso, o objetivo principal deste trabalho foi estabelecer um procedimento eficiente para producao de cardiomiocitos atraves de reprogramacao de celulas-tronco mesenquimais de tecido adiposo. Inicialmente foram construidos vetores lentivirais contendo promotores de genes expressos especificamente em cardiomiocitos, ATP2a2, GJA1, MYH6, MYL2 e NPPA, dirigindo a expressao bicistronica dos genes neoR e GFP, a fim de permitir tanto o acompanhamento da reprogramacao em cardiomiocito quanto a selecao destas celulas para sua amplificacao e caracterizacao. A reprogramacao foi realizada com 5Æ-azacitidina, seguida por eletroporacao com o plasmideo pOKS2a, que expressa os genes Oct4, Klf4 e Sox2. Observou-se que a expressao de GFP iniciou-se com a transfeccao do pOKS2a e persistiu, na maioria dos casos, durante alguns dias. A quantificacao de celulas GFP+ ao longo da reprogramacao indica que menos de 0,015 % das celulas estavam ativadas, geralmente, nos primeiros dias apos a transfeccao. As celulas GFP+ foram selecionadas com G418 para buscar marcadores moleculares de cardiomiocitos atraves de RT-PCR e imunocitoquimica. Tanto os marcadores genicos como os marcadores proteicos caracteristicos de cardiomiocitos estavam presentes nas celulas selecionadas, com algumas variacoes entre elas. O conjunto destes resultados indica que as celulas reprogramadas tinham caracteristicas de um cardiomiocito, no entanto, a variacao morfologica e de marcadores moleculares indicam que ha necessidade de caracterizacoes adicionais para valida-las como cardiomiocitoBV UNIFESP: Teses e dissertaçõe

Trypanosoma cruzi extracellular amastigotes trigger the protein kinase D1-cortactin-actin pathway during cell invasion

Trypanosoma cruzi extracellular amastigotes (EAs) display unique mechanisms for cell invasion that are highly dependent on host actin filaments. Protein kinase D1 (PKD1) phosphorylates and modulates the activity of cortactin, a key regulator of actin dynamics. We evaluated the role of host cortactin and PKD1 in actin filament dynamics during HeLa cell invasion by EAs. Host cortactin, PKD1 and actin are recruited by EAs based on experiments in fixed and live cells by time lapse confocal microscopy. EAs trigger PKD1 and extracellular signal-regulated kinase 1/2 activation, but not Src family kinases, and selectively phosphorylate cortactin. Heat-killed EAs and non-infective epimastigotes both triggered distinct host responses and did not recruit the molecules studied herein. EA invasion was influenced by depletion or overexpression of host cortactin and PKD1, respectively, suggesting the involvement of both proteins in this event. Collectively, these results show new host cell mechanisms subverted during EA internalization into non-phagocytic cells

Role of religion, spirituality, and faith in assisted reproduction

Purpose: The purpose of this study is to evaluate the impact of the patient’s faith, religion, and spirituality on the outcomes of intracytoplasmic sperm injection (ICSI) cycles. Materials and methods: Eight hundred and seventy-seven patients received a questionnaire containing information on faith, religiosity, and spirituality and the results of the questionnaires were correlated with ICSI outcomes. Patients stated to be Catholic (n = 476), spiritists (n = 93), Evangelical (n = 118), and other religion (n = 32), and 78 did not identify with any religious group. Results: A significant increase in fertilization, high-quality embryos, and pregnancy rate was found among Spiritists and Evangelicals. Patients who included the infertility diagnosis and treatment in their prayers showed an increased pregnancy rate, and those who reported their faith to be affected by the infertility diagnosis presented a decreased high-quality embryos rate. The high-quality embryos rate was increased among patients who answered that their faith contributed to their decision to undergo infertility treatment. The cycle’s cancelation was negatively correlated with the frequency of religious meetings, and the frequency of prayers was positively correlated with the response to ovarian stimulation. Finally, belief in treatment success positively influenced the embryo quality. Conclusion: The findings suggest that spirituality plays a role in adjusting the psychological aspects of an infertile patient

