New approaches to improve the cord blood unit hematopoietic progenitor cell content

Cord blood is a well-established alternative to bone marrow and peripheral blood stem cell transplantation. To this day, over 400 000 unrelated donor cord blood units have been stored in cord blood banks worldwide. To enable successful cord blood transplantation, recent efforts have been focused on finding ways to increase the hematopoietic progenitor cell content of cord blood units. In this study, factors that may improve the selection and quality of cord blood collections for banking were identified. In 167 consecutive cord blood units collected from healthy full-term neonates and processed at a national cord blood bank, mean platelet volume (MPV) correlated with the numbers of cord blood unit hematopoietic progenitors (CD34+ cells and colony-forming units); this is a novel finding. Mean platelet volume can be thought to represent general hematopoietic activity, as newly formed platelets have been reported to be large. Stress during delivery is hypothesized to lead to the mobilization of hematopoietic progenitor cells through cytokine stimulation. Accordingly, low-normal umbilical arterial pH, thought to be associated with perinatal stress, correlated with high cord blood unit CD34+ cell and colony-forming unit numbers. The associations were closer in vaginal deliveries than in Cesarean sections. Vaginal delivery entails specific physiological changes, which may also affect the hematopoietic system. Thus, different factors may predict cord blood hematopoietic progenitor cell numbers in the two modes of delivery. Theoretical models were created to enable the use of platelet characteristics (mean platelet volume) and perinatal factors (umbilical arterial pH and placental weight) in the selection of cord blood collections with high hematopoietic progenitor cell counts. These observations could thus be implemented as a part of the evaluation of cord blood collections for banking. The quality of cord blood units has been the focus of several recent studies. However, hemostasis activation during cord blood collection is scarcely evaluated in cord blood banks. In this study, hemostasis activation was assessed with prothrombin activation fragment 1+2 (F1+2), a direct indicator of thrombin generation, and platelet factor 4 (PF4), indicating platelet activation. Altogether three sample series were collected during the set-up of the cord blood bank as well as after changes in personnel and collection equipment. The activation decreased from the first to the subsequent series, which were collected with the bank fully in operation and following international standards, and was at a level similar to that previously reported for healthy neonates. As hemostasis activation may have unwanted effects on cord blood cell contents, it should be minimized. The assessment of hemostasis activation could be implemented as a part of process control in cord blood banks. Culture assays provide information about the hematopoietic potential of the cord blood unit. In processed cord blood units prior to freezing, megakaryocytic colony growth was evaluated in semisolid cultures with a novel scoring system. Three investigators analyzed the colony assays, and the scores were highly concordant. With such scoring systems, the growth potential of various cord blood cell lineages can be assessed. In addition, erythroid cells were observed in liquid cultures of cryostored and thawed, unseparated cord blood units without exogenous erythropoietin. This was hypothesized to be due to the erythropoietic effect of thrombopoietin, endogenous erythropoietin production, and diverse cell-cell interactions in the culture. This observation underscores the complex interactions of cytokines and supporting cells in the heterogeneous cell population of the thawed cord blood unit.

Direct bone marrow HSC transplantation enhances local engraftment at the expense of systemic engraftment in NSG mice

Direct bone marrow (BM) injection has been proposed as a strategy to bypass homing inefficiencies associated with intravenous (IV) hematopoietic stem cell (HSC) transplantation. Despite physical delivery into the BM cavity, many donor cells are rapidly redistributed by vascular perfusion, perhaps compromising efficacy. Anchoring donor cells to 3-dimensional (3D) multicellular spheroids, formed from mesenchymal stem/stromal cells (MSC) might improve direct BM transplantation. To test this hypothesis, relevant combinations of human umbilical cord blood-derived CD34(+) cells and BM-derived MSC were transplanted into NOD/SCID gamma (NSG) mice using either IV or intrafemoral (IF) routes. IF transplantation resulted in higher human CD45(+) and CD34(+) cell engraftment within injected femurs relative to distal femurs regardless of cell combination, but did not improve overall CD45(+) engraftment at 8 weeks. Analysis within individual mice revealed that despite engraftment reaching near saturation within the injected femur, engraftment at distal hematopoietic sites including peripheral blood, spleen and non-injected femur, could be poor. Our data suggest that the retention of human HSC within the BM following direct BM injection enhances local chimerism at the expense of systemic chimerism in this xenogeneic model.

