Phenotypic Markers and Functional Regulators of Myelomonocytic Cells

In this chapter, there is a description of hematopoietic stem cells, maturation curve and their differentiation into myeloid cells, including phenotypes and transcription factors involved in this process. Further, we discuss myeloid maturation curve from myeloid precursor, monoblast, premonocyte to monocytes, and also monocytes subsets regarding their CD14 and CD16 expressions and related functions in health and disease. In addition, we reason about the differentiation from monocytes either in dendritic cells or in macrophages in vitro using differential growth factors; these cells are differentiated from those found in vivo being named as monocyte-derived cells. Furthermore, we explore distinguished phenotype of monocytes, macrophages, and dendritic cells monocyte-derived in vitro, using confocal microscopy and flow cytometry, in order to display morphological and phenotypic differences among them

Vascular endothelial growth factor-A enhances indoleamine 2,3-dioxygenase expression by dendritic cells and subsequently impacts lymphocyte proliferation

Dendritic cells (DCs) are antigen (Ag)-presenting cells that activate and stimulate effective immune responses by T cells, but can also act as negative regulators of these responses and thus play important roles in immune regulation. Pro-angiogenic vascular endothelial growth factor (VEGF) has been shown to cause defective DC differentiation and maturation. Previous studies have demonstrated that the addition of VEGF to DC cultures renders these cells weak stimulators of Ag-specific T cells due to the inhibitory effects mediated by VEGF receptor 1 (VEGFR1) and/or VEGFR2 signalling. As the enzyme indoleamine 2,3-dioxygenase (IDO) is recognised as an important negative regulator of immune responses, this study aimed to investigate whether VEGF affects the expression of IDO by DCs and whether VEGF-matured DCs acquire a suppressor phenotype. Our results are the first to demonstrate that VEGF increases the expression and activity of IDO in DCs, which has a suppressive effect on Ag-specific and mitogen-stimulated lymphocyte proliferation. These mechanisms have broad implications for the study of immunological responses and tolerance under conditions as diverse as cancer, graft rejection and autoimmunity.SBIBHIAEInst Israelita Ensino & Pesquisa Albert Einstein, Ctr Pesquisa Expt, São Paulo, BrazilHosp Israelita Albert Einstein, Inst Cerebro, Ctr Pesquisa Expt, São Paulo, BrazilUniv São Paulo, Fac Med, Dept Pediat, São Paulo, BrazilWeb of Scienc

Lymphoid Hematopoiesis and Lymphocytes Differentiation and Maturation

Lymphocytes belong to the lymphoid lineage and are considered as divergent from other blood cells lineages as those from the myeloid or erythroid lineage. Lymphoid hematopoiesis is not trivial, because although lymphocytes are found in the bloodstream and their precursor originates in the bone marrow, they mainly belong to the separate lymphatic system, which interacts with the blood circulation. We will discuss B cell differentiation in the bone marrow and the later stages of maturation in secondary lymphoid tissues, besides the B cell profiles in interfollicular, perifollicular, and follicular areas. In addition, we will also discuss T-cell precursor and natural killer cells derivation in the marrow. Furthermore, we will also discuss T-cell precursor migration to thymus, differentiation, rearrangement, thymic selection, involved transcription factors, and, finally, T-cell profiles and subsets in secondary lymphoid organs. We will provide flow cytometry plots showing strategies to identify and characterize NK, T and B lymphocytes and their subsets in circulation. Furthermore, we will provide illustrations to help the reader to understand and visualize the information provide over the chapter. Furthermore, the comprehension about lymphocytes and their contribution to the immune response will favor their application in developmental hematology and immunology. These topics are very important for the continuous development of knowledge

Common molecular pathways involved in human CD133+/CD34+ progenitor cell expansion and cancer

<p>Abstract</p> <p>Background</p> <p>Uncovering the molecular mechanism underlying expansion of hematopoietic stem and progenitor cells is critical to extend current therapeutic applications and to understand how its deregulation relates to leukemia. The characterization of genes commonly relevant to stem/progenitor cell expansion and tumor development should facilitate the identification of novel therapeutic targets in cancer.</p> <p>Methods</p> <p>CD34+/CD133+ progenitor cells were purified from human umbilical cord blood and expanded <it>in vitro</it>. Correlated molecular changes were analyzed by gene expression profiling using microarrays covering up to 55,000 transcripts. Genes regulated during progenitor cell expansion were identified and functionally classified. Aberrant expression of such genes in cancer was indicated by <it>in silico </it>SAGE. Differential expression of selected genes was assessed by real-time PCR in hematopoietic cells from chronic myeloid leukemia patients and healthy individuals.</p> <p>Results</p> <p>Several genes and signaling pathways not previously associated with <it>ex vivo </it>expansion of CD133+/CD34+ cells were identified, most of which associated with cancer. Regulation of MEK/ERK and Hedgehog signaling genes in addition to numerous proto-oncogenes was detected during conditions of enhanced progenitor cell expansion. Quantitative real-time PCR analysis confirmed down-regulation of several newly described cancer-associated genes in CD133+/CD34+ cells, including <it>DOCK4 </it>and <it>SPARCL1 </it>tumor suppressors, and parallel results were verified when comparing their expression in cells from chronic myeloid leukemia patients</p> <p>Conclusion</p> <p>Our findings reveal potential molecular targets for oncogenic transformation in CD133+/CD34+ cells and strengthen the link between deregulation of stem/progenitor cell expansion and the malignant process.</p