CXCR3 chemokine receptor guides Trypanosoma cruzi-specific T-cells triggered by DNA/adenovirus ASP2 vaccine to heart tissue after challenge

Submitted by Sandra Infurna ([email protected]) on 2020-03-24T19:09:05Z No. of bitstreams: 1 JoselilannesVieira_etal_IOC_2019.pdf: 4012278 bytes, checksum: 37989d2af0935e8d6a3149a8a2d217f5 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2020-03-24T19:22:19Z (GMT) No. of bitstreams: 1 JoselilannesVieira_etal_IOC_2019.pdf: 4012278 bytes, checksum: 37989d2af0935e8d6a3149a8a2d217f5 (MD5)Made available in DSpace on 2020-03-24T19:22:19Z (GMT). No. of bitstreams: 1 JoselilannesVieira_etal_IOC_2019.pdf: 4012278 bytes, checksum: 37989d2af0935e8d6a3149a8a2d217f5 (MD5) Previous issue date: 2019Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Biociências. Santos, SP, Brasil.Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Biofísica. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Biofísica. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Biociências. Santos, SP, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia das Interações. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil / University of Massachusetts Medical School. Division of Infectious Diseases and Immunology. Worcester, USA.Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil / Universidade Federal de São Paulo. Departamento de Biociências. Santos, SP, Brasil.CD8+ T lymphocytes play an important role in controlling infections by intracellular pathogens. Chemokines and their receptors are crucial for the migration of CD8+ T-lymphocytes, which are the main IFNγ producers and cytotoxic effectors cells. Although the participation of chemokine ligands and receptors has been largely explored in viral infection, much less is known in infection by Trypanosoma cruzi, the causative agent of Chagas disease. After T. cruzi infection, CXCR3 chemokine receptor is highly expressed on the surface of CD8+ T-lymphocytes. Here, we hypothesized that CXCR3 is a key molecule for migration of parasite-specific CD8+ T-cells towards infected tissues, where they may play their effector activities. Using a model of induction of resistance to highly susceptible A/Sn mice using an ASP2-carrying DNA/adenovirus prime-boost strategy, we showed that CXCR3 expression was upregulated on CD8+ T-cells, which selectively migrated towards its ligands CXCL9 and CXCL10. Anti-CXCR3 administration reversed the vaccine-induced resistance to T. cruzi infection in a way associated with hampered cytotoxic activity and increased proapoptotic markers on the H2KK-restricted TEWETGQI-specific CD8+ T-cells. Furthermore, CXCR3 receptor critically guided TEWETGQI-specific effector CD8+ T-cells to the infected heart tissue that express CXCL9 and CXCL10. Overall, our study pointed CXCR3 and its ligands as key molecules to drive T. cruzi-specific effector CD8+ T-cells into the infected heart tissue. The unveiling of the process driving cell migration and colonization of infected tissues by pathogen-specific effector T-cells is a crucial requirement to the development of vaccine strategies

Lipidomic profile as a noninvasive tool to predict endometrial receptivity

For the present study we asked whether the endometrial fluid lipidomic may be a useful approach to predict endometrial receptivity in freeze-all cycles. For this case-control study, endometrial fluid samples were collected from 41 patients undergoing freeze-all cycles. Samples were split depending on the pregnancy outcome: positive group (n = 24) and negative group (n = 17). Data were acquired by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were applied. A list of potential biomarker ion ratios was obtained and the values were used to build a receiver operating characteristic (ROC) curve to predict pregnancy success. The lipid categories were attributed by LIPID MAPS database. Ion ratios were established according to their correlations and used for the analysis. The PCA showed a tendency of separation between the studied groups, whereas the PLS-DA was able to clearly distinguish them. Fifteen ratios (13 hyper-represented in the negative and two hyper-represented in the positive group) were selected according to their importance for model prediction. These ratios were used to build the ROC curve, which presented an area under curve of 84.0% (95%CI: 69.2-97.4%; p = 0.009). These findings suggest that lipidomic profiling of endometrial fluid may be a valuable tool for identifying the time interval comprising the window of implantation.86214515