Cyclin dependent protein kinases as drug targets – potential in cardiovascular diseases

Complications of atherosclerosis such as myocardial infarction and stroke are the primary cause of death in Western societies. The development of atherosclerotic lesions is a complex process, including endothelial cell dysfunction, inflammation, extracellular matrix alteration and vascular smooth muscle cell (VSMC) proliferation and migration. Various cell cycle regulatory proteins control VSMC proliferation. Protein kinases called cyclin dependent kinases (CDKs) play a major role in regulation of cell cycle progression. At specific phases of the cell cycle, CDKs pair with cyclins to become catalytically active and phosphorylate numerous substrates contributing to cell cycle progression. CDKs are also regulated by cyclin dependent kinase inhibitors, activating and inhibitory phosphorylation, proteolysis and transcription factors. This tight regulation of cell cycle is essential; thus its deregulation is connected to the development of cancer and other proliferative disorders such as atherosclerosis and restenosis as well as neurodegenerative diseases. Proteins of the cell cycle provide potential and attractive targets for drug development. Consequently, various low molecular weight CDK inhibitors have been identified and are in clinical development. Tylophorine is a phenanthroindolizidine alkaloid, which has been shown to inhibit the growth of several human cancer cell lines. It was used in Ayurvedic medicine to treat inflammatory disorders. The aim of this study was to investigate the effect of tylophorine on human umbilical vein smooth muscle cell (HUVSMC) proliferation, cell cycle progression and the expression of various cell cycle regulatory proteins in order to confirm the findings made with tylophorine in rat cells. We used several methods to determine our hypothesis, including cell proliferation assay, western blot and flow cytometric cell cycle distribution analysis. We demonstrated by cell proliferation assay that tylophorine inhibits HUVSMC proliferation dose-dependently with an IC50 value of 164 nM ± 50. Western blot analysis was used to determine the effect of tylophorine on expression of cell cycle regulatory proteins. Tylophorine downregulates cyclin D1 and p21 expression levels. The results of tylophorine’s effect on phosphorylation sites of p53 were not consistent. More sensitive methods are required in order to completely determine this effect. We used flow cytometric cell cycle analysis to investigate whether tylophorine interferes with cell cycle progression and arrests cells in a specific cell cycle phase. Tylophorine was shown to induce the accumulation of asynchronized HUVSMCs in S phase. Tylophorine has a significant effect on cell cycle, but its role as cell cycle regulator in treatment of vascular proliferative diseases and cancer requires more experiments in vitro and in vivo.

Mother-Infant Antibodies to Pneumococcal Polysaccharides and Proteins : Transfer and Persistence of Maternal Antibodies and Development of Vaccine-Induced and Naturally Acquired Antibodies in Infants