Platelet-derived exosomes induce endothelial cell apoptosis through peroxynitrite generation: experimental evidence for a novel mechanism of septic vascular dysfunction

Abstract\ud \ud \ud \ud Introduction\ud \ud Several studies link hematological dysfunction to severity of sepsis. Previously we showed that platelet-derived microparticles from septic patients induce vascular cell apoptosis through the NADPH oxidase-dependent release of superoxide. We sought to further characterize the microparticle-dependent vascular injury pathway.\ud \ud \ud \ud Methods\ud \ud During septic shock there is increased generation of thrombin, TNF-α and nitric oxide (NO). Human platelets were exposed for 1 hour to the NO donor diethylamine-NONOate (0.5 μM), lipopolysaccharide (LPS; 100 ng/ml), TNF-α (40 ng/ml), or thrombin (5 IU/ml). Microparticles were recovered through filtration and ultracentrifugation and analyzed by electron microscopy, flow cytometry or Western blotting for protein identification. Redox activity was characterized by lucigenin (5 μM) or coelenterazine (5 μM) luminescence and by 4,5-diaminofluorescein (10 mM) and 2',7'-dichlorofluorescein (10 mM) fluorescence. Endothelial cell apoptosis was detected by phosphatidylserine exposure and by measurement of caspase-3 activity with an enzyme-linked immunoassay.\ud \ud \ud \ud Results\ud \ud Size, morphology, high exposure of the tetraspanins CD9, CD63, and CD81, together with low phosphatidylserine, showed that platelets exposed to NONOate and LPS, but not to TNF-α or thrombin, generate microparticles similar to those recovered from septic patients, and characterize them as exosomes. Luminescence and fluorescence studies, and the use of specific inhibitors, revealed concomitant superoxide and NO generation. Western blots showed the presence of NO synthase II (but not isoforms I or III) and of the NADPH oxidase subunits p22phox, protein disulfide isomerase and Nox. Endothelial cells exposed to the exosomes underwent apoptosis and caspase-3 activation, which were inhibited by NO synthase inhibitors or by a superoxide dismutase mimetic and totally blocked by urate (1 mM), suggesting a role for the peroxynitrite radical. None of these redox properties and proapoptotic effects was evident in microparticles recovered from platelets exposed to thrombin or TNF-α.\ud \ud \ud \ud Conclusion\ud \ud We showed that, in sepsis, NO and bacterial elements are responsible for type-specific platelet-derived exosome generation. Those exosomes have an active role in vascular signaling as redox-active particles that can induce endothelial cell caspase-3 activation and apoptosis by generating superoxide, NO and peroxynitrite. Thus, exosomes must be considered for further developments in understanding and treating vascular dysfunction in sepsis.LRL and MJ have research grants from Fundação de Amparo a Pesquisa do Estado de São Paulo – FAPESP. MJ received a research grant from Sociedade Beneficente Israelita-Brasileira Hospital Albert Einstein.LRL and MJ have research grants from Fundação de Amparo a Pesquisa do Estado de São Paulo – FAPESP. MJ received a research grant from Sociedade Beneficente IsraelitaBrasileira Hospital Albert Einstein

Identification of Novel Immunoregulatory Molecules in Human Thymic Regulatory CD4+CD25+ T Cells by Phage Display

Thymic CD4+CD25+ cells play an important role in immune regulation and are continuously developed in the thymus as an independent lineage. How these cells are generated, what are their multiple pathways of suppressive activity and which are their specific markers are questions that remain unanswered. To identify molecules involved in the function and development of human CD4+CD25+ T regulatory cells we targeted thymic CD4+CD25+ cells by peptide phage display. A phage library containing random peptides was screened ex vivo for binding to human thymic CD4+CD25+ T cells. After four rounds of selection on CD4+CD25+ enriched populations of thymocytes, we sequenced several phage displayed peptides and selected one with identity to the Vitamin D Receptor (VDR). We confirmed the binding of the VDR phage to active Vitamin D in vitro, as well as the higher expression of VDR in CD4+CD25+ cells. We suggest that differential expression of VDR on natural Tregs may be related to the relevance of Vitamin D in function and ontogeny of these cells