Symptomless nasopharyngeal carriage of Streptococcus pneumoniae (pneumococcus) is very common in young children. Occasionally the carriage proceeds into mild mucosal diseases, such as sinusitis or acute otitis media, or into serious life-threatening diseases, such as pneumonia, sepsis or meningitis. Each year, up to one million children less than five years of age worldwide die of invasive pneumococcal diseases (IPD). Especially in the low-income countries IPD is a leading health problem in infants; 75% of all IPD cases occur before one year of age. This stresses the need of increased protection against pneumococcus in infancy. Anti-pneumococcal antibodies form an important component in the defence against pneumococcal infection. Maternal immunisation and early infant immunisation are two possible ways by which potentially protective antibody concentrations against pneumococci could be achieved in early infancy. The aim of this thesis is to increase the knowledge of antibody mediated protection against pneumococcal disease in infants and young children. We investigated the transfer of maternal anti-pneumococcal antibodies from Filipino mothers to their infants, the persistence of the transferred antibodies in the infants, the immunogenicity of the 23-valent pneumococcal polysaccharide vaccine (PPV) in infants and the response of the children to a second dose of PPV at three years of age. We also investigated the development of antibodies to pneumococcal protein antigens in relation to culture-confirmed pneumococcal carriage in infants. Serum samples were collected from the mothers, the umbilical cords and from the infants at young age as well as at three years of age. The samples were used to determine the antibody concentrations to pneumococcal serotypes 1, 5, 6B, 14, 18C and 19F, as well as to the pneumococcal proteins PspA, PsaA, Ply, PspC, PhtD, PhtDC and LytC by the enzyme immunoassay. The findings of the present study confirm previously obtained results and add to the global knowledge of responses to PPV in young children. Immunising pregnant women with PPV provides the infants with increased concentrations of pneumococcal polysaccharide antibodies. Of the six serotypes examined, serotypes 1 and 5 were immunogenic already in infants. At three years of age, the children responded well to the second dose of PPV suggesting that maternal and early infant immunisations might not induce hyporesponsiveness to polysaccharide antigens after subsequent immunisations. The anti-protein antibody findings provide useful information for the development of pneumococcal protein vaccines. All six proteins studied were immunogenic in infancy and the development of anti-protein antibodies started early in life in relation to pneumococcal carriage.

Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis

Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NF kappa B pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NF kappa B-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression.

Prudentia Iuris, 2007, n° 62-63 (número completo)

Editorial -- Las Segundas Jornadas sobre Ley Natural llevadas a cabo en la Facultad de Derecho de la Pontificia Universidad Católica Argentina -- Presentación y estudio preliminar / Gabriel Limodio -- Habermas / Eduardo Martín Quesada -- Iusnaturalismo vs. iuspositivismo (un alegato iusnaturalista) / Rodolfo L. Vigo -- Derecho Natural y Ciencia Jurídica / Carlos I. Massini Correas -- Ley natural y pluralismo cultural / Félix Adolfo Lamas -- La laicidad del derecho y la Encíclica “Deus caritas est” / Carlos Raúl Sanz -- Universalismo de derechos y asimilacionismo / Juan Cianciardo -- Ley natural y multiculturalismo: verdad y diálogo / Daniel Alejandro Herrera -- La falacia del Particularismo: sobre las condiciones de posibilidad de una Ética Global / Raúl Madrid Ramírez -- Homenaje a la Dra. María Josefa Méndez Costa. Encuentro Interuniversitario de Derecho de Familia. “Familia y Matrimonio hoy” -- Matrimonio y familia / Gabriel Limodio -- Palabras de la homenajeada / María Josefa Méndez Costa -- El concepto jurídico de familia. Matrimonio y familia / Catalina Elsa Arias de Ronchietto -- El principio jurídico de matrimonialidad y las políticas públicas. La familia: cordón umbilical de la humanidad / Catalina Elsa Arias de Ronchietto -- El concepto de familia, su juricidad y la querida profesora María Josefa Méndez Costa. Insinuaciones jurídicas de una tarde inolvidable / Úrsula Basset -- El consentimiento matrimonial sobre la necesidad de que sea prestado por un hombre y una mujer / Eduardo Sambrizzi -- Familia, matrimonio y la legislación sobre educación / Jorge Nicolás Lafferriere -- I Congreso de evangelización de la cultura -- Católicos y vida pública en América Latina / Guzmán M. Carriquiry Lecour -- Nota bibliográfica

Diário da Constituinte [gravação de vídeo] : [programa n. 176]

Sessão da Assembleia Nacional Constituinte, presidida pelo Deputado Ulysses Guimarães (PMDB-SP), abordou diversos temas: criação de blocos partidários, situação da segurança pública, a conservação da contribuição do SENAC, SESC, SESI, SENAI e o problema da nacionalização do solo brasileiro. A Deputada Raquel Cândido (PFL-RO) declara que o subsolo do Brasil é o sangue da Nação, por isso a Constituição deve definir o que é uma empresa nacional e estabelecer uma política minerária séria. O Deputado Ademir Andrade (PMDB-PA) relata que o atual Código de Mineração privilegia as grandes empresas mineradoras e multinacionais e defende a criação de uma política de concessões que democratize a exploração da riqueza mineral. Dois grupos de constituintes, chamados de Interpartidário e Grupo dos 32 (tritna e dois), trabalham juntos para buscar o consenso nos pontos mais polêmicos da Constituição. Eles irão elaborar um documento que será enviado ao Relator Bernardo Cabral (PMDB-AM). Em um dia de conversa foi possível chegar a um acordo em várias propostas : criação de um sistema único de saúde, desapropriação urbana paga com títulos do tesouro, criação de um conselho nacional de comunicações. Em relação à Reforma Agrária, o Deputado Euclides Scalco (PMDB-PR) informa sobre consenso em pontos como a função social da propriedade, a desapropriação pelo poder público, o uso de títulos da dívida agrária e a imissão de posse. O Senador Virgílio Távora (PDS-CE) declara que a redação precisa ainda de maleabilidade política para atender os interesses da reforma agrária e deixar preservado a defesa dos direitos primordiais da propriedade. O Presidente da Assembleia Nacional Constituinte (ANC), Deputado Ulysses Guimarães (PMDB-SP) quer o entendimento na elaboração da nova Carta e é para isso que irá dedicar todo o seu tempo.

Diário da Constituinte [gravação de vídeo] : [programa n. 502 ]

A constituinte começa a votar as mudanças no poder judiciário. Portadores do vírus HIV levam apelo ao Congresso. O presidente da Associação Brasileira Interdisciplinar da AIDS, Herbert de Souza, se reuniu com líderes de diversos partidos e apresentou um projeto de lei que puni com a prisão o doador que fizer doação de sangue sabendo que é portador do vírus HIV. Ele sugeriu também que atividades hemoterápicas sejam exercidas sem fins lucrativos, além de punição aos hospitais que se recusarem a atender pessoas soropositivas. A constituinte começou a votar todo o capítulo sobre o poder judiciário. A constituinte começou a votar o capítulo do Poder Judiciário. Existem algumas mudanças nesse capítulo como: o habeas data, mandado de injunção e o mandato de segurança coletivo. Alguns constituintes querem outras mudanças. Como o constituinte Paulo Pimentel que quer alterar o atual projeto de constituição no artigo em que se cria o Conselho Nacional de Justiça. Para ele esse conselho deveria ser suprimido. A sessão começou com a votação do substitutivo de Centrão sobre Poder Judiciário. O texto foi aprovado sem maiores discussões, graças a um acordo de lideranças. Foram 359 votos a favor e 10 contra. A primeira emenda a ser votada foi a do constituinte Oswaldo Trevisan que propõe a federalização da justiça. Mas a emenda acabou sendo rejeitada. Foi aprovada a emenda que propõe que o ingresso na carreira judiciária aconteça por meio do cargo de juiz substituto e de concurso público. Essa proposta foi feita pelos deputados Nelson Aguiar e Konder Reis. A sessão continuará ao longo da noite, sendo que quase todas as emendas que tratam do Poder Judiciário poderão ser votadas.

A Proteomic Study on Cell Cycle Progression of Endothelium Exposed to Tumor Conditioned Medium and the Possible Role of Cyclin D-1/E

This study was designed to comprehensively analyze the differential expression of proteins from human umbilical vein endothelial cells (HUVECs) exposed to tumor conditioned medium (TCM) and to identify the key regulator in the cell cycle progression. The HUVECs were exposed to TCM from breast carcinoma cell line MDA-MB-231, then their cell cycle distribution was measured by flow cytometer (FCM). The role of protein in cell cycle progression was detected via two-dimensional polyacrylamide gel electrophoresis (2-DE) and western blotting. Following the stimulation of TCM, HUVECs showed a more cells in the S phase than did the negative control group (ECGF-free medium with 20% FBS), but the HUVECs' level was similar to the positive control group (medium with 25 mug/ml ECGF and 20% FBS). Increased expression of cyclin D-1/E and some changes in other related proteins occurred after incubation with TCM. From our results, we can conclude that breast carcinoma cell line MDA-MB-231 may secrete soluble pro-angiogenic factors that induce the HUVEC angiogenic switch, during which the expression of cell cycle regulator cyclin D-1/E increases and related proteins play an important role in this process.

The Mitogenic and Anti-Apoptotic Activity of Tumor Conditioned Medium on Endothelium

This study was designed to observe the effect of tumor conditioned medium (TCM) on the proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to TCM from breast carcinoma cell line MDA-MB-231, then we measured their proliferation, apoptosis and cell cycle distribution by MTT and flow cytometery (FCM). Following the stimulation of TCM, HUVECs showed higher pro-mitogenic and anti-apoptotic ability than did the negative control group (ECGF-free medium with 20% FBS), but a similar ability to the positive control group (medium with ECGF and 20% FBS). From these results, we can conclude that breast carcinoma cell line MDA-MB-231 could secret soluble pro-angiogenic factors that induce HUVEC angiogenic switching, including cell cycle progression, proliferation and growth. The role and character of these factors remain to be further studied.

Diário da Constituinte [gravação de vídeo] : [programa n. 651]

No 1º Turno de votações os constituintes proibiram o comércio de sangue no pais, se valer o que já foi aprovado o Estado será o responsável pela coleta e doação de todo sangue no Brasil. Os constituintes explicam porque é importante controlar o sangue. Mostra um dos hemocentros de Brasília, onde todo o sangue é controlado de acordo com padrões internacionais. Já está na gráfica o parecer do Relator Bernardo Cabral sobre as 1844 emendas entregues no 2º Turno, que foi resultado de 10 dias de trabalhos intensos dos Relatores. Os artigos que mais receberam emendas foram os que tratam: da aposentadoria dos professores (40 propostas de alterações), estabilidade dos servidores públicos (27 propostas de alterações), inelegibilidade de parentes (22 propostas de alterações) e a questão da desapropriação de terras produtivas (19 propostas de alterações)

Diário da Constituinte [gravação de vídeo] : [programa n. 750]

Trata da questão da comercialização e doação de sangue e de órgãos. Uma Lei Complementar será criada para que a doação seja de livre e espontânea vontade.

Manifestação solicitada pelo TCU à CONOF/CD acerca de relatório preliminar, relativo à consulta sobre a aplicação da Lei Complementar nº 141, de 2012, formulada pela Comissão de Seguridade Social e Família/CD : TC 046.061/2012‐6

Avalia o relatório elaborado pelo TCU acerca da consulta formulada pela CSSF/CD(TC 046.061/2012‐6). Procura a pacificação de entendimento no âmbito do Congresso Nacional.

Diário da Constituinte [gravação de vídeo] : [programa n. 714]

A Constituinte proibiu o comércio de sangue e aprovou o novo Sistema único de Saúde. Foi votado o titulo das Disposições Transitórias. Foram decididos artigos e emendas tais como: o confisco de bens resultantes de crimes ilícitos, o destino da ilha de Fenando de Noronha, tabelamento dos juros, validade dos decretos leis, anistia dos débitos com a Receita Federal, direito do povo de escolher o sistema de governo.

Diário da Constituinte [gravação de vídeo] : [programa n. 581]

Iminência de acordo de Líderes para votação de direitos dos índios. Consenso acerca da posse de terras imemoriais, da demarcação de terras, bem como da dependência de autorização expressa do Congresso Nacional para realização de atividades de mineração em área indígena. Divergência quanto ao destino dos índios aculturados. Destinação de 18% de recursos orçamentários da União, 25% de recursos de Estados e Municípios para financiamento do ensino público no Brasil. Transplante de órgãos e comercialização de sangue e derivados serão objetos de legislação ordinária, conforme decidido pela Assembleia Nacional Constituinte. Avanços no reconhecimento oficial da união estável e da mãe solteira como entidades familiares, da igualdade entre homem e mulher no casamento e da inclusão dos direitos da criança no capítulo sobre Família na Constituição da Nova República.