Predictors of high-quality cord blood units

BACKGROUNDAnalysis of umbilical cord blood (UCB) transplants shows a correlation between engraftment and total number of infused cells. Thus, it is worth evaluating what maternal and neonatal characteristics and collection techniques may affect the quality of UCB units. STUDY DESIGN AND METHODSA cross-sectional study was performed with 7897 donors sequentially selected in three health care institutions in Brazil from October 2004 to March 2012, in which both quantitative and qualitative approaches were applied. All donors were considered suitable for cord blood collection. RESULTSThe maternal and neonatal characteristics and techniques of collection that influenced the total number of nucleated cells (TNCsp<0.001) were type of delivery, newborn weight and sex, and institution of UCB collection. The TNC count was associated with gestational age (p=0.008), type of delivery (p<0.001), newborn sex (p<0.001), newborn weight (p<0.001), and UCB collection technique (p=0.003). Center B presented the largest number of nucleated cells in its results (p<0.001), followed by Center A (p=0.001). Other characteristics, such as maternal age, were analyzed but were not relevant for the nucleated cell number. CONCLUSIONThis study provides elements for a model that allows an efficient selection of UCB donors, prioritizing candidates who have a better chance to lead to an optimized use of cord blood cells units.Univ Fed Sao Paulo UNIFESP, Escola Paulista Enfermagem, Sao Paulo, BrazilInst Nacl Cardiol, Rio De Janeiro, BrazilInst Israelita Ensino & Pesquisa Albert Einstein, Sao Paulo, BrazilHosp Israelita Albert Einstein, Dept Hemoterapia, Sao Paulo, BrazilUniv Fed Sao Paulo UNIFESP, Escola Paulista Enfermagem, Sao Paulo, BrazilWeb of Scienc

Gait speed, balance and functional capacity in a sample of community-dwelling older adults

Introduction: Falls in older people is an important public health concern since they are responsible for a high number of hospitalizations, health complications, disability, and death. Gait speed has been identified as a predictor of health state in elderly populations and it is related to falls and functional capacity. The aim of this study was to identify the risk of falling in a sample of Portuguese older adults living in the community and to investigate the associations between gait speed, balance, and functionality. Methods: This was a cross-sectional study. Assessment included gait speed (GS) with 4-meter walk test; balance with the Berg Balance Scale (BBS); functional capacity with the Composite Physical Function Scale (CPF). Descriptive and correlational statistics were performed to analyze data. Results: 46 community-dwelling older adults (32 women; 14 men) aged 77 ± 9 years participated in our study. Mean value for GS was 1.17 ± 0.37 m/s which is normal for this population. For BBS and CPF median was 52 and 19, respectively. BBS results revealed a risk of falling off 43% and functional capacity of our participants was at moderate levels. The study of correlations between variables also showed positive associations between GS and BBS (R = 0.631; p = 0.00) and between GS and CPF (R = 0.605; p = 0.00). Conclusions: Positive associations between GS and balance and between GS and functional capacity highlight the role of GS in the assessment of fall risk and functional capacity since it is a simple and easy test to perform.info:eu-repo/semantics/publishedVersio

Inflammation and circulating endothelial progenitor cells in patients with coronary artery disease and residual platelet reactivity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Albert Einstein HospitalUniversidade de São Paulo Faculdade de Medicina Hospital das ClínicasDuke University Medical Center Duke Clinical Research InstituteUniversidade Federal de São Paulo (UNIFESP), Escola Paulista de Medicina (EPM)UNIFESP, EPMFAPESP: 05/57710-3SciEL

Engineered chimeras reveal the structural basis of hexacoordination in globins: A case study of neuroglobin and myoglobin

Background Myoglobin (Mb) and neuroglobin (Ngb) are representative members of pentacoordinated and bis-histidyl, hexacoordinated globins. In spite of their low sequence identity, they show surprisingly similar three-dimensional folds. The ability of Ngb to form a hexacoordinated bis-histidyl complex with the distal HisE7 has a strong impact on ligand affinity. The factors governing such different behaviors have not been completely understood yet, even though they are extremely relevant to establish structure-function relationships within the globin superfamily.Fil: Boron, Carlos Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Capece, Luciana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pennacchietti, Francesca. Università di Parma; ItaliaFil: Wetzler, Diana Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Bruno, Stefano. Università di Parma; ItaliaFil: Abbruzzetti, Stefania. Università di Parma; ItaliaFil: Chisari, Lucía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Luque, F. Javier. Universidad de Barcelona; EspañaFil: Viappiani, Cristiano. Università di Parma; ItaliaFil: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; ArgentinaFil: Nadra, Alejandro Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin